Plasma For Bio-Decontamination, Medicine and Food Security

Plasma for Bio-Decontamination, Medicine
and Food Security

NATO Science for Peace and Security Series
This Series presents the results of scientific meetings supported under the NATO
Programme: Science for Peace and Security (SPS).
The NATO SPS Programme supports meetings in the following Key Priority areas:
(1) Defence Against Terrorism; (2) Countering other Threats to Security and (3) NATO,
Partner and Mediterranean Dialogue Country Priorities. The types of meeting supported
are generally Advanced Study Institutes and Advanced Research Workshops. The
NATO SPS Series collects together the results of these meetings. The meetings are
co-organized by scientists from NATO countries and scientists from NATOs Partner
or Mediterranean Dialogue countries. The observations and recommendations made
at the meetings, as well as the contents of the volumes in the Series, reflect those of
participants and contributors only; they should not necessarily be regarded as reflecting
NATO views or policy.
Advanced Study Institutes (ASI) are high-level tutorial courses to convey the latest
developments in a subject to an advanced-level audience
Advanced Research Workshops (ARW) are expert meetings where an intense but
informal exchange of views at the frontiers of a subject aims at identifying directions for
future action
Following a transformation of the programme in 2006 the Series has been re-named and
re-organised. Recent volumes on topics not related to security, which result from meet-
ings supported under the programme earlier, may be found in the NATO Science Series.
The Series is published by IOS Press, Amsterdam, and Springer, Dordrecht, in conjunction
with the NATO Emerging Security Challenges Division.
Sub-Series
A. Chemistry and Biology Springer

B. Physics and Biophysics Springer
C. Environmental Security Springer
D. Information and Communication Security IOS Press
E. Human and Societal Dynamics IOS Press
http://www.nato.int/science
http://www.springer.com
http://www.iospress.nl
Series A: Chemistry and Biology

Plasma for Bio-Decontamination,
Medicine and Food Security
edited by
Zdenko Machala
Comenius University
Bratislava, Slovakia
Karol Hensel
Comenius University
and
Yuri Akishev
SRC RF Triniti, Troitsk
Moscow Region, Russia
Published in Cooperation with NATO Emerging Security Challenges Division

Proceedings of the NATO Advanced Research Workshop on
Plasma for Bio-Decontamination, Medicine and Food Security
Demnovsk Dolina, Slovakia
1518 March 2011
Library of Congress Control Number: 2011945683
ISBN 978-94-007-2909-4 (PB)

ISBN 978-94-007-2851-6 (HB)
ISBN 978-94-007-2852-3 (e-book)
DOI 10.1007/978-94-007-2852-3
Published by Springer,
P.O. Box 17, 3300 AA Dordrecht, The Netherlands.
www.springer.com
Printed on acid-free paper
All Rights Reserved

Springer Science+Business Media B.V. 2012
No part of this work may be reproduced, stored in a retrieval system, or transmitted
in any form or by any means, electronic, mechanical, photocopying, microfilming,
recording or otherwise, without written permission from the Publisher, with the
exception of any material supplied specifically for the purpose of being entered and
executed on a computer system, for exclusive use by the purchaser of the work.
Preface
Plasmas, especially non-thermal plasmas maintained close to room temperature at

normal atmospheric pressure, have recently found many breakthrough applications
in biology, medicine, and security. Plasmas can efficiently kill bacteria, yeasts and
molds and other hazardous microorganisms, including potential bio-terrorism
agents, even spores and biofilms that are generally very difficult to inactivate by
traditional methods, which are in addition non-friendly for the environment. Cold
plasmas generated by electrical discharges can be employed for bio-decontamination
and sterilization of surfaces, medical instruments, water, air, food, even of living
tissues without causing their damage and other side effects, and represents a great
potential in medicine and defense against terrorism. The sterilizing effect of plasma
treatment can be attributed to several active agents, including the UV radiation,
electric field, charged particles, generated radicals and reactive species, providing in
total synergic mechanisms of bio-inactivation.
Direct or indirect plasma interaction with living cells of microorganisms or even
humans is a new quickly developing field issuing in many bio-medical in vivo appli-
cations, e.g. for the treatment of skin diseases and foot ulcer. Cold plasma can also
stop bleeding, making it effective in some surgical procedures and in treating intes-
tinal ulcers and persistent nosebleeds. Enhanced blood coagulation by plasma in
conjunction with its excellent aseptic properties, as well as proved success in wound
healing, open up new possibilities in military and defense applications. Plasma
treatment also allows cell manipulations, their removal and targeted transfer into the
injured area, which could also be used to accelerate wound healing. Plasma induced
apoptosis (programmed cell death) of melanoma or other tumor cells in vivo and
in vitro is being successfully tested, which brings forth a great potential for cancer
treatment. Besides, plasma enables painless treatment of dental caries or root canal
disinfection and other dental applications.
However, plasma induced biomedical processes are still mostly regarded as an
efficient black box. Deeper understanding in elementary mechanisms of plasma-
cell interaction, synergies of different mechanisms, as well as knowledge on the
microorganism resistance to plasma active agents due to the cell reparation, is nec-
essary to develop in order to efficiently apply plasma in biomedicine. There is no
v
vi Preface
doubt that multidisciplinary approach of plasma physicists, microbiologists,

medical doctors and engineers is required in this area.
NATO Advanced Research Workshop (ARW) Plasma for bio-decontamination,
medicine and food security held in beautiful Jasn mountain resort in Demnovsk
dolina, Slovakia, on 1518 March 2011 became a perfect place for such scientific
and social gathering. The workshop addressed various social, scientific and techni-
cal aspects of such complex and challenging problem as plasma in biomedicine
and other fields of human activity. It hosted 52 participants from 16 countries,
including the world-wide key players in the area of plasma decontamination and
medicine.
The scientific program of NATO ARW comprised 9 key lectures, 26 oral presen-
tations, 19 posters, and panel discussion, divided into several topical blocks: Bio-
decontamination, Biofilms, Food security and decontamination, Plasma interactions
with cells and DNA, Wound healing and medical applications, Electric fields and
plasma sources, UV irradiation and excilamps.
The workshop was very successful, inspiring and stimulating for opening new
horizons for science as well as for pushing the novel scientific results into revolu-
tionary applications in environmental protection, food security and medicine, even-
tually resulting in everyday engineering and clinical practices. The only shadow of the
event has been recently cast by the shocking news of a sudden death of one of our
participants and author of one article in this book, Mykola Guivan. We are so sorry
to have lost an expert, a colleague, and a friend.
This book is a compilation of selected reviewed manuscripts issuing from the
presentations at the NATO ARW Plasma for bio-decontamination, medicine and
food security. All contributions passed through a tough peer-review process. The
text is categorized to six major topics, although many articles hit more than one
topic:
1. Plasma bio-decontamination, water chemistry and effects on cells
2. Plasma biofilm inactivation and dentistry applications
3. Plasma-based UV sterilization
4. Plasma tissue treatment and wound healing
5. Plasma and electric fields in medicine
6. Plasma for food security
This volume, in addition to well-known textbooks such as Plasma Chemistry
(Fridman, Cambridge University Press, 2008) or the preceding NATO book Plasma
Assisted Decontamination of Biological and Chemical Agents (Geri and Fridman,
Springer, 2008) has own value because it provides a complimentary and compre-
hensive overview of current research activities in bio-decontamination, medicine
and food security assisted by plasma.
At the final panel discussion, the workshop participants concluded that plasma
physicists, chemists, biologists, medical doctors and engineers have to learn each
others languages to foster their tight co-operation and offer their achievements to
the industry and higher authorities. Only combination of deep fundamental researches
in plasmas and microbiology with clinical tests can lead to success. An emphasis
Preface vii
must be given to the implementation of plasma applications in food technology and

clinical practices. In a spirit of the workshop and in the terminology of microbiolo-
gists we wrapped up: regardless to our different backgrounds we should act like
different members of the biofilm (a very resistant microbial structure where vari-
ous microorganisms mutually help each other to survive and to develop).
Last but not least, the editors would like to appreciate the contributing scientists,
researchers and students who traveled to Slovakia from around the world and made
this workshop scientifically solid and socially warm. We would also like to recog-
nize our colleagues and students from the Faculty of Mathematics, Physics and
Informatics, Comenius University in Bratislava who contributed to the smooth
organization of the event and provided the technical and IT support, especially
Mrio Janda. Our sincere gratitude goes to all peer reviewers of the manuscripts
submitted for this volume who spent a tremendous amount of their time and efforts
to ensure the highest possible quality of the contributions, namely:
Pavel Baroch, Kurt Becker, Claudia Bender, Ronny Brandenburg, Graciella Brelles-
Mario, Valeriy Chernyak, Yves Creyghton, Danil Dobrynin, Svetlana Ermolaeva,
Irina Filatova, Alexander Fridman, Mykola Guivan, Jos Hueso Martos, Georg
Isbary, Mrio Janda, Chunqi Jiang, Kevin Keener, Juergen Kolb, Spencer Kuo, Jan-
Wilm Lackmann, Deanna Lacoste, Juergen Lademann, Peter Luk, Petr Luke,
Jerzy Mizeraczyk, Akira Mizuno, Emmanuel Odic, Joanna Pawat, Oleg Petrov,
Jozef Rhe, Eric Robert, Gilbert Shama, Libua ikurov, Joao Santos Sousa,
Victor Tarasenko, Ionut Topala, Vyacheslav Tsiolko, Victor Vasilets, Thomas von
Woedtke, Klaus-Dieter Weltmann, Qingsong Yu, and Weidong Zhu.
At last, we acknowledge NATO for its generous support of the NATO ARW on
Plasma bio-decontamination and for its support of this publication.
Zdenko Machala, Karol Hensel and Yuri Akishev, the editors.
Contents
Preface .............................................................................................................. v
List of Corresponding Authors ...................................................................... xv
Part I Plasma Bio-decontamination, Water Chemistry

and Effects on Cells
1 Atmospheric Pressure Plasmas for Decontamination

of Complex Medical Devices .................................................................. 3
Klaus-Dieter Weltmann, Jrn Winter, Martin Polak, Jrg Ehlbeck,
and Thomas von Woedtke
2 Characterization of Damage to Bacteria and Bio-macromolecules
Caused by (V)UV Radiation and Particles Generated
by a Microscale Atmospheric Pressure Plasma Jet ............................. 17
Jan-Wilm Lackmann, Simon Schneider, Franz Narberhaus,
Jan Benedikt, and Julia E. Bandow
3 Bio-decontamination of Water and Surfaces by DC
Discharges in Atmospheric Air .............................................................. 31
Zdenko Machala, Barbora Tarabov, Michal Pelach, Zuzana ipoldov,
Karol Hensel, Mrio Janda, and Libua ikurov
4 Biological Decontamination Using Pulsed Filamentary
Microplasma Jet ...................................................................................... 45
Ramasamy Pothiraja, Jan-Wilm Lackmann, Gernot Keil,
Nikita Bibinov, and Peter Awakowicz
5 The Fungal Spores Survival Under
the Low-Temperature Plasma ................................................................ 57
Hana Soukov, V. Scholtz, J. Julk, and D. Savick
ix
x Contents
6 Plasma-Liquid Interactions: Chemistry

and Antimicrobial Effects ...................................................................... 67
Thomas von Woedtke, Katrin Oehmigen,
Ronny Brandenburg, Tom Hoder, Christian Wilke,
Marcel Hhnel, and Klaus-Dieter Weltmann
7 Damages of Biological Components in Bacteria
and Bacteriophages Exposed to Atmospheric
Non-thermal Plasma ............................................................................... 79
Akira Mizuno and Hachiro Yasuda
8 Investigations of Bacterial Inactivation and DNA Fragmentation
Induced by Flowing Humid Argon Post-discharge .............................. 93
Emmanuel Odic, S. Limam, M.J. Kirkpatrick, B. Dodet,
S. Salamitou, and M.S. DuBow
9 DNA Oxidation by Reactive Oxygen Species Produced
by Atmospheric Pressure Microplasmas .............................................. 107
Joao Santos Sousa, Pierre-Marie Girard, Evelyne Sage,
Jean-Luc Ravanat, and Vincent Puech
10 Optical Emission Spectroscopic Evaluation of Different
Microwave Plasma Discharges and Its Potential
Application for Sterilization Processes.................................................. 121
Jos L. Hueso, Vctor J. Rico, ngel Yanguas-Gil,
Jos Cotrino, and Agustn R. Gonzlez-Elipe
Part II Plasma Biofilm Inactivation and Dentistry Applications
11 Battling Bacterial Biofilms with Gas Discharge Plasma ..................... 135

Anna Zelaya, Kurt Vandervoort, and Graciela Brelles-Mario
12 Inactivation of Microorganisms in Model Biofilms
by an Atmospheric Pressure Pulsed Non-thermal Plasma.................. 149
Yuri Akishev, N. Trushkin, M. Grushin, A. Petryakov, V. Karalnik,
E. Kobzev, V. Kholodenko, V. Chugunov, G. Kireev, Yu. Rakitsky,
and I. Irkhina
13 Low Temperature Atmospheric Argon Plasma: Diagnostics
and Medical Applications ....................................................................... 163
Svetlana Ermolaeva, Oleg Petrov, Nailya Zigangirova,
Mikhail Vasiliev, Elena Sysolyatina, Sergei Antipov,
Maxim Alyapyshev, Natalia Kolkova, Andrei Mukhachev,
Boris Naroditsky, Tetsuji Shimizu, Anatoly Grigoriev,
Gregor Morfill, Vladimir Fortov, and Alexander Gintsburg
Contents xi
14 A Sub-microsecond Pulsed Plasma Jet for Endodontic

Biofilm Disinfection................................................................................. 179
Chunqi Jiang, Christoph Schaudinn, David E. Jaramillo,
Martin A. Gundersen, and J. William Costerton
15 Medical Plasma in Dentistry: A Future Therapy
for Peri-implantitis .................................................................................. 191
Ina Koban, Lukasz Jablonowski, Axel Kramer,
Klaus-Dieter Weltmann, and Thomas Kocher
16 Inactivation of Candida Strains in Planktonic and Biofilm
Forms Using a Direct Current, Atmospheric-Pressure
Cold Plasma Micro-Jet ........................................................................... 201
Wei-Dong Zhu, Peng Sun, Yi Sun, Shuang Yu, Haiyan Wu,
Wei Liu, Jue Zhang, and Jing Fang
17 Non-thermal Atmospheric Plasma Treatment for Deactivation
of Oral Bacteria and Improvement of Dental Composite
Restoration............................................................................................... 215
Qing Song Yu, H. Li, A.C. Ritts, B. Yang, M. Chen, L. Hong,
C. Xu, X. Yao, and Y. Wang
Part III Plasma-Based UV Sterilization
18 Features of the Sterilization by VUV/UV Irradiation

of Low-Pressure Discharge Plasma ....................................................... 231
Vyacheslav V. Tsiolko
19 Applications of Excilamps in Microbiological and Medical
Investigations ........................................................................................... 251
Victor F. Tarasenko, E.A. Sosnin, O.S. Zhdanova,
and E.P. Krasnozhenov
20 Xenon Iodide Exciplex Lamp as an Efficient Source
for the UV Surface Cleaning and Water Decontamination................. 265
Mykola Guivan, H. Motomura, and M. Jinno
Part IV Plasma Tissue Treatment and Wound Healing
21 Antisepsis of the Skin by Treatment with Tissue-Tolerable

Plasma (TTP): Risk Assessment and Perspectives............................... 281
Jrgen Lademann, Heike Richter, Alexa Patzelt, Martina C. Meinke,
Joachim W. Fluhr, Axel Kramer, Klaus-Dieter Weltmann,
and Olaf Lademann
xii Contents
22 Cold Microsecond Spark Discharge Plasma Production

of Active Species and Their Delivery into Tissue ................................. 293
Danil Dobrynin, Gregory Fridman, Gary Friedman,
and Alexander Fridman
23 Surface Dielectric Barrier Discharge Jet for Skin Disinfection .......... 301
Yves Creyghton, Rogier Meijer, Paul Verweij, Frank van der Zanden,
and Paul Leenders
24 Cold Atmospheric Plasma for Clinical Purposes: Promising
Results in Patients and Future Applications ........................................ 311
Georg Isbary
25 Tissue Tolerable Plasma and Polihexanide: Are Synergistic
Effects Possible to Promote Healing of Chronic wounds?
In Vivo and In Vitro Results .................................................................. 321
Claudia P. Bender, Nils-Olaf Hbner, Klaus-Dieter Weltmann,
Christian Scharf, and Axel Kramer
26 Helium Atmospheric Pressure Plasma Jet: Diagnostics
and Application for Burned Wounds Healing ...................................... 335
Ionut Topala and Andrei Nastuta
27 Non-equilibrium Air Plasma for Wound Bleeding Control ................ 347
Spencer P. Kuo, Cheng-Yen Chen, Chuan-Shun Lin,
and Shu-Hsing Chiang
Part V Plasma and Electric Fields in Medicine
28 Subcellular Biological Effects of Nanosecond Pulsed

Electric Fields .......................................................................................... 361
Juergen F. Kolb and Michael Stacey
29 First Achievements and Opportunities for Cancer Treatment
Using Non-thermal Plasma .................................................................... 381
Eric Robert, Marc Vandamme, Julien Sobilo, Vanessa Sarron,
Delphine Ries, Sbastien Dozias, Laura Brulle,
Stphanie Lerondel, Alain Le Pape, and Jean Michel Pouvesle
30 Nitric Oxide Plasma Sources for Bio-decontamination
and Plasma Therapy ............................................................................... 393
Victor N. Vasilets and Anatoly B. Shekhter
31 Generation of Focused Shock Waves in Water
for Biomedical Applications ................................................................... 403
Petr Luke, Pavel unka, Petr Hoffer, Vitaliy Stelmashuk,
Ji Bene, Pavla Poukov, Marie Zadinov, and Jan Zeman
Contents xiii
32 DBD Plasma Assisted Silver Functionalization

of Surgical Meshes .................................................................................. 417
Jozef Rhe, Hana Polkov, Eva Jonov, Markta Hudcov,
Miroslav Zahoran, and Petr Nasadil
Part VI Plasma for Food Security
33 Prospects for Treating Foods with Cold Atmospheric

Gas Plasmas ............................................................................................. 433
Gilbert Shama and Michael G. Kong
34 Decontamination of Bacillus subtilis Spores in a Sealed
Package Using a Non-thermal Plasma System ..................................... 445
Kevin M. Keener, J.L. Jensen, V.P. Valdramidis, E. Byrne,
J. Connolly, J.P. Mosnier, and P.J. Cullen
35 Impact of Atmospheric Plasma Generated by a DBD Device
on Quality-Related Attributes of Abate Fetel Pear Fruit ................ 457
Annachiara Berardinelli, Lucia Vannini, Luigi Ragni,
and M. Elisabetta Guerzoni
36 Fungicidal Effects of Plasma and Radio-Wave Pre-treatments
on Seeds of Grain Crops and Legumes ................................................. 469
Irina Filatova, Viktor Azharonok, Alexander Shik,
Alexandra Antoniuk, and Natalia Terletskaya
Subject Index ................................................................................................... 481

List of Corresponding Authors
Yuri Akishev Low Temperature Plasma Department, SRC RF TRINITI, Troitsk,

Moscow region, Russia
Julia E. Bandow Department of Microbial Biology, Ruhr-University Bochum,
Bochum, Germany
Claudia P. Bender Institute of Hygiene and Environmental Medicine, University
Medicine Greifswald, Greifswald, Germany
Annachiara Berardinelli Agricultural Economics and Engineering Department,
University of Bologna, Cesena, Italy
Graciela Brelles-Mario Biological Sciences Department, California State
Polytechnic University, Pomona, CA, USA
Yves Creyghton TNO Thin Film Technology, Eindhoven, The Netherlands
Danil Dobrynin Electrical and Computer Engineering Department, Drexel
University, Philadelphia, PA, USA
Svetlana Ermolaeva Gamaleya Institute of Epidemiology and Microbiology,
Moscow, Russia
Irina Filatova Laboratory of Physics of Plasma Accelerators, The State Scientific
Institution B.I. Stepanov Institute of Physics of The National Academy of Sciences
of Belarus, Minsk, Belarus
Mykola Guivan Department of Quantum Electronics, Uzhgorod National
University, Uzhgorod, Ukraine
Jos L. Hueso Instituto de Ciencia de Materiales de Sevilla, Avda Americo
Vespucio, Seville, Spain
Departamento de Qumica Inorgnica, CSIC-University of Sevilla, Seville, Spain
Georg Isbary Department of Dermatology, Allergology and Environmental
Medicine, Hospital Munich, Munich, Germany
xv
xvi List of Corresponding Authors
Chunqi Jiang Department of Electrical Engineering Electrophysics, Viterbi

School of Engineering, University of Southern California, Los Angeles, CA, USA
Kevin M. Keener Department of Food Science, Purdue University, West Lafayette,
IN, USA
Ina Koban Unit of Periodontology, Policlinics for Restorative Dentistry,
Periodontology and Endodontology, Ernst-Moritz-Anrdt University, Greifswald,
Germany
Juergen F. Kolb Leibniz Institute for Plasma Science and Technology e.V. (INP
Greifswald), Greifswald, Germany
Spencer P. Kuo Department of Electrical and Computer Engineering, Polytechnic
Institute of New York University, Brooklyn, NY, USA
Jrgen Lademann Department of Dermatology and Allergology, Charit
Universittsmedizin Berlin, Berlin, Germany
Petr Luke Institute of Plasma Physics, Academy of Sciences of the Czech
Republic, Prague, Czech Republic
Zdenko Machala Division of Environmental Physics, Faculty of Mathematics,
Physics and Informatics, Comenius University, Bratislava, Slovakia
Akira Mizuno Department of Environmental and Life Sciences, Toyohashi
University of Technology, Toyohashi, Japan
Emmanuel Odic E3S Department of Power and Energy Systems, SUPELEC,
Gif-sur-Yvette Cedex, France
Ramasamy Pothiraja Institute for Electrical Engineering and Plasma Technology,
Ruhr-Universitt Bochum, Bochum, Germany
Jozef Rhe Faculty of Science, Masaryk University, Brno, Czech Republic
Department of Experimental Physics, Comenius University, Bratislava, Slovakia
Eric Robert GREMI, CNRS-PolytechOrlans, Orleans Cedex 2, France
Gilbert Shama Department of Chemical Engineering, Loughborough University,
Loughborough, Leics, UK
Joao Santos Sousa Laboratoire de Physique des Gaz et des Plasmas (LPGP),
Centre National de la Recherche Scientifique (CNRS) and Universit Paris-Sud,
Orsay, France
Instituto de Plasmas e Fuso Nuclear Laboratrio Associado, Instituto Superior
Tcnico, Lisboa, Portugal
Hana Soukov Department of Computing and Control Engineering, Institute of
Chemical Technology in Prague, Praha, Czech Republic
List of Corresponding Authors xvii
Victor F. Tarasenko Laboratory of Optical Radiation, High Current Electronics

Institute, Tomsk, Russian Federation
Ionut Topala Plasma Physics Laboratory, Faculty of Physics, Alexandru Ioan
Cuza University of Iasi, Iasi, Romania
Vyacheslav V. Tsiolko Department of Gas Electronics, Institute of Physics NAS of
Ukraine, Kiev, Ukraine
Victor N. Vasilets Institute for Energy Problems of Chemical Physics, Russian
Academy of Sciences, Chernogolovka, Moscow region, Russia
Klaus-Dieter Weltmann Leibniz Institute for Plasma Science and Technology e.
V. (INP Greifswald), Greifswald, Germany
Thomas von Woedtke Leibniz Institute for Plasma Science and Technology e.
V. (INP Greifswald), Greifswald, Germany
Qing Song Yu Center for Surface Science and Plasma Technology, Department of
Mechanical and Aerospace Engineering, University of Missouri, Columbia, MO,
USA
Wei-Dong Zhu Department of Applied Science and Technology, Saint Peters
College, Jersey City, NJ, USA
Part I
Plasma Bio-Decontamination,
Water Chemistry and Effects on Cells
Chapter 1
Atmospheric Pressure Plasmas
for Decontamination of Complex
Medical Devices
Klaus-Dieter Weltmann, Jrn Winter, Martin Polak, Jrg Ehlbeck,

and Thomas von Woedtke
Abstract Atmospheric pressure plasma sources produce a multiplicity of different

antimicrobial agents and are applicable to even complicated geometries as well as
to heat sensitive materials. Thus, atmospheric pressure plasmas have a huge poten-
tial for the decontamination of even complex medical devices like central venous
catheters and endoscopes. In this paper we present practicable realizations of atmo-
spheric pressure plasma sources, namely plasma jet, dielectric barrier discharge and
microwave driven discharge that are able to penetrate fine lumen or are adaptable to
difficult geometries. Furthermore, the antimicrobial efficacy of these sources is
given for one example setup in each case.
1.1 Introduction
Recent improvements in medical science mostly go along with the enhancement or

the new development of diagnostic and therapeutic devices. In dependence on their
field of application the assembly of these devices can become quite complex, which
means that a multiplicity of different materials are used. Furthermore, sensitive
electronical or mechanical components can be implemented as well. Endoscopes or
central venous catheters are prominent examples for such complex medical devices.
K.-D. Weltmann (*) J. Winter M. Polak J. Ehlbeck T. von Woedtke

Leibniz Institute for Plasma Science and Technology e. V. (INP Greifswald),
Felix-Hausdorff-Str. 2, 17489 Greifswald, Germany
e-mail: weltmann@inp-greifswald.de
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 3
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_1, Springer Science+Business Media B.V. 2012
4 K.-D. Weltmann et al.
Typically, these devices are intended to be applied in contact with or even inside the
human body. Hence, stringent hygienic standards must be kept in order to avoid
device associated infections. Sterility, which means a state of being free from viable
microorganisms, is generally one of the key requirements in the preparation process
of a complex medical device. But also the sufficient decontamination from other
substances e.g. pyrogens must be considered. The most effective sterilization method
is still the use of hot steam with a temperature of 121C or 134C [1]. However, the
hot steam sterilization of catheters and endoscopes is restricted due to their imple-
mented heat sensitive materials. Furthermore, fever provoking bacterial endotoxins
(pyrogens) are not removed by this process.
Low temperature sterilization methods like the low temperature steam steriliza-
tion with formaldehyde (NTDF) and the use of microbicidal gases e.g. ethylene
oxide (ETO) or hydrogen peroxide are commercially applied to reprocess heat sen-
sitive devices nowadays. Despite the advantage of avoiding thermal damage of the
medical product these methods have different disadvantages. Most of the used
microbicidal gases are highly toxic and carcinogenic so that special requirements
are necessary to guarantee sterilization personnels safety. Furthermore, these gases
strongly penetrate the materials of the medical devices. This results in long outgas-
times until a sterilized device can be used again. Another disadvantage appears if
the low-temperature sterilization process uses alternating pressure techniques or
generally works in the low-pressure regime. In both cases the pressure sensitivity of
the medical device must be considered.
Because of difficult to predict irritation especially of polymeric materials as well
as very high safety requirements, gamma or electron-beam irradiation, respectively,
are also not practicable sterilization methods in several cases.
An alternative decontamination method for complex medical devices is the use
of atmospheric pressure plasmas [2], where a multiplicity of different antimicrobial
agents interacts with the biological contaminant. These agents are radicals and
chemical products e.g. atomic oxygen (O), hydroxyl (OH), reactive oxygen (ROS)
and reactive nitrogen species (RNS), high energy UV radiation, charged particles,
alternating electric fields, heat as well as physical and chemical etch processes.
Until now many investigations have been made to distinguish what plasma agent
exactly dominates the microbiological inactivation [35]. In fact, this is an impor-
tant but also sophisticated task since it depends sensitively on the plasma source and
the experimental conditions. However, most of the investigations show that the
combination of all plasma agents have much more effect compared to the efficacy
of one single component. But not only the pure antimicrobial effect makes atmo-
spheric pressure plasma interesting for the decontamination of complex medical
devices. Moreover, their ability to etch and degenerate dangerous bacterial endotox-
ins and their ability to penetrate small cavities opens up new fields of application in
the medical decontamination sector.
During the last decade great efforts have been made in inventing new plasma
sources and adapt them to the decontamination specific conditions. Three exam-
ples, on which this paper is focused on, are the atmospheric pressure plasma jet
(called APPJ according to [6]), the dielectric barrier discharge (DBD) and the
1 Atmospheric Pressure Plasmas for Decontamination of Complex Medical Devices 5
microwave driven discharge (MDD). In comparison with the APPJ and the DBD,
which are classified as non-thermal plasmas with moderate gas temperature (close
to room temperature) the MDD is indeed a low temperature but thermal plasma [7, 8].
Hence, its gas temperature is typically much higher than room temperature and can
exceed 5,000 K. However, using thermal plasmas in remote mode, where the
plasma itself does not reach the contaminated object, but the produced radiation,
radicals and chemical compounds, enables the treatment of even heat sensitive
devices.
Usually, for plasma-based techniques to inactivate and/or remove microorgan-
isms the term sterilization is used. However, a sterilization method has to meet
strict and well-defined requirements. Therefore, we prefer the use of the term
decontamination or bio-decontamination meaning a general inactivation or
removal of unwanted biological contaminants, especially microorganisms [9].
In this contribution, practicable realizations of atmospheric pressure plasma
sources for decontamination and their antimicrobial efficacy are presented. Since
catheters and endoscopes are prominent examples for the group of complex medical
devices only plasma sources that are basically adaptable to the specific device prop-
erties like geometry or heat sensitivity are considered here. The microbicidal efficacy
is determined either on commercially produced catheters or on polytetrafluoroethyl-
ene (PTFE) test tubes, which are adequate to the biopsy channels used in real
endoscopes.
1.2 Materials and Methods
In this study three different atmospheric pressure plasma sources are used. The
APPJ is a 27.12 MHz RF-driven source with a mean power of 20 W [8, 10, 11].
It consists of a nozzle made of ceramics with an inner diameter at the nozzle outlet
of about 7 mm. In the center of the nozzle a needle electrode is axial situated and
coupled with the RF-voltage; the grounded electrode is a ring shaped structure
directly at the outlet of the nozzle. Typical gas flow rates are in the range of up to
20 standard liters per minute (slm) of argon. In this configuration the filamentary
discharge is ignited inside the nozzle and the excited, diffuse shining gas leaves the
nozzle and can be used for surface treatment. These kinds of jets can be arranged in
different arrays. Here, the jets are mounted in a ring-like structure or a T-type nozzle
is used to treat medical devices e.g. catheters (Figs. 1.4 and 1.5).
To apply a dielectric barrier discharge (DBD) towards the inner surfaces of medi-
cal devices, two different setups were used. The first works with an inner electrode
as shown in Fig. 1.1. Here, the medical device is simulated by an alternate use of
PTFE tube (d = 2 mm) and metal tubes (d = 2 mm). This is a typical material combi-
nation e.g. to connect the biopsy channel with the control unit in endoscopes. To
ignite a discharge inside the tubes an inner electrode (d = 0.2 mm) is completely
covered with a dielectric (quartz glass, d = 1.2 mm) and introduced into the tubes.
The metal tubes work as grounded electrodes, the inner electrode is connected with
Fig. 1.1 Electrode arrangement to treat the inner surfaces of a combination of PTFE tubes and
metals with a DBD
Fig. 1.2 Modified PTFE tube with bifilar helix electrode configuration to ignite a DBD inside
the tube containing (1) powered electrode, (2) grounded electrode, (3) outer tube, (4) inner tube,
(5) discharge, (6) power supply, (7) electrical circuit for measuring the consumed power
up to 15 kV sinusoidal voltage with a frequency in the kHz range. The gas flow is
kept constant at 1 slm argon.
The second setup to generate a discharge inside a long flexible PTFE tube is
based on a bifilar helix electrode configuration as presented in Fig. 1.2.
The modified tube consists of an inner tube (4) and an outer tube (3) concentrically
aligned. Intermediate, a powered (1) and a grounded (2) electrode are arranged equidis-
tant twisted around the inner tube. The distance between the electrodes is in the range
of mm, the geometry of the electrodes is variable. The inner diameter of the modified
tubes is 2 mm whereas the complete wall thickness is about 1 mm. The gas flow is typi-
cally in the range of 12 slm argon with up to 400 standard cubic centimeters per
minute (sccm) nitrogen admixture and up to 50 sccm oxygen admixture. To ignite the
discharge an alternating voltage of some kHz with amplitude up to 11 kV is applied.
Furthermore, a microwave driven discharge was utilized to decontaminate the
inside and the outside of medical device test specimens. This device works at
2.45 GHz and the consumed power is up to 1.5 kW. Accordingly, the gas tempera-
ture is in the range of some thousand Kelvin with gas fluxes up to 20 slm of com-
pressed (dry) air. The distance between the microwave torch and the contaminated
test specimens is about 25 cm connected via a metal tube which cooled the plasma
activated gas (see Fig. 1.3). Hence, the gas temperature of the used exhaust gas is
about 150C. The metal tube is connected to a simple process chamber where the
contaminated test specimens, 1 m long PTFE tubes with inner diameter of 2 mm and
an outer diameter of 3 mm, are mounted. During the exhaust gas propagation
Fig. 1.3 Schematic

illustration of the microwave
driven discharge in
combination with the process
chamber to decontaminate
test specimen as substitutes
for medical devices
through the process chamber the gas temperature further reduces. So, bacterial
inactivation induced by hot gas treatment can be obviated. The process chamber
was kept closed for 30 min, afterwards the exhaust gas was pumped down.
1.3 Results and Discussion
1.3.1 Atmospheric Pressure Plasma Jet
APPJs are very easy to handle discharge setups. Since these plasma jets are really tiny
sources of some cm length it is possible to arrange them in a very tight manner. So there
is a potential treatment of the outer surface of many different medical devices with
varying diameters from some mm up to several cm. Depending on the diameter of the
medical device the amount of APPJs has to be adjusted to guarantee a homogeneous
treatment of the whole surface. In Fig. 1.4ad some feasible arrangements of these
plasma jets are displayed. E.g. it is possible to build a ring-like alignment with different
diameters to treat catheters as shown in Fig. 1.4d.
Another method to homogeneously treat the outer surface of catheters is the use of
special plasma guiding discharge heads like the T-type head shown in Fig. 1.5. The
advantage of this setup is the use of only one source. Nevertheless, a homogeneous
treatment of the outer surface of small diameter medical devices is possible. To test the
inactivation rates of the plasma jets with T-type discharge head, catheters were divided
into six sections (each 6 cm long). Each section was contaminated with suspension of
Staphylococcus aureus, whereas the last section was kept untreated as a reference sam-
ple. The results for a gas flow of 20 slm with and without 0.25% admixture of air are
shown in Fig. 1.6. After treatment with both gas mixtures some sections of the catheter
are free of viable micro organisms (indicated in blue as minimum value). However, the
Fig. 1.4 Different plasma jet arrays for homogeneous treatment of the outer surface of medical
devices [8]
Fig. 1.5 Application of T-type plasma jet head towards catheters as representation for medical
devices [8]
treatment with air admixture results in a higher amount of microorganism-free sections,

whereby the median is at the detection limit. Also the maximum values (indicated in red)
show a higher inactivation rate of S. aureus for admixing air.
In conclusion, APPJs are on the one hand easy to handle and therefore easy to
apply to medical devices. They are very tiny which enables complex arrangements
of these jets to treat complex medical devices. Furthermore, APPJs are not limited
towards special surface material and it is possible to treat even material combina-
tions or large cavities as shown in Fig. 1.7. Also the antibacterial property of these
plasma jets is proven for lots of different microorganisms. All these advantages
show the reasonable use of APPJs to decontaminate medical devices.
Fig. 1.6 Inactivation rates of vegetative S. aureus in logarithmic scale for different gas mixtures
and amount of treatment repetition
Fig. 1.7 Direct treatment of material combinations (PTFE and metal tube) and large cavities with
a plasma jet [11]
On the other hand these discharges typically generate small plasma plumes. To treat
large areas special arrangements or discharge heads have to be invented. Moreover,
APPJs need high gas flows in the range of some slm. Especially, the high gas flow and
therefore the high costs are the limitation for some industrial applications.
1.3.2 Dielectric Barrier Discharge
Dielectric barrier discharges are to a maximum size adaptable to even complicated geo-
metrical structures. In Fig. 1.8a DBD in argon at atmospheric pressure is generated
inside a thin tube with an outer diameter of 2 mm using a high voltage driven inner
electrode. The material of the tube alternates between metal and PTFE in accor-
dance to the setup displayed in Fig. 1.1, whereas the length of the metal and the
PTFE tube section is 5 cm, respectively. This demonstrates that plasma treatment
of even difficult material junctions in combination with complicated geometry is
technically possible.
Fig. 1.8 Ignition of a DBD inside a tube with an outer diameter of 2 mm and different materials
Fig. 1.9 DBD in pure argon inside a commercially produced endoscopy biopsy channel [12]
In Fig. 1.9 the ignition of a DBD in pure argon inside a commercially produced
endoscopy biopsy channel is shown. Therefore, a silica glass capillary tube with an
implemented high voltage connected metal wire electrode is inserted into the biopsy
channel. The metal wire fortification at the outside of the tube acts as grounded
electrode. After flushing the biopsy channel with argon and applying a high voltage
signal atmospheric pressure plasma is generated inside the tube. The advantage of
this configuration is that the still existing fortification of the endoscope can easily be
used as grounded electrode. Hence, only an effortless modification on the endo-
scope is necessary to ignite a DBD. Beside this advantage the mechanical insertion
of the high voltage driven electrode into the biopsy channel can potentially lead to
damages of the inner tube wall or to small scratches, which enhances the attachment
of bacteria and other contaminants.
To avoid this disadvantage another plasma generation concept must be applied.
One technical realization is the bifilar helix electrode configuration as it is displayed
in Fig. 1.10. Here, a PTFE tube with a wall thickness of 0.5 mm is helically sur-
rounded by a pair of isolated copper electrodes.
For plasma ignition the tube is permanently flushed by a mixture of 1.5 slm
argon and 20 sccm nitrogen and a sinusoidal voltage of 20 kVpp with a frequency
of 7 kHz is applied. For endoscope implementation such high voltage amplitude
Fig. 1.10 Technical realization of a bifilar helix discharge (a) PTFE tube with a helically arranged
pair of isolated copper electrodes (b) plasma generation at atmospheric pressure in the same tube,
gas mixture: 1.5 slm argon +20 sccm nitrogen, supply voltage and frequency: 20 kVpp and 7 kHz
5 carrier gas: 1.5 slm argon

exposure time: 10 min
surviving colony forming units
median (N=3)
4 minimum surviving CFU
log10 (CFU/sample)
maximum surviving CFU
2
detection limit
0
pure argon 20 sccm N2 10 sccm N2 0.5 slm air
+ 2 sccm O2
gas admixture
Fig. 1.11 Microbicidal efficacy of a DBD, generated by using a bifilar helix electrode configura-
tion. The surviving CFU per sample for the inactivation of B. atrophaeus spores and 0.3% BSA is
displayed for a gas flow rate of 1.5 slm pure argon and three different admixtures of nitrogen and
oxygen
might be critical for the sensitive electronics implemented into most endoscopes
(e.g. video-chip at the distal end). However, by changing the electrode shape and
reducing the isolation thickness comparable discharges with peak-to-peak voltages
below 4 kV can be generated.
The advantage of the bifilar electrode configuration is the simple plasma ignition
and the maintained tube flexibility, which is important for implementation as biopsy
channels in real endoscopes. Furthermore, different gas mixtures are selectable in
this setup.
This has an influence on the inactivation of bacteria as Fig. 1.11 indicates. The
inner tube walls (tube length: 30 cm) are contaminated with Bacillus atrophaeus
spores using a contamination method described in [13]. Since the contamination of

real biopsy channels always includes not only bacteria but also proteins, 0.3%
bovine serum albumin (BSA) is admixed to the contaminating spore suspension.
After 10 min of plasma treatment an initially sterile suspension has been pumped
through the tube for 20 min at a flow rate of 30 ml min1 in order to recover the
surviving bacterial spores. By means of proliferation assays the number of surviv-
ing spores is determined. For pure argon plasma using a gas flow rate of 1.5 slm a
median number of surviving colony forming units (CFU) of 2.4 log10 is obtained.
By admixing small amounts of nitrogen or nitrogen and oxygen the median num-
ber of CFU slightly decreases. For a high amount of nitrogen and oxygen (0.5 slm
air admixture) the number of surviving microorganisms significantly increases up
to 4.5 log10.
As discussed above, the advantage of this setup is that it requires no mechanical
insertion of tubes or wires into the biopsy channel to ignite plasma. However, to
apply this configuration inside a commercially manufactured endoscope, the bifilar
helix electrode design must be implemented. This might lead to higher production
costs on the one hand. But on the other hand it might simplify the conditioning of
complex medical devices.
In conclusion, DBDs generated at atmospheric pressure have a huge potential for
the decontamination of complex medical devices. In particular, this is due to their
good antimicrobial efficacy and their adaptability on even difficult geometries.
1.3.3 Microwave Driven Discharge
Compared to plasma jets and DBDs microwave driven discharges are mostly free
of electrodes. The temperature inside the discharge is typically in the range of
some thousand Kelvin and therefore not suitable to decontaminate heat sensitive
medical devices in direct contact (see Fig. 1.12). Here, the afterglow plasma the
exhaust gas or the plasma activated gas was used to inactivate B. atrophaeus
spores.
The test specimens were 1 m PTFE tubes with an inner diameter of 2 mm and an
outer diameter of 3 mm. The inner walls of the tubes were contaminated by rinsing
a B. atrophaeus spores solution with about 108 CFU/ml for 15 min through the
tube [13]. The outside of the tubes was spot contaminated with 100 ml suspension
of about 107 CFU/ml. The contaminated tubes were fixed in a 1.1 m long process
chamber with inner diameter of 5 cm. The process chamber was connected to the
discharge via a metal tube which cooled the plasma activated gas down to 150C.
Inside the process chamber the temperature of the gas is further reduced.
Measurements with a mass spectrometer (model: GSD 301 O1, Pfeiffer Vacuum,
Germany) on the outlet of the metal tube showed different reactive nitrogen and
oxygen species as active components.
Fig. 1.12 Photo of a microwave driven discharge used in this work
Fig. 1.13 Inactivation results of B. atrophaeus spores after 30 min exposure time of plasma acti-
vated gas generated by a microwave driven discharge
The inactivation results for the in- and outside of the contaminated tubes after
plasma activated gas treatment for 30 min exposure time are shown in Fig. 1.13.
Obviously, the plasma activated gas shows high inactivation rates of 4.5 log10 CFU
for the inner tube walls and of 4 log10 for the outer tube walls. These results were
achieved without any alternating pressure techniques. The plasma activated gas
reaches the inside of the tube per diffusion.
In conclusion, microwave driven discharges show huge capability for decon-
tamination of medical devices. Because of the electrodeless ignition and the use of
plasma activated gas they are capable to treat even complex devices. The plasma
activated gas can penetrate into small cavities and is therefore especially interest-
ing for medical devices with fine lumina e.g. endoscopes or catheters. The device
presented in this paper uses air as process gas which in fact is very cost effective.
This autarkic property of microwave driven discharges allows an easy implementation

into existing sterilization apparatus and processes.
1.4 Conclusion
Medical devices have a complex shape and are composed of heat sensitive compo-
nents. Thus, only a small choice of conventional sterilization processes like gas
sterilization can be applied to reprocess them. Since these methods work with dan-
gerous and health hazardous chemicals, special safety conditions and/or desorption
times have to be considered. Alternatively, atmospheric pressure plasma sources
can be used to decontaminate medical devices. In this paper we presented three dif-
ferent plasma sources, namely the plasma jet, the dielectric barrier discharge and the
microwave driven discharge. Each source has its advantages and disadvantages
depending on the field of application. Plasma jets are most capable for fine lumina
and cavities and show high inactivation rates for short exposure times. Since plasma
jets are very small sources, they can be arranged in lots of different configuration to
treat large areas in short times. DBDs are advantageous concerning their spatial
dimensions. They can be designed for nearly every configuration, as shown in this
paper with the bifilar helix electrode arrangement, and work with very small process
gas fluxes. DBDs can be used in direct or indirect mode and in combination with
their high inactivation rates for short exposure times DBDs are very versatile for
decontamination of complex medical devices. The microwave driven discharges
commonly work with high electrical power and therefore generate high gas tem-
peratures. This makes most of them improper for direct treatment of medical devices.
However, the produced plasma activated gas can be used in indirect mode. The
activated gas penetrates even small lumina within short times and shows high inac-
tivation rates. This makes microwave driven discharges especially interesting for
complete sterilization of complex medical devices.
In a modern view of microbiological safety of medical products, there are no
longer processes needed for final sterilization or decontamination of the finished
product but techniques which can be introduced into methods of production as well
as reprocessing to produce a device which is safe for the designated use. The main
advantage of atmospheric-pressure plasma-based decontamination techniques is the
possibility to adapt it to special product as well as process requirements. This is the
main chance to use plasma into the medical and pharmaceutical practice [9].
Acknowledgments The work was founded by the German Federal Ministry of Education and
Research (BMBF), project name: PLASMOSE Plasmagesttzte Oberflchenmodifizierung
mittels modularer selektiver Plasmaquelle, contract number 13N8666 and: ENDOPLAS
Inaktivierende Mikroplasmen zur Sterilisierung im Lumen von medizinischen Instrumenten, contract
number 13N9320. The authors thankfully acknowledge U. Schnabel and L. Kantz for microbiological
assistance, Dr. M. Stieber and Dr. R. Brandenburg for fruitful discussions.
References
1. Pfug IJ (1990) Microbiology and engineering of sterilization processes, 7th edn. Environmental
Sterilization Laboratory, Minniapolis
2. Ehlbeck J, Schnabel U, Polak M, Winter J, Von Woedtke Th, Brandenburg R, von dem Hagen
T, Weltmann K-D (2011) Low temperature atmospheric pressure plasma sources for microbial
decontamination. J Phys D Appl Phys 44:013002
3. Laroussi M (2005) Low-temperature plasma-based sterilization: overview and state of the art.
Plasma Processes Polym 2:391400
4. Gaunt LF, Beggs CB, Georghiou GE (2006) Bactericidal action of the reactive species pro-
duced by gas-discharge nonthermal plasma at atmospheric pressure: a review. IEEE Trans
Plasma Sci 34:12571269
5. Dobrynin D, Fridman G, Friedman G, Fridman A (2009) Physical and biological mechanisms
of direct plasma interaction with living tissue. New J Phys 11:115020
6. Schuetze A, Yeong JY, Babayan SE, Park J, Selwyn GS, Hicks RF (1998) The atmospheric-
pressure plasma jet: a review and comparison to other plasma sources. IEEE Trans Plasma Sci
26(6):16851694
7. Tendero C, Tixier C, Tristant P, Desmaison J, Leprince P (2006) Atmospheric pressure plasmas:
a review. Spectrochim Acta B 61:230
8. Ehlbeck J, Brandenburg R, von Woedtke T, Krohmann U, Stieber M, Weltmann K-D (2008)
PLASMOSE antimicrobial effects of modular atmospheric plasma sources. GMS Kranken-
haushyg Interdiszip 3(1):212
9. von Woedtke Th, Kramer A, Weltmann K-D (2008) Plasma sterilization: what are the condi-
tions to meet this claim? Plasma Processes Polym 5:534539
10. Brandenburg R, Ehlbeck J, Stieber M, von Woedtke Th, Zeymer J, Schlter O, Weltmann K-D
(2007) Antimicrobial treatment of heat sensitive materials by means of atmospheric pressure
rf-driven plasma jet. Contrib Plasma Phys 47:7279
11. Weltmann K-D, Brandenburg R, von Woedtke T, Ehlbeck J, Foest R, Stieber M, Kindel E
(2008) Antimicrobial treatment of heat sensitive products by miniaturized atmospheric pres-
sure plasma jets (APPJs). J Phys D Appl Phys 41:194008
12. Schnabel U, Maucher T, Khnlein J, Volkwein W, Niquet R, Trick I, Stieber M, Mller M,
Werner H-P, Ehlbeck J, Oehr C, Weltmann K-D (2011) Multicentre trials for decontamination
of fine-lumen PTFE tubes loaded with bacterial endospores by low and atmospheric pressure.
Plasma Processes Polym 8: DOI: 10.1002/ppap.201000206 (early view online)
13. Maucher T, Schnabel U, Volkwein W, Khnlein J, Winter J, Weltmann K-D, Trick I, Oehr C
(2011) Assembly of standardized test specimen for microbial quantification of plasma steril-
ization processes of fine PTFE tubes as used in thermo sensitive medical devices like flexible
endoscopes. Plasma Processes Polym 8:200207
Chapter 2
Characterization of Damage to Bacteria
and Bio-macromolecules Caused by (V)UV
Radiation and Particles Generated by
a Microscale Atmospheric Pressure Plasma Jet
Jan-Wilm Lackmann, Simon Schneider, Franz Narberhaus, Jan Benedikt,

and Julia E. Bandow
Abstract Atmospheric pressure plasma jets effectively inactivate bacteria on surfaces

including infected tissues. This is due to the combined effects of (V)UV radiation,
reactive oxygen and nitrogen species, ions, and high electric fields. A well-character-
ized microscale atmospheric pressure plasma jet (m-APPJ) operated with He/O2 gas
mixture has been modified so that (V)UV radiation and heavy reactive particles (mainly
O3 molecules and O atoms) emitted from the plasma source can be separated effec-
tively. The separation is achieved by an additional lateral He flow, which diverts the
heavy particles from the jet axis. The new jet geometry is called X-Jet. Separation of
different plasma components allows studying their effects on living cells and bio-
macromolecules separately. First, the effectiveness of the separation of different plasma
components was demonstrated by treatment of monolayers of vegetative Bacillus sub-
tilis cells. To characterize effects on nucleic acids, dried plasmid DNA and total cellular
RNA were treated with the separated plasma components. Dried bovine serum albumin
was used to study etching effects of (V)UV radiation and heavy particles on proteins.
We found that heavy particles emitted from the X-Jet kill vegetative cells more effec-
tively than the (V)UV radiation from this type of plasma source. All bio-macromolecules
investigated, DNA, RNA, and proteins, are affected by plasma treatment. DNA
exposed to the (V)UV-channel of the jet seems to be prone to thymine dimer formation
not only in vitro but also in vivo as indicated by induction of the photolyase in
Escherichia coli, while DNA strand breaks occur under both jet channels. Heavy
particles seem more effective in degrading RNA and in etching protein in vitro.
J.-W. Lackmann F. Narberhaus J.E. Bandow (*)

Microbial Biology, Department for Biology and Biotechnology,
Ruhr University Bochum, Universittsstr. 150, 44801 Bochum, Germany
e-mail: julia.bandow@rub.de
S. Schneider J. Benedikt
Coupled Plasma-Solid State Systems, Department for Physics and Astronomy,
Ruhr University Bochum,
Universittsstr. 150, 44801 Bochum, Germany
18 J.-W. Lackmann et al.
2.1 Introduction
Atmospheric pressure plasmas are known to be capable of inactivating bacteria. The

inactivation is, however, usually characterized with regards to its effectiveness,
while the exact inactivation mechanisms and the role of different reactive species
(oxygen radicals, metastables, UV photons, ions) and possible synergistic mecha-
nisms among them are not well understood. We use a radio frequency driven atmo-
spheric pressure plasma jet operated with He gas or He and a small O2 gas admixture
(<1%) to treat vegetative E. coli and B. subtilis cells as well as bio-macromolecules
that are essential for life to gain detailed knowledge about the plasma-cell interac-
tion. E. coli is a Gram-negative bacterium that is commonly found in the lower
intestine of warm-blooded organisms and B. subtilis is a Gram-positive model
organism typically living in the soil. The plasma is generated in a 30 mm long chan-
nel with a square cross section with an area of 1 1 mm.2 This channel is formed by
two electrodes and two glass plates. A He gas flow of 1.4 slm (standard liter per
minute) with admixture of 0.6% of O2 and an rf-voltage of around 230 VRMS are
used. This source is very well characterized by optical emission spectroscopy, mass
spectrometry, and two-photon absorption laser induced fluorescence (TALIF). More
details about this source and its diagnostics can be found elsewhere [1, 2]. This
plasma source very effectively produces reactive oxygen species (ROS). The dis-
sociation degree of O2 is up to 20% and the densities of O atoms or O3 molecules
exceed 1015 cm3. Additionally, plasma simulations of He/O2 plasma at atmospheric
pressure indicate the presence of singlet delta O2(a1Dg) metastables with similar
density [3]. Moreover, it is possible to suppress or enhance the flux of the selected
species in the effluent by proper selection of operation parameters. However, the
interference of reactive radical species and radiation complicates the evaluation of
data on inactivation efficiencies and mechanisms with respect to the dependence on
both effects. To study these dependencies separately, we have modified the nozzle
of the jet in order to separate all reactive species from plasma radiation. The geom-
etry of the modified jet, the effectiveness of this separation, and the treatment of
bacteria and bio-macromolecules are discussed in the following.
2.2 Separation of Plasma Radiation

and Heavy Reactive Particles
The concept of separation of plasma radiation and reactive particles is based on the
fact that convection dominates the transport of heavy particles. Typical diffusion
times through the channel are orders of magnitude longer than the average residence
time of particles within the jet. For that reason the gas flow emanating from the jet
axis can be diverted by a second lateral He gas flow as long as laminar flow condi-
tions are preserved. A structure of two gas channels crossing each other in a
45-degree angle has been constructed to maintain well-controlled conditions and to
2 Characterization of Damage to Bacteria and Bio-macromolecules Caused 19
Fig. 2.1 Geometry of the X-Jet (a) Photograph of an atmospheric pressure microplasma jet in He/
O2 gas mixture with an additional He flow crossing the plasma effluent. The additional helium flow
steers the flow of radical species into a side channel. (V)UV radiation propagates along the line of
sight with the plasma through the helium atmosphere and exits the jet through the direct channel.
(b) Fluid dynamics simulation of the flow in the X-Jet. Arrows correspond to gas velocity and the
grey shading represents the flow of reactive species (e.g. O atoms). This simulation illustrates the
separation of (V)UV and heavy reactive particles
prevent admixture of surrounding gas into the gas flow (Fig. 2.1a). The modified
microplasma jet is called X-Jet. A simulation of heavy particle density confirms that
the particles are diverted into the side channel as shown in Fig. 2.1b. It is a 2D
simulation of the flow of helium (no slip boundary condition at the wall, flow 1.4 slm
in both channels) and the concentration of some representative reactive particles
from the plasma. The reactive particles react in the gas phase with a time constant
of 0.12 ms (reaction time of O with 0.2% O2 in He), it is assumed that they have a
surface reaction probability equal to 1 (approximated by setting the concentration at
the wall to 0), and that they have a binary diffusion coefficient of D = 1 cm2 s1.
The velocity field (white arrows) and the color-coded concentration of reactive
particles (from blue to red) are shown. An absolutely calibrated emission spectrum
of the jet has been measured in the past down to the wavelength of 115 nm, the
cutoff limit of the MgF2 window [4]. Two atomic oxygen emission lines at 115
(1D1D0) and 130 nm (3P3S0) dominate the spectrum. Additionally, weak emission
due to parts of the Schumann-Runge bands of O2 and a weak H line at 120 nm
(2P2S0) have been observed. We also expect that the emission spectrum contains
the He*2 excimer continuum in the 58100 nm range and a strong atomic oxygen
line at 98 nm since these emission features have been observed by other authors in
atmospheric pressure plasmas with helium [5].
It was checked that less than 0.7% of radiation between 115 and 875 nm is detect-
able on the axis of the side channel. We expect the same for the VUV radiation below
115 nm. Some reactive or excited particles can diffuse from the plasma effluent into
the direct channel when the same gas flows are used in the direct and side channels.
However, these particles will not reach the center of the direct channel after the
crossing point due to the slow diffusion and will therefore not reach the treated
surface. The flow pattern close to the surface makes sure that only the particles close
to the axis of a gas channel reaches the surface as we have shown previously [6].
Hence radiation is effectively separated from radical species. Both plasma radiation
and heavy reactive particles (mainly O3 molecules or O radicals) can be now used
separately for the treatment. The effects of (V)UV radiation treatment, reactive oxy-
gen species treatment and combined treatment of both plasma components on E. coli
and B. subtilis cells can be studied with this device.
2.3 Impact of Plasma and Its Components

on Vegetative B. subtilis Cells
The performance of the X-Jet was tested by treating a B. subtilis layer. The substrates,
LB agar plates with B. subtilis monolayers, were prepared as follows. B. subtilis 168
was grown for 18 h overnight at 37C in liquid LB medium [7]. The cultures were
diluted to an optical density of 0.1 at 500 nm and were applied by a 1 s spray pulse
onto LB agar plates. Before plasma treatment, the plates were incubated for 2 h at
37C. After plasma treatment, the sample plates were incubated overnight for 18 h
at 37C to allow survivors to multiply. Plasma treatment of agar plates prior to the
application of cells had no effect on bacterial growth and the pH of the medium did
not change during plasma treatment.
The agar plates were treated in air as ambient atmosphere. The jet-to-substrate
distance was adjusted to 4 mm with the direct channel parallel to the surface nor-
mal (Fig. 2.2). First, the effect of the combined treatment was tested without an
additional He flow. A typical dose-effect relationship was observed. Extending
treatment time resulted in increasing inhibition zones. When the additional He flux
through the angular channel was activated, two separate inhibition zones appeared.
A small inhibition zone approximately 2 mm in diameter formed directly under the
direct channel due to exposure to (V)UV radiation (position B in Fig. 2.2), whereas
heavy reactive species were diverted from the jet axis and caused a larger second
inhibition zone (position C). No inhibition zone(s) were observed after a 1-min
plasma treatment with either the (V)UV or particle channel indicating higher
efficiency of the combined treatment. Similar effects were observed when treating
E. coli [6].
The inhibition zone under the direct channel was larger than the 2 mm diameter
of the irradiated area. Most probably, the ambient air diffuses into the effluent under
the direct channel. The (V)UV radiation dissociates O2 molecules from air and inac-
tivation occurs on larger than expected area.
The X-Jet (1.4 slm He with 0.6% O2 and URMS = 230 V) with additional He flow
(1.4 slm) through the side channel was used for all following studies in this article.
The jet was positioned as indicated in Fig. 2.2b for the treatment with plasma radiation
Fig. 2.2 Inhibition zones after treatment of B. subtilis without and with additional He flow. The
position of the sample relative to the X-Jet is indicated on the left side with the areas treated by the
direct and the side channels indicated with letters. Without an additional He flow (a) particles and
UV migrate through the direct channel onto the plate with B. subtilis at position A. When the He
flow is turned on (b) only (V)UV propagates through the direct channel at position B, whereas
particles are steered into the side channel and treat the plate at position C
only (we will call this (V)UV channel) and it was turned by 45 to make the side
channel perpendicular to the surface for the treatment with heavy reactive particles
only (we will call this particle channel). The jet-substrate distance was always 4 mm
and the gas flow from the channel, which was not used, was blocked in such a way
that it could not reach the treated surface.
2.4 Plasma Impact on Biological Macromolecules
In addition to treatment of living bacterial cells, the impact of plasma and its compo-
nents on bio-macromolecules is investigated. Like all organisms, bacterial cells consist
of a lot of different macromolecules that are essential for life. In bacteria the genetic
information is stored in the nucleoid in form of double stranded DNA. In case of E. coli
and B. subtilis, the genetic information is located on a single circular chromosome.
Genes are transcribed into single stranded RNAs, which are used as templates for pro-
tein synthesis in a process called translation. Proteins fulfill structural and enzymatic
functions and are therefore the main drivers of cellular processes. Disruption of this
complex machinery that produces enzymes based on genetic information unequivocally

limits growth and in most cases will even lead to cell death. By themselves, DNA-,
RNA-, or protein damage could each play the critical role in bacterial inactivation
through plasma or its components. Therefore, we study the influence of plasma on
individual bio-macromolecules, although in cells exposed to plasma all macro-
molecules can be expected to be subject to damage to some degree.
2.4.1 DNA Damage by Plasma
Plasma is capable of inducing single and double strand breaks in plasmid DNA in
liquid solution [8]. We demonstrate here that plasma also leads to the formation of
multimers in dried plasmid DNA and the formation of thymine dimers in DNA.
Aliquots of commercially available plasmid DNA (5 mg pUC18 plasmid DNA
from Fermentas, St. Leon-Rot, Germany) dried onto glass slides under vacuum
(spot size < 2 mm) were treated for 15 min either with the (V)UV channel or the
reactive particle channel of the plasma effluent. The treatment was performed under
a He atmosphere to exclude admixture of oxygen or nitrogen molecules from ambi-
ent air, which could result in absorption of the (V)UV radiation, generation of new
radicals in dissociation of ambient molecules, and destruction of plasma generated
radicals in reactions with ambient molecules. After the treatment, nucleotides were
removed from the glass slides by washing with DNase-free water and analyzed via
agarose gel electrophoresis [7] (Fig. 2.3). Aliquots dried on glass but not treated
with plasma served as control. pUC18 has a size of around 2.7 kilo base pairs (kb).
After gel electrophoresis, two distinct bands could be observed in the untreated
control, which is typical for plasmid DNA.
Circular DNA can either be in a supercoiled (lower band, around 1.8 kb) or
relaxed (upper band, around 3.4 kb) state. Plasmid DNA is isolated from bacterial
cells in the supercoiled state when there is no damage to its backbone structure at
any position. The relaxed state indicates a break in one of the two DNA strands. In
this state the plasmid is still intact. In the control sample, most of the plasmid DNA
is supercoiled as indicated by the higher band intensity of the 1.8 kb band. Only a
low amount of plasmid DNA is relaxed. These single-strand breaks most likely
occur during the drying process or when washing the DNA off the glass slides. After
treatment with the (V)UV component, the intensity of the supercoiled plasmid band
dropped visibly while a new band at a higher molecular weight of around 7 kb
appeared. According to the molecular weight standard, the new band most likely
contains plasmid dimers.
It is known that UV radiation can lead to multimer formation. UV radiation
induces DNA strand breaks and is also capable of cross-linking nucleotides from
different plasmids together [9]. Multimer formation could not be observed when
treating plasmid DNA with plasma in liquid solution [8].
Most likely, the plasmids have to be in direct contact with each other to facili-
tate multimer formation, which is only the case at very high molecule density.
Fig. 2.3 Plasmid DNA treated with the (V)UV or particle channel of the X-Jet. pUC18 plasmid
spotted on glass slides, treated with the (V)UV or particle channel of the X-Jet and analyzed via
agarose gel electrophoresis. The control was spotted on glass slides but not treated. The molecular
weight standard (10 kb DNA ladder, Fermentas, St. Leon-Rot, Germany) is commercially avail-
able. kb: kilo base pairs. Exposure times, brightness, and contrast were modified individually for
the different test conditions to optimize band visibility
After treatment with the particle channel, no dimer formation was observed.
However, a third band was detected ranging in size between the band of the super-
coiled and relaxed plasmid DNA. This band likely corresponds to linearized plas-
mid DNA indicating the introduction of double strand breaks into the DNA. It was
previously shown by OConnell et al. [8] that double strand breaks in plasmid DNA
correlate with the applied oxygen admixture in He plasma and, therefore, the amount
of emitted oxygen radicals. Our results corroborate this observation since we dem-
onstrate that plasma-generated reactive particles (including atomic O), without
additional UV radiation, are capable of inducing double strand breaks. Plasma treat-
ment is capable of modifying DNA by inducing DNA strand breaks as well as cross-
linking DNA. This highly artificial test system with high amounts of dried plasmid
DNA modified by plasma treatment suggests an industrial application for atmo-
spheric plasmas in inactivation of DNA contaminations on surfaces. Future experi-
ments will show to what extent DNA cross-linking and introduction of strand breaks
correlate with bacterial inactivation. They both could play a key role as they each
impair DNA replication and disrupt transcription.
To further investigate the impact of plasma on DNA, an 18-thymine oligomer
was treated with the different effluent channels and investigated by Fourier trans-
form infrared (FTIR) spectroscopy (Fig. 2.4a). Purified oligomers of 18 thymines
(dT18) were purchased (Thermo Scientific, Bonn, Germany) and dissolved in
A. dest. at a concentration of 0.5 mM. 20 ml aliquots were spotted on glass slides,
dried, and treated with the (V)UV or particle channel of the X-Jet in a He atmo-
sphere. Samples were measured by FTIR spectroscopy before and after treatment
and difference spectra were calculated.
Fig. 2.4 FTIR difference spectra of dT18 (a) and thymine dimer formation (b) difference absorption
spectra were calculated by subtracting the pre-plasma treatment absorption spectra from the post-
plasma treatment absorption spectra. Wave numbers for C=C and CC bonds were taken from [10]
Samples treated with the (V)UV channel for 5 min show an intensity loss around
a wave number of 1,600 cm1. Furthermore, an intensity increase is observed in the
CC fingerprint region of thymine at wave numbers less than 800 cm1. On the other
hand, samples treated for 5 min with the particle channel showed no difference in
intensities at wave numbers between 2,000 and 400 cm1. An intensity decrease at a
wave number around 1,600 cm1 indicates loss of C=C double bonds, while at the
same time, an intensity increase in the finger print region between 600 and 400 cm1
indicates the formation of CC single bonds. Both the decrease in C=C double
bonds and an increase of CC single bonds in (V)UV-treated dT18 samples, are in
accordance with thymine dimer formation (Fig. 2.4b), a well-known UV-induced
type of DNA damage. The C=C double bonds of two adjacent thymines are broken
and a cyclic butane ring consisting of four CC single bonds is formed, linking the
neighboring nucleotides covalently [11].
Thymine dimer formation is a type of DNA damage, which commonly occurs in
nature. It interrupts DNA replication and is, for instance, one of the reasons for sun-
burn and skin cancer [12]. Several bacterial cells feature highly specific DNA repair
systems, the photolyases. These enzymes use the visible part of the spectrum of sunlight
to reconstitute the double bonds [13]. In E. coli, the photolyase system is encoded by
phrB, a gene only expressed in the presence of thymine dimers [14]. This specific gene
regulation allows us to use a reporter gene fusion consisting of the regulatory region
of the phrB gene and the reporter gene lacZ encoding an enzyme, b-galactosidase, whose
enzymatic activity serves as a surrogate read-out for the gene expression level. Enzyme
activity is typically measured by conversion of a colorless substrate, e.g. 5-bromo-
4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal), into a dye. The phrB-lacZ
fusion provides a means to investigate the relative level of thymine dimer formation in
Fig. 2.5 phrB-lacZ fusion treated with the UV or the particle channel overnight. E. coli mutants
were plated on LB agar plates containing X-gal as substrate and were treated with plasma compo-
nents overnight. Controls were treated simultaneously with gas only. Blue color (area marked by
dotted line) indicates expression of phrB. Only the plate treated overnight with the (V)UV-channel
shows blue color directly under the jet nozzle (details of agar plates were magnified). Color figure
available in online version
the living bacterial cell, in vivo, by comparing b-galactosidase activity under control
growth conditions and after plasma treatment.
An E. coli mutant carrying the reporter gene fusion was treated with the different
X-Jet channels (Fig. 2.5) and gene expression, and, therefore, indirectly thymine
dimer levels, were monitored by the amount of X-gal converted into a blue dye [15].
To this end E. coli DH5a cells carrying the phrB reporter gene fusion in addition
to the phrB gene were incubated overnight at 37C in LB medium with tetracycline
to select for the reporter plasmid. In order to achieve a monolayer of bacterial cells
on LB agar plates containing X-gal and tetracycline, the overnight culture was
diluted to an optical density of 0.1 at 580 nm and applied to the plates in a 1-s spray.
These LB agar plates were incubated for 2 h at 37C before treatment with short
plasma pulses (1 s plasma every 180 s) overnight at room temperature. This mild
long-time treatment ensures continuous but non-lethal exposure to plasma-caused
stress, requiring the cells to cope with the inflicted damages by inducing repair
mechanisms, while still allowing cell proliferation. Either the (V)UV or particle
channel was used for treatment and an additional control was treated with He/O2 gas
mixture only. LB agar plates were photographed (Fig. 2.5). Formation of the blue
color indicates b-galactosidase activity and, therefore, presence of thymine dimers.
The blue dye was only observed in case of treatment with the (V)UV channel, but
not after exposure to the particle channel or under control conditions. These results
indicate the formation of thymine dimers in vivo specifically by (V)UV radiation.
We can conclude from our experiments that plasma can damage DNA both
in vitro and in vivo. Whereas in vitro tests using isolated bio-macromolecules offer
Fig. 2.6 B. subtilis total RNA before and after treatment. The control sample (C) was treated with
He/O2 gas only. The other samples were treated for 2 min with the (V)UV channel or the particle
channel of the X-Jet
controlled test conditions, it is paramount to compare such experiments with in vivo

conditions relevant for the living cell. We showed that different plasma components
play different roles in DNA damage. With the help of the X-Jet, we were able to
demonstrate the formation of DNA multimers by cross-linking in vitro. Thymine
dimer formation by UV radiation emitted from atmospheric pressure plasma
occurred in vitro as well as in vivo. Particles, emitted from the same plasma, did not
cause thymine dimer formation. In the future, we will further investigate the extent
of DNA cross-linking in vivo and characterize the effects of DNA treatment with the
particle channel.
2.4.2 Plasma Damage to RNA
Little is known about plasma-induced damage to RNA molecules although this may
be another mechanism relevant for bacterial inactivation. RNA is transcribed from
a DNA template and can itself serve as a template for protein synthesis. Stable RNA
molecules play a key role as part of ribosomes, the protein synthesis factories of the
cell. To analyze the effect plasma has on RNA, B. subtilis total RNA, containing
approximately 90% stable RNA molecules, was extracted from bacterial overnight
cultures [16]. 10 mg aliquots were spotted on RNase-free glass slides and treated
with the X-Jet channels for 2 min. After treatment, RNA was washed off the glass
slides with RNase-free water and analyzed via RNA-optimized agarose gel-electro-
phoresis [16] (Fig. 2.6). The control was also dried on glass slides but only treated
with He/O2 gas instead of plasma for 2 min. The two distinct bands in the control
lane are the stable 23S and 16S rRNAs that make up most of the total RNA in a cell.
Intensities of these bands decreased during plasma exposure.
In contrast to the DNA treatment experiment, no RNA multimer formation was
observed after treatment with the UV or the particle channel. Under both treatment
conditions band intensity decreased, with the effect being much more pronounced
after treatment with the particle channel. Possible explanations could be RNA
damage by induction of single strand breaks or plasma-based etching due to

particles in the effluent of the plasma jet. We hope to shed more light on the modi-
fications of RNA by plasma with future experiments.
2.4.3 Protein Etching with the X-Jet
When RNA is translated into an amino acid chain, this chain forms secondary and
tertiary structures to finally become a functional protein. Proteins are highly diverse and
fulfill many essential cellular functions. Destroying the functionality of a single essential
protein species is sufficient to compromise cell viability. Therefore, protein damage is
discussed as a major inactivation mechanism [17]. One well-characterized impact of
plasma on protein is etching, the removal of matter by plasma treatment [18].
BSA aliquots were applied to Si-wafers and dried under vacuum to create a plain
protein layer with a thickness of few micrometers. These layers were exposed to the
(V)UV channel or the reactive particle channel of the X-Jet (same conditions as
above) for 10 min. The treatment was performed in a controlled He atmosphere with
a jet-to-substrate distance of 4 mm. The etching profiles were determined by profi-
lometry. In this measurement, a sharp tip scans the surface topography along a
straight line, which, in this case, was a line over the symmetry axis of the etched
structure. Etching of a BSA layer, a biological model sample, was compared to the
etching of a non-biological model plasma polymer layer, a so-called hydrogenated
amorphous carbon (a-C:H) film. A 300 nm a-C:H film was produced in low pressure
CH4 plasma. The a-C:H film had an atomic hydrogen content of 45% and a density
of around 1 g cm3 [6]. The etching profile of the a-C:H film has a bell shape with a
full width at half maximum around 2 mm and with a maximum etching rate of about
30 nm/min (Fig. 2.7a). This profile corresponds perfectly to a flux of atomic oxygen
to the surface as predicted by a 2D fluid simulation of particle transport and reaction
kinetics in the effluent [6].
Similar etching profiles with the same etching rates were observed when treating
the BSA protein layer and the a-C:H film as shown in Fig. 2.7a. The profiles indi-
cate that heavy reactive particles in the plasma effluent (likely oxygen atoms) can
etch protein layers effectively and the etching mechanism does not depend strongly
on the layer structure of the sample. The measured BSA profile has much higher
roughness than the a-C:H film. This likely has two reasons, namely the inhomoge-
neity occurring in the drying process and the formation of cracks in the part of the
layer exposed to the reactive particles, which were detected by optical microscopy
(Fig. 2.7b). These cracks could be caused by tensile stress in the layer, which appears
during the etching as a result of the shrinking of the layer structure.
Both the a-C:H film and the BSA layer were also exposed to the (V)UV compo-
nent of the plasma effluent. No changes in the a-C:H film thickness or structure
were observed on the time scale of 20 min. In case of BSA, sample roughness was
increased after 20 min of (V)UV treatment suggesting minor damage of the upper
layer of the protein coat (data not shown).
Fig. 2.7 BSA and a-C:H etching with the particle channel of the X-Jet. (a) A BSA layer or an
a-C:H film were treated with the particle channel of the X-Jet under He atmosphere for 10 and
4 min, respectively. Etching rates per minute were calculated. Measured curves (4,000 points/mm)
were smoothed using a Savitzky-Golay algorithm set to 700 points. (b) An optical microscope
image shows the BSA layer after treatment with the particle channel
2.5 Conclusions
With the new X-Jet design of an atmospheric pressure plasma jet operated in He/O2
gas mixture, plasma radiation and heavy reactive particles in the effluent can be
separated effectively. The X-Jet allows to study separately the effects of (V)UV
plasma radiation and reactive particles on living cells and biological matter.
We were able to demonstrate that both plasma components inactivate vegetative
bacterial cells. DNA treatment with the (V)UV channel resulted in thymine dimer
formation both in vitro and in the living cell. Treatment with the reactive particle
channel led to introduction of DNA single and double strand breaks in vitro. We
further observed damage to stable RNA molecules in vitro after treatment with both
the (V)UV and the particle channel. Exposure of protein to the reactive particles
showed an etching rate for biological macromolecules comparable to that of an
a-C:H film. In vivo studies to investigate plasma effects on RNA and proteins inside
of vegetative bacterial cells are underway.
References
1. Ellerweg D, Benedikt J, von Keudell A, Knake N, Schulz-von der Gathen V (2010)

Characterization of the effluent of a He/O2 microscale atmospheric pressure plasma jet by
quantitative molecular beam mass spectrometry. New J Phys 12:013021
2. Knake N, Reuter S, Niemi N, Schulz-von der Gathen V, Winter V (2008) Absolute atomic
oxygen density distributions in the effluent of a microscale atmospheric pressure plasma jet.
J Phys D 41:194006
3. Liu D-X, Rong M-Z, Wang X-H, Iza F, Kong MG, Bruggeman P (2010) Main species and
physicochemical processes in cold atmospheric-pressure He + O2 plasmas. Plasma Processes
Polym 7:846
4. Bahre H, Lange H, Schultz-von der Gathen V, Foest R (2011) Vacuum ultraviolet (VUV) emis-
sion of an atmospheric pressure plasma jet (mAPPJ) operated in helium-oxygen mixtures in
ambient air. Acta Technica 56:T199
5. Kurunczi P, Lopez J, Shah H, Becker K (2001) Excimer formation in high-pressure microhol-
low cathode discharge plasmas in helium initiated by low energy electron collisions. Int J Mass
Spectrom 205:277283
6. Schneider S, Lackmann J-W, Narberhaus F, Bandow JE, Benedikt J (2011) Separation of
plasma radiation and reactive particles in the effluent of a He/O2 atmospheric pressure plasma
jet. J Phys D 44:295201
7. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn.
Cold Spring Harbor Laboratory Press, New York
8. OConnell D, Cox LJ, Hyland WB, McMahon SJ, Reuter S, Graham WG, Gans T, Currell FJ
(2011) Cold atmospheric pressure plasma jet interactions with plasmid DNA. Appl Phys Lett
98:043701
9. Pospisilova S, Kypr J (1997) UV light-induced crosslinking of the complementary strands of
plasmid pUC19 DNA restriction fragments. Photochem Photobiol 65:945948
10. Hesse M, Meier H, Zeeh B (2007) Spectroscopic methods in organic chemistry, 2nd edn.
Thieme, Stuttgart
11. Setlow RB (1966) Cyclobutane-type pyrimidine dimers in polynucleotides. Science 153:379386
12. Matsumura Y, Ananthaswamy HN (2004) Toxic effects of ultraviolet radiation on the skin.
Toxicol Appl Pharmacol 195:298308
13. Hearst JE (1995) The structure of photolyase: using photon energy for DNA repair. Science
268:18661872
14. Goosen N, Moolenaar GF (2008) Repair of UV damage in bacteria. DNA Repair 7:353379
15. Miller J (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press,
New York
16. Waldminghaus T, Fippinger A, Alfsmann J, Narberhaus F (2005) RNA thermometers are
common in a- and g-proteobacteria. Biol Chem 386:12791286
17. Bernard C, Leduc A, Barbeau J, Saoudi B, Yahia LH, De Crescenzo G (2006) Validation of
cold plasma treatment for protein inactivation: a surface plasmon resonance-based biosensor
study. J Phys D 39:3470
18. Rossi F, Kylian O, Rauscher H, Gilliland D, Sirghi L (2008) Use of a low-pressure plasma
discharge for the decontamination and sterilization of medical devices. Pure Appl Chem
80:19391951
Chapter 3
Bio-Decontamination of Water and Surfaces
by DC Discharges in Atmospheric Air
Zdenko Machala, Barbora Tarabov, Michal Pelach,

Zuzana ipoldov, Karol Hensel, Mrio Janda, and Libua ikurov
Abstract Two types of DC-driven atmospheric air discharges, including a streamer

corona and a transient spark with short high current pulses of limited energy, were
employed for bio-decontamination of water and various surfaces (agar plates, plas-
tic foils, human teeth) contaminated by bacteria or spores (Salmonella typhimurium,
Bacillus cereus). Both discharges generate cold non-equilibrium plasma. The dis-
charges combined with the electro-spraying of the treated water through the
needle electrode lead to fast and efficient bio-decontamination. Experiments com-
paring direct and indirect plasma effects, oxidation stress measurements in the cell
membranes, and chemical changes induced in the treated water enable assessment
of the plasma agents being responsible for microbial inactivation. Radicals and
reactive oxygen species seem to be dominant biocidal agents, although deeper under-
standing of the plasma-induced water chemistry and of the temporal evolution of the
bio-inactivation processes is needed.
3.1 Introduction
Cold (nonequilibrium) plasmas at atmospheric pressure find recently numerous biologi-

cal and bio-medical applications thanks to their reactive nature. Atmospheric pressure
plasmas applied for sterilization and bio-decontamination are mostly generated by
Z. Machala (*) K. Hensel M. Janda

Division of Environmental Physics, Faculty of Mathematics, Physics and Informatics,
Comenius University, Mlynsk dolina F2, 84248 Bratislava, Slovakia
e-mail: machala@fmph.uniba.sk
B. Tarabov M. Pelach Z. ipoldov L. ikurov
Division of Biomedical Physics, Faculty of Mathematics, Physics and Informatics,
Comenius University, Mlynsk dolina F2, 84248 Bratislava, Slovakia
32 Z. Machala et al.
various plasma jets, usually in rare gases (He or Ar) with/without admixtures of O2
(or H2O). The plasma jets are typically blown into the ambient air where the rare gas
plasma entrains air components. Air plasmas at atmospheric pressure have addi-
tional advantages of no need of special gases and an easy application in ambient
environment. Various atmospheric air plasma discharges have been tested for bio-
decontamination: DC [14], AC [5], dielectric barrier (DBD) [613], RF [14], and
pulsed discharges [8, 15, 16]. The plasmas can be also generated directly in water
or on water-air boundary [1, 9, 10, 17, 18], which is of great interest for water
decontamination. Atmospheric plasmas have been tested on a large variety of
prokaryotic microorganisms, such as bacteria, spores, viruses, and some eukaryotic
yeasts, fungi and microalgae, resulting in partial disinfection (12 log reduction of
microbial population) up to complete sterilization. Interaction of the plasmas with
the tissues of higher organisms including humans and plasma applications for skin
disinfection, wound healing, blood coagulation, dentistry, surgery, inducing apoptosis
pathways for cancer treatment, etc. are targeted by a revolutionary novel discipline
plasma medicine.
In bio-decontamination by plasma, it is crucial to understand the role of various
mechanisms involved. The significant mechanisms depend on the plasma composi-
tion (gas), temperature, treated microorganisms and the environment (air, water,
surfaces, etc.). In atmospheric pressure plasmas, the major role is typically attrib-
uted to radicals and reactive oxygen species (ROS, e.g. OH, O, O3) [1, 2, 6, 1012,
1416, 1820] and to charged particles, especially O2 [7, 21] affecting the cell
membranes. UV radiation plays a role only if photons in UV C germicide region
(220280 nm) or in vacuum UV are produced [11, 18]. In cold air discharges
(corona, DBDs, pulsed discharges), UV C or VUV are usually not generated, so
radicals and ROS are identified as the dominant bio-inactivation agents [1, 6, 9, 10,
15, 20, 22, 23].
Our former bio-decontamination investigations with DC discharges in atmo-
spheric air with water were in agreement with this statement and demonstrated the
dominant role of radicals and ROS [24, 25]. In this paper, the biocidal effects of two
atmospheric air plasma sources treating water and surfaces are investigated positive
DC streamer corona (SC) and transient spark (TS). Despite DC applied voltage,
these discharges have a pulsed character with nanosecond repetitive pulses. The
pulsed plasmas are especially convenient for penetration into topographically non-
uniform surfaces and cavities, which is applicable e.g. in teeth root canal disinfection
[6, 8, 16, 26].
The effects of SC and TS on selected bacteria in water solutions and on various
surfaces were tested. Bio-decontamination of water is important for waste water
cleaning or drinking water disinfection and is involved in all bio-medical applica-
tions and food technology, since cells and most foods contain water. Plasma decon-
tamination of various surfaces is important in medicine, e.g. for the sterilization of
endoscopes, implants and other heat sensitive materials, and in dentistry for cavities
or dental plaque treatment [27, 28] or root canal disinfection [6, 8, 16, 26].
We focus on the identification of the dominant plasma agents in bio-inactivation
by coupling the electrical characteristics, emission spectra, and biocidal effects of
3 Bio-Decontamination of Water and Surfaces by DC Discharges in Atmospheric Air 33
the studied discharges in various regimes with the measurements of the oxidative
stress induced in microbial cells. Comparing direct with indirect plasma effects
enables further separation of various biocidal plasma agents (electric field, charged
particles, neutral active species, UV radiation).
3.2.1 Discharge Experimental Set-Up
The experimental setup for fundamental investigations of the DC discharges in

point-to-plane geometry, with a high voltage (HV) syringe hollow needle elec-
trode enabling water flowing through the discharge zone and a plane or mesh
electrode is depicted in Fig. 3.1. The inter-electrode spacing was varied 510 mm.
A positive DC high voltage was applied through the ballast resistor R (20 MW for
SC and ~5 MW for TS). The discharge voltage was measured by a high voltage
probe Tektronix P6015A. The discharge current was measured: on a 50 W (SC) or
1 W (TS) resistor and by a Rogowski current monitor PEARSON 2877. The cur-
rent and voltage signals were processed by a digitizing 200 MHz oscilloscope
Tektronix TDS 2024. The discharges were photo-documented with a digital cam-
era Olympus E410.
The emission spectroscopy optical system comprised a dual fibre-optic compact
spectrometer Ocean Optics SD2000 with CCD detector for fast scanning in the UV
and VIS-NIR regions (200500 and 5001,050 nm, resolution 0.61.2 nm), filters,
fused silica lenses, irises, and fibre optics. The discharge setup was placed in the
Faraday cage together with the optical components mounted on translation stages,
which enabled lateral and vertical scanning capabilities.
The bio-decontamination effects of the DC discharges were tested on flowing
water and on direct/indirect treatment of the contaminated solid surfaces (agar
plates, plastic foils, human teeth).
3.2.2 Flowing Water Discharges with Electrostatic Spraying
The discharge set-up shown in Fig. 3.1 enabled the contaminated water to flow
directly through the high voltage hollow needle electrode, and so through the
corona active region in its proximity. The effect of electrostatic spraying occurred
when the high voltage was applied on the needle electrode [29]. Figure 3.2 shows
the photographs of the electro-spray of water with SC and TS discharges. These
experiments were repeated 510 times for each discharge type at various operation
parameters.
Fig. 3.1 Experimental setup for DC discharges, with a high voltage hollow needle electrode
enabling water flowing through the discharge zone and a plane or mesh electrode
Fig. 3.2 Photographs of the electro-spray of water in 8 mm gap, water flow rate 0.5 mL/
min, exposure time 1/10 s: (a) electro-spray with SC, 6.5 kV, (b) transition SC-TS, 7.8 kV,
( c) TS, 9 kV
3.2.3 Agar Surface Direct and Indirect Exposure to Plasma
We compared direct and indirect plasma effects on contaminated solid agar sur-
faces. A needle electrode was placed about 1 cm above the agar surface in the
centre of the Petri dish and the SC or TS was applied for various treatment times
(5 s2 min). In direct treatment, the agar was grounded with a wire. Indirect plasma
effects on the contaminated agar were tested by placing the grounded mesh elec-
trode ~2 mm above the agar; this shielded the electric field and expectantly trapped
the charged particles, letting but neutral particles and partial UV light to reach the
Fig. 3.3 Schematics of electrode arrangements for (a) direct and (b, c) indirect plasma treatment
of contaminated agar plates. (b) Mesh electrode ~2 mm above agar surface trapped the charged
particles and shielded the electric field. (c) Quartz (or MgF2) window only transmitted light from
the discharge (including UV)
agar surface. Previously we also tested an indirect exposure to UV (and vacuum

UV) light from the discharge only by placing a quartz (or MgF2) window onto the
agar surface. [25]
Figure 3.3 schematically depicts these arrangements (including that with quartz
window). The agar in the direct treatment (Fig. 3.3a) represents a certain electrical
resistance between the discharge and the ground, its typical value was 12 kW
depending on the water content. In indirect set-ups (Fig. 3.3b, c), a small resistor r
was inserted between the mesh and the ground to simulate the agars resistance. Its
exact value was set empirically from case to case to make the discharge pulses of
about the same amplitude and shape as in the direct treatment on agar. This ensured
the same discharge properties in all three set-ups.
3.2.4 Polypropylene Foil and Teeth Surfaces
The plastic and teeth surfaces were treated by positive and negative corona only. TS
resulted in the material damage after a few minutes of treatment. Figure 3.4 shows
the photographs of plastic foils and extracted human teeth (provided by volunteers)
treated by positive and negative DC corona. The foils and the teeth samples were
placed on the plate grounded electrode and the high voltage was applied to the needle
5 mm above them. We compared treatment of dry and moist foils. The teeth were
always treated moist (physiological solution) to mimic their natural environment.
3.2.5 Bacteria and Their Cultivation
Bio-decontamination effects of investigated DC discharges were tested on selected

Gram-negative bacteria Salmonella typhimurium (Salmonella enterica, serovar
Typhimurium, strains TA 98 and 100) or Gram-positive Bacillus cereus, either
suspended in deionised water, tap water and saline (physiological) solution with
initial populations 106107 colony forming units per mL (CFU/mL), or directly
spread on the solid nutrient medium (agar, Roth Ltd.) on Petri dishes, about 105
Fig. 3.4 Photographs of contaminated plastic (a, d) and tooth (b, c, e, f) samples with atmospheric
pressure air DC discharges: 1st row positive streamer corona, 2nd row negative corona (Trichel
pulses)
per dish. Sterile polypropylene foils and extracted human teeth were contaminated
by B. cereus spores (20 mL drop with about 104105 spores).
The microbial cultivation was carried out in a sterile environment in the follow-
ing steps: an overnight bacterial culture was first prepared in a shaker with sterile
liquid nutrient. A hot sterile nutrient medium agar was poured into sterile Petri
dishes, on which the bacteria were grown, and solidified. Cultivated bacteria in the
liquid nutrient were compared with McFarland turbidity scale to assess their initial
population per mL. They were then diluted in water to obtain desired concentra-
tions. The plasma experiments were performed with both discharges, at various
parameters and treatment times and repeated 510 times. Three to five Petri dishes
from each sample were taken for statistical evaluation. These were incubated during
1224 h in a thermostat at 37C. The grown CFUs on the treated and reference
samples were counted and evaluated.
3.2.6 Measurements of the Oxidative Stress
Interaction of ROS with the bacterial cell membranes results in the peroxidation of
membrane lipids. The final product of lipoperoxidation is malondialdehyde (MDA),
quantifiable by spectrophotometry after the reaction with thiobarbituric acid (TBA)
at 90100C [30]. This method of thiobarbituric acid reactive substances (TBARS)
was applied to measure the oxidative stress induced in bacteria in distilled water
exposed to SC and TS similar to [24, 25]. We assigned the TBARS concentrations
from the absorbance of MDA at 532 by using Lambert-Beers law with the absorp-
tion coefficient 1.57 105 mol1 L cm1 [30].
3.3.1 Applied DC Discharges
Two types of DC discharges of both polarities operating in atmospheric air with

water were investigated: a streamer corona (SC), and a transient spark (TS). These
discharges generate non-equilibrium plasmas inducing various chemical and bio-
logical effects important in bio-decontamination. Their electrical parameters and
emission spectra were documented in detail in our previous works [3133]. The
typical voltage and current waveforms of SC and TS discharges in 10 mm gap (with
electro-spray of water) are shown in Fig. 3.5. The SC regime generates cold plasmas
(~300 K).
When a positive high voltage of a few kV is applied to the point electrode, SC
appears, typical with small current pulses of streamers (~10 mA) with a repeti-
tive frequency of 1030 kHz, during which the discharge voltage remains fairly
constant. As the positive voltage is further increased (to ~12 kV in 10 mm gap),
the streamers establish a conductive channel that gradually heats, thus enhanc-
ing the reduced electric field E/N, which eventually leads to a spark breakdown
with excessive current pulse. In our case, the spark pulse current is limited by
the ballast resistor R that drops the voltage as the current increases, and the
capacity C between the electrodes that is small (order of 10 pF). C is a sum of
the internal capacity of the discharge gap and the capacities of the high voltage
cable and the voltage probe. Thus, when the sparks forms, it is only transient
since the discharged energy is small (0.11 mJ). After the pulse, C is recharged
by a growing potential on the stressed electrode and triggers a new pulse. This
transient spark becomes then a repetitive streamer-to spark transition discharge,
with each spark pulse (~1 A) preceded by one or a sequence of streamer pulses.
The repetitive frequency of pulses is 0.510 kHz, and increases with the applied
voltage. Thanks to the very short pulse duration (~10100 ns) given by the small
C and a limiting R, the plasma cannot reach equilibrium conditions and remains
at relatively low gas temperature (~5001,500 K), depending on frequency, i.e.
dissipated power.
With the negative applied voltage, SC typical with Trichel pulses with
smaller amplitudes (~0.3 mA) but higher frequencies up to 1 MHz appear.
When the voltage is increases, Trichel pulses merge and establish a pulseless
regime with a constant current (~0.3 mA) and constant voltage. These corona
regimes generate cold plasmas (~300 K). Upon further voltage increase, this
pulseless glow corona eventually transits to the negative TS with properties similar
to positive TS.
Optical emission spectroscopy of TS described in detail in [31] showed the presence
of atomic O, N and H, OH radicals, and the N2+ ions. Part of O radicals reacts with
air O2 and form ozone O3. There was practically no UV C radiation detected from
SC and TS.
a 25 b 20
18
20 16
U [kV] U [kV]
14
U [kV] / I [mA]
I [mA] I [A]
U [kV] / I [A]
12
15
10
8
10
6
4
5 2
0
0 2
100 0 100 200 300 400 40 20 0 20 40 60 80 100
t [ns] t [ns]
Fig. 3.5 Typical voltage and current waveforms of positive (a) SC and (b) TS discharges with
electro-spray of water, 10 mm gap
3.3.2 Flowing Water Treatment Through the Stressed Electrode
The water solution contaminated by bacteria (B. cereus and S. typhimurium) flew
directly through the high voltage hollow needle electrode, and so through the plasma
active zone in its proximity. In addition, the effect of electrostatic spraying substan-
tially improved the efficiency of bio-decontamination compared to our previous
set-ups for water treatment [24].
The efficiency of transient spark was higher than of streamer corona. TS decreased
the initial microbial population roughly by 3 logs and SC by 1 log. We use a new
parameter E-value (Joule per treated water volume and one log reduction of microbial
population) to express the combined energy requirements and efficiency of the pro-
cess. Streamer corona is far less energy-demanding than TS, as shown in Fig. 3.6.
The temperature of the treated water did not change in SC and was increased by
maximum 10 K in TS. The lethal heat effect of the discharges to bacteria can be
excluded.
3.3.3 Chemical Changes Induced in Water
The chemical effects induced in the plasma treated water were measured by pH and
conductivity probes and spectrophotometric method for H2O2. Depending on the
initial conductivity of the treated water (1, 500, 1,000 mS/cm, physiologic solution
~14 mS/cm) and the plasma parameters, we observe a pH decrease from 57 down
to 35 and an increase of conductivity (from 1 up to 1,000, from 500 up to 1,300 mS/cm).
The measured peroxides (H2O2) reached up to 500 mM. pH decrease is probably due
to the nitric acid formation. However, additional tests showed that the nitric acid
solution of the same pH does not lead to the same biocidal effects. In agreement
with [34], it is clear that acid environment in synergy with plasma agents leads to
the bacterial inactivation. In addition, we suppose an interaction of nitrites and
peroxides at lowered pH; this has to be further studied with using buffers.
Efficiency Evalue
100
E-value [J/ml.log reduction]

95 100
Efficiency [%]
90
10
85
80
1
75
TS+ SC+ TS- SC-
Fig. 3.6 Comparison of inactivation efficiency of B. cereus in saline solution with E-value for
positive and negative TS and SC. Medians with 1st and 3rd quartiles as error bars. A typical num-
ber of repeated experimental sets was 10
3.3.4 Oxidative Stress Induced in Bacteria
Figure 3.7 shows the inactivation efficiencies for both positive discharges applied to
the electro-sprayed water with the measured concentrations gains Dc(TBARs) of
TBARs for B. cereus and S. typhimurium. The same bacterial samples were irradiated
by biocidal UV C radiation (Hg lamp, 254 nm, 1 min) for comparison. Concentrations
c(TBARs) correlated with the inactivation efficiencies of the discharges.
UV C radiation induced almost no Dc(TBARS) despite its efficiency was very
high. Obviously, UV dominant biocidal mechanism is not peroxidation of cell mem-
branes. On the contrary, SC and TS plasma treatments significantly enhanced the
TBARS concentration. This indicates that oxidations of cell membranes by reactive
oxygen species ROS are important in microbial inactivation.
3.3.5 Direct vs. Indirect Plasma Treatment
A comparison of direct and indirect plasma effects on contaminated (S. typhimurium)

agar surfaces was aimed at separation of various biocidal plasma agents. In indirect
exposure, a grounded mesh filtered the charged particles and electric field from
neutral radicals and excited species. The corresponding electrode arrangements
were shown in Fig. 3.3. Figure 3.8 shows the photographs of the contaminated
agar surfaces after direct and indirect TS treatment. The effects of plasma on
contaminated agar are visible as dark voids.
Both direct plasma and indirect exposures to neutral reactive species caused
apparent bio-decontamination (voids). The areas of inactivated voids were measured
by processing the Petri dish photographs in ImageJ software and statistically evaluated.
Bacillus cereus Salmonella typhimurium

n=6 n=6 n=5 100 0.16 n=8 n=8 n=7 100
0.08
98 0.14 98
Efficiency 96 Efficiency 96
0.12
c(TBARS) [mol/l]
c(TBARS) [mol/l]
0.06 94 94
Efficiency [%]
Efficiency [%]
92 0.10 92
90 90
0.04 0.08
88 88
86 0.06 86
0.02 84 0.04 84
82 82
0.02
80 80
0.00
78 0.00 78
TS+ SC+ UV TS+ SC+ UV
Fig. 3.7 TBARS concentration gains (with respect to control) and decontamination efficiencies of
B. cereus and S. typhimurium in water treated by SC and TS with electro-spray, compared with
30 s exposure to UV C (Medians with the 1st and 3rd quartiles as error bars; n is the number of
repeated experimental sets)
Fig. 3.8 Photographs of contaminated agar surfaces with area inactivated by direct (upper row)
and indirect (lower row) exposure to TS for various exposure times. 60 s (far right) is not in the
same scale
The heat effect of TS on contaminated agar is possible in the very small area
under the discharge. TS usually resulted in a tiny hole (12 mm diameter) of dried
agar. Nevertheless, the area of decontamination (void) was always much larger than
this tiny hole in its centre. The void kept the ambient temperature after treatment, so
we can neglect the heat effect on bacteria. In the indirect treatment, the TS heat was
lead out through the mesh electrode which was not in the direct contact with the
agar surface: the heat effect can be thus excluded.
Figure 3.9 shows the inactivated area (normalized through three experimental
series) for direct and indirect exposure for short treatment times (515 s). The
inactivated area increases with the exposure time. The direct exposure is much
direct
95%
normalized inactivated area [a.u.] 3 indirect
75%
median
25%
2 5%
0
5 10 15
treatment time [s]
Fig. 3.9 Normalized inactivated area as a function of treatment time for direct and indirect exposures
to TS. Medians, 1st and 3rd quartiles, and 5th and 95th percentiles; statistics of 3 repeated experimental
series, 2 3 5 samples each
stronger for 5 s, which indicates that charged particles are important at the beginning
of the treatment. However, at 15 s the direct and indirect effects become more
similar. At 60 s treatment, there is very little difference between the direct and
indirect exposures with both discharges, indicating a crucial role of reactive neu-
tral species.
Exposure to the UV light only transmitted by quartz or MgF2 windows demon-
strated no visible decontamination. This correlates with the emission spectra of SC
and TS lacking UV C or VUV light.
3.3.6 Polypropylene Foil and Teeth Surface Treatment
Plastic foils (dry and moist) and human teeth surfaces (moist) contaminated with
B. cereus spores were treated by positive and negative streamer corona. Figure 3.10
shows the results in efficiency vs. energy graphs. The positive SC was more effective
and more energetic on dry plastic samples, whereas the negative pulsed corona was
more efficient on moist ones with about the same energy consumption. Negative pulse-
less corona was largely more energetic (~247 J) despite it provided the best sporicidal
efficiency (~93%). The experiments with moist teeth surfaces showed that the efficien-
cies in 3 and 5 min exposure times were almost the same (within the error bars). This
suggests that in this set-up, it does not make sense to extend the treatment time. Positive
streamer corona was the most efficient and negative Trichel pulse corona slightly less
Fig. 3.10 Decontamination efficiency vs. energy (medians with first and third quartiles as error
bars): dry or moist plastic foils 2 min treatment (left, 3 experimental sets with 46 samples each)
and moist teeth 3 and 5 min treatment (right, 3 experimental sets with 57 samples each)
efficient and less energetic. These preliminary findings are now being verified in the
treatment of teeth contaminated by Streptococcus mutans biofilms.
3.4 Conclusions
Bio-decontamination of water and surfaces contaminated by bacteria and spores

(Salmonella typhimurium, Bacillus cereus) was tested in two DC discharges in atmo-
spheric pressure air: streamer corona and transient spark. Both were found very efficient
when the treated water was electro-sprayed directly through the high voltage needle
electrode and thus through the active discharge zone. The role of reactive oxygen species
(O, OH, O2, O3) was confirmed by the absorption spectroscopic detection of the products
of cell membrane oxidation stress (TBARs method).
The comparisons of direct and indirect exposure of contaminated agar surfaces
to SC and TS enabled separation of various biocidal agents. Both direct exposure to
plasma and indirect exposure to active neutral species only had almost the same
effect on bacteria at exposure times from 60 s, whereas direct plasma effect was
stronger at very short exposures (<15 s). The detailed mechanism has to be further
studied, so far it seems that charged particles play a role in very short exposures and
neutral species (such as ROS) are crucial at longer exposures. Separated UV radia-
tion from SC and TS demonstrated no significant biocidal effect.
Decontamination of bacterial spores by positive and negative corona on plastic
foils and teeth surfaces provided satisfying results, taking into account that spores
are extremely resistant to adverse conditions. The next step currently being perfor-
med is the treatment of teeth surfaces contaminated by Streptococcus mutans bio-
films in cavities that naturally cause a dental plaque.
In summary, we demonstrated that cold atmospheric air DC discharges can be

efficiently used for bio-decontamination of water and various surfaces. Radicals and
reactive oxygen species seem to be dominant biocidal agents, although understand-
ing the plasma-induced water chemistry and the temporal evolution of the bio-
decontamination mechanisms requires further research.
Acknowledgments Effort sponsored by Slovak grant agency VEGA 1/0668/11 and 1/0711/09,
Slovak Research and Development Agency APVV SK-CZ-0179-09. The human teeth were pro-
vided by patients of Dr. O. ipoldov. We thank P. Luke (IPP Prague) for his motivating ideas, and
I. Jedlovsk and B. Pongrc (FMFI Bratislava) for assistance.
References
1. Akishev Yu, Grushin M, Karalnik V, Trushkin N, Kholodenko V, Chugunov V, Kobzev E,

Zhirkova N, Irkhina I, Kireev G (2008) Atmospheric-pressure, nonthermal plasma sterilization
of microorganisms in liquids and on surfaces. Pure Appl Chem 80:1953
2. Sigmond RS, Kurdelova B, Kurdel M (1999) Action of corona discharges on bacteria and
spores. Czech J Phys 49:405
3. Scholtz V, Julk J, Kha V (2010) The microbicidal effect of low-temperature plasma generated
by corona discharge: comparison of various microorganisms on an agar surface or in aqueous
suspension. Plasma Processes Polym 7:237243
4. Dobrynin D, Arjunan K, Fridman A, Friedman G, Morss Clyne A (2011) Direct and controllable
nitric oxide delivery into biological media and living cells by a pin-to hole spark discharge
(PHD) plasma. J Phys D Appl Phys 44:075201 (10pp)
5. Kuo SP, Tarasenko O, Chang J, Popovic S, Chen CY, Fan HW, Scott A, Lahiani M, Alusta P,
Drake JD, Nikolic M (2009) Contribution of a portable air plasma torch to rapid blood coagu-
lation as a method of preventing bleeding. New J Phys 11:115016
6. Deng X, Shi J, Kong MG (2006) Physical mechanisms of inactivation of Bacillus subtilis
spores using cold atmospheric plasmas. IEEE Trans Plasma Sci 34:1310
7. Fridman G, Brooks AD, Balasubramanian M, Fridman A, Gutsol A, Vasilets VN, Ayan H,
Friedman HG (2007) Comparison of direct and indirect effects of non-thermal atmospheric-
pressure plasma on bacteria. Plasma Processes Polym 4:370
8. Ayan H, Staack D, Fridman G, Gutsol A, Muhkin Y, Starikovskii A, Fridman A, Friedman G
(2009) Application of nanosecond-pulsed dielectric barrier discharge for biomedical treatment
of topographically non-uniform surfaces. J Phys D Appl Phys 42:125202
9. Tang YZ, Lu XP, Laroussi M, Dobbs FC (2008) Sublethal and killing effects of atmospheric-
pressure, nonthermal plasma on eukaryotic microalgae in aqueous media. Plasma Processes
Polym 5:552
10. Qiong T, Wenju J, Zhang Y, Zhishan Y, Mariana LT (2009) Inactivation of dinoflagellate
Scrippsiella trochoidea in synthetic ballast water by reactive species generated from dielectric
barrier discharges. J Phys D Appl Phys 42:095203
11. Laroussi M, Leipold F (2004) Evaluation of the roles of reactive species, heat, and UV radia-
tion in the inactivation of bacterial cells by air plasmas at atmospheric pressure. Int J Mass
Spectrom 233:81
12. Yasuda H, Hashimoto M, Rahman M, Takashima A, Mizuno A (2008) States of biological
components in bacteria and bacteriophages during inactivation by atmospheric dielectric bar-
rier discharges. Plasma Processes Polym 5:615
13. Dobrynin D, Fridman G, Mukhin YV, Wynosky-Dolfi MA, Rieger J, Rest RF, Gutsol AF,
Fridman A (2010) Cold plasma inactivation of Bacillus cereus and Bacillus anthracis
(Anthrax) Spores. IEEE Trans Plasma Sci 39:18781883
14. Montie TC, Kelly-Wintenberg K, Roth JR (2000) An overview of research using the one
atmosphere uniform glow discharge plasma (OAUGDP) for sterilization of surfaces and
materials. IEEE Trans Plasma Sci 28:41
15. Pointu AM, Ricard A, Dodet B, Odic E, Larbre J, Ganciu M (2005) Production of active
species in N2O2 flowing post-discharges at atmospheric pressure for sterilization. J Phys
D Appl Phys 38:1905
16. Jiang C, Chen MT, Gorur A, Schaudinn C, Jaramillo DE, Costerton JW, Sedghizadeh PP,
Vernier PT, Gundersen MA (2009) Nanosecond pulsed plasma dental probe. Plasma Processes
Polym 6:479
17. Mizuno A, Hori Y (1988) Destruction of living cells by pulsed high-voltage application.
IEEE Trans Ind Appl 24:387
18. Luke P, lupek M, Babick V, unka P (2008) Ultraviolet radiation from the pulsed corona
discharge in water. Plasma Sources Sci Technol 17:024012
19. Brandenburg R, Ehlbeck J, Stieber M, Woedtke Tv, Zeymer J, Schluter O, Weltmann KD
(2007) Antimicrobial treatment of heat sensitive materials by means of atmospheric pressure
rf-driven plasma jet. Contrib Plasma Phys 47:72
20. Gweon B, Kim DB, Moon SY, Choe W (2009) Escherichia coli deactivation study controlling
the atmospheric pressure plasma discharge conditions. Curr Appl Phys 9:625
22. Lu X, Ye T, Cao Y, Sun Z, Xiong Q, Tang Z, Xiong Z, Hu J, Jiang Z, Pan Y (2008) The roles
of the various plasma agents in the inactivation of bacteria. J Appl Phys 104:053309
23. Laroussi M (2005) Low temperature plasma-based sterilization: Overview and state-of-the-art.
24. Machala Z, Jedlovsk I, Chldekov L, Pongrc B, Giertl D, Janda M, ikurov L, Polic P
(2009) DC discharges in atmospheric air for bio-decontamination - spectroscopic methods for
mechanism identification. Eur Phys J D 54:195
25. Machala Z, Chldekov L, Pelach M (2010) Plasma agents in bio-decontamination by dc
discharges in atmospheric air. J Phys D Appl Phys 43:222001
26. Lu X, Cao Y, Yang P, Xiong Q, Xiong Z, Xian Y, Pan Y (2009) An RC plasma device for
sterilization of root canal of teeth. IEEE Trans Plasma Sci 37:668673
27. Sladek REJ, Stoffels E, Walraven R, Tielbeek PJA, Koolhoven RA (2004) Plasma treatment of
dental cavities: a feasibility study. IEEE Trans Plasma Sci 32:15401543
28. Yu QS, Huang C, Hsieh F, Huff HE, Duan YX (2007) Bacterial inactivation using a low-
temperature atmospheric plasma brush sustained with argon gas. J Biomed Mater Res B Appl
Biomater 80:211219
29. Borra J-P, Ehouarn P, Boulaud D (2004) Electrohydrodynamic atomisation of water stabilised
by glow discharge - operating range and droplet properties. J Aerosol Sci 35:1313
30. Bachowski GJ, Pintar TJ, Girotti AW (1991) Photosensitized lipid-peroxidation and enzyme
inactivation by membrane-bound merocyanine-540 - reaction-mechanisms in the absence and
presence of ascorbate. Photochem Photobiol 53:481
31. Machala Z, Janda M, Hensel K, Jedlovsk I, Letinsk L, Foltin V, Martiovit V, Morvov M
(2007) Emission spectroscopy of atmospheric pressure plasmas for bio-medical and environ-
mental applications. J Mol Spectrosc 243:194
32. Machala Z, Jedlovsk I, Martiovit V (2008) DC discharges in atmospheric air and their
transitions. IEEE Trans Plasma Sci 36:918
33. Janda M, Martiovit V, Machala Z (2011) Transient spark: a dc-driven repetitively pulsed
discharge and its control by electric circuit parameters. Plasma Sources Sci Technol
20:035015
34. Oehmingen K, Hahnel M, Brandenburg R, Wilke Ch, Weltmann KD, von Woedtke T (2010)
The role of acidification for antimicrobial activity of atmospheric pressure plasma in liquids.
Chapter 4
Biological Decontamination Using Pulsed
Filamentary Microplasma Jet
Ramasamy Pothiraja, Jan-Wilm Lackmann, Gernot Keil, Nikita Bibinov,

and Peter Awakowicz
Abstract Microplasma jet for the generation of pulsed filamentary discharge at

atmospheric pressure has been devised for biological decontamination as well as
for modification of surface properties. Long plasma-filament is generated inside
a quartz tube and characterized using optical emission spectroscopy, current
voltage measurements, numerical simulations and microphotography. Efficiency
of our plasma source for the decontamination on inner surface of the tube as well
as on objects placed in proximity of plasma effluent is studied. Escherichia coli
(Gram-negative bacteria) and spores of Bacillus atrophaeus (Gram-positive bac-
teria) are used for the decontamination studies. Decontamination of Bacillus
atrophaeus endospores, which are layered on PET polymer material, and placed
in the proximity of plasma effluent, shows the mean logarithmic bacterial reduc-
tion of 3.67 for the treatment time of 120 s. Inactivation of Escherichia coli
coated on inner surface of the tube shows the mean logarithmic bacterial reduc-
tion of about 5 for the treatment time of 30 s. In addition to this, inhibition stud-
ies of bacteria coated on agar plate are also carried out. It shows plasma effluent
generated in our plasma source is very effective for the inhibition of bacterial
colonization.
R. Pothiraja (*) N. Bibinov P. Awakowicz

Institute for Electrical Engineering and Plasma Technology, Ruhr-Universitt Bochum,
44801 Bochum, Germany
e-mail: pothiraja@aept.rub.de
J.-W. Lackmann
Microbial Biology, Department for Biology and Biotechnology,
Ruhr University Bochum, Universittsstr. 150, 44801 Bochum, Germany
G. Keil
KHS GmbH, 55543 Bad Kreuznach, Germany
46 R. Pothiraja et al.
4.1 Introduction
Atmospheric pressure cold plasmas are very useful for the production of UV
photons, excited atoms/molecules, radicals and charged species. Although these
active species can also be produced in low pressure plasmas as well as in ther-
mal plasmas, using atmospheric pressure cold plasmas has many advantages.
Since it is operated at atmospheric pressure, there is no need for any vacuum
equipment and makes the device portable. In addition to this, despite the high
temperature of the electrons, the average gas temperature is close to room tem-
perature because of the low energetic ions; hence, suitable for the treatment of
temperature sensitive objects without affecting their bulk properties. In this
regard, plenty of research efforts are being made in order to understand and
optimize the atmospheric pressure cold plasma for the production of various
specific active species for different specific applications. One of the most impor-
tant and exciting applications of these plasmas are in the field of biomedicine
for cancer treatment [1], sterilization [2], dental bleaching [3], blood coagula-
tion [4], and wound healing [5]. As for the sterilization of microorganisms is
concerned, many sterilization techniques already exist, however, they either
involve exposure to toxic chemicals such as ethylene oxide or involve subject-
ing the specimen to high temperatures, as in the case of autoclaving. This is not
suitable for many heat sensitive and biological materials, especially for medical
and food package applications.
Low temperature plasmas operating at atmospheric pressure are particularly
well suited for treatment of these sensitive materials. In this regard, several
research groups have developed various plasma sources with a range of power
supply and electrode configurations; and studied efficiency their plasma source
towards inactivation or sterilization of various microorganisms including
Escherichia coli (E. coli) and spores of Bacillus atrophaeus (B. atrophaeus), and
studied inactivation or sterilization mechanism. For example, Laroussi et al. used
resistive-barrier discharge (RBD) to study the decontamination and its mecha-
nism [6]. Park et al. used microwave induced argon plasma for the sterilization
studies [7]. Fridman et al. used DBD based plasma source and studied the effect
of direct plasma and indirect plasma on sterilization [8]. Kong et al. used DBD
based plasma plume for the inactivation of spores [9]. Machala et al. used positive
DC discharge in water spray for the decontamination of bacteria [10]. Weltmann
et al. used plasma jet with RF power supply to study the effectiveness of plasma
against wound pathogens [11]. We have used pulsed positive filamentary plasma
source operating in coaxial DBD principle for the inactivation of E. coli (Gram-
negative bacteria) and spores of B. atrophaeus (Gram-positive bacteria). Here, we
report the plasma source configuration, plasma characterization using optical
emission spectroscopy, inactivation of B. atrophaeus and E. coli, and possible
mechanism of inactivation.
4 Biological Decontamination Using Pulsed Filamentary Microplasma Jet 47
4.2 Experimental and Simulation Details
4.2.1 Experimental Setup
Experimental setup used for the biological decontamination studies, and methods
used for plasma characterization are similar to the one reported previously [12].
Hence, only a brief description is given here. Our microplasma jet consists of a
tungsten electrode with the diameter of 1.6 mm. One end of this electrode is
sharpened to an angle of 30, while the other end of the electrode is connected
to a high voltage generator. The output voltage and the pulse frequency of this
generator can be controlled and varied from 0 to 20 kV and from 4 to 500 kHz,
respectively. Each high voltage pulse exhibits a sequential profile with damped
oscillations. A tube made of quartz with the inner diameter of 6 mm, is used in
order to test the decontamination of the tube. The outer diameter of the tube is
8 mm. The tungsten electrode is placed coaxially inside this quartz tube
(Fig. 4.1). A copper tube is used as a grounded electrode. Length of the grounded
electrode is about 40 mm. It is placed coaxially on the outer surface of the quartz
tube and is separated from the inner electrode. Hence, the quartz tube also acts
as dielectric in between the electrodes. The distance between the driven elec-
trode and the grounded electrode can be varied, and for the present studies, it is
kept as about 50 mm. Because of its transparency in UV-Vis region, the quartz
tube facilitates the plasma characterization through emission spectroscopy. A
void is present in the grounded electrode, through which information of the fila-
ment generated inside the quartz tube in the grounded electrode region is
obtained. Argon is used as the working gas during decontamination studies. The
flow rate of argon is kept as 2.4 slm. Nitrogen (about 1 sccm) is admixed with
argon during diagnostic studies.
Relatively and absolutely calibrated echelle (ESA 3000) and Ocean Optics
(QE65000) spectrometers are used to obtain the emission spectra [13]. The solid
angle of the optical fiber used in echelle spectrometer amounts to 0.012 sr. The
diameter of entrance hole of diaphragm is 1 mm. Spectral resolution of the echelle
spectrometer amounts to Dl = 0.015 nm at l = 200 nm and Dl = 0.060 nm at
l = 800 nm. The groove density of echelle grating, g = 75 grooves per mm. The
optical resolution of Ocean Optics spectrometer is ~0.147.7 nm. A Pearson cur-
rent monitor (model, 6585; output, 1V = 1A) is used for plasma current measure-
ment, which is mounted around the cable connecting the generator and the tungsten
electrode. The output of the current monitor is connected to an oscilloscope
(LeCroy 9314A). The actual voltage applied for plasma generation is measured by
connecting the output of the generator to the oscilloscope through a voltage divider
with the dividing factor of 2,000. The pulse frequency is fixed as 22 kHz for all the
experiments. For the plasma volume determination, a high speed sensitive camera
(PCO sensicam qe) is used.
Fig. 4.1 Schematic view of experimental setup
4.2.2 Determination of Gas Temperature
Gas temperature in active plasma volume is one of the important parameters,

because of its influence on gas density in plasma and on the rate constants of chemi-
cal reactions. The rotational temperature of diatomic molecules is considered as the
gas temperature, since the rotational and the translational degrees of freedom have
equal temperatures because of very fast rotational relaxation at atmospheric pres-
sure. For the determination of gas temperature, the rotational intensity distribution
in the emission of neutral nitrogen molecule N2(CB, 00) is used. The emission
spectrum is measured perpendicular to the axis of the plasma filament, as shown in
Fig. 4.1. Since the spectral resolution of our echelle spectrometer is not high enough
to determine the intensities of the separate rotational lines in the emission spectrum
of neutral nitrogen molecules, the rotational temperature is determined by a fitting
procedure. For this purpose, we calculate the intensity distribution in the emission
of N2(CB, 00) (l = 337.1 nm) for different values of rotational temperature using
the program code developed for this purpose [14]. By comparing the measured
emission spectra with the calculated spectra for various rotational temperatures, we
determine the actual rotational temperature of nitrogen molecule with an inaccuracy
of 30 K (Fig. 4.2).
Molecular emissions N2(CB) can be excited not only by electron impact, but
by collisions with argon metastable atoms also. In the latter case, rotational dis-
tribution in the emission spectrum of these excited molecules is described by the
equilibrium distribution with the temperature of approximately 2,000 K [15].
This effect is not taken into account in our studies because of the following rea-
sons: (1) short lifetime of argon metastables in our experimental conditions and
therefore lower probability of excitation of N2 by collisions with metastables and
(2) fast rotational relaxation of diatomic molecular excited states at atmospheric
pressure.
Fig. 4.2 Determination of

gas temperature by
comparing the simulated
emission of N2(CB, 00)
Intensity (a.u)
with the experimentally
measured emission spectrum Experimental
Simulated for 580 K
334 335 336 337

Wavelength (nm)
4.2.3 Determination of EVDF, Rate Constants

and Electron Density
The relative intensity of N2(CB, 00) with respect to the intensity of N2+(BX,
00) is used for the determination of electron velocity distribution function (EVDF)
by including the contribution of excitation of nitrogen molecules by argon meta-
stables. For this purpose, the relative intensity of N2(CB, 00) with respect to the
intensity of N2+(BX, 00) is simulated for various EVDFs for our experimental
conditions by numerical solution of the Boltzmann equation in local approximation
and varied electric field applying the program code EEDF developed by
Napartovich et al. [16]. Finally, by comparing the experimentally determined
I + I N+ (B- X)
relative intensities ( N2 (B- X) ) with the simulated relative intensities ( 2 )
I N2 (C - B) I N2 (C - B)
for various EVDFs, the actual EVDF is determined.
Using the normalized EVDF and the known collisional cross section sexc (cm2)
for electron impact excitation [17], we calculate the rate constants k (cm3s1) for
electron impact excitation of N2, using the Eq. 4.1:
2C
k = 4p 2 fv ( E ) E s exc ( E ) dE, (4.1)
0 me
where, me is the mass of electron (g), E is the kinetic energy of electrons (eV) and
C = 1.602 1012 erg eV1.
The electron density (ne, cm3) is determined using the Eq. 4.2 from the measured
absolute intensity of N2(CB, 00) emission ( I N2 (C - B) , photcm3s1), nitrogen den-
sity ([N2], cm3), the electron impact excitation rate constant for N2(CB, 00) emis-
sion ( kN2 (C) , cm3s1), contribution of excitation of N2(C) by collision with argon
metastables ( K Ar
N 2 (C) ) [12], contributions of the quenching of N2(C) by argon ( Q N 2 (C) ),
met
the plasma volume (Vp, cm3), value of fraction of time in which plasma is active (tf),
and the geometrical factor (gf), which relates the total quantity of photons produced
in the plasma source to the observed quantity of photons by the emission spectrom-
eter. To determine this geometrical factor, we consider the actual volume of plasma,
from which spectrometer receives the photons as well as the distance between the
active plasma volume and the entry point of optical fiber.
I N2 (C - B)
ne = (4.2)
[N 2 ](kN2 (C) + K NAr2met(C) )Q N2 (C) Vp t f g f
4.2.4 Biological Sample Preparation and Analysis
For the E. coli decontamination experiments, the E. coli DH5a strain is incubated
over night in LB media at 37C [18]. It is diluted to an optical density of 0.5 at
580 nm; 50 ml of this solution is spread onto agar plates of diameter 9 cm. Hard agar
plates are made with 30 g of Agar-Agar per liter media, while soft agar plates are
made with 15 g of Agar-Agar per liter media. After plating, the agar plates with the
bacteria are incubated for an additional 2 h at 37C. After plasma treatment, it is
incubated at 25C for 3 days. From the visible change on agar plate due to the
plasma treatment, the inhibition zone is determined. For the decontamination exper-
iment of E. coli coated tube, 5 105 cells are coated inside the tube. After plasma
treatment, the tube is washed and the survival cells are counted by usual method
[18]. For all experiments, reference samples are made and are analyzed along with
plasma treated samples.
For the decontamination of spores of B. atrophaeus, first set of experiments are
carried out with about 300 spores placed on a polyethyleneterephthalate (PET)
polymeric plate in a circular area of a few mm diameter. The second set of experi-
ments are carried out by placing about 4,700 spores placed on each PET plate in a
circular area with a few mm diameter. A standard procedure reported elsewhere is
used for the samples preparation and analysis [18].
4.3.1 Plasma Generation and Characterization
Long plasma filaments are generated as positive leader like discharge inside a tube in
pure argon as the working gas (Fig. 4.3). Decontamination experiments are carried
out at this condition. However, during plasma diagnostic studies, nitrogen is admixed
with argon. Plasma parameters are determined at two regions in the filament; one at
Fig. 4.3 Current and voltage 6 0.8

profiles during the pulsed Applied voltage
Applied voltage (kV)

positive filamentary 3 0.6
Current (A)
discharge. The pulse
0 0.4
frequency is 22 kHz. The
voltage frequency of each 3 0.2
pulse sequence is around Current
200 kHz 6 0.0
9 0.2
0.0 2.0x105 4.0x105
Time (s)
the midpoint in between the powered electrode and grounded electrode, and the
second point is at the middle region of the grounded electrode.
Gas temperature in active plasma is about 600 K. Since the discharge duration is
in the range of 100200 ns, the actual temperature will be much less than this value,
especially in the effluent of plasma. This factor gives the possibility to use this plasma
source for decontaminating the temperature sensitive materials. The power dissi-
pated is about 12 W during the discharge. The reduced electric field close to spike of
driven electrode is high (about 1,700 Td) compared to this in grounded region (about
900 Td). High reduced electric field indicates that argon ionization reaction is more
probable than the argon metastables formation reaction. The electron density in both
measured points is comparable with each other, and is in the order of 1011 cm3. This
indicates that the average ion density in active plasma is in the order of 1011 cm3,
which can play important role especially in direct plasma treatment.
4.3.2 Microorganisms Decontamination Studies
The E. coli layered agar plates are placed at 11 cm coaxially away from the grounded
electrode. The exit point of effluent in the tube is placed at the centre of the agar
plate. The distance between the effluent exit point of the tube and the agar plate is
about 2 cm. This inhibition studies are carried out for E. coli on soft agar plates as
well as on hard agar plates. Even when the treatment is carried out for 10 s, the
inhibition zone has the diameter of 1.2 cm (Fig. 4.4). As the treatment time is
increased, the area of inhibition zone also increased (Fig. 4.4). The prolonged treat-
ment of agar plates leads to the reduction of transparency of agar plates, at the area
of direct contact of effluent. This could be because of drying [8] and deformation of
agar structure due to plasma (effluent) induced chemical structural deformation
reaction. Chemically, agar is a polymer made up of subunits of the sugar galactose,
and is a component of the cell walls of several species of red algae. Algae itself is
known to undergo degradation in plasma [19]. Specifically, sugar galactose is known
to undergo photo chemical reaction [20], and argon plasma is known to produce
high energetic photons [21].
Diameter of inhibition zone (cm)

6
4
Hard agar plate
3 Soft agar plate
2
1
0 150 300 450 600
Treatment time (sec)
Fig. 4.4 Images of inhibition zone generated after plasma treatment of E. coli coated agar plates (left);
plot of inhibition zone with respect to plasma treatment time on hard and soft agar plates (right).
Studies on the decontamination of inner surface of the tube are also carried
out by coating E. coli on inner surface of the quartz tube, which is a part of our
experimental set up, from the point close to the spike till the exit point of the
effluent. In other words, E. coli is coated in the effluent region, grounded region
and the region between electrodes. Analysis of plasma treated tube shows the
logarithmic reduction of colony forming units is about 5 with the bacterial sur-
vival ratio is 0.0038%. This survival could be due the fact that some traces of
microorganisms are present in the powered electrode region, and this region is
not in contact with plasma.
Encouraged by the efficiency of decontamination of vegetative microorganism, we
have carried out efficiency studies of our plasma source for the inactivation of spore.
For this purpose, spores of B. atrophaeus are used. In order to find out the possibility
of decontamination of temperature sensitive materials, these endospores are layered
on PET polymer material. All decontamination experiments of spores are carried out
in the effluent of plasma generated in our plasma source. It is carried out in a similar
way as carried out for E. coli inhibition studies, but with the distance between the exit
point of effluent in tube and PET plate is about 1 cm. First set of experiments are
started with less number of spores (about 300) layered in a circular area within diam-
eter of a few mm on a PET plate. Analysis of plasma effluent treated plates shows that
within the treatment time of 70 s, all spores are inactivated. Hence the second set
experiments are carried out with high number of spores (about 4,700) layered in a
circular area with diameter of a few mm on PET plate. Analysis of plasma effluent
treated samples shows the complete inactivation of spore in 2 min. In other words, the
logarithmic reduction of spores is about 3.67 for the plasma effluent treatment time of
2 min (Fig. 4.5). Repetition of all the experiments shows good reproducibility.
4.3.3 Possible Inactivation Mechanism
The microorganism E. coli, which are coated inside the tube in the region between
the electrodes as well as in the region of grounded electrode, are in direct contact
with the plasma during the treatment. In this case, constituent of plasma, especially
Fig. 4.5 Plot of mean 4

logarithmic bacterial
Mean logarithmic bacterial reduction

reduction with respect to ~4700 spores
plasma treatment time for ~300 spores
spores of B. atrophaeus on 3
Complete inactivation
PET plates
0
0 30 60 90 120 150
Treatment time (sec)
charged species, should have played important role in inactivating the E. coli [22]
along with photons. Since the charged species are not in direct contact with E. Coli
coated in the effluent region of the tube, their influence in inactivation of E. Coli in
this region is very less. Hence in this (effluent) region, photons produced in the
plasma and in the plasma effluent could have played important role. Also, it is con-
sidered that argon metastables played important role in inactivating the E. coli both
in active plasma region as well as in effluent region of treatment [23].
For the E. coli inhibition studies and the B. atrophaeus inactivation studies,
samples are placed in atmospheric air. These samples are treated with plasma efflu-
ent. Hence, the contribution of charged species for the inactivation will be very less
compared to the inactivation of E. coli coated tube through direct plasma. Argon
plasma, at atmospheric pressure conditions, emits photons mainly in Vacuum
Ultraviolet (VUV) and near infrared (NIR) spectral regions. The photons in NIR
region do not have enough energy to inactivate the microorganism. The VUV pho-
tons produced, as shown in the Scheme 4.1, in plasma and effluents should have
played important role in inactivation. In addition to this, oxygen in air, which is in
contact with the effluent, should have played important role as shown in the
Scheme 4.1,
The argon excimer (Ar2*) in triplet state has enough life time to reach air,
which is occupied between the sample and the exit point of plasma. This excimer
will produce VUV photons (wavelength, ~128 nm) through spontaneous emission.
This photon itself has enough energy to inactivate the microorganism, especially
E. coli, through photochemical reaction. In addition to this, this photon can dis-
sociate oxygen molecules into two oxygen atoms. This atomic oxygen can react
with microorganism through oxidation reaction and inactivate the microorgan-
isms, E. coli and B. atrophaeus [24]. The atomic oxygen can also react with
molecular oxygen to produce ozone molecule. The produced ozone molecule can
also inactivate the microorganism through oxidation reaction. By these ways,
microorganisms placed in atmospheric air could have decontaminated in the argon
Scheme 4.1 Plasma

chemical reactions involved
in the production of argon
excimer, VUV photons,
atomic oxygen and ozone
molecules
plasma effluent. Systematic experimental analysis of inactivation mechanism in

our plasma source is under the progress.
4.4 Summary
Atmospheric pressure pulsed filamentary plasma source with argon working gas is
used for the decontamination studies. The efficiency of our plasma source for the
decontamination on inner surface of the tube as well as on objects placed in proxim-
ity of plasma effluent is studied. E. coli and spores of B. atrophaeus are used for the
decontamination studies. Inhibition studies of E. coli on agar plates are also carried
out. It shows that plasma effluent generated in our plasma source is very effective
for the inhibition of bacterial colonization. Decontamination studies of B. atropha-
eus endospores, which are layered on PET polymer material and placed in the prox-
imity of plasma effluent, show the mean logarithmic spores reduction of about 3.7
(complete inactivation) for the treatment time of 120 s. Decontamination studies of
E. coli coated on inner surface of the tube show the mean logarithmic bacterial
reduction of about 5 for the treatment time of 30 s. As a part of inactivation mecha-
nistic study, plasma is characterized using optical emission spectroscopy, current
voltage measurements, numerical simulations and microphotography. During the
characterization of plasma, nitrogen is admixed with argon and its emission is used
for determination of gas temperature, EVDF and electron density. Electron density
is in the order of 1011 cm3. The reduced electric field is in the range of 9001,700
Td. At this condition, argon ionization reaction is more probable than the argon
metastable formation reaction.
References
1. Zhang X, Li M, Zhou R, Feng K, Yang S (2008) Ablation of liver cancer cells in vitro by a
plasma needle. Appl Phys Lett 93:021502
2. Uhm HS, Lim JP, Li SZ (2007) Sterilization of bacterial endospores by an atmospheric-
pressure argon plasma jet. Appl Phys Lett 90:261501
4. Vargo JJ (2004) Clinical applications of the argon plasma coagulator. Gastrointest Endosc
59:8188
5. Nosenko T, Shimizu T, Morfill GE (2009) Designing plasmas for chronic wound disinfection.
New J Phys 11:115013
6. Laroussi M, Alexeff I, Richardson JP, Dyer FF (2002) The resistive barrier discharge. IEEE
Trans Plasma Sci 30:158159
7. Lee MH, Park BJ, Jin SC, Kim D, Han I, Kim J, Hyun SO, Chung K-H, Park J-C (2009)
Removal and sterilization of biofilms and planktonic bacteria by microwave-induced argon
plasma at atmospheric pressure. New J Phys 11:115022
8. Fridman G, Brooks AD, Balasubramanian M, Fridman A, Gutsol A, Vasilets VN, Ayan H,
Friedman G (2007) Comparison of direct and indirect effects of non-thermal atmospheric-
pressure plasma on bacteria. Plasma Processes Polym 4:370375
spores using cold atmospheric plasmas. IEEE Trans Plasma Sci 34:13101316
10. Machala Z, Chldekov L, Pelach M (2010) Plasma agents in bio-decontamination by dc dis-
charges in atmospheric air. J Phys D Appl Phys 43:222001
11. Daeschlein G, Tv W, Kindel E, Brandenburg R, Weltmann K-D, Jnger M (2010) Antibacterial
activity of an atmospheric pressure plasma jet against relevant wound pathogens in vitro on a
mimulated wound environment. Plasma Processes Polym 7:224230
12. Pothiraja R, Bibinov N, Awakowicz P (2010) Pulsed corona plasma source characterization for
film deposition on the inner surface of tubes. J Phys D Appl Phys 43:495201
13. Bibinov N, Halfmann H, Awakowicz P, Wiesemann K (2007) Relative and absolute intensity
calibrations of a modern broadband echelle spectrometer. Meas Sci Technol 18:13271337
14. Bibinov NK, Fateev AA, Wiesemann K (2001) On the influence of metastable reactions on
rotational temperatures in dielectric barrier discharges in He-N2 mixtures. J Phys D Appl Phys
34:18191826
15. Nguyen TD, Sadeghi N (1983) Rotational and vibrational distributions of N2(C 3Pu) excited by
state-selected Ar(3P2) and Ar(3P0) metastable atoms. Chem Phys 79:4155
16. Stefanovc I, Bibinov NK, Deryugin AA, Vinogradov IP, Napartovich AP, Wiesemann K
(2001) Kinetics of ozone and nitric oxides in dielectric barrier discharges in O2/NOx and N2/O2/
NOx mixtures. Plasma Sources Sci Technol 10:406416
17. Itikawa Y (2006) Cross sections for electron collisions with nitrogen molecules. J Phys Chem
Ref Data 35:3153
18. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, vol 13, 3rd edn.
Cold Spring Harbor Laboratory Press, New York
19. Avramidis G, Stwe B, Wascher R, Bellmann M, Wieneke S, von Tiedemann A, Vil W (2010)
Fungicidal effects of an atmospheric pressure gas discharge and degradation mechanisms. Surf
CoatTechnol 205:S405S408
20. Binkley RW, Binkley WW (1972) Photochemical reactions of carbohydrates. Carbohydr Res
23:283288
21. Sobottka A, Drler L, Lenk M, Prager L, Buchmeiser MR (2010) An open argon dielectric
barrier discharge VUV-source. Plasma Processes Polym 7:650656
22. Seo YS, Mohamed A-AH, Woo KC, Lee HW, Lee JK, Kim KT (2010) Comparative studies of
atmospheric pressure plasma characteristics between He and Ar working gases for steriliza-
tion. IEEE Trans Plasma Sci 38:29542962
23. Xu G, Zhang G, Shi X, Ma Y, Wang N, Li Y (2009) Bacteria inactivation using DBD plasma
jet in atmospheric pressure argon. Plasma Sci Technol 11:8388
24. Lim J-P, Uhma HS, Li S-Z (2007) Influence of oxygen in atmospheric-pressure argon plasma
jet on sterilization of Bacillus atrophaeous spores. Phys Plasmas 14:093504
Chapter 5
The Fungal Spores Survival Under
the Low-Temperature Plasma
Hana Soukov, V. Scholtz, J. Julk, and D. Savick
Abstract This paper presents an experimental apparatus for the decontamination

and sterilization of water suspension of fungal spores. The fungicidal effect of sta-
bilized positive and negative corona discharges on four fungal species Aspergillus
oryzae, Clacosporium sphaerospermum, Penicillium crustosum and Alternaria
sp. was studied. Simultaneously, the slower growing of exposed fungal spores was
observed. The obtained results are substantially different in comparison with those
of the analogous experiments performed with bacteria. It may be concluded that
fungi are more resistant to the low-temperature plasma.
5.1 Introduction
There are numerous works describing the biological effects of low-temperature

plasma generated in electrical discharges, devoted mainly to the killing of prokary-
otic bacteria (see reviews [13]) or various applications in human medicine [4]. In
regard to fungi, only a preliminary announcement exists in the publication [5], from
H. Soukov (*)
Department of Computing and Control Engineering, Institute of Chemical Technology in Prague,
Technick 5, 166 28, Praha, Czech Republic
e-mail: hana.souskova@vscht.cz
V. Scholtz
Department of Physics and Measurements, Institute of Chemical Technology in Prague
Technick 5, 166 28 Praha, Czech Republic
J. Julk
Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University in
Prague, Studnikova 7, 128 00 Praha, Czech Republic
D. Savick
Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology,
Institute of Chemical Technology in Prague, Technick 5, 166 28 Praha, Czech Republic
58 H. Soukov et al.
which results the growth inhibition of Penicillium digitatum after two days long
corona discharge exposure, and there is an Akishevs [6] mention of the inactivation
of Aspergillus niger and Candida lipolytica on agar surface after exposure to the
plasma jet device. Soukov [7] studied the decontamination of water suspension of
fungal spores by corona discharge in detail. The possible application of plasma
sterilization may be useful e.g. for the treatment of fruits surface, preventing the
mould overgrow and bacterial putrefaction. Another possible motivation for search-
ing for the decontamination methods based on low-temperature plasma is e.g. the
subvention of The European Union for the application of low-temperature plasma
to the superficial decontamination of table eggs in connection with its plan of hen
breeding transformation, more details may be found in [8] and [9].
The mechanisms of the cells inactivation, using corona discharge, are not reliably
determined and are still open for discussion; the research in this field is advancing.
The synergic action of reactive and/or charged particles, as NOX, atomic oxygen O,
singlet oxygen 1O2, ozone O3, superoxide anion O2, hydroxyl radical OH etc.,
surrounding the cells, appears to be the most probable cause of cell wall or cell
membrane damage. To some extent, rising UV radiation can also bear certain impact.
We did not follow the presence of these particles, described in detail in, e.g. [10].
5.2 The Method of Research and Microorganisms Used
In the previous work of Scholtz et al. [11], the decontamination features of corona
discharge on the water suspension of different types of bacteria were studied (cocci,
rod shaped, gram-positive, gram-negative, extremophile). The total inactivation of
all types of bacteria by corona discharge occurred after 4 min.
The main aim of this work was to verify the devitalisation sensitivity of eukary-
otic microorganisms (fungi) to low-temperature plasma as a basic presumption of
the use of the non-thermal plasma for decontamination, and eventually sterilisation.
The other aim of the work was to compare the two types of corona discharge and to
compare their known effects on bacteria and their effects on micromycete spores.
The tolerance of four different non-pathogenic micromycetes from four different
genera was studied. The fungal species were obtained from The Department of
Biochemistry and Microbiology (DBM), Institute of Chemical Technology in
Prague. The genera Penicillium, Cladosporium and Alternaria massively occur in
the environment during summer time and can cause many allergic reactions.
1. Penicillium crustosum (DBM 4159)
2. Aspergillus oryzae (DBM 4002)
3. Cladosporium sphaerospermum (DBM 4282)
4. Alternaria sp. (DBM 4004)
The low temperature plasma was generated using the previously described sim-
ple apparatus of an open-air type [12]. The discharge burns on the point electrode,
represented by the tip of a syringe needle connected with a serial resistance of
5 The Fungal Spores Survival Under the Low-Temperature Plasma 59
Decontamination
High
Inoculation
voltage
20 M
corona Cultivation
Resistor
sample Evaluation
Fig. 5.1 The block diagram of the method used. The experimental arrangement of inactivation of
microbial suspension
20 MW to the positive or the negative pole of direct current high voltage supply.
The ground electrode was realized by the surface of water suspension of microor-
ganisms connected with an immersed platinum wire to the negative or the positive
pole of the supply. The discharge voltage was set to 9.7 kV and the distance
between the tip of the needle and water surface was adjusted to cca 3 mm to
get the discharge current of 400 mA. Due to the serial resistance in used apparatus,
the positive point-to-plane discharge burns in the regime of transient spark corona
see e.g. [13], the negative point-to-plane discharge burns in the regime of glow
corona discharge, see e.g. [14]. Some electric characteristic of used discharges are
described in [15].
All exposures were performed under laminar flow of HEPA-filtered air to prevent
the airborne contamination; the air-conditioning of the laboratory controlled the
ambient conditions.
The stock suspensions of fungal spores were prepared immediately before the
exposure to the discharge. The fungal spores were harvested using a bacteriological
loop and suspended in sterile physiological saline. Figure 5.1 shows the arrange-
ment of experiments. The 0.5 ml of the suspension was pipetted into the sterile
wells of a dot plate and exposed to the positive and the negative corona discharge
for time intervals which varied from 5 to 30 min. Following the exposure, the con-
tent of each well was diluted, spread onto the surface of YGC agar (Yeast, Glucose,
Chloramfenicol) and after 34 day cultivation at 24C, the number of the survived
colonies (CFU colony forming units) was counted. The initial concentrations of
the suspensions were also determined by the cultivation method under the hereinbe-
fore conditions.
Every result was confirmed by another two experiments. The uncertainty is pre-
sented as error bars in graphs.
Finally, to compare the growth of exposed and non-exposed spores following
experiment was done. From the stock suspension, 0.5 ml was pipetted into sterile
wells and exposed for 15 min to the positive corona discharge. Subsequently, the
exposed and non-exposed spores were cultivated under the above-mentioned condi-
tions. In regular 6-h intervals, the growing and sporulation were observed.
60 H. Soukov et al.
5.3 Results
The effect of both types of corona discharges on spores of Cladosporium sphaero-

spermum was found to be approximately the same. The number of surviving cells
decreased from the initial concentration of 106 cfu ml1 in the first 10 min of expo-
sure to 105 cfu ml1 and then rapidly declined to total inactivation within the next
10 min. The spores were totally inactivated by both the negative and the positive
corona discharge in 20 min. See Fig. 5.2.
The effect of both types of corona discharge on spores of Alternaria sp. seems to
be approximately the same, except the total inactivation of the spores occurred
already after 15 min. See Fig. 5.3.
Cladosporium sphaerospermum
1E+07
negative
1E+06 1500000
276000
concentration [cfu/ml]
250000 positive
1E+05 114000
90000
1E+04
1E+03
1E+02 100
1E+01 10
1E+00
0 5 10 15 20 25 30
exposure time [min]
Fig. 5.2 The number of survived C. sphaerospermum spores after exposure to the corona
discharge
Alternaria sp.
1E+07
1480000 940000 negative
1E+06 438000
positive
386000
1E+05 314000
52000
1E+04
7100
2000
1E+03
1E+02
22
1E+01
2
1E+00
0 5 10 15 20
exposure time [min]
Fig. 5.3 The number of survived Alternaria sp. spores after exposure to the corona discharge
Penicillium crustosum
1E+05
negative
12500 7600 positive
1E+04 4900
1E+03 2500 4600

790
500
1E+02
40
1E+01
1E+00
0 5 10 15 20 25 30
exposure time [min]
Fig. 5.4 The number of survived P. crustosum spores after exposure to the corona discharge
Aspergillus oryzae
1E+06
negative
136000
1E+05 28000 positive
25000
1E+04
1900
8000
1E+03
600
1E+02 108
1E+01
1E+00
0 5 10 15 20 25 30
exposure time [min]
Fig. 5.5 The number of survived A. oryzae spores after exposure to the corona discharge
The total inactivation of Penicillium spores occurred after 25 min, but only by the
impact of positive corona discharge. The negative corona discharge did not cause
the total inactivation even after 30 min of exposure. The positive corona discharge
has a greater effect than the negative one; it is shown in Fig. 5.4.
The fungal spores of Aspergillus oryzae appeared to be the most resistant in
comparison to the other species of fungi. The number of the survived spores fell by
three orders of magnitude, but the total inactivation did not occur even after 30 min
of exposure. Both types of corona discharge showed a comparable low effect of
devitalisation. It is shown in Fig. 5.5.
The dynamics of micromycete growth after exposure to a sublethal dose of
plasma was interesting: the surviving exposed fungal spores grew visibly slower in
62 H. Soukov et al.
Fig. 5.6 The dynamics of micromycete growth and sporulation of exposed and non-exposed
fungal spores (A. Orzyae, P. crustosum and C. sphaeerospermum)
comparison with the non-exposed ones (see Fig. 5.6). The first colonies appearance
in exposed micromycetes was delayed by 35 h for Cladosporium, by 45 h for
Aspergillus and even by 65 h with for Penicillium as compared with the non-exposed
cultures. On the other hand, the time interval between growth appearance and
sporulation was shortened substantially in cultures of the spores exposed to dis-
charge. The numbers of fungal colonies on the agar surface were in each case
approximately 50 cfu ml1, so that the above-mentioned differences cannot be attrib-
uted to different growing conditions. The micromycetes exposed (A. orzyae, P. crus-
tosum and C. sphaeerospermum) sporulated faster than the non-exposed ones. In the
previous works about bacteria and yeast [11], no similar effects were observed.
The dynamics of growth of Alternaria sp. is shown in Fig. 5.7. While all the non-
exposed spores germinated within 72 h, the exposed ones germinated even after 134 h
after inoculation. The fungus Alternaria sp. sporulated very fast and no delay in
sporulation between non-exposed and exposed but survived spores were observed.
The aim of Fig. 5.7 is to present only the time delay between the sporulation of
the exposed and unexposed spores. It would be very difficult to gain the same initial
number of CFU on the both Petri dishes; nevertheless, their numbers were very
close and do not affect the growth. From this graph, it is possible to derive the
hypothesis, that after exposure to a sublethal dose of plasma, some spores are killed,
some survive without harm and some are partially harmed and can germinate again
after revitalisation. The germination of the partially harmed spores, however, took
longer time than the germination of the intact or unexposed spores.
Alternaria sp.
140
120
100
80
CFU
60
40
20
0
72 134
0 20 40 60 80 100 120 140
Time of growth [hours]
Nonexposed spores Exposed spores
Fig. 5.7 Dynamics of growth of Alternaria sp. spores
5.4 Discussion
Comparing the results presented here with those obtained with bacteria, it may be
concluded that bacteria are more susceptible to the low-temperature plasma than
fungi. Using the less effective negative corona discharge, the complete inactivation
of Escherichia coli and Staphylococcus aureus was achieved after two and 4 min of
exposure, respectively [10]. The bacterial spores of Geobacillus stearothermophilus
seem to be also more susceptible to the exposure than the fungal ones [2].
The higher resistance of fungi is probably caused by the encapsulation of fungal
spores, in comparison with the unprotected cell membrane of vegetative bacteria.
The fungal cell wall is composed of a polysaccharide-based three-dimensional
network. The construction of the common central core of the cell wall is composed
mainly of branched b-1,3-glucan and chitin. Genomic as well as drug studies have
shown that the death of the fungus can result from the inhibition of the syntheses of
cell wall polysaccharide.
According to [16], the cell wall is now seen as a dynamic structure that is
continuously changing as a result of the modification of culture conditions and
environmental stresses.
Because of its essential biological role, unique biochemistry and structural
organization and the absence in mammalian cells of most of its constitutive compo-
nents, the cell wall is an attractive target for the development of new antifungal
agents. Inhibition of the fungal cell synthesis, when the cell integrity pathway is
64 H. Soukov et al.
affected, evokes compensatory reactions. This leads to an increase in one component

in fungal cell while the synthesis of another component is perturbed.
Such compensatory reactions explain why antifungal therapy with cell wall
inhibitors is difficult, but also reflect the immense dynamics of the synthesis of cell
wall components. This is a possible explanation of the effect of the stepwise mecha-
nisms of spores inactivation with partially reversible disruptions in the structure of
spores, which retards or inhibits the germination.
The fungicidal effect of the low-temperature plasma generated by the positive
and negative corona discharge differs for various species. The total inactivation of
fungal spores occurs in 15 min with Alternaria sp. and in 20 min with Cladosporium
sphaerospermum, whereas the Aspergillus oryzae spores were not completely inac-
tivated even after 30 min of exposure. The spores of Penicillium crustosum have
different susceptibility to various corona discharges.
For now we cannot explain these differences, neither between positive or negative
corona treatments, nor among particular genera. The variable microbicidal efficiency
of various discharge types was previously documented but not explained [17]. It is
probably caused by the different nature of reactive particles produced by particular
discharge type, which have not been determined in detail yet.
Different susceptibility to plasma treatment was previously observed also for
bacteria and fungi, namely Escherichia coli, Staphylococcus aureus and Candida
albicans. [11]. These differences, however, were found to depend on experimental
conditions: during exposition on the surface of agar media, C. albicans appeared to
be the most sensitive, whereas the comparable susceptibility of bacteria was some-
what lower. On the contrary, if exposition was conducted in aqueous suspensions,
the Gram-negative E. coli was inhibited within 2 min and the Gram-positive
S. aureus within 4 min, whereas 30 min was necessary to inhibit the yeast C. albi-
cans. This suggests, that the sensibility is not governed by the composition of cell
envelope: if so, the Gram-positive bacteria with a thick cell wall should be the most
resistant, the Gram-negative ones intermediately and the yeast lacking the cell wall
should be the most susceptible.
Concerning fungi, the influence if different cell wall composition cannot be
excluded, but the only trend observable so far, is related to the colouring of spores:
the darker is the spores colour, the higher is its susceptibility to corona discharges.
The spores of Cladosporium and Alternaria were black coloured and the total inac-
tivation after the exposure to both the types of discharge occurred within 15 or
20 min respectively. The spores of Penicillium were grey-green coloured and the
total inactivation occurred in 25 min, but only by the impact of positive corona dis-
charge. The spores of Aspergillus were yellow and the total inactivation did not
occur even after 30 min of exposure by both the discharge types. This finding, how-
ever, contradicts to the statements of microbiologists, who consider the dark dyes
and pigments as the protective elements of fungal spores.
The exposure to plasma retards the consecutive fungal growth: while the
non-exposed spores grow within 4570 h, the exposed ones need 80135 h. This
stress also enhances the sporulation in cultures grown from exposed spores. The
possible explanation of this phenomenon is the stress or damage of fungal spores
caused by the low-temperature plasma, acting either on the envelope or the genome
of exposed spores, the reparation of which needs some time as compared with the
unexposed ones. The shorter time between the growth and sporulation of exposed
spores may be explained by the effects of stress on the spores which consider the envi-
ronment as unfavourable and by the quick sporulation try to change the living place.
5.5 Conclusion
The article presents the results of the study of fungicidal effects of negative and
positive stabilized corona discharge. The results show that the low-temperature
plasma created by corona discharge decreases the number of the survival spores
with all the fungal species tested. A 10-min-long exposure did not lead to any sig-
nificant decrease of their number, which declined approximately by one decade.
After a longer exposure, the decrease of the survival spores was more significant
and the differences among the species were perceptible.
The positive and negative corona discharges show almost the same impact on
Cladosporium sphaerospermum and Alternaria sp., whereas somewhat different
impact was observed for Penicillium crustosum and partially for Aspergillus oryzae.
We are not able to explain these differences so far.
In general, the fungal spores showed higher resistance to corona discharge expo-
sure than other microorganisms. So in possible application of low-temperature
plasma decontamination the test of fungal spores can not be omitted.
The higher effectiveness of positive corona discharge was proved with bacteria
and yeast, but this phenomenon was not confirmed with fungal spores.
The method used is in the stage of a fundamental research and its application to
larger samples or its practical use requires further experiments.
Acknowledgments This work has been supported by grants No. MSM R 6046137306, MSM
R 0021620806 and SVV-2010-26 0506.
References
1. Laroussi M (2005) Low temperature plasma-based sterilization: overview and state-of-the-art.

2. Scholtz V, Julk J, Kha V, Mosinger J (2008) Decontamination effects of low-temperature
plasma generated by corona discharge. Part I: an overview. Prague Med Rep 108:115127
3. Moreau M, Orange N, Feuilloley MGJ (2008) Non-thermal plasma technologies: new tools for
bio-decontamination. Biotechnol Adv 26:610617
4. Fridman G, Friedman G, Gutsol A, Shekhter AB, Vasilets VN, Fridman A (2008) Applied
plasma medicine. Plasma Processes Polym 5:503533
5. Scholtz V (2005) Corona discharge influence on micro-organisms. Probl Atomic Sci Technol
10(1):190191
66 H. Soukov et al.
6. Akishev Y, Grushin M, Karalnik V, Trushkin N, Kholodenko V, Chugunov V, Kobzev E,

Zhirkova N, Irkhina I, Kireev G (2008) Atmospheric-pressure, nonthermal plasma sterilization
7. Soukov H (2010) Psoben kornovho vboje na spory mikromycet. Bachelors theses,
Institute of Chemical Technology in Prague
8. Vannini L, Montanari C, Berardinelli A, Ragni L, Sirri F, Guerzoni ME (2009) Assessment of
the efficacy of a low-temperature gas plasma prototype for superficial decontamination of table
eggs. In: Proceedings of WPSA XIX European symposium on the quality of poultry meat,
Finland, pp 18
9. Berardinelli A, Ragni L, Vannini L, Montanatri C, Sirri F, Guarniery A, Guerzoni ME (2009)
Atmospheric-pressure gas plasma for decontamination of food products. In: Proceedings of
XXXIII CIOSTA CIGR V conference 2009, Reggio di Calabria, pp 7377
10. Chang J-S, Lawless PA, Yamamoto T (1991) Corona discharge processes. IEEE Trans Plasma
Sci 19:11521166
11. Scholtz V, Julk J, Kha V (2010) The microbicidal effect of low-temperature plasma gener-
ated by corona discharge: comparison of various microorganisms on an agar surface or in
aqueous suspension. Plasma Processes Polym 7:237243
12. Julk J, Kha V, Scholtz V (2006) Corona discharge: a simple method of its generation and
study of its bactericidal properties. Czech J Phys 56:B1333B1338
13. Machala Z, Chldekov L, Pelach M (2010) Plasma agents in bio-decontamination by dc dis-
charges in atmospheric air. J Phys D Appl Phys 43:222001
14. Akishev YS, Grushin ME, Kochetov IV, Napartovich AP, Pankin MV, Trushkin NI (2000)
Transition of a multipin negative corona in atmospheric air to a glow discharge. Plasma Phys
Rep 26:172178
15. Hork P, Khun J (2010) Impedance-stabilized positive corona discharge and its decontamina-
tion properties. J Phys Conf Ser 223:012006
16. Latg JP (2007) The cell wall: a carbohydrate armour for the fungi cell. Mol Microbiol
66(2):279290
17. Scholtz V, Kommov L, Julk J (2011) The influence of parameters of stabilized corona
discharges on its germicidal effects. Acta Phys Pol A 119:803806
Chapter 6
Plasma-Liquid Interactions: Chemistry
and Antimicrobial Effects
Thomas von Woedtke, Katrin Oehmigen, Ronny Brandenburg, Tom Hoder,

Christian Wilke, Marcel Hhnel, and Klaus-Dieter Weltmann
Abstract Plasma-induced inactivation of bacteria in aqueous liquids is supported

by acidic pH and accompanied by generation of low-molecular chemical species
which are detected as nitrate, nitrite and hydrogen peroxide. To get more insight
into mechanisms of change of liquid composition by plasma treatment as well as
transmission of bactericidal plasma effects into aqueous liquids, bactericidal effects
under different plasma-treatment conditions and liquid analytics are combined with
theoretical considerations to focus possible reaction channels of plasma-water
interactions.
6.1 Introduction
At the present state of knowledge, inactivation of bacteria in aqueous liquids by

plasma treatment is accompanied by several changes of liquid composition which
are more or less necessary to enable or to support antimicrobial effects. Under atmo-
spheric air conditions, acidification as well as generation of low-molecular chemical
species which are detectable as nitrate (NO3), nitrite (NO2) and hydrogen peroxide
(H2O2) has been identified as results of plasma-liquid interaction. It was also dem-
onstrated recently that acidification alone is not to induce comparable bactericidal
efficacy independent on plasma treatment [15].
T. von Woedtke (*) K. Oehmigen R. Brandenburg T. Hoder C. Wilke M. Hhnel

K.-D. Weltmann
e-mail: woedtke@inp-greifswald.de; weltmann@inp-greifswald.de
68 T. von Woedtke et al.
Fig. 6.1 Possible reaction channels of plasma/gas-liquid interactions [5]
Based on plasma diagnostics as well as gas-phase and liquid analytics, a general

and wide-range spectrum of possible chemical reactions can be hypothesized
(Fig. 6.1). But, it is not known in detail which reactions are playing dominating or
supporting roles for acidification, chemical species generation and, last but not least,
bacteria inactivation. The intention of the work presented here is to contribute to this
ongoing discussion and to focus in particular the question of liquid acidification and
its role for bacteria inactivation.
6.2 Experimental
Plasma treatment of liquids was realized using a surface dielectric barrier discharge
(surface-DBD) arrangement which was described in detail elsewhere [46]. Plasma
is generated on the surface of an electrode array. For liquid treatment in atmospheric
air conditions, this array was mounted by a special construction into the upper shell
of a Petri dish (60 mm diameter) in that way that a constant distance of 5 mm
between the high-voltage electrode surface (35 mm diameter; surface area 9.6 cm2)
and the surface of a liquid sample in the lower shell of the Petri dish (55 mm diam-
eter; surface area 23.8 cm2) can be adjusted (Fig. 6.2, left).
For liquid treatment in argon atmosphere at atmospheric pressure conditions, the
electrode array is integrated into a special housing which was constructed in that
way that it can be mounted on the lower shell of a 55-mm petri dish forming a reaction
6 Plasma-Liquid Interactions: Chemistry and Antimicrobial Effects 69
Fig. 6.2 Experimental set-up of surface dielectric barrier discharge arrangements for liquid treat-
ment in atmospheric air (left) and in argon atmosphere (right)
chamber sealing off the outside (Fig. 6.2, right). By a special gas supply channel,
this chamber can be flooded with argon gas (gas flow 0.5 slm). Corresponding to the
other experimental setup (Fig. 6.2, left), a constant distance of 5 mm between high-
voltage electrode surface and surface of liquid sample can be adjusted. As power
source a commercial Fourier synthesis pulse generator was used. In all experiments
at ambient air conditions, a pulsed sinusoidal voltage of 10 kVpeak (20 kHz) with a
0.413/1.223 s plasma-on/plasma-off time was used. For experiments in argon atmo-
sphere, a pulsed sinusoidal voltage of 3 kVpeak (40 kHz) with a 0.413/1.223 s plasma-on/
plasma-off time was used.
As test liquids, distilled water as well as sodium chloride solution (NaCl solu-
tion; NaCl 0.85%; 8.5 g NaCl per 1,000 ml water) have been used. No outgassing
procedure was performed before plasma treatment of liquids.
As test microorganism Escherichia coli NTCC 10538, kindly provided by
Institute of Hygiene and Environmental Medicine, Ernst Moritz Arndt University
Greifswald, Germany, was used. Overnight culture of E. coli was diluted using NaCl
solution, to get concentrations of 107108 colony forming units per milliliter (cfu ml1;
stock suspension).
For plasma treatment of liquids containing suspended microorganisms, 5 ml of
E. coli stock suspension were treated with the DBD plasma for different times up to
30 min. The number of viable microorganisms (cfu ml1) was estimated by the
surface spread plate count method using aliquots of serial dilutions of microorgan-
ism suspensions in NaCl solution according to the European Pharmacopoeia.
Detection limit of this procedure was 10 cfu ml1. Serial dilution of microorganism
suspensions served also as an effective procedure to neutralize the bactericidal
activity of reactive species contained in the plasma-treated liquid.
Inactivation kinetics of microorganisms is depicted in semi-logarithmic plots.
If the number of microorganisms fell below the detection limit, i.e. no viable
microorganisms have been found, for clearness these values in the graphs are set at
5 cfu ml1. Three trials (mean of n = 2 each) were done with similar concentrations
of E. coli suspensions (107108 cfu ml1). The kinetics of these three trials showed
the same inactivation effects for E. coli.
For plasma treatment of aqueous liquids without bacteria, 5 ml of distilled water
or sodium chloride solution (0.85%) were used.
pH measurements were done using a microprocessor pH meter pH 196
(WTW, Weilheim, Germany; measuring range/precision: 0.0014.00/ 0.01) with
a semi-micro pH-electrode (4.5 mm diameter; SENTEK P13, Sentek Ltd., UK).
For photometric measurements, UV/VIS Spectrophotometer SPECORD S 600
(analytic jena GmbH, Jena, Germany) was used. The photometer has an accuracy
of measurement of 0.5 nm in the UV range and 0.3 nm in the VIS range. The
spectral resolution is 1.21.3 nm. Nitrite and nitrate concentrations were estimated
by color forming reactions using commercially available test kits (Spectroquant,
Merck). Nitrite reacts with sulfanilic acid and N-(1-naphthyl)-ethylen diamine
hydrochloride via azo sulfanilic acid to a magenta colored azo dye whose absorp-
tion at 525 nm was measured. Nitrate reaction with 2,6-dimethylphenol gives,
after a reaction time of 10 min 4-nitro-2,6-dimethylphenol, an orange colored
product, whose absorption was measured at 340 nm. Hydrogen peroxide detection
based on the reaction of titanyl sulfate to yellow-colored peroxotitanyl sulfate,
which was detected at 405 nm.
For direct photometric analysis, total absorption spectra have been recorded from
200 up to 1,000 nm.
According to the actual state of knowledge, biological as well as chemical plasma

effects in liquids are a result of complex interactions at the plasma/gas-liquid interface
and subsequent reactions in the liquid volume. As it was demonstrated recently,
inactivation of bacteria suspensions by surface-DBD in an atmospheric air environ-
ment was possible in non-buffered liquids, only, because acidification seems to be a
necessary but not sufficient precondition for antibacterial effectivity [2, 4]. Even, if
some ideas of possible mechanisms of action have been discussed already, it is not
clear yet which reaction mechanisms are really responsible both for acidification
and antibacterial activity.
As another result of surface-DBD treatment of liquids in atmospheric air, con-
tinuously increasing H2O2 and NO3 concentrations as well as temporarily increas-
ing NO2 concentrations dependent on treatment time have been found [4].
Consequently, it was concluded that low-molecular nitrogen as well as oxygen-
containing chemical species should play a key role for these effects. The short-term
increase of nitrite as well as its relatively low concentration compared to nitrate in
parallel to decreasing pH dependent on plasma treatment time could be explained
by the fact that, under acidic conditions, a disproportionation of HNO2 into HNO3
and nitric oxide (NO) takes place which is generally accelerated by heating and
concentration increase [7]:
3HNO2 HNO3 2 NO H2O (6.1)
8,0
7,0
6,0
5,0
pH
4,0
3,0
2,0 pH in water (argon)

pH in water (air)
1,0
pH in NaCl solution (air)
0,0
0 5 10 15 20 25 30
treatment time [min]
Fig. 6.3 Change of pH in 5 ml water dependent on surface-DBD plasma treatment time in argon
atmosphere () and in atmospheric air (), and sodium chloride solution in atmospheric air () [5]
Because no liquid temperature increase was measured resulting from plasma

treatment, in this system this reaction should be dependent on concentration. Based
on these facts it was assumed that nitric acid (HNO3) is the main cause for acidifica-
tion. This hypothesis was based additionally on a comparison of molar nitrate con-
centrations and molar concentrations of acidity-causing protons (H+) deductible
from pH. Both concentrations were found to be equimolar [4]. An identical line of
argument was used by Ikawa et al. to conclude that acidification by plasma expo-
sure is caused by the dissolution of nitrogen oxides generated from ambient air by
the plasma [2].
However, HNO3 is a strong acid with a pKa of 1.3 [7]. That means that continu-
ously increasing generation of nitric acid must result in continuously decreasing pH
dependent on plasma treatment time. However, measurements of pH in water as
well as NaCl solution resulted in a stabilization of pH around 3 within 30 min
surface-DBD treatment under atmospheric air conditions (Fig. 6.3).
Consequently, if HNO3 is really responsible for acidification, liquids should, in
fact, become much more acidic than actually observed. On the other hand, for
HNO2, pKa values between 3.2 and 2.8 are given in literature [811]. This means,
that nitrous acid below a pH value of about 3 for the most part is undissociated, i.e.
protons are bounded on nitrite and, consequently, a further pH decrease is not pos-
sible even if more HNO2 is added or generated. This corresponds to the measure-
ments demonstrated in Fig. 6.3.
Consequently, in contrast to the hypothesis that acidification of plasma-treated
liquids is caused mainly by HNO3 generation, the arguments presented here give
rise to the assumption that HNO2 is playing the lead in this process.
This was supported by another test where both distilled water and NaCl solution
have been treated for 30 min by surface-DBD plasma in atmospheric air, and
Fig. 6.4 Absorption spectra of nitrous acid (- - -), of 30 min plasma treated water (. . .) and of 30 min
plasma treated sodium chloride (NaCl) solution ( ) [5]
UV-VIS spectra of the liquids were recorded in the range between 200 and 1,000 nm
(Fig. 6.4). Absorption maximum of nitrate or nitric acid, respectively, has been
found at 305 nm [5]. In plasma-treated liquids, a distinct absorption maximum was
found at 227 nm which has been shown to be identical to the absorption maximum
of a nitrous acid (HNO2) solution at pH 2.8 which is another indication for the pre-
dominating role of HNO2 for acidification. However, at least partial occurrence of
nitrate cannot be excluded completely, particularly because nitrite partially will dis-
proportionate at higher concentrations (see Eq. 6.1). Moreover, nitrate has another
absorption maximum at 220 nm, and therefore a partial superimposition of the
227 nm peak by traces of nitrate is possible.
Nevertheless, this cannot explain the high nitrate compared to nitrite concentra-
tions in 5 ml liquid detected after 30 min surface-DBD treatment under atmospheric
air conditions as reported in our recent study [4].
The detection of nitrate was based on a color forming reaction whereby nitrate
reacts with 2,6-dimethylphenol to form the orange colored 4-nitro-2,6-dimethylphenol
within a reaction time of 10 min:
(6.2)
To use this reaction to detect nitrate concentration it must be a quantitative

reaction, i.e. complete content of nitrate in the liquid sample investigated must react
with the indicator substance. But, as mentioned already, under acidic conditions a
disproportionation of NO2 into NO3 takes place (see Eq. 6.1).
If NO3 as the product of this reaction is consumed by an indicator, the equilib-
rium of this reaction is moving to this product with the result that by and by the
complete nitrite is transformed into nitrate resulting in high nitrate concentrations as
detected. Because nitrite and nitrate have been measured in different aliquots of the
plasma-treated liquid, this transformation of nitrite forced by the analytics of nitrate
was not noticed. Only with direct UV-VIS spectrometry of the liquids (see Fig. 6.4)
without color forming and analyte consuming reactions this artifact can be
avoided.
Moreover, from a chemical point of view, also a direct reaction of the NO2 radi-
cal with the phenol ring of 2,6-dimethylphenol is conceivable:
(6.3)
NO2 is a result of reaction of NO evolving from disproportion of HNO2 into

HNO3 (see Eq. 6.1), with oxygen (O2) which is present in a non-degassed liquid in
atmospheric air environment:
(6.4)
Consequently, the detection of those nitrate concentrations after surface-DBD

treatment of liquid under atmospheric air conditions as reported earlier, seems to be
an artifact caused by the analytical method using a color forming reaction. Additional
and more sophisticated analytical methods which are not based on analyte-consum-
ing color forming reactions like ion chromatography will give more detailed and
reliable results about the ratio of nitrate and nitrite in plasma treated liquids.
A second question is related to the role of NO3, NO2 or H2O2 for bactericidal
effects. One indirect way to find an answer was the treatment of liquids as well as
bacteria suspensions by surface-DBD under comparable atmospheric pressure con-
ditions but in argon atmosphere. For this experiment, a specially sealed reaction
chamber was used (see Fig. 6.2, right). Before the plasma was ignited, the chamber
was flooded by argon gas (gas flow 0.5 slm) for 5 min to guarantee a nearly com-
plete displacement of air.
Compared to the results with surface-DBD treatment at atmospheric air, nearly
no bacteria inactivation was detected after DBD-plasma treatment of E. coli suspen-
sion in argon atmosphere even though a non-buffered NaCl solution was used
(Fig. 6.5).
As it was demonstrated in Fig. 6.3, there was also an acidification of the liquid
leading to a final pH of about 4 after 30 min. However, detection of nitrogen and
oxygen containing chemical species (detected as NO3, NO2 and H2O2 using color
forming reactions) resulted in a slight concentration increase of H2O2 equivalents up
109
number of viable microorganisms

108
107
(cfu . ml -1) 106
105
104 argon
103
ambient air
102
101
detection limit
100
0 5 10 15 20 25 30
plasma treatment time [min]
Fig. 6.5 Inactivation kinetics of 5 ml E. coli suspensions in non-buffered NaCl solution dependent
on surface-DBD plasma treatment time in argon () an ambient air atmosphere ()
to 6 mg l1 in 5 ml liquid within 30 min, only (data not shown). In the same liquid
volume, a H2O2 equivalent concentration of 18 mg l1 was measured after 30 min
surface-DBD treatment in atmospheric air [4]. But, neither NO3 nor NO2 related
concentrations were detectable if the liquid was surface-DBD treated in an argon
atmosphere. The last fact is not surprising because of the lack of air in this test
system. However, mechanisms of H2O2 generation as well as acidification have to
be investigated in more detail. One possible explanation is plasma-induced water
dissociation which has been discussed as a result of plasma generation directly in
water [12].
According to the results presented here, reactive species containing both nitro-
gen and oxygen seem to be necessary for antimicrobial effectivity because such
species could not be detected after plasma treatment in argon atmosphere. Nitrate or
nitrous acid, respectively, has been identified to play a key role in acidification.
There are some reports in literature about more or less distinct antimicrobial activity
of nitrite under acidic conditions whereas it is hypothesized that not nitrite itself but
other reactive species arising from further reactions are responsible for the antimi-
crobial activity [1315].
In this context it has to be taken into consideration that plasma-generation in
argon has been realized using lower energy input compared to air (3 kV vs. 10 kV
ignition voltage). This was inevitable because, according to the Paschen curves
higher ignition voltage is needed to generate plasma in air compared to argon. On
the other hand, the use of 10 kV ignition voltage in argon atmosphere was not pos-
sible in this experimental setup because of intensive spark generation. However,
because the surface-DBD plasma was not in direct contact with the bacteria contain-
ing liquid, from our point of view bactericidal effects of different electric fields
should be unlikely.
On the one hand as it was mentioned already, spontaneous disproportionation of

nitrous acid into nitric acid is accompanied by the formation of NO (see Eq. 6.1).
NO is well known as important biological transmitter and modulator as well as
effector molecule of the nonspecific immune response. NO is reported to have sev-
eral cytotoxic effects. However, it is believed that these effects are not due to direct
action of NO but mediated by its oxidation products. Besides NO2 (see Eq. 6.4)
such an oxidation product is peroxynitrate (ONOO) or the corresponding weak
peroxynitrous acid (OHOOH; pKa 6.8) [1618]. Both ONOOH and ONOO are
described in the literature as strong oxidants oxidizing lipids, fatty acids and pro-
teins directly [1921].
On the other hand, in acidic media in the presence of H2O2 a rapid direct forma-
tion of peroxynitrous acid (ONOOH) from HNO2 is reported [19, 22, 23]:
HNO2 + H2O2 ONOOH + H2O (6.5)
However, these are exactly the same conditions as are created in liquids resulting
from surface-DBD treatment at atmospheric air. Therefore it was assumed that
ONOOH or ONOO, respectively, could be mainly responsible for bactericidal
effects in plasma-treated liquid. To prove, if this species really is formed in liquids
treated with surface-DBD in atmospheric air, absorption spectrum of 30 min plasma
treated NaCl solution was investigated in detail (Fig. 6.4, inlet).
According to Daiber and Ullrich, ONOO or ONOOH, respectively, have an
absorption maximum at 302 nm [19]. Besides the distinct peak at 227 nm which was
assigned to HNO2, another but very small maximum was identified at 302 nm which
can be identified to be caused by low concentrations of ONOOH. Because the half-
life of ONOOH is reported to be very short [16, 23], this makes it very difficult to
generate and analyze this substance in a reproducible way. Nevertheless, because of
its high reactivity as oxidizing agent, even low concentrations could be high enough
to start lethal reactions. Because the substance is consumed during such interac-
tions, this could accelerate a further generation of OHOOH according to Eq. 6.5.
6.4 Summary and Conclusion
Based on a recent study describing antibacterial effectivity as well as acidification

and generation of nitrogen and oxygen containing chemical species in liquids which
are treated with a surface-DBD under atmospheric air conditions [4], the aim of this
work was to get more insight into possible reactions which are playing dominating
or supporting roles for acidification, chemical species generation and, last but not
least, bacteria inactivation resulting from plasma-liquid interactions.
It was demonstrated that acidification is mainly caused by HNO2 generation in
liquids, at least if the plasma treatment is realized in an atmospheric air environ-
ment. Surprisingly, a lower but distinct acidification results of liquid treatment under
argon atmosphere, too, where no nitrite or nitrate could be detected. Consequently,
there must be alternative reaction channels leading to acid generation without nitrogen
containing chemical species. This needs more investigation. Recently, Friedman

raised the very interesting hypothesis of a very strong plasma acid which is pos-
sibly based on some kind of reactive oxygen species, only [24]. It would be interesting
to continue this discussion.
The identification of reactive species which are responsible for antimicrobial
effectivity is much more complicated. By comparison of bactericidal effect of liquid
treatment under atmospheric air conditions with that under argon atmosphere, it
could be concluded only that, similar to acidification these reactions must be closely
connected to nitrite generation. Based on theoretical considerations and direct
photometric analysis of plasma-treated liquid, peroxynitrite (ONOO) or peroxyni-
trous acid (ONOOH), respectively, have been hypothesized to play a key role for
antimicrobial activity of plasma-treated liquids were both nitrogen and oxygen
containing chemical species are generated. But, because of its very low stability,
analytics of ONOO/ONOOH is very complicated. Therefore, this hypothesis
cannot be clarified finally and needs further work. However, because it is reported
that under acidic conditions ONOOH is generated directly from HNO2 and H2O2
(see Eq. 6.5), this could be one explanation for the supporting role of acidic pH for
bactericidal efficacy of surface-DBD plasma treatment of liquids under atmospheric
air conditions.
In general, the detailed detection of reactive species in plasma-treated liquids is
on of the most important forthcoming tasks. The possible artifact in nitrate analytics
discussed in this study should call the attention to a general problem. Conventional
and established methods of quantitative liquid analytics which are mainly based on
color forming reactions of the substances to be detected with indicator molecules
seems to be not specific to that extent as it is needed for detailed clarification of
plasma-liquid interaction mechanisms. As another example, detection of hydrogen
peroxide using the reaction of titanyl sulfate to yellow-colored peroxotitanyl sulfate
can record not only hydrogen peroxide itself but some other species which trigger
peroxidating reactions. On the one hand, various such reactive oxygen species like
the superoxide anion radical (O2) are hypothesized to play dominating roles in
plasma-liquid interaction and for biological effects transmitted in plasma-treated
liquids, respectively. Therefore, it is absolutely necessary to identify it exactly. On
the other hand, these species are in most cases difficult to produce in vitro and to use
it to test its cross-reactivity with other peroxidizing agents.
Consequently, from our point of view the key to success with further insight into
detailed mechanisms of plasma-liquid interactions is a highly sophisticated liquid
analytics. This is a main precondition not only for further understanding of details
of reaction mechanisms of plasma interaction with cells and tissue but also to open
the new and promising field of plasma pharmacy, i.e. the plasma supported genera-
tion and/or optimization of reactive agents containing liquids.
Acknowledgements This study was realized within the joint research project Campus
PlasmaMed supported by the German Federal Ministry of Education and Research (grants
no. 13 N9779 and 13 N11188).
References
1. Satoh K, MacGregor J, Anderson JG, Woolsey GA, Fouracre RA (2007) Pulsed-plasma disin-
fection of water containing Escherichia coli. Jpn J Appl Phys 46:11371141
2. Ikawa S, Kitano K, Hamaguchi S (2009) Effects of pH on bacterial inactivation on aqueous
solutions due to low-temperature atmospheric pressure plasma application. Plasma Processes
Polym 6:3342
3. Liu F, Sun P, Bai N, Tian Y, Zhou H, Wei S, Zhou Y, Zhang J, Zhu W, Becker K, Fang J (2010)
Inactivation of bacteria in an aqueous environment by a direct-current, cold-atmospheric-
pressure air plasma microjet. Plasma Processes Polym 7:231236
4. Oehmigen K, Hhnel M, Brandenburg R, Wilke Ch, Weltmann K-D, von Woedtke Th (2010)
5. Oehmigen K, Winter J, Wilke Ch, Brandenburg R, Hhnel M, Weltmann K-D, von Woedtke
Th (2011) Estimation of possible mechanisms of Escherichia coli inactivation by plasma
treated sodium chloride solution. Plasma Processes Polym 8:904913
6. Hhnel M, von Woedtke Th, Weltmann K-D (2010) Influence of the air humidity on the reduc-
tion of Bacillus spores in a defined environment at atmospheric pressure using a dielectric
barrier surface discharge. Plasma Processes Polym 7:244249
7. Holleman AF, Wiberg E, Wiberg N (2007) Lehrbuch der anorganischen Chemie. de Gruyter,
Berlin (in German)
8. Butler R, Feelisch M (2008) Therapeutic uses of inorganic nitrite and nitrate: from the past to
the future. Circulation 117:21512159
9. Lundberg JO, Weitzberg E, Gladwin MT (2008) The nitrate-nitrite-nitric oxide pathway in
physiology and therapeutics. Nat Rev Drug Discov 7:156167
10. Riordan E, Minogue N, Healy D, ODriscoll P, Sodeau JR (2005) Spectroscopic and optimiza-
tion modeling study of nitrous acid in aqueous solution. J Phys Chem A 109:779786
11. Anastasio C, Chu L (2009) Photochemistry of nitrous acid (HONO) and nitrous acidium ion
(H2ONO+) in aqueous solution and ice. Environ Sci Technol 43:11081114
12. Dodet B, Odic E, Goldman A, Goldman M, Renard D (2005) Hydrogen peroxide formation by
discharges in argon/water vapour mixtures at atmospheric pressure. J Adv Oxid Technol
8:9197
13. Dykhiuzen RS, Fraser A, McKenzie H, Golden M, Leifert C, Benjamin N (1998) Heliobacter
pylori is killed by nitrite under acidic conditions. Gut 42:334337
14. Wullt M, Odenholt I, Walder M (2003) Activity off three disinfectants and acidified nitrite
against Clostridium difficile spores. Infect Control Hosp Epidemiol 24:765768
15. Rao A, Jump RLP, Pultz NJ, Pultz MJ, Donskey CJ (2006) In vitro killing of nosocomial
pathogens by acid and acidified nitrite. Antimicrob Agents Chemother 50:39013904
16. Krncke K-D, Fehsel K, Kolb-Bachofen V (1997) Nitric oxide: cytotoxicity versus cytoprotection
how, why, when, and where? Nitric Oxide 1:107120
17. Wink DA, Mitchell JB (1998) Chemical biology of nitric oxide: insights into regulatory, cyto-
toxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med 25:434456
18. Pacher P, Beckman JS, Liaudet L (2007) Nitric oxide and peroxynitrite in health and disease.
Physiol Rev 87:315424
19. Daiber A, Ullrich V (2006) Radikalchemie im Organismus. Stickstoffmonoxid, Superoxid und
Peroxynitrit. Chemie in unserer Zeit 6:366375 (in German)
20. Radi R, Beckmann JS, Bush KM, Freeman BA (1991) Peroxynitrite-induced membrane lipid
peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys
288:481487
21. Koppenol WH, Moreno JJ, Pryor WA, Ischiropoulos H, Beckman JS (1992) Peroxinitrite, a
cloaked oxidant formed by nitric oxide and superoxide. Chem Res Toxicol 5:834842
22. Klebanoff SJ (1993) Reactive nitrogen intermediates and antimicrobial activity: role of nitrite.
Free Radic Biol Med 14:351360
23. Lobachev VL, Rudakov ES (2006) The chemistry of peroxynitrite. Reaction mechanisms and
kinetics. Russ Chem Rev 75:375396
24. Friedman G (2010) Plasma pharmacology. In: Proceedings of the 3rd international conference
on plasma medicine (ICPM-3), Greifswald, Germany, September 1924 2010
Chapter 7
Damages of Biological Components in Bacteria
and Bacteriophages Exposed to Atmospheric
Non-thermal Plasma
Akira Mizuno and Hachiro Yasuda
Abstract Mechanism of inactivation of bio-particles exposed to dielectric barrier

discharge, DBD, has been studied using E. coli and bacteriophages. States of differ-
ent biological components were monitored during the course of inactivation.
Analysis of green fluorescent protein, GFP, introduced into E.coli cells proved that
Non-thermal Plasma, NTP causes a prominent protein damages without cutting
peptide bonds. We have developed a biological assay which evaluates in vitro DNA
damage of the bacteriophages. Bacteriophage l having double stranded DNA was
exposed to DBD, then DNA was purified and subjected to in vitro DNA packaging
reactions. The re-packaged phages consist of the DNA from discharged phages and
brand-new coat proteins. Survival curves of the re-packaged phages showed
extremely large D value (D = 25 s) compared to the previous D value (D = 3 s) from
the discharged phages. The results indicate that DNA damage hardly contributed to
the inactivation, and the damage in coat proteins is responsible for inactivation of
the phages. M13 phages having single stranded DNA were also examined with the
same manner. In this case, damage to DNA was as severe as that of the coat
proteins.
7.1 Introduction
Emergence of the next pandemic influenza, avian flu (H5N1) or swine flu (H1N1),
and drug resistant bacteria are of concern for public health. Non-thermal atmospheric
pressure plasma is effective for dealing with such menaces caused by bio-particles
(BPs) because, in principle, it can decontaminate both the surface of materials [1, 2]
A. Mizuno (*) H. Yasuda

Department of Environmental and Life Sciences, Toyohashi University of Technology,
Tempaku-cho, 4418580 Toyohashi, Japan
e-mail: mizuno@ens.tut.ac.jp
80 A. Mizuno and H. Yasuda
and room air [3, 4] at low gas temperature without using bactericides. Though the
study of plasma decontamination is expanding [59], the mechanism of inactivation
of BPs are still to be studied. In this study, we have evaluated the damages to E. coli,
B. subtilis exposed to dielectric barrier discharges [10]. GFP (green fluorescent
protein) in E. coli can be damaged quickly by the exposure to the DBD (dielectric
barrier discharge), and the intensity of GFP and survivability shows similar trend
against the exposure time. Turbidity of B. subtilis solution decreases quickly with
the exposure to the DBD. We have also developed a biological assay to evaluate
DNA-specific damage in bacteriophages treated with DBD. It was found that l
phage DNA was strong against the plasma application in comparison with coat
proteins. Phages having single stranded DNA, however, can be damaged as sensi-
tive as its coat protein.
7.2 Experimental
7.2.1 Collection and Destruction of BPs

by Electrostatic Precipitation
BPs are commonly existing around us, and electrostatic precipitation can collect
suspended BPs in air with high efficiency and low pressure drop. In the meantime,
corona discharges in ESPs generate radicals and other oxidative particles. Those are
effective to destroy BPs. Previous experimental work indicated effective destruction
of yeast cell on the collection electrode of a corona discharge system [11]. We have
tested the destruction effect of a corona discharge generated in the needle-plate
system. Bacteriophage MS2 can also be destroyed, with improved efficiency when
the phages are in wet condition.
Film or culture media can be used as the collecting electrode. Figure 7.1a illus-
trates the ESP used in this study. The needle electrode with DC high voltage was
placed above the YPD (Yeast extract Peptone Dextrose) medium, and corona dis-
charge was generated between them. Distance between the needle electrode and
YPD medium was 40 mm. The ESP was placed in a box and a fan was set at the top
of the box to introduce the air from the upper side and the BPs are collected on the
YPD medium. Different voltage was tested (030 kV) on each fresh YPD plate.
Collected samples were incubated at 30C for 48 h for the colony forming.
Figure 7.1b shows the effect of ESP on the BPs collection. Particles in room air
were collected on YPD medium for 1 min. Positive and negative voltages were exam-
ined in this experiment. The result shows that, for both polarities, the number of
collected bacteria increased with applied voltage. However, the positive corona dis-
charge shows the higher efficiency than the negative corona. The highest collection
efficiency was observed with applied voltage of 25 kV. This efficiency suggesting
that collection of bacteria was enhanced by about 50 times compared with non-
applied discharge BPs amount. In addition, influence of applied voltage and distance
7 Damages of Biological Components in Bacteria 81
Fig. 7.1 Collection of suspended bio-particles in air using electrostatic precipitation. (a) An elec-
trostatic precipitator for collecting bio-particles in air. (b) Number of colony due to collected
bio-particles using the ESP
Fig. 7.2 Radial distribution of the colonies collected by electrostatic precipitation. X-axis: relative
density of colonies. Y-axis: distance from the center (just under the needle electrode). (a) Positive
corona (8 and 13 kV). (b) Negative corona (8 and 13 kV)
of electrodes on collection profiles of BPs was noticed, as shown in Fig. 7.2. In this
case, the distance between the needle electrode and the surface of YDP media was
10 mm. This small distance was selected to measure the effect of radial distance
more clearly. Relative density of the colonies was measured for comparison. This
was calculated by measuring the colonies in a concentric circle of 9 mm width, and
divided by total number of colonies. Close to the center of the plate, just under the
high voltage electrode, the relative colony density increased. However, above a cer-
tain applied voltage, the relative colony density at the center decreased. The total
number of colonies (Fig. 7.1b) also decreased with very high voltage. At 30 kV, the
number of collected bacteria decreased. Due to ionization of corona discharge, radi-
cals such as O, OH are generated, and from these short-lived radicals, long lived
oxidative species are generated. From mass spectroscopy, it is known that corona
discharge generates stable neutral O3 molecules, and ions such as O3(H2O)
Fig. 7.3 Plaque forming unit

of MS2 phage on culture
media exposed to negative
corona discharge
Fig. 7.4 The dielectric barrier discharge
n,NO3(H2O) n, O2(H2O)n, in negative corona and NH4+(H2O) n in positive corona

[12]. The decrease in colonies is due to lethal effect of these species generated by the
corona discharge. It should be noted that, with the silver needle, ozone generation
can be reduced in positive corona by at least 1/3 compared with that in negative
corona. This is the reason for larger number of colonies in the positive corona.
Figure 7.3 shows the Plaque Forming Unit, PFU, of MS2 phage, exposed to the
negative corona discharge with the electrode spacing of 30 mm and 15 kV. In this
case, the MS2 phages were placed on a culture medium. PFU decreased with the
exposure time.
7.2.2 Dielectric Barrier Discharge for Study the Mechanism

of BP Destruction
Figure 7.4 illustrates the dielectric barrier discharge (DBD) reactor used in this
study. Stainless steel mesh (diameter: 50 mmj, 20 mesh (separation between adja-
cent wire of the mesh = 1/20 in.)) and aluminum plate were used as high voltage and
GND electrodes respectively. Teflon sheet (2 mm-thick) was set on the high voltage
electrode as a dielectric barrier. A high voltage AC power supply (Kasuga-denki
AGF-010) was used to generate the uniform filamentous streamers in the
atmospheric air gap (3 mm). All of the discharge experiments in this report were
done in a fixed electrical condition except operating time. The peak to peak voltage,
the frequency of the applied voltage and the input power were 40kVp-p, 2 kHz and
88 W, respectively. A piece of polyethylene terephthalate (PET) film (90 mm 30 mm,
0.1 mm thickness) was soaked in 0.1% gelatin for 5 min and air dried for 24 h under
UV light. 20 ml of sample solution was spotted and widely spread to 34 mm2 on the
PET film and immediately applied the atmospheric plasma for intended time. After
the DBD application, the sample solution was recovered into a microtube with addi-
tional washing by 100 ml of distilled water on the surface of the PET film. The
recovered samples were rendered for experiments of survival estimation, fluores-
cence measurement, protein analysis, DNA analysis and microscopic observation.
Green Fluorescent Protein, GFP, coding plasmid pGLO (5.4 kb in size) was
purchased from Bio-Rad Co. Inc. and transfected to E.coli MV1184. The transformants,
E.coli MV1184(pGLO), were propagated by shaking at 37C for over night in 20 ml
of LB medium supplemented with 5 mM L-arabinose which induces and accumu-
lates GFP in the cells. The cells were harvested by centrifugation at 5,000 g for
5 min, washed and finally suspended in 3 ml of 100 mM Tris HCl(pH8.0).
Bacteriophage lambda (lCI857Sam7) was induced from l lisogen, E.coli M65.
The E.coli cells were inoculated to 200 ml of LB medium in a 1 L flask and shaken
at 32C. When OD650 (optical density at 650 nm wavelength) reached to 0.5, cul-
tivation temperature was quickly shifted to 42C and shaken for 20 min and then
shaken for 3 h at 40C. The cells were harvested by centrifugation at 8,000 g for
5 min and resuspended with 10 ml of SM buffer. The cells were lysed by adding
0.1 ml of chloroform and 10 ml of 2 mg/ml of pancreatic DNase and gentle shaking
at 37C for 20 min. The cell lysate was centrifuged at 10,000 g for 10 min and
recovered the supernatant. Further purification of the lphage in the lysate by step-
wise CsCl density gradient centrifugation and CsCl equilibrium density gradient
centrifugation was performed according to the literature protocol [13] except that
the phage was finally dialyzed against 100 mM Tris HCl(pH8.0), 1 mM MgCl2.
Purified DNA from E.coli MV1184 (pGLO) was obtained by serial extraction of
the cells with phenol, phenol-chloroform, and chloroform as described in [6]. After
precipitation with ethanol, the DNA was dissolved in 100 mM Tris HCl(pH8.0),
1 mM EDTA.
GFP was purified from E.coli MV1184(pGLO) following the protocols of cell
lysis and affinity column chromatography using hydrophobic resin from Bio-Rad
Co. Inc. The DBD treated cells were serially diluted with SM buffer and plated on
LB plates. After incubation at 37C for 30 h, colonies on the plates were counted.
Titration of the DBD treated lphage was done as follows. As indicator cells,
E.coli Y-mel was cultivated in LB medium at 32C for over night. 0.1 ml of the seri-
ally diluted lphage solution was mixed with 0.1 ml of the indicator cell suspension
and kept at 37C for 10 min. Three milliliter of LB soft agar kept at 45C was added
to the mixture of infection and poured on a LB plate. Phage plaques on the plates
were counted after incubation at 40C for 30 h.
All of the survival measurement experiments were duplicated independently and
the mean value was adopted.
7.3.1 Sterilization of E.coli
In the application of atmospheric DBD (dielectric barrier discharge) for bacterial

sterilization, the cells in water solution (wet samples) was used. The sample solu-
tion was spread on the PET film widely, keeping the thickness of the water layer less
than 0.1 mm. This wet-samples did not evaporate significantly during the DBD
application which suggested the cells were kept in low and harmless temperature.
All the wet samples for DBD application contained 100 mM Tris HCl(pH8.0) to
avoid rapid dropping down the initial neutral pH. The phenomena of increasing
acidity of the samples may be mainly caused by NOX gas produced in the air field of
the discharge. NOx will form nitric acid or nitrous acid after dissolving in water. In
our buffering conditions, neutral pH of the samples were guaranteed until 30 s dis-
charge, but 40 s discharge led the sample solution to acidic around pH 4.
Figure 7.5 shows the survival curve of DBD treated E.coli MV1184(pGLO)
cells. 2 log10 reduction was seen at 30 s DBD treatment and 5 log10 reduction was
achieved after 40 s of the treatment. The curve showed a characteristic of multi-
slope lines. The D value of the first slope (010 s), the second slope (1030 s) and
the third slope (3040 s) were about 10, 35 and 4 s, respectively. The steep decrease
between 30 and 40 s might be due to acidification of the solution, generating H-O-O
radicals from O2 [14].
7.3.2 Damages of Cellular Protein and DNA
Figure 7.5 also indicates the fluorescent intensity of GFP in E.coli MV1184(pGLO)
cells after time-lapse treatment with DBD. The intensity decreased with increasing
the time of DBD treatment, and complete inactivation of the GFP function was seen
at 40 s of the treatment. The exhibited results recall some correlation to the cell
survival. Pouring alkali solution to the bleached samples did not improve their fluo-
rescence though normal (non-discharged) GFP inactivated in acid solution exhib-
ited full recovery of the fluorescence by neutralization with alkali. It was suggested
that the bleaching of GFP by DBD treatment seemed to be caused by its irreversible
denaturation or chemical modification or degradation.
Figure 7.6 shows the SDS (sodium dodecyl sulfate) polyacrylamide gel electro-
phoresis of E.coli MV1184(pGLO) subjected to the atmospheric DBD. Cells were
lysed and fractionated in a 14% gel before staining with Coomassie Brilliant Blue
(CBB). Lane M is a protein standards marker, lane 1 is purified GFP, lane 2 is non-
induced E.coli MV1184 (pGLO), Lanes 38 represent the cells treated with DBD
for 0, 5, 10, 20, 30, 40 s, respectively. The arrow indicates GFP.
The thick band (indicated by the arrow) of the lane 38 corresponds to GFP. GFP
and many other intrinsic proteins of E.coli were not degraded significantly. Even in
Fig. 7.5 Survival rate of the E.coli after the DBD treatment. DBD dielectric barrier discharge,
CFU colony forming unit
Fig. 7.6 SDS polyacrylamide gel electrophoresis of E.coli MV1184(pGLO) subjected to the
atmospheric DBD. Cells were lysed and fractionated in a 14% gel before staining with coomassie
brilliant blue (CBB). Lane M is a protein standards marker. Lane 1 is purified GFP. Lane 2 is non-
induced E.coli MV1184 (pGLO). Lanes 38 represent the cells treated with DBD for 0, 5, 10, 20,
30, 40 s, respectively. The arrow indicates GFP
the 40 s discharge treated sample (lane 8), considerable amount of full length GFP
was remaining though its fluorescence was completely lost (Fig. 7.5). These results
suggest that the cause of the GFP bleaching by DBD treatment was irreversible
denaturation of its tertiary structure or chemical modification by oxidation or reduc-
tion but not the degradation of the peptide bonds.
Figure 7.7a and b shows the analysis of DNA from the DBD treated cells by
agarose gel electrophoresis. The chromosome DNA was slightly cut by sharing
force of mixing before separation with electrophoresis. In Fig. 7.7a, the chromo-
somal DNA was separated into two different positions on the 0.3% agarose gel. One
stayed near the sample well which did not enter the gel matrix and the other
migrated to the position slightly larger than the l phage DNA (48.5 kb). The pattern
of the chromosomal DNA bands did not change until 30 s of the DBD treatment.
Fig. 7.7 Agarose gel electrophoresis of the cellular DNA from E.coli MV1184 (pGLO) treated by
the DBD. (a) DNA was extracted and separated in 0.3% gel before staining with ethidium bromide.
Lane M1 is hind digests of lDNA, Lane M2 is monomeric lDNA. Lanes 16 represent the DNA
from the cells treated with DBD for 0, 5, 10, 20, 30, and 40 s, respectively. Arrows indicate chro-
mosome DNA. (b) Plasmid DNA fractionated in 0.8% gel. Lane M represents the purified pGLO
DNA. Lanes 16 represent the plasmid DNA fraction from the cells treated with DBD for 0, 5, 10,
20, 30, and 40 s, respectively. sc, oc and l represent super-coiled, open circler, and linear form,
respectively. (c) Plasmid DNA fractionated in 0.8% gel from the purified DNA subjected to DBD.
Lane M represents the purified pGLO DNA. Lanes 16 represent the naked DNA samples treated
with DBD for 0, 5, 10, 20, 30, and 40 s, respectively
Some degradation was seen in the 40 s treated sample which seems to be caused by
increased acidity of the sample because DNA is labile to acid. It is concluded that
the chromosomal DNA did not degraded remarkably during inactivation of E.coli
cells by DBD treatment. Bright materials at the bottom of the gel are ribosomal
RNA. Similarly to the DNA, influence of the DBD treatment to the ribosomal RNA
was not detected. Plasmid DNA is a good reporter molecule about DNA degradation
because only a nick introduction changes its topological structure from super-coiled
to relaxed form. Figure 7.7b shows the plasmid DNA (pGLO) fraction separated
from the DBD treated cells on 0.8% agarose gel electrophoresis. The bands at lower,
upper and middle positions correspond to super-coiled, relaxed (open circular) and
linear form of the plasmid molecules, respectively. The appearance of three types of
molecular form represents some degradation of the plasmid DNA has occurred, but
it seems to be caused by cellular intrinsic DNase during recovery of the samples
because non-discharged sample (0 s sample) also contained the three types of the
DNA (Fig. 7.7b, lane l control). The pattern of the plasmid DNA bands did not
change until 30 s of the DBD treatment which means that the nick caused by the
discharge was introduced scarcely until 30 s of the DBD treatment to the cells
(Fig. 7.7b, lane 25). The degradation of the plasmid seen in lane 6 may be caused
by acidic effect. Figure 7.7c shows the plasmid DNA fractions from the DBD
Fig. 7.8 E.coli cells which produce GFP with and without the exposure to the DBD. (a)(c) fluo-
rescent of GFP (d)(f) fluorescent of DNA by addingYOYO-1
applied purified E.coli DNA solution (naked DNA). With the lapse of discharged
time, super-coiled molecules have decreased and relaxed molecules have increased
(Fig. 7.7c, lane 26). The rational change of the isomeric form of the plasmid DNA
indicates the nick introducing activity was present in the DBD treatment to the
naked DNA. The inertness of the DBD for the DNA inside the bacterial cells
(Fig. 7.7b) may be caused by the protective effect of gram negative bacterial cell
envelope (inner membrane, outer membrane, periplasm and cell wall). In any case,
DNA destroying activity of the DBD treatment was very small, though it should be
reminded that subtle changes in DNA may largely affect to cell viability.
Images of E. coli cells which produce GFP were shown in Fig. 7.8 with and with-
out the exposure to the DBD. Originally the fluorescent was very bright, and the cells
were clearly observed (Fig. 7.8a). With the exposure to DBD, the cells gradually
changed into faint images because of the bleaching of GFP (Fig. 7.8b, and c). The
observation agrees well to the results of fluorescent spectroscopy. Figure 7.8d, and e
show images of the DBD applied cells stained with YOYO-1(1,1-((4,4,7,7-
Tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-
oxazole)-2-methylidene)quinolinium tetraiodide), a fluorescent dye binds specifically
to double stranded DNA. The restored blight images of the 30 s DBD treated cells
stained with YOYO-1 (Fig. 7.8d) means that considerable amount of chromosomal
DNA was remaining inside the cell without extreme degradation; this is consistent
with the results from DNA analysis by electrophoresis. The cells over-sterilized with
80 s discharge showed tight aggregation. Those cell wall might have been destroyed
and interconnected with each other (Fig. 7.8e). Figure 7.8f shows the YOYO-1
stained host MV1184 cells which do not produce GFP. Only a fraction of the cells
Fig. 7.9 Inactivation profile of the l phage
were strongly stained and other major fractions were stained weakly. The bright cells
seem to be dead ones because YOYO-1 does not intrude easily inside the living
healthy cells. Increasing of the rate of brightly stained cells by DBD treatment
(Fig. 7.8d) strongly suggests that cell wall and cell membrane are damaged and
destroyed, at least locally or in a small manner, during the sterilization.
7.3.3 Bacteriophage Inactivation
Damages on the cell membrane have been thought to be essential for bacterial ster-
ilization by low temperature plasma. Bacteriophages usually do not have membrane
and consist of only proteins and nucleic acids. It is interesting to investigate the
effect of the plasma to membrane free bacteriophages.
Figure 7.9 shows the inactivation profile of the lphage after time-lapse treatment
with the DBD. Number of infectious phage decreased quickly and complete inacti-
vation was achieved by 30 s discharge treatment. The rate of inactivation was higher
than that of E. coli in the same electrical conditions of DBD. The profile exhibited
a characteristic of a single slope curve and the D-value was about 5 s.
Figure 7.10a shows the analysis of proteins from the DBD treated lphages by
SDS polyacrylamide gel electrophoresis. lphage has about 20 genes of coat proteins,
but only two major proteins were detectable on the gel (Figure 7.10a lane 14). These
proteins degraded rapidly comparing to the E. coli cellular proteins and could not
detect in the 30 s discharged sample. Though the degradation rate of the proteins was
slow comparing to the inactivation rate of the phage, the time of the disappearance of
protein and the completion of the inactivation coincided. The exposure of phage to
the solution of outer environment might lead to the high sensitivity of the protein to
the DBD action. Inactivation of the phage proteins involved in binding to the cell
surface receptor may contribute largely to the decrease of infectious phage.
Fig. 7.10 Analysis of protein and DNA from bacteriophage l subjected to the DBD. (a) SDS polyacryl-
amide gel electrophoresis of bacteriophage l subjected to the atmospheric DBD. Bacteriophagel was
lysed and fractionated in a 14% gel before staining with CBB. Lane M is a protein standards marker. Lane
0 represents a large amount of purified l phage. Lanes 16 represent the proteins from the phages treated
with DBD for 0, 5, 10, 20, 30, 40 s. (b) A 0.3% agarose gel electrophoresis of DNA from bacteriophage
l subjected to the atmospheric DBD. Lane M1 is monomeric DNA. Lane M2 is hind digests of lDNA.
Lanes 16 represent the DNA from phages treated with DBD for 0, 5, 10, 20, 30, 40 s
Fig. 7.11 The method to estimate the DNA-specific damage in plasma applied l phages
Figure 7.10b shows the analysis of DNA from the DBD treated l phages by 0.3%
agarose gel electrophoresis. The phage DNA degraded faster comparing to the
E. coli cellular DNA and could not detect in the 40 s discharged sample, but the
degradation was slow when compared to the inactivation rate of the phage. Here
also, the protective function of the cellular membrane from the attack of discharge
to the DNA was suggested.
7.3.4 Evaluation of DNA Damage
Figure 7.11 illustrates the method to estimate the DNA-specific damage in plasma
applied lphages. Putative damage is introduced in both protein and DNA of the
Fig. 7.12 Plaque forming unit of lphage and M13 phage subjected to the DBD. After the DBD
exposure, the double stranded DNA oflphages were extracted and re-packaged, or the single
stranded DNA of M13 phages were extracted and transfected to E. coli. (a) PFU curves of plasma
treated l phages, and the re-packaged phages. (b) PFU of the DBD treated M13 phages and
Relative PFU curves obtained from the transfection of recovered single stranded DNA after the
exposure to the DBD
plasma treated phages (Phage A). DNA is extracted from Phage A and packaged
in vitro to form newly packaged phages (Phage B). Phage B does not have protein
damage and carries only DNA damage originated from Phage A. Therefore, all of the
inactivation factors in Phage B originate DNA damage brought from Phage A.
Because re-packaging procedure of lphage usually decreases the efficiency of infec-
tion, absolute number of active phages (phage titer) in Phage A and Phage B can not
be compared directly. Comparison of the normalized survival curves from Phage A
and Phage B enables to evaluate the DNA damage and the protein damage.
Figure 7.12a shows the relative PFU (plaque forming unit) curves obtained from
the re-packaged lphages and the plasma treated phages. These PFU curves were
normalized with each control sample (0 s samples) to give the PFU value of 100. The
profile exhibited the characteristic of a single slope curve until 20 s discharge and
the D value was about 25 s. Large D value of the re-packaged phages means very
slow decrease of infective phages until 20 s. The results indicate that the DNA dam-
age introduced by plasma was very small and did not accumulate prominently with
increase of the discharge time. It seems that all of the initial damage for inactivation
in phages introduced by plasma was protein damage, and DNA damage was marked
on the phages already inactivated by protein damage. So, it is concluded that inacti-
vation of lphage by atmospheric DBD was attributed to the damage of coat pro-
teins. The damage of lDNA was negligible in the early stage of the inactivation.
The damage responsible for phage inactivation can be recognized only when the
assay of phage viability was carried out. Therefore, the damage for inactivation may
not be directly correlated to the amount of the molecular damage. For example,
molecular damage introduced in the binding protein which interacts with phage
receptors on E. coli cell surface may largely affect to the inactivation. Moreover, it
should be counted that cells have a highly developed DNA repair system. Molecular
damage of DNA might have been repaired effectively than that of proteins.
The bio-assay of DNA damage has enough reproducibility when the strains of
phage and host cell were fixed. This assay can be applied to not only plasma inactiva-
tion but also any type of inactivation step of bacteriophages. Application of the DNA
damage assay to other plasma sources or different sets of bacteriophages and host
cells may bring valuable insights into the mechanism of plasma inactivation.
For comparison, damages to M13 phages were evaluated using the same proce-
dure. After the exposure to DBD, the DNAs (single stranded) were extracted, and
were transfected to E. coli to measure the infection rate. The result as shown in
Fig. 7.12b indicated that the infection ability did not recover. The comparison shows
that double-stranded DNA is stronger against the exposure to DBD, as the DNA
repairment system of the host cell works if the damage remains on one strand.
7.4 Conclusion
Application of low temperature atmospheric plasma (atmospheric DBD) for the

sterilization of bacteria and bacteriophage was investigated. Both E. coli and lphage
were inactivated effectively by DBD exposure. Damages of cell membrane, proteins
and DNA were detected but not significantly during the sterilization. We found that
some proteins (GFP, lcoat protein) were irreversibly inactivated quickly by dena-
turation or chemical modification such as oxidation or reduction but not degrada-
tion. Not only the membrane destruction or DNA damage, but the quick inactivation
of proteins may have an important role in sterilization by DBD.
Comparison between l and M13 phages was made and found that single stranded
DNA is vulnerable to the DBD exposure. This could be due to the function of the
DNA repairment system of the cell so that minor damage of double stranded DNA
is fixed.
References
1. Montie C, Kelly-Wintenberg K, Roth JR (2002) An overview of research using the one atmo-
sphere uniform glow discharge plasma (OAUGDP) for sterilization of surfaces and materials.
IEEE Trans Plasma Sci 28:41
2. Laroussi M, Alexeff I, Kang WL (2000) Biological decontamination by nonthermal plasmas.
3. Herrmann HW, Henins I, Park J, Selwyn GS (1999) Decontamination of chemical and biologi-
cal warfare (CBW) agents using an atmospheric pressure plasma jet (APPJ). Phys Plasmas
6:2284
4. Gallagher MJ Jr, Vaze N, Gangoli S, Vasilets VN, Gutsol AF, Milovanova TN, Anandan S,
Murasko DM, Fridman AA (2007) Rapid inactivation of airborne bacteria using atmospheric
pressure dielectric barrier grating discharge. IEEE Trans Plasma Sci 35:1501
5. Yasuda H, Hashimoto M, Rahman MM, Takashima K, Mizuno A (2008) Analysis of the inac-
tivation mechanism of bacteriophage by Atmospheric barrier discharge. Plasma Processes
Polym 5:615
6. Sladek REJ, Stoffels E (2005) Deactivation of Escherichia coli by the plasma needle. J Phys D
Appl Phys 38:1716
7. Moisan M, Barbeau J, Moreau S, Pelletier J, Tabrizian M, Yahia LH (2001) Low-temperature
sterilization using gas plasmas: a review of the experiments and an analysis of the inactivation
mechanisms. Int J Pharm 226:1
8. Deng XT, Shi JJ, Chen HL, Kong MG (2007) Bacterial inactivation using atmospheric pres-
sure single pin electrode microplasma jet with a ground ring. Appl Phys Lett 90:013903
9. Sato T, Doi A, Urayama T, Nakatani T, Miyahara T (2007) Inactivation of Escherichia Coli by
a coaxial microwave plasma flow. IEEE Trans Ind Appl 43:1159
10. Tanino M, Xilu W, Takashima K, Katsura S, Mizuno A (2007) Sterilization using dielectric
barrier discharge at atmospheric pressure. Int J Plasma Environ Sci Technol 1:102
11. Abdel-Salam M, Nakano M, Mizuno A (2008) Culturing of cells as influenced by exposure to
AC and DC fields. J Phys Conf Ser 142:012020
12. Nagato K (1999) Chemical composition of atmospheric ions. J IEEJ 23(1):3743
13. Davis LG, Dibner MD, Battery JF (1986) Methods in molecular biology. Elsevier, New York
14. Ikawa S, Kitano K, Hamaguchi S (2010) Effects of pH on bacterial inactivation in aqueous
Polym 7(1):3342
Chapter 8
Investigations of Bacterial Inactivation
and DNA Fragmentation Induced by Flowing
Humid Argon Post-discharge
Emmanuel Odic, S. Limam, M.J. Kirkpatrick, B. Dodet,

S. Salamitou, and M.S. DuBow
Abstract Bio-contaminated surfaces were exposed to an atmospheric pressure

flowing post-discharge, i.e. without direct contact of the plasma with the surface.
The non-thermal plasma source was a dielectric barrier discharge. Using humid
argon as a feed gas, a reduction of six orders of magnitude of survivors could be
obtained for Escherichia coli. An investigation of bacterial inactivation mechanisms
during the plasma induced treatment was conducted. For this purpose, DNA (plas-
mid and genomic DNA in aqueous solution) degradation by the plasma process was
studied, assuming that the bacterial inactivation is obtained when the bacterial DNA
is fragmented. According to the operating conditions (feed gas, reactor geometry
and discharge input power), DNA fragmentation was evaluated in correlation with
aqueous phase hydrogen peroxide concentration measurements. It appears that
hydrogen peroxide is not the only factor responsible for DNA fragmentation and
that short-lived species produced by water dissociation are major contributors.
8.1 Introduction
Non-thermal plasma technologies have been heavily investigated during the last
decade for biomedical applications including surface decontamination of thermally
sensitive materials [13]. Several techniques were studied with different excitation
sources operating in different gases at low pressure [4, 5] and atmospheric pressure
[59]. The dielectric barrier discharge (DBD) process under investigation operates at
E. Odic (*) S. Limam M.J. Kirkpatrick B. Dodet

E3S Department of Power and Energy Systems, SUPELEC,
3, rue Joliot-Curie, F-91192 Gif-sur-Yvette Cedex, France
e-mail: emmanuel.odic@supelec.fr
S. Salamitou M.S. DuBow
Institut de Gntique et Microbiologie, Universit Paris-Sud 11,
F-91405 Orsay, France
94 E. Odic et al.
atmospheric pressure in humid argon. Argon is preferred to air as a feed gas in order
to avoid the formation of ozone and nitrogen oxides (and nitric acid if water is present
[9]) which may damage the treated surface. Furthermore, water dissociation,
producing highly oxidative species, is much more efficient in the presence of argon
in the discharge. The contaminated surfaces to be treated are exposed to a non
emissive flowing post-discharge, i.e. to the discharge products, without direct contact
of the plasma (here in a filamentary regime) with the surface. Such a remote exposure
mode was chosen in order to obtain a homogeneous surface treatment and to minimize
surface material degradation. During the interaction of the discharge with a microor-
ganism, DNA can be released from cells [10] and damaged [11]. In the present work,
inactivation of a model planktonic microorganism and degradation of DNA mole-
cules in solution were investigated in separate experiments in correlation with the
nature of the active species transported by the flowing post-discharge.
8.2.1 Discharge Reactors and Biological Sample

Treatment Devices
For most of the experiments, the DBD reactor (reactor 1 in Fig. 8.1) consisted of a
stainless steel rod (2 mm diameter) centered in a dielectric Pyrex tube (6 and 3 mm
external and internal diameters respectively) externally covered with a 24 mm length
stainless steel mesh (allowing optical emission spectroscopy) connected to ground.
The inner electrode was connected to an AC (530 kHz) high voltage power supply
(0.510 W discharge input power). The DBD reactor was fed with humid argon (at
room temperature, 0.52 L/min). The outlet of the discharge tube was connected to
the treatment vessel in which biological samples were exposed to the flowing post-
discharge. More precisely, biological samples were placed 5 mm from the tube
outlet, 15 mm from the discharge zone itself.
In a second reactor geometry (reactor 2, see Fig. 8.1), the electrode system consisted
of two circular plane electrodes (brass) separated by an alumina (Al2O3) dielectric disc
(2 mm thickness and 60 mm diameter). The same high voltage power supply as for reac-
tor 1 was used; the high voltage electrode and the grounded electrode were 30 and
26 mm diameter respectively. Due to the size difference between the metallic electrodes,
the higher electric field region is located along the triple line (gas/metal/dielectric)
corresponding to the circumference of the smaller diameter plane electrode (grounded
electrode). Surface discharges then propagate on the alumina surface on one side of the
ceramic disc. A Pyrex dish, onto which the biological samples were deposited, was
placed facing this surface discharge (i.e. the lower side of the Al2O3 disc in Fig. 8.1). The
distance between the biological samples and the surface discharge was fixed to 30 mm.
Arcal 1 grade argon from Air Liquide was used (O2 < 0.5 ppm, N2 < 1 ppm, H2O
< 1 ppm) in all experiments. The gas flow was controlled using Brooks Sho-rate rotameters.
8 Investigations of Bacterial Inactivation and DNA Fragmentation 95
Feed gas
HV power HV power
Feed gas
supply supply
Pyrex tube
Al2O3disc
NTP source: DBD
volume discharge NTP source: DBD
surface discharge
Gas outlet Gas outlet Treatment vessel

Bio samples
Gas outlet
Treatment vessel
Bio samples
Reactor 1 Reactor 2
Fig. 8.1 DBD reactors and biological sample treatment devices
Water vapor was added to argon by bubbling through distilled water in a gas sparging
bottle at room temperature. Water vapor content was adjusted by mixing dry argon and
water saturated (at room temperature) argon, while keeping total flow rate constant.
8.2.2 Stable Discharge Products
Hydrogen peroxide, oxygen and hydrogen are end products of water dissociation.
Gas phase chemical measurements of hydrogen and oxygen were simultaneously
made using an Agilent 6890A gas chromatograph (GC) with a Restek 100/120
Shincarbon-ST 2 m 1 mm column and a thermal conductivity detector. Argon was
used as the GC carrier gas. Hydrogen peroxide was collected by absorption in dis-
tilled water (Henrys Law constant = 105 M/atm), in the form of water droplets or
water film, submitted to the plasma post-discharge for various exposure times.
Using absorption spectroscopy (Perkin Elmer Lambda 15 spectrometer) of hydro-
gen peroxide-vanadate complex at ~430 nm, liquid phase measurement of hydrogen
peroxide concentration was made. It should be noted that the ppm units for hydro-
gen and oxygen are in the gas phase (volume/volume), and should not be confused
with the ppm units for hydrogen peroxide in liquid phase (weight/weight).
8.2.3 Microorganisms Exposure
The bacteria strain used to evaluate the surface decontamination activity of the
flowing humid argon post-discharge was Escherichia coli (Gram-negative bacterium,
96 E. Odic et al.
Enterobacteriaceae family of gamma-proteobacteria; strain DH1: ATTC 33849).

Bacteria suspended in 10 mL droplets (~108 bacteria/mL in droplets of 20% Vol. LB/
distilled water solution) were spotted onto four sterile glass slides (12 mm diameter
cover glass slides), which were then placed 5 mm below the reactor outlet and treated
by the discharge effluent gas. Each slide then was contaminated with about 106 bac-
teria. Prior to plasma treatment, a 10 min humid argon purge (2 L/min) of the treat-
ment vessel (with samples inside) was made. An extended exposure of the samples
to the flowing gas mixture with plasma off was systematically done, corresponding
to each treatment time, so providing the control samples. Once the plasma treatment
was achieved, bacterial samples were then collected by ten successive rinses with a
10 mL of 20/80 Vol. LB/distilled water solution. The obtained 100 mL suspension
was then diluted to 1 mL with the same solution. According to the expected results,
serial dilutions of the suspension could be done before plating 100 mL of the suspen-
sion on agar plates for direct CFU counting (30300 colonies per Petri dish). When
high decontamination efficiency was expected, the 1 mL suspension was not diluted
but was directly filtered on a Millipore 0.45 mm filter which was then plated on an
agar plate. Colony counting was done after 24 h of incubation at 37C.
8.2.4 DNA Exposure
DNA solutions in the form of 1020 mL droplets were spotted onto sterile glass
slides and were then exposed to the plasma treatment in the same way as had the
bacteria samples. Genomic DNA (100 mg/mL denatured salmon sperm DNA) and
plasmid (50 mg/mL PET9SnI: 4,285 bp circular double stranded DNA) were used
as model compounds. After treatment, according to the experimental conditions, the
sample could be dried or not. DNA was collected by four successive rinses with
10 mL distilled water droplets. The resulting solution was then analyzed. Agarose
gel (Tris Acetate EDTA TAE buffer solution pH8) electrophoresis was used to sepa-
rate DNA molecules according to their molecular weight and 3-D structure. Before
deposition of the DNA solution (20 mL) in the wells, 2 mL of negatively charged
loading buffer solution were added. After migration, the agarose gel was immerged
for 15 min in an ethidium bromide (EtBr intercalated into DNA) solution (0.5 mg/
mL) for DNA migration band visualization by UV light.
8.3.1 Non-thermal Plasma Source Characterization
The major stable products of water dissociation were measured in the flowing post-
discharge (O2 and H2 concentration in the discharge effluent using GC-TCD technique)
a.u.
9
8
7
6
5
4
3
2
1
0
200 300 400 500 600 700 800 900
wavelength (nm)
Fig. 8.2 Discharge emission spectrum for 50% RH at room temperature. Light collected by an optical
fiber, through the Pyrex wall and counter electrode (stainless steel mesh). Reactor 1: 3 W/2 L/min
of reactor 1 while optical emission spectroscopy was used in order to evaluate the OH
emission (in the discharge since the post-discharge is non emissive). Flow rate and
input power were maintained constant, 2 L/min and 3 W respectively, when the water
vapor content in argon was decreased at room temperature from 80% RH down to
near dry conditions (the elimination of trace water could not be obtained without
heating the device). An example of obtained emission spectra (OH* at 308 nm) is
presented in Fig. 8.2 and further results in Fig. 8.3.
The first major result found was that the smaller the humidity, the greater the OH
emission, down to a concentration range of 150800 ppm H2O, after which the
emission began to decrease (although never reaching zero due to trace water; the
lowest water vapor concentration presented in Fig. 8.3 corresponds to about 30 ppm).
The rise in OH emission with water content for very low humidity is due to a rising
production rate of total OH, while the decrease for even higher levels of water con-
tent is due to increased quenching of excited OH by water vapor, leading to greater
proportion of OH existing in the ground state.
This quenching reaction of OH*(A2S+) by water molecule can be found in litera-
ture [12] where the following mechanism was proposed:
Ar* ( p ) + H O Ar ( p ) + OH (A ) + H
4
2
3 * 2 +
(8.1)
( ) (
OH* A 2 + + H 2 O OH X 2 + H 2 O ) (8.2)
The range of 150800 ppm water content is close to the humidity for obtaining
the maximum emission by excited OH found in [12] (~200 ppm). Of course, emis-
sion of excited OH is not a direct measure of total hydroxyl radical concentration
nor of its evolution with water vapor content. The production of hydrogen and
98 E. Odic et al.
250 1
ppm H2
ppm O2
max OH emission (normalized)

200 max OH 0,8
150 0,6
H2, O2 ppm
100 0,4
50 0,2
0 0
0 5000 10000 15000 20000 25000 30000
ppm H2O
Fig. 8.3 Maximum normalized OH emission (308 nm) in the discharge zone, and hydrogen
oxygen concentration measured in the discharge effluent vs. water vapor content in argon. Reactor
1: 3 W/2 L/min
oxygen rises with humidity from dry conditions to ~2,00010,000 ppm H2O and
then slowly decreases. As mentioned above, direct measurement of total OH con-
centration has not been made but the production of hydrogen peroxide was studied.
Hydrogen peroxide formation results from:
OH + OH + Ar H 2 O2 + Ar (8.3)
HO2 + HO2 H 2 O2 + O2 (8.4)
HO2 + HO2 + Ar H 2 O2 + O2 + Ar (8.5)
Details about the kinetics can be found in [13].

Using the same discharge source, the concentration of hydrogen peroxide col-
lected in water droplets submitted to the discharge effluent was seen to increase
with water vapor content, whatever the flow rate, until reaching a plateau, whose
level depends on the input power (or energy density in J/L) [14]. In all cases, hydro-
gen and oxygen are always produced in much greater (at least one order) quantities
than hydrogen peroxide [13]. However, the hydrogen peroxide concentration plateau
level reached at given input power value as water vapor content increases suggests
that above a critical value of water vapor concentration, production of total OH
stabilizes. This assumption would agree with the stoichiometry inherent in reaction
(8.1), meaning that above this point, excited argon, and not water vapor, becomes
the limiting reactant in reaction (8.1). This critical value might then correspond to a
maximum production of total OH (and correspondingly H), leading to the inflection
in H2 production seen in Fig. 8.3. This implies that the observed decay in H2
production may come from increased quenching of energetic electrons and argon
metastable by increasing water content.
8.3.2 Bacteria Inactivation Tests
The same humid argon discharge effluent was investigated for the purpose of surface
decontamination. Glass slides contaminated with E.coli (~106 bacteria suspended in
a 10 mL water droplet) were exposed to the discharge effluent in comparable condi-
tions (2.6 W 2 L/min 95% RH in argon) and for increasing treatment time. In
order to estimate the loss in decontamination efficiency which should be caused by
longer transfer time of the activated species (short lived species such as OH and HO2
radicals and excited Ar* atoms) from the plasma source, a 525 mm long PFA tube
(6 mm ID) could be inserted between the outlet of the DBD tube and the biological
samples. Figure 8.4 shows the results (survival curve, i.e. number of survivors vs.
exposure time) obtained in both conditions: samples exposed to the flowing post-
discharge (1) directly at the DBD tube outlet (15 mm from the discharge source) and
(2) at the outlet of the PFA tube (540 mm from the discharge source). A reduction of
six orders of magnitude of E. coli population was achieved within 15 and 40 min for
the short distance and long distance experiments, respectively.
N (E. coli )
1,E+07
controls
1,E+06
1,E+05
540 mm from source

1,E+04
1,E+03
15 mm from source
1,E+02
1,E+01
1,E+00
1,E-01
0 5 10 15 20 25 30 35 40 45
Treatment time (min)
Fig. 8.4 E. coli survivors vs. exposure duration to DBD flowing post-discharge. Reactor 1: 2.6 W
2 L/min humid argon
100 E. Odic et al.
70 20
H2O2 15 mm from plasma source
18
60 H2O2 540 mm from plasma source
16
H2O2 in aqueous phase ppm
Water film volume loss %

contact with H2O2/Ar gas mixture -plasma off
50 14
Vol. loss 15 mm from plasma source
12
40 Vol. loss 540 mm from plasma source
10
30
8
20 6
4
10
2
0 0
0 2 4 6 8 10
Treatment time (min)
Fig. 8.5 Evolution of hydrogen peroxide concentration in a 3 mL distilled water film submitted
to a humid argon flowing post-discharge (reactor 1: 2.6 W 2 L/min humid argon); grey diamonds
at 10 min correspond to the hydrogen peroxide concentration measured in a 3 mL distilled water
film submitted to a flowing hydrogen peroxide/argon gas mixture (2 L/min) through reactor 1
(discharge off)
In these conditions, the production of hydrogen peroxide was separately measured

by exposing a water film (3 mL distilled water) to the discharge effluent for both
source/tube outlet distances (i.e. 15 and 540 mm from the discharge). The results
obtained, presented in Fig. 8.5, where the hydrogen peroxide concentration in the
water film is plotted as a function of the exposure time, indicate that about the same
amount of hydrogen peroxide is found directly at the outlet of the discharge tube
and at the outlet of the PFA tube (in series with the DBD tube). For both cases, a loss
in water film volume was observed (see Fig. 8.5), increasing with treatment time
and temperature. This evaporation was slightly higher for the shortest distance
between samples and discharge; at 10 min running time, an increase in gas tempera-
ture at the discharge tube outlet (15 mm from the source) of 6C was measured, and
evaporation was higher (~8.5% volume loss) than 540 mm from de source (5%
volume loss). As a consequence, hydrogen peroxide concentration was also higher
(the boiling point of hydrogen peroxide is 150.2C at atmospheric pressure).
In order to simulate hydrogen peroxide production by the humid argon dis-
charge, measurements were performed at the tube outlet (discharge off) when
bubbling argon through hydrogen peroxide solutions (various concentrations
obtained by dilutions from non-stabilized H2O2 30% Vol. CAS 95313). Figure 8.5
shows that comparable H2O2 concentrations in the water film could be observed
after an exposure time of 10 min, by contact with either the flowing discharge
effluent (2 L/min) or with a flowing argon/H2O2 gas mixture (2 L/min) obtained
when bubbling argon in a 3.1% Vol. H2O2 solution (grey diamonds at 10 min in
Fig. 8.5). Since the oxidative properties of hydrogen peroxide can lead to a pos-
sible decontamination effect, bacteria samples were submitted to this argon/H2O2
mixture (2 L/min) for 10 min. The results (0.4 log reduction see grey diamonds
in Fig. 8.4 at 10 min) are very close to those observed with the 10 min plasma
treatment with exposure at the outlet of the long PFA tube (0.38 log reduction),
but far inferior to those observed with the 10 min plasma treatment with direct
exposure at the DBD tube outlet (4.3 log reduction). It can then be assumed that
the surface decontamination effect observed (1) for a long distance from the
source is due to hydrogen peroxide and (2) for a short distance from the source
could be due to the combination of hydrogen peroxide and short-lived species
produced by the discharge. In the latter case, the action of hydrogen peroxide can-
not be neglected since, as shown in Fig. 8.5, its concentration in the water phase
is increasing faster for short distance from the source, even if gas phase produc-
tion of hydrogen peroxide remains constant, because of water evaporation. This
phenomenon will be drastically increased in the case of water droplets (in which
bacteria are suspended) as opposed to 3 mL water films. A second remark is that
at 2 L/min, the transit time of active species from the discharge to the surface
(15 mm from the source) was estimated to 25 ms. The interaction of short lived
species can thus not be neglected. This point was also investigated through the
study of DNA degradation using reactors 1 and 2.
8.3.3 DNA Degradation Tests
When a microorganism is not viable, this doesnt mean that its DNA has been dam-
aged. But if its DNA is strongly damaged, the microorganism is dead. This basic
consideration first motivated the study of the interaction of solutions of DNA (here,
both genomic DNA and plasmid) with the effluent of an argon discharge in order to
investigate the inactivation mechanisms induced by this plasma treatment.
A first set of experiments was conducted with reactor 2 for which gas flux was
not forced from the plasma zone to the contaminated surface; active species reached
the DNA contaminated surface through diffusion, possibly enhanced by the ionic
wind [15]. Distilled water droplets (10 mL) containing genomic DNA (1 mg) were
submitted to the effluent of a humid argon surface discharge during 10 min for three
discharge input power values: 2.5, 7.5 and 10 W. After collection, the treated DNA
solutions were analyzed by the gel electrophoresis technique (Fig. 8.6).
Lanes 3, 6 and 9 are controls. Lanes 1 and 2, obtained for the lower input power
(2.5 W), exhibit a smeared appearance, evidence of a partial degradation of DNA.
When the discharge input power is increased (7.5 W: lanes 4 and 5), the initial
DNA migration band disappears (complete degradation of DNA) and the smear is
shifted toward lower molecular weight. For the maximum input power (10 W:
lanes 7 and 8), the DNA is highly fragmented as shown by the weak smear in the
very low molecular weight migration region. In order to measure the hydrogen
peroxide concentration in the exposed droplets, the same experiments were per-
formed with distilled water instead of DNA solutions. As illustrated by Fig. 8.7
(reactor 2 data points and trend line), hydrogen peroxide concentration in water
droplets linearly increases with input energy (increasing input power for identical
102 E. Odic et al.
Fig. 8.6 Genomic DNA agarose gel electrophoresis after different treatment conditions. Lanes
19: 10 min. flowing post-discharge treatment (reactor 2: 2.510 W 0.5 L/min humid argon).
Lanes 1017: 10 min. incubation in H2O2 sol (S = samples, C = controls, migration from the top to
the bottom, .i.e. from high molecular weight to low molecular weight)
exposure time) and a 250 ppm maximum concentration is obtained for 10 W. In

order to evaluate the DNA degradation by hydrogen peroxide, the same quantity of
genomic DNA was incubated for 10 min in H2O2 solutions of increasing concentra-
tion (from 100 to 250 ppm). The electrophoresis gel of Fig. 8.6 proves that, even
for the higher hydrogen peroxide concentration (250 ppm: lane 16), this treatment
does not result in a polynucleotide chains fragmentation. The comparison with the
plasma treatment leading to the same water droplet H2O2 concentration (10 W,
250 ppm: lanes 7 and 8) clearly indicates that direct contact with the produced
hydrogen peroxide is not the mechanism responsible for DNA degradation during
the plasma treatment. As for E. coli treatment, the role played by short lived spe-
cies must be considered.
Similar experiments were performed with reactor 1, from which the transport of
active species from the discharge to the surface is forced by the feed gas flow. The
gas flow was reduced to 0.7 L/min in order to lower the water evaporation observed
during E. coli treatment experiments (2 L/min). Figures 8.8 and 8.7 (reactor 1 data
points and trend line) present the results obtained for genomic DNA degradation
and water droplet H2O2 concentration respectively.
P (W) - Reactor 2
0 2 4 6 8 10 12
400
H2O2 in aqueous phase ppm
350
300
250
200
reactor 1
reactor 2
150
100
50
0
0 0,5 1 1,5 2 2,5 3
P (W) - Reactor 1
Fig. 8.7 Hydrogen peroxide concentration measured in 10 mL distilled water droplets after
10 min exposure time to the discharge effluent for increasing discharge input power. Reactor 1:
0.52.5 W 0.7 L/min humid argon. Reactor 2: 110 W 0.5 L/min humid argon
For the lowest value of input power, the DNA migration band first appears as
slightly smeared (0.5 W: lanes 1 and 2). Increasing the discharge power to 1 and
1.5 W leads to similar results (lanes 4, 5, 7, 8), but when a 2.5 W value is reached,
both the initial DNA migration band and smear have disappeared (lane 10), indicat-
ing an almost total fragmentation of genomic DNA.
Numerical simulation and experimental results previously reported [13] showed
that decreasing the gas flow rate in reactor 1 and thus increasing the residence time
in the discharge (which corresponds to an increase in the specific energy density and
also gas temperature), led to increased decomposition of the formed H2O2 for the
higher input power values. As a matter of fact, in Fig. 8.7 (reactor 1 data points and
trend line), hydrogen peroxide concentration vs. input power goes through a maxi-
mum value (~350 ppm) and then decreases. By comparing the results of Figs. 8.7
and 8.8, it can be seen that for the maximum H2O2 concentration, corresponding to
a 11.5 W input power range, the DNA degradation is low. However, when com-
plete DNA fragmentation is achieved (2.5 W), the H2O2 concentration is slightly
below 200 ppm, the same concentration which was found for a 0.5 W input power,
and for which DNA degradation was minimal. This indicates that a mechanism
other than reaction with hydrogen peroxide is needed to fully explain the observed
DNA damage, likely reaction with hydroxyl radical. Furthermore, an experiment of
10 min of incubation of DNA in H2O2 solutions did not show significant degradation
of DNA (Fig. 8.8 lanes 12 and 14).
Aside from oxidation by hydrogen peroxide, DNA degradation was then studied
with a lower molecular weight DNA: PET9SnI, a circular double stranded DNA. Its
three conformations can be seen in lane 1 of the gel electrophoresis photograph of
104 E. Odic et al.
Fig. 8.8 Genomic DNA agarose gel electrophoresis after different treatment conditions. Lanes
111: 10 min. flowing post-discharge treatment (reactor 1: 0.52.5 W 0.7 L/min humid argon).
Lanes 1215: 10 min. incubation in H2O2 sol (S = samples, C = controls, migration from the top to
the bottom, .i.e. from high molecular weight to low molecular weight)
Fig. 8.9: nicked open-circular (one strand cut) conformation, relaxed circular con-
formation and supercoiled conformation (covalently closed-circular 3D structure
resulting in a compact form). Plasmid solution (50 mg/mL) droplets (20 mL) were
exposed to the discharge effluent of reactor 1. For a constant input power (1.3 W),
the feed gas composition and flow rate were modified. Treatment times were
extended to 20 min. Submitted to a dry argon discharge effluent (lanes 36), the
migration band corresponding to the nicked open-circular conformation of plasmid
disappears, while the relaxed and supercoiled conformation migration bands are
strongly diminished in intensity; a smear is observed in the low molecular weight
region. It is worth noting that the effect is less pronounced for the lower flow rate
(0.2 L/min: lanes 5 and 6), probably caused by the increased transfer time for short-
lived species (mainly Ar* in this case). In the same conditions of flow rate, treat-
ment time and input power, dry argon was replaced by humid argon. A complete
fragmentation of plasmid was obtained, as can be seen in lanes 7 and 8 where neither
migration bands nor any smear can be observed. It can then be assumed that water
dissociation products are the main species responsible for DNA degradation, even if
excited argon may contribute (e.g. reacting with liquid water).
Fig. 8.9 Plasmid PET9SnI agarose gel electrophoresis after 20 min. flowing post-discharge treat-
ment (lanes 38). Reactor 1: 1.3 W 0.20.7 L/min dry and humid argon (M = molecular weight
marker, S = samples, C = controls, migration from the top to the bottom, .i.e. from high molecular
weight to low molecular weight)
8.4 Conclusion
The treatment of contaminated surfaces by non-emissive flowing humid argon post-

discharge was investigated. A reduction of six orders of magnitude of E. coli popula-
tion was achieved: (1) within 15 min when the contaminated surface was directly
exposed to the discharge tube outlet (2) within 40 min when exposed to the outlet of a
525 mm long PFA tube inserted in series with the discharge tube exit. While the long
distance activity of the discharge effluent could be attributed to the action of the hydro-
gen peroxide formed by the discharge, short distance efficiency suggests the contribu-
tion of short-lived species. Similar experiments carried out on two model DNA
(genomic DNA and plasmid) led to their complete degradation when water vapor was
added to argon in the discharge. Results also demonstrated that direct contact with the
produced hydrogen peroxide was not the mechanism responsible for DNA degrada-
tion during the plasma treatment. Short-lived species produced by water dissociation
(implying Ar* in the discharge), such as OH and HO2 radicals could be the major
species accountable for DNA fragmentation and fast E. coli inactivation.
106 E. Odic et al.
References
1. Lee HW, Park GY, Seo YS, Im YH, Shim SB, Lee HJ (2011) Modelling of atmospheric
pressure plasmas for biomedical applications. J Phys D Appl Phys 44:053001 (27pp)
2. Kong MG, Groesen G, Morfill G, Nosenko T, Shimizu T, van Dijk J, Zimmermann JL (2009)
Plasma medicine: an introductory review. New J Phys 11:115012
3. Laroussi M (2009) Low-temperature plasmas for medicine? IEEE Trans Plasma Sci 37(6)
4. Boudam MK, Saoudi B, Moisan M, Ricard A (2007) Characterization of the flowing after-
glows of an N2-O2 reduced pressure discharge: setting the operating conditions to achieve a
dominant late afterglow and correlating the NOb UV intensity variation with the N and O atom
densities. J Phys D: Appl Phys 40:1694
5. Sarrette J-P, Cousty S, Merbahi N, Ngre-Salvayr A, Clment F (2010) Observation of
antibacterial effects obtained at atmospheric and reduced pressures in afterglow conditions.
Eur Phys J Appl Phys 49:13108
6. Villeger S, Cousty S, Ricard A, Sixou M (2003) Sterilization of E. Coli bacterium in a flowing
N2-O2 post-discharge reactor. J Phys D: Appl Phys 36:6062
7. Kamgang-Youbi G, Herry J-M, Meylheuc T, Brisset J-L, Bellon-Fontaine M-N, Doubla A,
Naitali M (2009) Microbial inactivation using plasma-activated water obtained by gliding elec-
tric discharges. Lett Appl Microbiol 48(1):1318
8. Pointu AM, Ricard A, Odic E, Ganciu M (2008) Nitrogen atmospheric pressure post dis-
charges for surface biological decontamination inside small diameter tubes. Plasma Processes
Polym 5(6):559
9. Oehmigen K, Hhnel M, Brandenburg R, Wilke Ch, Weltmann K-D, von Woedtke Th (2010)
Plasma Processes Polym Special Issue: Plasma Medicine 7(34):250257
10. Rahman M, Tanino M, Hashimoto M, Nakano M, Yasuda H, Takashima K, Mizuno A (2008)
Fundamental study on quasi-real-time detection of airborne bio-particles using discharge
plasma. Thin Solid Films 516:66996703
11. Yasuda H, Miura T, Kurita H, Takashima K, Mizuno A (2010) Biological evaluation of DNA
damage in bacteriophages inactivated by atmospheric pressure cold plasma. Plasma Processes
Polym Special Issue: Plasma Medicine 7(34):301308
12. Hibert C, Gaurand I, Motret O, Pouvesle JM (1999) [OH(X)] measurements by resonant
absorption spectroscopy in a pulsed dielectric barrier discharge. J Appl Phys 85(10):7070
13. Kirkpatrick MJ, Dodet B, Odic E (2007) Atmospheric pressure humid argon DBD plasma for
the application of sterilization measurement and simulation of hydrogen, oxygen, and hydro-
gen peroxide formation. Int J Plasma Environ Sci Technol 1(1):96101
14. Dodet B, Odic E, Goldman A, Goldman M, Renard D (2005) Hydrogen peroxide formation by
discharges in argon/water vapor mixtures at atmospheric pressure. J Adv Oxid Technol
8(1):9197
15. Dong B, Bauchire JM, Pouvesle JM, Magnier P, Hong D (2008) Experimental study of a DBD
surface discharge for the active control of subsonic airflow. J Phys D: Appl Phys 41:155201
Chapter 9
DNA Oxidation by Reactive Oxygen Species
Produced by Atmospheric Pressure
Microplasmas
Joao Santos Sousa, Pierre-Marie Girard, Evelyne Sage,

Jean-Luc Ravanat, and Vincent Puech
Abstract Arrays of microcathode sustained discharges (MCSDs) have been

developed for the production of high fluxes of singlet delta oxygen (SDO) and ozone
(O3) at atmospheric pressure. SDO and O3 densities higher than 1017 and 1016 cm3,
respectively, have been efficiently produced and transported over distances longer
than 50 cm. These arrays of MCSDs have been optimized to supply well-quantified
and tunable fluxes of either SDO or O3. This plasma source has been found to be very
useful for examining the reactivity of these reactive oxygen species with biological
components. Preliminary results indicate that both SDO and O3 are able to oxidize
DNA, originating great damages in DNA such as single- and double-strand breaks
and base oxidation. It has been observed that while all bases of DNA are almost
indifferently and quite effectively oxidized by O3, SDO reacts mainly with guanine.
J.S. Sousa (*)

Laboratoire de Physique des Gaz et des Plasmas (LPGP), Centre National de la Recherche
Scientifique (CNRS) and Univ. Paris-Sud,
Orsay, France
Instituto de Plasmas e Fuso Nuclear Laboratrio Associado,
Instituto Superior Tcnico, Lisboa, Portugal
e-mail: joao.santos-sousa@u-psud.fr
P.-M. Girard E. Sage
Laboratoire de Biologie des Radiations, CNRS & Institut Curie,
Orsay, France
J.-L. Ravanat
Laboratoire des Lsions des Acides Nucliques, CEA & Univ. Joseph Fourier,
Grenoble, France
V. Puech
Laboratoire de Physique des Gaz et des Plasmas, CNRS & Univ. Paris-Sud,
Orsay, France
108 J.S. Sousa et al.
Reactive oxygen species (ROS) are well known to play an important role in several
biological systems, and generate oxidative damage to a variety of cellular components
[14]. If the amount of oxidative damage overcomes the repair capacity of the cell,
this can ultimately lead to cell death, which is very important to take into account
for biomedical applications of plasmas. Therefore, fundamental studies are essen-
tial to determine firstly the nature of plasma-generated ROS, and secondly the abil-
ity of those ROS to damage biomolecules. In this context, we have developed arrays
of microcathode sustained discharges (MCSDs) for the production of ROS at atmo-
spheric pressure. Recently, we have demonstrated that due to their remarkable sta-
bility, MCSDs can be operated in a continuous basis to produce DC glow discharges
in He/O2 mixtures, free from the glow-to-arc transition, at high gas pressure, with
low values of the reduced electric field (510 Td) and gas temperature (300400 K)
[5]. We have also shown that MCSDs are very effective in producing large amounts
of singlet delta oxygen (SDO) and ozone (O3) at atmospheric pressure [6]. In fact,
SDO densities higher than 1017 cm3 have been efficiently produced and transported
over distances longer than 50 cm, providing SDO fluxes greater than 100 mmol/h
[7]. Furthermore, O3 densities up to 1016 cm3 have also been obtained. More impor-
tantly, it has been shown that the density ratio of SDO to O3 can be finely and easily
tuned in the range 103 to 10+5 through the values of discharge current, gas flow and
mixtures (especially by adjusting the O2 and NO partial pressures) [6]. As so, these
arrays of MCSDs, by allowing a controlled production of either SDO or O3 at atmo-
spheric pressure, are ideal tools for studying in detail the reactivity of these ROS
towards biological components.
In the present paper, we used the molecule of deoxyribonucleic acid (DNA) as a
tool to monitor oxidative damage induced by plasma-generated ROS. Preliminary
results of experiments concerning the use of arrays of MCSDs as a plasma source
for biological studies concerning DNA oxidation are presented and discussed.
9.1 Experimental Setup
In order to study the reactivity of SDO and O3 towards DNA, aqueous solutions of
DNA were exposed to a gas flow of either SDO or O3. We have developed a
microplasma reactor using four MCSDs for the production of high SDO and O3
densities. As schematized in Fig. 9.1, each MCSD consists of a micro-hollow cathode
discharge (MHCD), a discharge concept first developed by Schoenbach et al. [8], and
a third 2.5 cm diameter planar electrode, positioned at 8 mm from the exit plane of
the MHCD. This 3-electrode configuration was initially proposed by Stark and
Schoenbach [9]. In our device, the MCSDs are powered from only one negative
power supply (for the four MHCDs cathodes) and from only one positive power
supply (for the four MCSDs anodes 2), through individual ballasting resistors. The
MHCDs anodes 1 are directly grounded. The distance between the individual
MHCDs is 8 mm. The MCSDs are operated in He/O2 mixtures (<4% O2) at atmo-
spheric pressure. It should be noted that to attain SDO densities higher than 1016 cm3,
low concentrations of NO (<200 ppm) have to be added into the gas mixture [57].
9 DNA Oxidation by Reactive Oxygen Species 109
Fig. 9.1 Schematic of a

Micro-Cathode Sustained
Discharge (MCSD)
Fig. 9.2 Schematic representation of the experimental setup
As represented in Fig. 9.2, after passing through the plasma reactor, the gas flow
is evacuated through a line including a ROS-DNA interaction cell. The interaction
between the plasma-generated ROS (in gas phase) and the DNA (in aqueous solu-
tion) is performed at 55 cm downstream from the last MCSD. An accurate quantifi-
cation of the fluxes of ROS reaching the biological solutions is imperative for a
better understanding of the mechanisms of DNA oxidation. As so, we have mea-
sured and monitored the SDO and O3 densities at the entrance and exit ports of the
interaction cell [7], using infrared optical emission spectroscopy and ultraviolet
absorption spectroscopy, respectively, as described in detail in [6].
Oxidation of DNA has been performed by continuously bubbling aqueous solu-
tions of DNA with a gas flow of either SDO or O3. The biological experiments have
been conducted as a function of time of exposition, and under two very different
experimental conditions with respect to SDO and O3 densities, as shown in Table 9.1.
Indeed, in condition A, SDO density is maximal (1.5 1016 cm3) and O3 density is below
the limit of detection (<2.0 1012 cm3), while in condition Z, O3 density is maximal
(7.0 1015 cm3) and SDO density is below the limit of detection (<3.0 1013 cm3).
As DNA degradation is very sensitive to pH changes [10], buffered solutions
(10 mM KH2PO4 or 10/1 mM Tris-HCl/EDTA, pH = 6.8) were used in order to
maintain a constant pH. While the damages to the DNA backbone were analyzed by
agarose gel electrophoresis [11], the products of oxidation were detected and quan-
tified using the accurate and sensitive high performance liquid chromatography tan-
dem mass spectrometry method (HPLC-EIS-MS/MS) [12]. More details on the
experimental setup and procedure can be found in [7].
Table 9.1 Main characteristics of the two experimental conditions used

for performing DNA oxidation experiments
Condition A Condition Z
He/O2/NO (in sccm) 8,000/80/1.4 8,000/300/0
Discharge current (in mA/MCSD) 3 2
SDO density (in cm3) ~ 1.5 1016 <3.0 1013
O3 density (in cm3) <2.0 1012 ~ 7.0 1015
9.2 DNA Oxidation
9.2.1 Plasmid DNA
To evaluate DNA damage induced by SDO and/or O3, we have monitored the topol-
ogy of a plasmid DNA, pcDNA3.1 (Invitrogen), upon ROS exposure. Initially
pcDNA3.1 is mainly supercoiled (cf. Fig. 9.3, lanes 0), but ROS-induced single
strand break(s) (SSB) and double strand break(s) (DSB) lead to opened circular
(or relaxed) DNA and linear DNA, respectively. Plasmid DNA (Ci = 5 mg/ml) diluted
in 200 ml of either 10/1 mM Tris-HCl/EDTA (pH = 6.8) or of H2O (Cf = 20 ng/ml)
was exposed to the afterglow gas flow for different periods of time (up to 8 min). As
previously mentioned, damage to the DNA backbone of pcDNA3.1 was analyzed
by agarose gel electrophoresis. The results presented in the next sections are pre-
liminary and only a qualitative analysis is done. For the moment, not enough data
have been collected to correlate the amount of damages on the DNA backbone to
the number of molecules of SDO and O3 reaching the interaction cell.
9.2.1.1 Ozone Versus Singlet Delta Oxygen as Oxidizing Agent
At first, plasmid DNA was exposed to an afterglow gas flow of either O3 or SDO in
buffered aqueous solutions (10/1 mM Tris-HCl/EDTA, pH = 6.8). As shown in
Fig. 9.3, both ROS cause damage to the backbone of plasmid DNA, as revealed by
the change in the supercoiling of pcDNA3.1 (from supercoiled to circular and lin-
ear forms). Moreover, the number of breaks in the backbone rises as a function of
exposure time. However, as for equivalent times of treatment the number of dam-
ages generated by O3 is significantly greater than that induced by SDO, O3 seems
to be much more effective than SDO on oxidizing plasmid DNA. Indeed, after
4 min of O3 exposure, no more plasmid DNA in the supercoiled form is detected,
showing that at least one single strand break has been generated per molecule.
Furthermore, after 1 min of O3 exposure, double strand breaks are also formed as
revealed by the presence of linear DNA (Fig. 9.3, top panel). In marked contrast,
even after 8 min exposure of plasmid DNA to SDO, supercoiled DNA molecules
are still observed on agarose gel, and linear DNA is not detected (Fig. 9.3, bottom
panel). These results suggest that O3 is very efficient at generating single strand
breaks, when compared to SDO, and that greater time of exposure increases the
Fig. 9.3 Digital photographs of agarose gels showing the effect of O3 (top panel) and SDO
(bottom panel) on DNA topology at different times of plasmid DNA exposition to a gas flow of
each reactive oxygen species
probability of two single strand breaks being formed opposite to each other. It
should be emphasized that for exposure times as long as 8 min only the gas flow of
O3 induces DSBs.
9.2.1.2 Influence of pH
Figures 9.4 and 9.5 highlight the importance of using buffered solutions in order to
study ROS-induced DNA oxidation. Indeed, similar experiments to those described
in Fig. 9.3 were conducted in H2O instead of Tris-HCl/EDTA. Two different sources
of water were used: a commercially available one (Analychrom, Fisher Scientific
Labosi, France), of chromatography grade (pH 6.8), and a home-made one
(pH 5.5), obtained by passing tap water through a 0.22 mm filter and further purify-
ing it using an ultra-pure water system (Purelab prima, ELGA). The initial pH of the
plasmid DNA solutions (before interaction with a gas flow of ROS; t = 0 min), as
well as their pH after 2 min of exposure, was measured with an indicator paper with
a precision of 0.25.
At first, we found that the amount of damages in the backbone tends to be con-
siderably higher in H2O (Figs. 9.4 and 9.5), when compared to the use of buffered
solutions of Tris-HCl/EDTA (Fig. 9.3), and that the time needed to reach the same
level of breaks is much shorter, for both gas flows of SDO and O3. This may be, at
least partly, explained by the fact that DNA concentration is kept constant in all
Fig. 9.4 Digital photographs

of agarose gels showing the
effect of O3 on DNA topology
at different times of
exposition to the gas flow
of plasmid DNA in H2O
solution at pH 6.8 (top panel)
and pH 5.5 (bottom panel)
Fig. 9.5 Digital photographs

of agarose gels showing the
effect of SDO on DNA
topology at different times
of exposition to the gas flow
of plasmid DNA in H2O
solution at pH 6.8 (top panel)
and pH 5.5 (bottom panel)
experiments, corresponding to approximately 6.9 1011 molecules of DNA per tube.

A solution of 10 mM Tris-HCl/EDTA corresponds to approximately 1.2 1018 mol-
ecules of Tris per tube. In other words, there is a large excess of Tris molecules
when compared to DNA molecules. As plasma-generated ROS can potentially
interact with Tris molecules, the use of this buffer at such concentration may strongly
minimize oxidation of plasmid DNA. In pure H2O, the probability of interaction
between plasma-generated ROS and DNA is, thus, much higher.
Furthermore, in contrast to the use of buffered aqueous solutions, whose pH
remained nearly stable around 7 even after 8 min of exposure to the afterglow gas
flow, the use of H 2O solutions resulted in a considerable decrease of pH to 4.
It should be noticed that this induced acidification was observed after only 2 min
of exposure, and regardless of the main ROS present in the afterglow gas flow.
Given our experimental conditions (closed and controlled atmospheric environ-
ment), reactive species from the gas phase have to be considered as the cause of
liquid acidification. Many factors could be involved in the observed decrease of pH.
Hydrogen peroxide (H2O2) generated in the liquid phase could have lead to the cre-
ation of acidic H3O+ ions by reactions with water molecules [13], consequentially
decreasing the pH. On the one hand, O3 decomposes in water creating hydroxyl
radicals (OH) [14], which, by recombination, can generate H2O2 [15]. On the other
hand, SDO by reacting with water molecules can also generate H2O2 [16, 17]. The
other possibility for decreasing pH in condition A might be the formation in the
liquid phase of nitrous acid (HNO2) and nitric acid (HNO3) via NO2 generated in
the gas phase from the NO that is added into the gas mixture [18, 19]. Moreover, we
cannot exclude that residual organic/inorganic compounds present in the H2O solu-
tions, even if at very low concentrations, may participate in the acidification of those
solutions when exposed to a gas flow of O3 or SDO. Nevertheless, the acidity of the
aqueous solution seems to also play a major role. Indeed, exposure of plasmid DNA
to O3 or SDO gas flows in H2O solutions with initial pH value of 5.5 (Figs. 9.4 and
9.5, bottom panels), instead of 6.8 (top panels), not only gives rise to circular single
stranded fragments but also to extensive broken plasmid DNA molecules (Figs. 9.4
and 9.5, t = 2 min). The fragmentation of the plasmid DNA is responsible for the
almost homogeneous continuum that is observed. These experiments show that the
characteristics of the liquid solutions used (notably their pH) affect the nature and
amount of DNA damages induced.
At last, it must be pointed out that few backbone damages do not mean few DNA
damages because of the possibility of direct oxidation of DNA bases (or even their
removal). As so, the analysis of the chemical modifications of DNA bases is essen-
tial for a better understanding of DNA oxidation.
9.2.2 Isolated DNA
In order to gain further insights into the mechanism of oxidation of isolated DNA by
SDO and O3, experiments have been performed concerning base chemical modifica-
tions. Buffered (10 mM KH2PO4, pH = 6.8) aqueous solutions (H2O and D2O) of
calf-thymus DNA (0.5 mg/mL in a total volume of 24 mL) were exposed to the
afterglow gas flow for various periods of time (up to 8 min). Calf-thymus DNA and
deuterated water (D2O) were obtained from Sigma (St. Louis, MO). Water was
deionized with a Millipore/Milli-Q system (Millipore, Molsheim, France). Prior to
the HPLC-EIS-MS/MS measurements, DNA digestions were performed as
previously described in [20, 21]. The main oxidation products of DNA bases
Adenine, Cytosine, Guanine, and Thymine are measured as 8-oxodAdo (8-oxo-7,
8-dihydro-2-deoxyadenosine), 5-OHdCyd (5-hydroxy-2-deoxycytidine),
8-oxodGuo (8-oxo-7,8-dihydro-2-deoxyguanosine), and DiolThy (cis- and
trans-5,6-dihydroxy-5,6-dihydrothymine), respectively.
9.2.2.1 Ozone as Oxidizing Agent
As shown in Fig. 9.6, the main oxidation products of all DNA bases were
obtained when bubbling the aqueous solutions of DNA with a gas flow of O3.
DNA oxidation by O3 was, thus, successfully achieved. For the case of Adenine
and Thymine, the oxidized nucleosides production increases almost linearly
with the number of O3 molecules reaching the DNA solution. A two fold increase
in the number of O3 molecules leads to almost a two fold increase in 8-oxoAdo
and DiolThy. In the case of Guanine and Cytosine, we observe a biphasic curve
indicating that above a certain amount of O3 molecules, the formation of the
oxidized nucleosides starts to saturate. The stabilization of the number of
detected oxidized nucleosides is not likely to be related to a reduction of their
production rate, but instead indicates that these main oxidation products
(5-OhdCyd and 8-oxodGuo) might also be re-oxidized by the O3 flow. Therefore,
there might be a balance between their formation and destruction by O3.
According to the literature [22], the oxidized nucleosides 5-OHdCyd and
8-oxodGuo are rather susceptible to oxidation, which could explain the satura-
tion-like curve observed for those products. However, one should take into
account that a stabilization of the number of detected oxidized nucleosides
could also result from a deficient digestion of DNA. In fact, the enzymes respon-
sible for DNA digestion are less efficient when the number of oxidized bases is
relatively high, which is the case of these experiments where the number of
modified bases is considerable (0.13% of total bases). Nevertheless, the all
data suggest that O3 is very effective on oxidizing DNA, and especially Cytosine
(3% of Cytosine bases were oxidized).
Fig. 9.6 Amount of oxidized nucleosides (5-OHdCyd, DiolThy, 8-oxodAdo, 8-oxodGuo) per
million correspondent bases (Cytosine, Thymine, Adenine, Guanine) as a function of the number
of O3 molecules. Aqueous solutions of calf-thymus DNA were exposed to an afterglow gas flow
of O3 molecules for different periods of time up to 8 min. Samples were analysed by HPLC-
EIS-MS/MS
9.2.2.2 Singlet Delta Oxygen as Oxidizing Agent
According to Figs. 9.7 and 9.8, SDO seems to be also able to induce DNA oxida-
tion. In fact, all DNA bases but Cytosine are effectively oxidized by the SDO gas
flow. These results are somewhat in contradiction with the literature. The biochemi-
cal studies on the SDO-mediated oxidation of DNA that have been so far published
indicate that Guanine is the only normal DNA base that reacts with SDO [23, 24],
as no evidence of oxidation has been found regarding the other three bases. However,
Fig. 9.7 Amount of oxidized nucleosides per million correspondent bases as a function of the num-
ber of SDO molecules. Aqueous solutions (H2O) of calf-thymus DNA were exposed to an afterglow
gas flow of SDO molecules for different periods of time up to 8 min. Samples were analysed by
HPLC-EIS-MS/MS
Fig. 9.8 Amount of oxidized nucleosides per million correspondent bases as a function of the
number of SDO molecules. Same protocol as in Fig. 9.5, except for the use of heavy water (D2O)
instead of ordinary deionized water (H2O)
unlike what has been observed for a gas flow of O3, the amount of 8-oxodAdo and
8-oxodGuo generated by the SDO gas flow rapidly reach a plateau suggesting that
these oxidation products can also interact with the SDO gas flow and be further
modified. Additionally, the enhancing effect of D2O was used in order to confirm
the SDO-mediated DNA oxidation, as the SDO lifetime in D2O is assessed to be
525 times longer than in H2O [25, 26]. This H-D isotope effect on the SDO lifetime
is a consequence of an electronic-vibrational radiationless deactivation of SDO,
where the electronic excitation energy of SDO is converted into vibrational energy
of terminal bonds of deactivating collision partners. As the rate constant of this
energy-transfer mechanism is much higher with O-H bonds (2,900 M1 s1) than
with O-D bonds (132 M1 s1) [26], this leads to a considerably higher SDO lifetime
in D2O solutions. SDO molecules are, therefore, much less de-excited while passing
through a D2O solution than a H2O solution. For similar gas flows of SDO bubbling
the aqueous solutions of DNA, the SDO concentration is, thus, higher in D2O solu-
tions. As a consequence, the probability of a DNA molecule being oxidated by SDO
in D2O solutions is also higher. Indeed, in heavy water, about 530 times more dam-
ages were induced, and the plateaux were reached more rapidly, correlating the
oxidized nucleosides formation to the presence of SDO.
9.2.2.3 Ozone Versus Singlet Delta Oxygen as Oxidizing Agent
As shown in Table 9.2, in the experimental conditions that have been used in this
work, O3 molecules are one to three orders of magnitude more efficient than SDO
molecules on oxidizing DNA bases. In this regard, it is important to notice that, unlike
O3, most SDO molecules deactivate by physical quenching in the aqueous solutions
before reacting with DNA. In fact, the SDO lifetime in aqueous solutions is estimated
to be nine orders of magnitude lower than in the gas phase (~106 s) [26]. As O3 seems
to be much more effective on oxidizing DNA than SDO (cf. Table 9.2), O3 could still
play a role in oxidizing DNA in experimental condition A, even if its density is below
the limit of detection. However, taking into account that between experimental condi-
tions A and Z, the O3 densities differ from at least near four orders of magnitude, one
can conclude that, for all bases but Cytosine, at least ten times more damages were
obtained in experimental condition A than those which could be induced by residual
O3. This is a good sign for either direct or indirect SDO activity.
Table 9.2 Number of detected oxidized nucleosides per million correspondent bases for
experimental conditions A and Z, and the respective ratio
A Z
O3-induced damage
SDO ~ 1.5 1016 cm3 SDO < 3.0 1013 cm3 (Z) to SDO-induced
O3 < 2.0 1012 cm3 O3 ~ 7.0 1015 cm3 damage (A) ratio
8-oxodAdo 20 3,385 ~170
8-oxodGuo 30 833 ~28
DiolThy 40 4,689 ~117
5-OHdCyd 14 29,706 ~2,122
It should be mentioned that we cannot completely rule-out the implication of

other reactive species than SDO and O3 in the formation of the studied DNA oxida-
tion products, even if they are present in much lower concentrations. In particular,
as the maximum density of SDO requires NO admixture [57], NO and its by-
products (i.e. NO2, NO3, N2O5, HNO3), even if in much lower concentration than
SDO, could also be, at least partly, responsible for the observed DNA oxidation in
condition A. In order to exclude any possibility of DNA oxidation by NO and its
by-products, some control experiments have been performed. Aqueous solutions of
DNA have been bubbled with a gas flow of He/NO (1% vol NO) without plasma
ignition, and with the afterglow gas of MHCDs and MCSDs operating in similar
experimental conditions to condition A but without O2 admixture, ie, in He/NO
mixtures (175 ppm NO), with He and NO flows of 8,000 and 1.4 sccm respectively,
and at 3 mA. So far, no evidence of DNA oxidation has been found, and the admix-
ture of O2 seems to be rather important. Nevertheless, additional work is necessary
to understand the chemistry induced by these two ROS on the gas and liquid phases,
and its consequences in the decomposition of DNA.
9.3 Conclusions
The experiments that have been conducted indicate that SDO and O3 are able to
induce DNA oxidation, generating various damages in DNA such as single- and
double-strand breaks and oxidized bases. In particular, the number of modified
bases was considerably high (0.13% of total bases). It has been observed that while
all bases of DNA are almost indifferently and quite effectively oxidized by O3, SDO
reacts mainly with Guanine. Moreover, O3 seems to be much more effective on
oxidizing DNA. Indeed, double-strand breaks only occurred when using gas flows
of O3. Besides that, the amount of oxidized bases was also much higher when O3
molecules interacted with the DNA solutions, compared to the use of gas flows of
SDO. The enhancing effect of heavy water (D2O) has been used to confirm the
SDO-mediated DNA oxidation. When using heavy water, not only the same trends
have been observed as when using H2O, but also from 5 to 30 times more damages
were induced, correlating, therefore, the oxidized nucleosides formation to the pres-
ence of SDO.
The results that have been obtained, even if preliminary, are very significant and
demonstrate that arrays of MCSDs are a quite promising plasma source. We have
shown that arrays of MCSDs are very suitable and useful tools for biological studies,
and, thus, likely to lead to new biomedical applications. In fact, in the context of the
new field of Plasma Medicine, our plasma source is unique. Indeed, in contrast to
other available sources of ROS, our arrays of MCSDs are able to supply well-quantified
and tunable fluxes of either SDO or O3. Nevertheless, there are still many open ques-
tions on the reactivity of ROS with DNA. Our experiments have shown that the
characteristics of the liquid solutions of DNA play an important role, affecting
the amount and nature of the damages induced by the plasma-generated ROS on the
mechanical and chemical structure of DNA. In fact, the chemistry in the interface of
the gas and liquid phases is very important but rather complicated. For a better under-
standing of the mechanism of ROS-mediated oxidation of DNA, efforts are to be
made to gain further insights into the chemistry of the liquid phase.
References
1. Packer L, Sies H (eds) (2000) Methods in enzymology, singlet oxygen, UV A and ozone.
Academic, New York
2. Wiseman H, Halliwell B (1996) Damage to DNA by reactive oxygen and nitrogen species: role
in inflammatory disease and progression to cancer. Biochem J 313:1729
3. Sies H (1997) Oxidative stress: oxidants and antioxidants. Exp Physiol 82:291295
4. Bandyopadhyay U, Das D, Banerjee RK (1999) Reactive oxygen species: oxidative damage
and pathogenesis. Curr Sci 77:658666
5. Sousa JS, Bauville G, Lacour B, Puech V, Touzeau M, Pitchford LC (2008) O2(a1g) pro-
duction at atmospheric pressure by microdischarge. Appl Phys Lett 93:011502-1011502-3
6. Sousa JS, Bauville G, Lacour B, Puech V, Touzeau M (2009) Atmospheric pressure generation
of O2(a1Dg) by microplasmas. Eur Phys J Appl Phys 47:22807-122807-7
7. Sousa JS, Bauville G, Lacour B, Puech V, Touzeau M, Ravanat JL (2010) DNA oxidation by
singlet delta oxygen produced by atmospheric pressure microdischarges. Appl Phys Lett
97:141502-1141502-3
8. Schoenbach KH, El-Habachi A, Shi W, Ciocca M (1997) High-pressure hollow cathode
discharges. Plasma Sources Sci Technol 6:468477
9. Stark RH, Schoenbach KH (1999) Direct current high-pressure glow discharges. J Appl Phys
85:20752080
10. Steenken S, Jovanovic SV (1997) How easily oxidizable is DNA? One-electron reduction poten-
tials of adenosine and guanosine radicals in aqueous solution. J Am Chem Soc 119:617618
11. Brody JR, Kern SE (2004) History and principles of conductive media for standard DNA
electrophoresis. Anal Biochem 333:113
12. Martinez GR, Ravanat JL, Cadet J, Medeiros MHG, Di Mascio P (2007) Spiroiminodihydantoin
nucleoside formation from 2-deoxyguanosine oxidation by [18O-labeled] singlet molecular
oxygen in aqueous solution. J Mass Spectrom 42:13261332
13. panel P, Diskin AM, Wang T, Smith D (2003) A SIFT study of the reactions of H3O+, NO+
and O2+ with hydrogen peroxide and peroxyacetic acid. Int J Mass Spectrom 228:269283
14. Glaze WH (1987) Drinking-water treatment with ozone. Environ Sci Technol 21:224230
15. Zellner R, Ewig F, Paschke R, Wagner G (1988) Pressure and temperature dependence of the
gas-phase recombination of hydroxyl radicals. J Phys Chem 92:41844190
16. Xu X, Muller RP, Goddard WA III (2002) The gas phase reaction of singlet dioxygen with
water: a water-catalyzed mechanism. Proc Natl Acad Sci USA 99:33763381
17. Wentworth P Jr, Jones LH, Wentworth AD, Zhu X, Larsen NA, Wilson IA, Xu X, Goddard WA
III, Janda KD, Eschenmoser A, Lerner RA (2001) Antibody catalysis of the oxidation of water.
Science 293:18061811
18. Oehmigen K, Hahnel M, Brandenburg R, Wilke Ch, Weltmann KD, von Woedtke Th (2010)
19. Chen CW, Lee HM, Chang MB (2008) Inactivation of aquatic microorganisms by low-
frequency AC discharges. IEEE Trans Plasma Sci 36:215219
20. Frelon S, Douki T, Ravanat JL, Pouget JP, Tornabene C, Cadet J (2000) High-performance
liquid chromatography-tandem mass spectrometry measurement of radiation-induced base
damage to isolated and cellular DNA. Chem Res Toxicol 13:10021010
21. Douki T, Court M, Sauvaigo S, Odin F, Cadet J (2000) Formation of the main UV-induced
thymine dimeric lesions within isolated and cellular DNA as measured by high performance
liquid chromatography-tandem mass spectrometry. J Biol Chem 275:1167811685
22. Douki T, Bretonniere Y, Cadet J (2000) Protection against radiation-induced degradation of
DNA bases by polyamines. Radiat Res 153:2935
23. Sies H, Menck CF (1992) Singlet oxygen induced DNA damage. Mutat Res 275:367375
24. Ravanat JL, Saint-Pierre C, Di Mascio P, Martinez GR, Medeiros MHG, Cadet J (2001)
Damage to isolated DNA mediated by singlet oxygen. Helv Chim Acta 84:37023709
25. Rodgers MAJ, Snowden PT (1982) Lifetime of oxygen (O2(1g)) in liquid water as determined
by time-resolved infrared luminescence measurements. J Am Chem Soc 104:55415543
26. Schweitzer C, Schmidt R (2003) Physical mechanisms of generation and deactivation of
singlet oxygen. Chem Rev 103:16851757
Chapter 10
Optical Emission Spectroscopic Evaluation
of Different Microwave Plasma
Discharges and Its Potential Application
for Sterilization Processes
Jos L. Hueso, Vctor J. Rico, ngel Yanguas-Gil,

Jos Cotrino, and Agustn R. Gonzlez-Elipe
Abstract The present work aims at studying different microwave flowing discharges
containing Ar and/or NO as alternative candidates to more extended N2 containing
plasma mixtures like N2-O2. Optical Emission Spectroscopy (OES) is used to dem-
onstrate the potential possibilities of these plasma mixtures to provide O* and UV
intermediate species demanded for sterilization purposes at low temperatures and
extended discharge gaps. Additionally, some plasma sterilization experiments with
Escherichia coli cultures are presented.
J.L. Hueso (*) V.J. Rico J. Cotrino A.R. Gonzlez-Elipe

Instituto de Ciencia de Materiales de Sevilla, Avda Americo Vespucio, 49,
41092 Sevilla, Spain
Departamento de Qumica Inorgnica, CSIC-University of Sevilla,
Avda Americo Vespucio, 49, 41092 Sevilla, Spain
Departamento de Fsica Atmica, Molecular y Nuclear de la Universidad
de Sevilla, Sevilla, Spain
e-mail: jlhueso@unizar.es
. Yanguas-Gil
Instituto de Ciencia de Materiales de Sevilla, Avda Americo Vespucio, 49,
41092 Sevilla, Spain
Departamento de Qumica Inorgnica, CSIC-University of Sevilla,
Avda Americo Vespucio, 49, 41092 Sevilla, Spain
Departamento de Fsica Atmica, Molecular y Nuclear de la Universidad
de Sevilla, Sevilla, Spain
Argonne National Laboratory, Argonne, IL, USA
122 J.L. Hueso et al.
10.1 Introduction
The application of plasma discharges for biomedical applications is one of the

most promising fields to be developed within the plasma research area [1].
Recent advances in sterilization of heat-sensitive medical tools, remediation of
bacteria, spores and other biomolecules or the improvement of cauterization of
wounds and coagulation of blood by means of indirect or direct intermediate
active species generated in plasmas are really encouraging [16]. One of the
main objectives to achieve is getting a better understanding of the fundamental
mechanisms of interaction between the plasma intermediate species and the cor-
responding biological interface. As part of this understanding process and
despite of being thoroughly studied for the last 20 years, the behavior of the
reactive species generated in the plasma discharge, their kinetics or power
absorption mechanisms under different experimental conditions are still very
important variables to be controlled. Optical Emission Spectroscopy (OES) is
considered as an excellent diagnostic tool for a wide variety of plasma pro-
cesses. This technique is non-intrusive and capable of detecting both neutral
and ionic excited species [7]. OES can also be used to measure temperatures and
relative populations, as long as the main parameters of the optical transitions
(i.e. transition probabilities or degeneracies and energies of the emitting levels)
are known.
In recent years, different type of discharges containing inert gases, N2O, O2, air,
N2, CO2, H2O2 or mixtures of them, have been considered for sterilization purposes
and in general terms, reasonable good results have been achieved [13, 6, 812]. In
all the cases, the UV radiation (200300 nm) coming from the glow discharges
seems to be one of the main factors for the sterilization activity of the plasmas. More
recent investigations have demonstrated the synergetic role of O atoms in conjunc-
tion with the UV photons [2, 1215]. Low/moderated pressure plasmas permit the
co-existence of UV photons and atomic atoms that has been reported as a synergetic
effect by Moisan et al. In contrast, under higher pressures and thus with higher col-
lision frequencies in the atmospheric conditions, a great part of the charged and
excited species are recombined with carrier gas atoms.
The aim of the present work consists of a thorough study of moderated-pres-
sure microwave (MW) plasma discharges containing NO to demonstrate the
potential possibilities of this molecule to provide the O and UV species demanded
for sterilization processes. Moreover, NO has been recently defined as an excel-
lent intermediate for anti-inflammatory and wound-healing processes and the
application of these plasma species to other regeneration treatments (i.e. angio-
genesis) can be envisaged [1, 16, 17]. The potential possibilities of Ar as carrier
gas in comparison with nitrogen [6, 14, 18] are also addressed in terms of plasma
length and density of afterglow active species. Finally, the in vitro sterilization
efficiency of Escherichia coli microorganisms under the exposure of selected
plasma mixtures is discussed.
10 Optical Emission Spectroscopic Evaluation of Different Microwave 123
10.2 Experimental Section
The dimensions of the quartz tube, terminated in a funnel-type enlargement, and the
stainless-steel cylinder chamber used for the experiments have been described else-
where [19]. It is coupled to the surface-wave (SW) surfatron which permits the
transformation of the electromagnetic power to the travelling wave and the propaga-
tion of the plasma discharge. The MW discharges are produced in a moderated
pressure range between 30 and 90 Torr, although the presented results are referred
to 45 Torr. Ar or N2 were fed through calibrated mass flow controllers as carrier
gases with a total flow of 50 cm3 min1. The concentration of NO and occasionally
O2 were kept constant at 3 103 and 2 104 ppm respectively. Axial profiles along
the x-axis of the quartz reactor at different points from the gap discharge have been
obtained for the main plasma mixtures. The OES spectra have been registered by
collecting the light with an optical fiber connected to a scanning monochromator
(Jobin-Ybon HR250) and a Hamamatsu photomultiplier (R928).
Escherichia coli DHa was grown in Lysogeny Broth (LB) liquid medium at
37C for 18 h. Prior to the plasma irradiation, the E. coli cells were extensively
washed with distilled water and the number of colony-forming units (CFU) was
determined by the viable count method to normalize the sterilization experiments.
This method was also used in the estimation of the survivor cells after the steriliza-
tion experiments. UV exposure experiments were carried out with a lamp emitting
at 254 nm. To properly compare the survival of E. coli after the different experi-
ments, total photon intensities measured by OES during the plasma experiments and
that of the UV lamp were used for normalization of results. Initial E. coli concentra-
tions were comprised between 1 108 and 3 108 CFU/mL (Fig. 10.1).
Fig. 10.1 Scheme of the experimental setup used for the OES study and the Escherichia coli
sterilization experiments: (a) mass flow controllers; (b) funnel-type quartz tube; (c) pressure
gauge; (d) MW power source; (e) surfatron launcher; (f) petri dish holder; (g) rotary pump/gas
outlet. (Right): digital photograph of the reactor
10.3 OES Study of Different Microwave Plasma Discharges
10.3.1 Ar-NO Discharges
Figure 10.3 shows a typical OES spectrum of an Ar/NO plasma mixture and
Table 10.1 summarizes the main contributions and their electronic transitions. The
Ar atomic lines from the 5p excited state in the 700850 nm region and the Second
Positive system of N2* in the region 300400 nm are the most intense features
detected. Ar* lines from lower (i.e., 4p) excited states and some O* atomic lines are
also observed in this figure. The presence of O* and N2* can be explained by the NO
decomposition (Fig. 10.2).
A more detailed examination of the UV region in Fig. 10.3 shows the emis-
sion of NO* bands corresponding to the b and g systems. These species are espe-
cially relevant for sterilization purposes according to previous works with
low-thermal plasmas. The following reactions may resume some of the most
probable mechanisms, including a first excitation step and a subsequent de-
excitation process:
Ar* + NO (X ) NO (A, B) NO (X )+ hn(a / b) (10.1)
NO (X )+ e NO (A, B) NO (X )+ hn (a / b ) (10.2)
N + O + M (M = N 2 ) NO (A, B)+ M (10.3)
where NO(X) represents the ground state of the NO molecule.

Reactions (10.1, 10.2, 10.3) are the most probable pathways for the NO* emis-
sion bands in the plasmas mixtures containing Ar. The systematic evaluation of the
intensity of the emission lines at different axial positions from the surfatron location
yielded a slightly decreasing tendency, as expected from previous experimental
results available in literature. By contrast, the profiles of NO* intensities surpris-
ingly follow the opposite tendency, i.e. intensities increase towards the end of the
glow zone (Fig. 10.4a).
Some of the Ar emission lines have been followed in order to obtain the Ar
excitation temperature. In this work a set of ten Ar emission lines with different
emitting levels have been measured. This set of lines, along with the properties of
the emitting levels (excitation energy, degeneracy and emission frequency) pro-
vides the information necessary to ascertain the relative populations of the excited
configurations of Argon. A Boltzmann-plot derived from those data is presented
in Fig. 10.4b: within the measurement errors (estimated to be of the order of a
15%), a linear relation appears between the logarithm of the intensity (modified
by the degeneracy of the emitting level and the emission frequency) and the energy
of the emitting level, so that the levels above the 4p excited configuration behave
as if they were in equilibrium with each other (see red-dotted linear trend for
upper-excited levels selected for calculations in Fig. 10.4b). Through the inverse
Table 10.1 Summary of main species detected by OES in the different plasma mixtures under
study [1921]
Species System Transition Position (nm)
N2 2nd Positive C3 B3 260400
NO b-System B2 X2 200300
g-System A2+ X2 260320
N2+ Principal 2
2 385395
Ar 4p and 5p lines 3s2p54p 3s2p54s 400440
3s2p55p 3s2p54s 700850
O Atomic lines 2s2p33p 2s2p33s 777; 782; 845
Fig. 10.2 Spectrum obtained by optical emission spectroscopy for an Ar/NO mixture (45 Torr,
60 W)
Fig. 10.3 Emission species

in the UV region for an
Ar-NO plasma discharge
Fig. 10.4 (a) Influence of the distance from the resonant cavity on the emission intensities of NOg
and NOb intermediates in the Ar+NO plasma discharge. (b) Plot-Boltzmann of the Ar levels mea-
sured by OES from line intensities (45 Torr, 60 W) for the Ar-NO mixture. Red dotted line shows
the upper excited states employed for the calculation of the excitation temperature
of the slope of this linear relation it is possible to obtain an excitation temperature

of the argon excited levels. This temperature was estimated to be 3,500 K in the
case of the Ar/NO mixture.
The excitation temperature derived from the Boltzmann-plot, however, must not
be confused with the electron kinetic temperature: the experimental values of this
parameter for surface-wave discharges are usually greater than 1 eV, and even at
atmospheric pressures electron temperatures over 0.5 eV have been experimentally
measured. Nevertheless, the analysis reveals that the excitation temperature of the
Ar species remains constant regardless of the axial position and that could be con-
sidered as a physical magnitude proportional to the electron kinetic temperature.
This is a common feature of low pressure discharges, as the collisions between par-
ticles are not high enough to ensure thermodynamic equilibrium of the different
species in the discharge [22, 23].
Therefore, the decreasing emission intensities of Ar, N2 and O2 species when
moving further from the surfatron are a consequence of the characteristic decreas-
ing profile of the electron density in surface-wave discharges. In SW plasmas the
electron density is known to have a maximum closer to the surface-wave launcher
and a minimum at the column end. As the populations of the excited states depend
directly on this parameter, it is reasonable to assume that their emission intensities
should fall when approaching the column end. For that reason, in principle it is
striking that the intensity of the NO* emission bands follows the opposite ten-
dency, increasing with the axial position contrary to the other species and the
electron density.
Herein, we can assume that the dissociation efficiency of NO is higher just
after the surfatron and become less important as we progressively move away.
Indeed, there seems to be a direct correlation between the electron density and
the position from the surface-wave launcher. We also assume that the NO (A,B)
creation pathways become predominant respect to the NO dissociation mecha-
nisms as we move along the glow discharge. The following reactions, may
explain the main dissociation pathways of NO and its relation with the electron
density [24]:
NO + e NO + + e + e (10.4)
Ar* + NO NO + + e + Ar (10.5)
NO + + e N + O (10.6)
Reactions (10.4, 10.5) are well known from the ionosphere chemistry and are
responsible for the production of the intermediate NO+ by electron impact ionization
(R5-6) or energy transfer from the Ar metastable states (R6). Depending on the posi-
tion of the glow discharge there is a competition between reactions (10.1, 10.2, 10.3)
for the production of NO and reactions (10.4, 10.5, 10.6) for its remediation. Recent
axial studies of MW Ar discharges at intermediate pressures have demonstrated the
rise of the electronic temperature at the end of the column [25, 26]. This fact can be
associated in our case with an increase of the recombination of molecular ions that
can contribute to the generation of NO* excited states through (R1-3) as we move
away from the surfatron launcher [2426]. From the point of view of the sterilization
efficiency, a higher concentration of NO* species at longer distances from the surfa-
tron launcher should ensure a higher sterilization efficiency even on surfaces not
directly exposed to the plasma. This difference supposes a clear advantage for the use
of Ar-NO for plasma sterilization.
10.3.2 N2-NO and N2-O2 Mixtures
The most common mixtures used in plasma based sterilization processes at low
pressures contain nitrogen as carrier gas. However, there are fewer studies in the
moderated pressure range considered in our case. The study of N2-NO and eventu-
ally N2-O2 will be mainly done for comparison purposes with the Ar-containing
mixtures. We find a first and clear distinction when we compare the OES general
spectra (Fig. 10.5) since the excited intermediates from the Second Positive System
of N2 are more intense than the others (Table 10.1). Conversely, there is no clear
signal from O* and only NO* intermediates from the NOg system are observed in the
presence of any of the other mixtures containing N2 as carrier gas (Fig. 10.6). This
fact is indicative of different excitation channels for Ar (atomic gas) and N2 (molec-
ular gas). In the case of N2 a great part of the energy transfer is consumed in the
excitation of vibrational levels. This fact might explain why only the less excited
levels of NO (i.e. NO(A)) are detected and why only the bands corresponding to the
NOg system are identified (Fig. 10.6a, b).
We underline reaction (10.7) as one of the main processes contributing to the
emission of NOg:
NO2* + NO(X) NO(A) NO(X) + hn(a ) (10.7)
Fig. 10.5 Spectrum obtained by OES for a N2/NO mixture (45 Torr, 60 W). The inset shows the
main transitions corresponding to the second positive system of N2
Fig. 10.6 Emission species in the UV region for: (a) N2-NO and (b) N2-O2 plasma discharges
In principle, the absence of the NOb system can be detrimental for sterilization
purposes in post-discharge configurations. Another major drawback accounts for
the limited length extension of the plasma discharge when N2 is present and the
abrupt decay of emission intensities as we move away from the surfatron launcher.
This difference supposes another clear advantage for the use of Ar-NO for plasma
sterilization. Not only the more active NOb species are formed with the mixture but
also a more effective exposure to UV emitters is ensured because of the higher
concentrations of NO* species existing at the end of the column. Nevertheless, the
presence of other chemical active species like long-live neutral O, especially in the
N2-O2 plasma mixture can not be ruled out for sterilization purposes [27, 28].
10.4 Escherichia coli Sterilization Experiments
The plasma mixtures containing Ar seem to be really promising candidates for ster-
ilization purposes and their use is not so extended and studied [6, 14, 18] as the
low-thermal plasmas with N2 as main carrier gas. For this reason, we carried out a
sterilization experiment with Escherichia coli bacteria after exposure to Ar, Ar-NO
and N2-O2 plasma discharges for increasing periods of time. The survival curves are
depicted in Fig. 10.7.
Both the D-values (Decimal value) defined as the time necessary to reduce the
original concentration of micro-organisms by one order of magnitude [3] and the
sterilization kinetics present different trends for the different gases. After 300 s of
exposure to single feed Ar, the number of colony-forming units was 6-log10 reduced.
The curve present single-slope kinetics and D-value of 54 s. For the binary mix-
tures, there is a clear bi-phasic deactivation mechanism represented by a double-
slope survival curve with an initial D-value inactivation time (D1 < 10 s) much
shorter than the second one (50 s < D2 < 90 s). This behaviour has been previously
reported in N2-O2 experiments at low pressures but not for Ar-NO discharges. D1-
values are almost identical for both mixtures, although global results are slightly
better for Ar-NO. In principle, we attribute the observed differences to the activity
of UV radiation. This hypothesis is reflected in the survival curve found after expo-
sure of the E. coli cultures to the UV lamp (Fig. 10.7). The obtained curve defines a
similar trend to those of the N2-O2 and Ar-NO plasmas, thus supporting the impor-
tance of the UV radiation in the sterilization processes. The relatively lower effi-
ciency found for the UV experiment suggests that besides the effect of the UV
photons, other factors play a certain role in the case of N2-O2 or Ar-NO plasmas.
Indeed, SEM images in Fig. 10.8 show disruption and cell lysis of E. coli cells
subjected to Ar-NO plasma treatment. If we compare the untreated bacteria (c.f.
Fig. 10.8a) with the irradiated ones (Fig. 10.8b), we observe serious morphological
changes both lengthwise and transversely in many individuals. Therefore it is prob-
able that an additional etching mechanism from the Ar* and O* intermediates are
also contributing to the erosion of cells as proven by previous authors. From the
survival curves, it also arises that UV photons are the main contributing factors to
the damage of the bacteria since the UV lamp and the binary mixtures containing
NO* species exhibit a similar bi-phasic deactivation mechanism. The mono-phasic
trend observed for Ar* must be attributed to etching processes thereby corroborating
the active role of UV species and the suitability of Ar-NO plasma discharges to
provide a good combination of UV and etching species. Likewise, in the case of the
N2-O2 plasma mixture, an analogous synergetic effect combining UV radiation and
erosion from O neutral species is expected as previously addressed by Moisans
Fig. 10.7 Survival curve of Escherichia coli irradiated by Ar, Ar+No and N2+O2 surface-wave
plasma discharges and an UV lamp. Initial E. coli concentrations were comprised between
1 1083 108 CFU/mL. Adapted from [11]
Fig. 10.8 SEM images of Escherichia coli: (a) as untreated control and (b) after irradiation
with an Ar+NO plasma for 10 min. The inset in (b) is represented in an enlarged scale. Reproduced
with permission from [11]
research group [27, 28]. These afterglow atomic O species can not be detected in
our system but can be active in the VUV region and stable for microseconds, thereby
providing enough energy to break C-C bonds from DNA strands or cause oxidative
irreversible damage to proteins, cytoplasmic membrane and genetic material.
10.5 Conclusions
Optical Emission Spectroscopy has demonstrated to be an excellent non-intrusive

diagnostic tool to evaluate the intermediate plasma species that may be contributing to
sterilization processes of E. coli. Ar-NO plasma discharges can generate a high
concentration of NO* (UV)-active emitters and Ar* and O* etching radicals that extend
up to much longer distances from the surfatron applicator than in the case of the
N2-NO counterparts. We put in evidence that the intensity of the NO* bands increases
at the end of the Ar-NO plasma while the NO destruction is maximum in the plasma
region close to the surfatron gap. We attribute this trend to the existence of two com-
peting reaction pathways where the electron density and the electronic temperature
are predominant at the beginning and the end of the plasma column, respectively.
Therefore, these factors should be taken into account for the design of sterilization
reactors in order to maximise the flux of active species. Likewise, it is shown that the
excitation of the vibrational and rotational levels of N2 limits its use as carrier gas and
constitutes a drawback for its implementation in sterilization processes of certain
dimensions or limited light of sight. In this sense, the use of a combined ternary
mixture of Ar-N2-O2 can be envisaged as a promising solution of compromise [14].
References
plasma medicine. Plasma Processes Polym 5(6):503533
2. Boudam MK, Moisan M (2010) Synergy effect of heat and UV photons on bacterial-spore
inactivation in an N-2-O-2 plasma-afterglow sterilizer. J Phys D Appl Phys 43(29):295202
Plasma Processes Polym 2(5):391400
4. Moisan M, Barbeau J, Pelletier J (2001) Plasma sterilization methods and mechanisms.
Vide-Sci Tech Et Appl 56(299):1528
mechanisms. Int J Pharm 226(12):121
6. Rossi F, Kylian O, Rauscher H, Hasiwa M, Gilliland D (2009) Low pressure plasma discharges
for the sterilization and decontamination of surfaces. New J Phys 11:115017
7. Machala Z, Janda M, Hensel K, Jedlovsky I, Lestinska L, Foltin V, Martisovits V, Morvova M
(2007) Emission spectroscopy of atmospheric pressure plasmas for bio-medical and environ-
mental applications. J Mol Spectrosc 243(2):194201
8. Moreau S, Moisan M, Tabrizian M, Barbeau J, Pelletier J, Ricard A, Yahia L (2000) Using the
flowing afterglow of a plasma to inactivate Bacillus subtilis spores: influence of the operating
conditions. J Appl Phys 88(2):11661174
9. Chau TT, Kao KC, Blank G, Madrid F (1996) Microwave plasmas for low-temperature dry
sterilization. Biomaterials 17(13):12731277
10. Sato T, Miyahara T, Doi A, Ochiai S, Urayama T, Nakatani T (2006) Sterilization
mechanism for Escherichia coli by plasma flow at atmospheric pressure. Appl Phys
Lett 89(7):073902
11. Hueso JL, Rico VJ, Frias JE, Cotrino J, Gonzalez-Elipe AR (2008) Ar+NO microwave plas-
mas for Escherichia coli sterilization. J Phys D Appl Phys 41(9):092002
12. Ricard A (2005) Optical spectroscopy on processing plasmas: cathode magnetron sputtering
and flowing post-discharges for elastomer activation and medical sterilization. Thin Solid
Films 475(12):15
13. Moreau M, Orange N, Brisset JL (2005) Application of electric discharges at atmospheric
pressure and ambient temperature for bio-decontamination. Ozone-Sci Eng 27(6):469473
14. Kylian O, Rossi F (2009) Sterilization and decontamination of medical instruments by low-
pressure plasma discharges: application of Ar/O-2/N-2 ternary mixture. J Phys D Appl Phys
42(8):085207
15. Villeger S, Sarrette JP, Ricard A (2005) Synergy between N and O atom action and substrate
surface temperature in a sterilization process using a flowing N-2-O-2 microwave post
discharge. Plasma Processes Polym 2(9):709714
16. Dobrynin D, Arjunan K, Fridman A, Friedman G, Clyne AM (2010) Direct and controllable
nitric oxide delivery into biological media and living cells by a pin-to-hole spark discharge
(PHD) plasma. J Phys D Appl Phys 44(7):075201
18. Stapelmann K, Kylian O, Denis B, Rossi F (2008) On the application of inductively coupled
plasma discharges sustained in Ar/O-2/N-2 ternary mixture for sterilization and decontamina-
tion of medical instruments. J Phys D Appl Phys 41(19):192005
19. Pearse RWB, Gaydon AG (1976) The identification of molecular spectra. Chapman & Hall,
New York
20. Hueso JL, Gonzalez-Flipe AR, Cotrino J, Caballero A (2005) Plasma chemistry of NO in
complex gas mixtures excited with a surfatron launcher. J Phys Chem A 109(22):49304938
21. Hueso JL, Gonzalez-Elipe AR, Cotrino J, Caballero A (2007) Removal of NO in NO/N-2,
NO/N-2/O-2, NO/CH4/N-2, and NO/CH4/O-2/N-2 systems by flowing microwave discharges.
J Phys Chem A 111(6):10571065
22. Yanguas-Gil A, Cotrino J, Alves LL (2005) An update of argon inelastic cross sections for
plasma discharges. J Phys D: Appl Phys 38(10):15881598
23. Yanguas-Gil A, Cotrino J, Gonzalez-Elipe AR (2004) Collisional radiative model of an argon
atmospheric capillary surface-wave discharge. Phys Plasmas 11(12):54975506
24. Dulaney JL, Biondi MA, Johnsen R (1987) Electron-temperature dependence of the recombi-
nation of electrons with No+ ions. Phys Rev A 36(3):13421350
25. Iordanova E (2010) Poly-diagnostic validation of spectroscopic methods: in-depth monitoring
of microwave induced plasmas. PhD Thesis, Eindhoven University of Technology, The
Netherlands
26. Jimenez-Diaz M (2010) Modelling of microwave induced plasmas: the interplay between
electromagnetism, plasma chemistry and transport. PhD Thesis, Eindhoven University of
Technology, The Netherlands
27. Boudam MK, Moisan M, Saoudi B, Popovici C, Gherardi N, Massines F (2006) Bacterial
spore inactivation by atmospheric-pressure plasmas in the presence or absence of UV photons
as obtained with the same gas mixture. J Phys D Appl Phys 39(16):34943507
28. Philip N, Saoudi B, Crevier MC, Moisan M, Barbeau J, Pelletier J (2002) The respective roles
of UV photons and oxygen atoms in plasma sterilization at reduced gas pressure: the case of
N-2-O-2 mixtures. IEEE Trans Plasma Sci 30(4):14291436
Part II
Plasma Biofilm Inactivation
and Dentistry Applications
Chapter 11
Battling Bacterial Biofilms with
Gas Discharge Plasma
Anna Zelaya, Kurt Vandervoort, and Graciela Brelles-Mario
Abstract Most studies dealing with growth and physiology of bacteria have been
carried out using free-living cells. However, most bacteria live in communities
referred to as biofilms where cooperative interactions among their members make
conventional methods of controlling microbial growth often ineffective. The use
of gas discharge plasmas represents an alternative to traditional decontamination/
sterilization methods. We studied biofilms using two organisms, Chromobacterium
violaceum and Pseudomonas aeruginosa. With the first organism we demon-
strated almost complete loss of cell culturability after a 5-min plasma treatment.
However, additional determinations showed that non-culturable cells were still
alive after short exposure times. We have recently reported the effect of plasma on
P. aeruginosa biofilms grown on borosilicate coupons. In this paper, we present
results for plasma treatments of 1-, 3-, and 7-day old P. aeruginosa biofilms grown
on polycarbonate or stainless-steel coupons. Results indicate nearly 100% of
biofilm inactivation after 5 min of exposure with similar inactivation kinetics
for 1-, 3-, and 7-day-old biofilms, and for both materials used. The inactivation
A. Zelaya
Biological Sciences Department, California State Polytechnic University,
3801 W. Temple Ave., Pomona, CA 91768, USA
K. Vandervoort
Physics Department, California State Polytechnic University,
Pomona, CA, USA
G. Brelles-Mario (*)
Biological Sciences Department, California State Polytechnic University,
Pomona, CA, USA
Center for Research and Development of Industrial Fermentations,
(CINDEFI, CCT LA PLATA-CONICET), Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, La Plata, Argentina
e-mail: gbrelles@csupomona.edu
136 A. Zelaya et al.
kinetics is similar for both organisms, suggesting that the method is useful regardless
of the type of biofilm. AFM images show changes in biofilm structure for various
plasma exposure times.
11.1 Introduction
A little more than a decade ago, microbiologists thought of microbes as cellular

entities living in isolation and growing apart. Most studies dealing with bacterial
physiology and growth have been carried out using free-living (planktonic) cells.
These studies have provided extensive information regarding the basic mecha-
nisms controlling the growth of individual bacteria. However, most microbes are
social and prefer to live as part of communities where, for good or for bad,
interactions take place [1]. A biofilm is an example of this type of community
where cooperative effects become important. Biofilms are microbial communities
that grow attached to a surface and embedded in a gummy-like matrix mostly
composed of exopolysaccharides together with proteins and excreted nucleic
acids. Biofilms are responsible for expensive and undesirable effects including
disease, biofouling, pipe plugging, dental plaque, and prosthetic device contami-
nation, just to mention a few.
Cooperative interactions among members of the biofilm make conventional
methods of controlling microbial growth often ineffective. Biofilms show
unusual resistance to nearly all forms of removal by drugs, chemicals or the
immune system. The classical use of heat and pressure in the autoclave is still a
valid and inexpensive method for many applications but it cannot be applied to
all situations, such as prosthetic devices within a patient, or to thermosensitive
materials. Some chemicals, such as ethylene oxide, allow low-temperature dis-
infection, but ethylene oxide is both mutagenic and carcinogenic. Other chemi-
cals, such as chlorine, pose an environmental hazard and some risks to human
health. Radiation can be used in some but not all the cases. Therefore, the prob-
lem of biofilm inactivation demands the development of alternative techniques
(reviewed in [1]).
Since the mid 1990s, when low temperature atmospheric pressure plasmas
became a hot research topic, investigations aimed at elucidating the effects of
plasmas on bacterial cells were initiated, mostly in the United States [2]. Most of
those studies were carried out with microorganisms in the free-living state or with
spores. However, there are fewer reports and most of them coming from our labora-
tory, about the use of plasma for biofilm disinfection or inactivation [1, 315].
Results on plasma-assisted biofilm inactivation are promising. However there are
still some concerns such as cell viability after plasma treatment that researchers
coming from different fields and working on this exciting interdisciplinary topic
should take into account before drawing conclusions [1, 9, 15].
11 Battling Bacterial Biofilms with Gas Discharge Plasma 137
Fig. 11.1 Transmission electron micrographs of C. violaceum cells detached from a 3-day old
biofilm after a 20-min plasma treatment (b) detail of the area indicated in (a) by an arrow
11.1.1 Previous Results from Our Laboratory
11.1.1.1 Chromobacterium violaceum
Our laboratory first studied biofilms produced by Chromobacterium violaceum, a

gram-negative bacterium commonly present in soil and water. Biofilms were grown
in polystyrene microtiter plates and subjected to plasma for different exposure
times. Cells were then detached from the biofilm, suspended, and dilutions plated in
order to count colony-forming units (CFU) after incubation. Results show that
99.6% of culturable cells were inactivated after a 5-min treatment [13, 6, 7, 9, 14].
Atomic force microscopy (AFM) images revealed sequential changes in cell mor-
phology occurring during plasma treatment [14]. Transmission electron microscopy
(TEM) images also showed changes in cell morphology. Figure 11.1 shows that
after a 20-min plasma treatment many cells are lysed and others show both cell wall
and cell membrane degradation (unpublished results). These results are in agreement
with AFM images previously reported [1, 9, 14]. Figure 11.2 displays AFM images
for C. violaceum biofilm-forming cells treated with plasma for different exposure
times. All images display 5 5 mm2 area scans of samples plasma-treated for 0 (a),
5 (b) and 60 min (c) and obtained from widely separated regions of the sample.
Figure 11.2a shows aggregates with the typical rod-shaped morphology of C. viola-
ceum cells of about 2 mm in length. After 60 min of plasma treatment (Fig. 11.2c),
images reveal broken or amorphous structures that are barely recognizable as bacte-
rial remnants. These results for the 60-min plasma-treated sample were universal in
that no recognizable intact bacterial cells in any of the 60-min images were ever
obtained. The 5-min plasma-treated samples show an intermediate type of cell dam-
age. There are some almost intact cells in aggregates with some damaged and some
undersized cells.
Fig. 11.2 AFM images of C. violaceum bacterial biofilm treated with gas discharge plasmas for
0 min (a), 5 min (b) and 60 min (c). The images are 5 5 mm scan areas
The AFM results clearly indicate that biofilm-forming bacteria go through

sequential morphological changes after plasma treatment. Bacterial cells go
through little change in cell morphology for the 5-min plasma treatment but incur
major cell damage for 60-min exposure times.
It was taken for granted at that time that the absence of colonies on the plates
implied that plasma was killing cells. However, we carried out viability tests both
by assessing the physiological state of the cells through ATP measurements
(BacTiter-GloTM microbial cell viability assay, Promega) and by a microscopic
approach that involved using two fluoresecent dyes (LIVE/DEAD BacLight
Bacterial Viability Kit, Promega), one that stains all cells green and one that stains
only dead cells red [9]. The combination of the physiological and metabolic deter-
minations, AFM, and fluorescence microscopy showed that non-culturable cells
were still alive after short plasma exposure times. These results indicated that via-
bility experiments are indispensable before drawing the conclusion that plasma kills
cells based solely on culturability [9].
11.1.1.2 Pseudomonas aeruginosa
We are presently studying plasma-mediated inactivation of Pseudomonas aeruginosa

biofilms. This bacterium is an opportunistic pathogen that preys on victims with com-
promised immune systems, patients on respirators, and causes infections of burned
tissue and colonization of catheters and medical devices. We have recently reported
the effect of plasma on P. aeruginosa strain PAO1 biofilms grown on borosilicate
coupons. Results indicated nearly 100% of biofilm inactivation after 5 min of plasma
exposure. The inactivation kinetics were similar for 1-, 3-, and 7-day old biofilms and
showed a rapid decline in the number of surviving cells followed by a much slower
decline. AFM images showed changes in biofilm structure for various plasma expo-
sure times. Micromechanical properties of biofilms were studied through force versus
distance curves. A decrease in cell adhesiveness to the abiotic surface and biofilm
thickness was reported [15]. Here we are reporting the effects of gas discharge plasma
on biofilms of different maturity levels grown on diverse abiotic surfaces.
11.2.1 Biofilm Growth
Pseudomonas aeruginosa 1-, 3-, and 7-day old biofilms were produced in batch
culture using the CDC biofilm reactor (BioSurface Tech., MT). The biofilms were
grown on polycarbonate and stainless-steel 12.7 mm diameter coupons in TSB
(Tryptic Soy Broth) at 37C with agitation. After the selected growth time, the
coupons were aseptically removed from the reactor and unbound bacteria were
removed by rinsing the coupons twice with sterile saline. Coupons were briefly air-
dried prior to being subjected to gas discharge plasma for various exposure times
(5, 10, 15, 30, and 60 min) under sterile conditions. A control without plasma treat-
ment (0-min exposure time) was included. After treatment, the coupons were placed
in a wet chamber and incubated with 50 mL of sterile saline for 10 min. Biofilms
were then scraped off the coupons and suspended in 1 mL of sterile saline, serially
diluted, and suspensions plated in duplicates on an agarized solid TSB medium.
Plates were incubated at 37C and evaluated for colony-forming-units (CFU) by
counting the colonies. Data (CFUs/mL) were transformed to percentages assigning
the control 100% of survival. Short-exposure time experiments (0, 1, 2, 3, and
5 min) were carried out as described above for 3-day old biofilms.
11.2.2 Plasma Generation and Conditions
The gas discharge plasma was produced using a commercially available inductively-
coupled Atomflo 300 reactor (Surfx Technologies, CA) that delivers an atmospheric
plasma jet [15]. The reactor consists of two perforated rectangular plates separated
by a gap 1.6-mm across. The upper aluminum electrode is connected to a 100-W RF
power supply (13.56 MHz), and the lower electrode is grounded. The size of the
plasma showerhead is 0.63 cm wide by 2.54 cm across. The average electron tem-
perature in the plasma is 1.5 eV and the average electron density in the plasma is
about 2 1011 cm3. The density of ground state N atoms is about 1 1015 cm3 at the
exit of the source and out to 5.0 mm. It starts to drop off thereafter. No significant
ion or electron density exists outside the source [11]. For the experiments, an atmo-
spheric-pressure plasma jet was generated using a He flow of 20.4 L/min, a second-
ary gas flow (N2) of 0.15 L/min, and an input power of 35 W. Both gases were
industrial grade. The plasma applicator was mounted such that the showerhead was
4 mm away from the biofilm.
11.2.3 Temperature Determination
To test the temperature reaching the coupon during plasma treatment, coupons of
either polycarbonate or stainless-steel were exposed to plasma previously equilibrated
for 5 min. A thermometer was placed directly on the coupon surface and the
temperature was monitored and recorded once a minute for 10 min. The maximum
temperature reached by the coupon was 35C, confirming that temperature is not
responsible for biofilm inactivation since P. aeruginosa is a human pathogen that
prefers to live a t 37C.
11.2.4 Atomic Force Microscopy (AFM)
Three-day old biofilms were grown on polycarbonate or stainless-steel coupons, treated

with plasma for 0, 5, 30 and 60 min, and processed as indicated above. The coupons
were rinsed with sterile saline twice; air-dried and AFM images were obtained in air in
contact mode using a Quesant Instruments Universal Scanning Probe Microscope.
Commercial silicon cantilevers from MikroMaschTM were employed with spring con-
stants from 0.1 to 0.5 N/m. For each coupon, ten widely separated regions were imaged
to obtain a representative sample and ensure reproducibility. Images consisted of 500
lines of 500 points per line for a total of 250,000 pixels of data.
To ascertain micromechanical properties of the biofilm, force-displacement curves
were obtained. The procedure consisted of bringing the AFM tip in contact with the
sample and then moving the sample upward a set distance while monitoring the deflec-
tion of the cantilever. At each sampling location where force-displacement curves were
obtained, the tip was brought in and out of contact at a rate of 0.5 Hz to the maximum
set sample deflection (0.6 mm) and the displacement curve was recorded upon the fifth
trial. This technique helped to reduce hysteresis that was often observed in the first few
trials. The process was then repeated so that at least five force-displacement curves
were recorded at each sampling location. For comparing samples with different plasma
treatments, all of the force-displacement data were recorded on the same day using the
same cantilever. This method ensured control for humidity and cantilever dependent
factors (such as spring constant) that can influence the shape of these curves [16].
11.2.5 Virulence Tests
A virulence test was developed by modifying a protocol designed for the model plant
Arabidopsis thaliana (Hong BY, 2010 personal communication). The mid-vein of
surface-sterilized leaves of Romaine lettuce was inoculated with a suspension of P.
aeruginosa cells obtained after treating biofilms with plasma as indicated in Sect. 11.2.1.
Unexposed P. aeruginosa was used as a positive control. A leaf injected with saline
only as well as a leaf that had not undergone any injections were also included as con-
trols. Leaves were then aseptically placed into UV sterilized- plastic bags. The bags
were sealed, laid flat at 37C and incubated for three additional days. Photos of leaves
were taken on the fourth day to obtain qualitative measurement of leaf damage.
The percentage of remaining culturable cells vs. plasma exposure time for stainless-steel
or polycarbonate-grown P. aeruginosa biofilms is shown in Fig. 11.3. The percent-
age of culturable cells at time 0 is 100% and corresponds to the control without
plasma treatment. The figures show that there is an obvious decrease in the percent-
age of culturable cells vs. time regardless of the biofilm age and the abiotic surface
used to grow the biofilm. A similar time-dependence behavior was reported for
P. aeruginosa biofilms grown on borosilicate coupons [15]. Seven-day old biofilms
behave in a similar way and do not seem to be more resilient than younger biofilms.
Similar results were also reported for C. violaceum biofilms grown for 4 or 7 days
on polystyrene microtiter plates [3, 6, 7, 9]. In the case of P. aeruginosa biofilms,
the decrease in the percentage of cells is even more dramatic since there are almost
no culturable cells after a 5-min treatment with plasma and most of the inactivation
occurs in less than 1 min of exposure to plasma (Fig. 11.3 insets b-2). However,
experiments with C. violaceum were carried out with a different plasma reactor and
although the spectrometry showed the same type of radicals that might be causing
cell inactivation (presence of NO g-bands around 250 nm and an OH band around
309 nm) [3], the actual amount of each radical may vary. The type of plasma used
for these experiments produces visible emission but a pretty insignificant amount of
UV since there is no detectable emission in the range of 200300 nm [11]. The
plasma conditions chosen for our experiments were those that maximized OH and
NO emissions and produced stable plasma [3]. The effect of temperature on biofilm
inactivation was ruled out as described in Sect. 11.2.3 and in reference [15].
Figure 11.4 displays typical AFM images for P. aeruginosa biofilms grown on
electropolished stainless-steel coupons and treated with plasma for different expo-
sure times, as indicated in Sect. 11.2.
For each 40 40 mm image, a cross section is included. For each image, a removal
of any overall background tilt was performed. This procedure involved subtracting
a plane determined from the average slope between the top and bottom edges and
right and left edges of the scan. There are no obvious qualitative differences that we
interpret from these images. From the cross sections of the images, the overall thick-
ness of the biofilm can be determined as the distance between the lowest features
(the flat surface of the stainless steel coupon) and the highest features (the peak of
the biofilm). Examining these differences yields biofilm thicknesses of ~700, 650,
and 500 nm for the 0, 5, and 30 min plasma-treated samples, respectively. A more
precise method for quantifying the surface topography of the biofilms was employed
by examining the average height of the surface features as relative to the lowest
point in the AFM scan (assigned the value of zero height). Using this method for the
ten regions sampled for each of the control, 5, and 30 min plasma-treated samples
yielded mean values of the average heights of 443, 415, and 401 nm, respectively.
Therefore, the trend of reduction of average height after a long period of plasma
treatment (30 min) was consistent with the reduction of biofilm thickness seen in the
images of Fig. 11.4 and the reduction in culturable cells in Fig. 11.3. However, there
a 1-day old biofilms
100
Percentage of culturable
90
80
70
60
cells
50
40
30
20
10
0
0 5 15 30 60
Exposure time (minutes)
stainless-steel coupons polycarbonate coupons
b 3-day old biofilms

100
90
80
70
60
cells
50
40
30
20
10
0
0 5 15 30 60
c 7-day old biofilms

100
90
80
70
60
cells
50
40
30
20
10
0
0 5 15 30 60
d-2 3-day old biofilms

100
90
80
70
60
cells
50
40
30
20
10
0
0 1 2 3 5
Exposure time (min)
Fig. 11.3 Percentage of culturable cells vs. plasma exposure time. Pseudomonas aeruginosa bio-
films were grown on either polycarbonate (orange) or stainless-steel (blue) for (a) 1 day; (b) 3 days
and (c) 7 days; and subjected to plasma for various exposure times and processed as indicated in
Sect. 11.2. Results are the average of at least four independent experiments. Platings for colony
counting were performed in duplicates. The bars, when present, represent the standard error of the
mean. In some cases, the standard error of the mean is not visible because it falls within the size of
the error bar. (b-2) Inset to part b. Short exposure times experiment
I II III
Fig. 11.4 AFM images of P. aeruginosa bacterial biofilm treated with gas discharge plasmas for
0 min (column I), 5 min (column II) and 30 min (column III). Cross sections of each 40 40 mm
scan are included, with the location of the cross section indicated by a horizontal line on the
image
0.8
Cantilever Displacement (m)
0.6
0.4
0.2
0
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0.2 0.4 0.6 0.8
0.2
Adhesive
Step 0.4
0.6
0.8
Sample Displacement (microns)
Fig. 11.5 P. aeruginosa biofilm force-displacement curve for the 30 min plasma-treated sample
on electropolished stainless steel coupon. Data for tip-sample approach (dashed line) and tip-
sample retraction (solid line) are shown. The negative displacement of the cantilever that occurred
due to tip adhesion to the biofilm upon retraction is designated as the adhesive step
was a wide variability in the average heights measured for each of the samples and
the reported mean values differed by less than one standard deviation.
Figure 11.5 shows a typical force displacement curve obtained on the P. aerugi-
nosa biofilm grown on stainless-steel coupons for the 30 min sample. The curve dis-
plays the same general features that were exhibited in all of our measured
force-displacement curves. Upon approach (dashed line), the tip encounters the sam-
ple surface at the origin of the graph, and deflects upward with a slope that increases.
Upon retraction (solid line), the tip roughly retraces the approaching curve with some
hysteresis. Upon further retraction (in the third quadrant of the graph), the tip adheres
to the surface until it breaks free, and the points retrace the approaching data along the
1.400
Displacement Curve Slope 1.200
1.000
0.800
0.600
0.400
0.200
0.000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Sample Region
Fig. 11.6 P. aeruginosa biofilm force-displacement curve slopes for 0 min (control, first ten bars)
and 30 min plasma-treated samples (last ten bars). The height of each bar on the graph corresponds
to the mean slope of five curves obtained for each region. Error bars represent plus and minus one
standard deviation about this mean
negative x axis of the graph. For the purposes of analysis, two sections of the curve
were considered. For approaching, the slope of the curve for positive sample displace-
ments up to 0.2 mm was determined. For significantly higher sample displacements,
the data are less reliable, since the optical detection of the cantilever deflection
becomes increasingly non-linear. Others have performed similar analyses on bacteria,
employing force-displacement curves over comparable scales [17, 18]. For retracting, the
height of the adhesive step, as indicated in Fig. 11.5, was measured.
The slope of the force-displacement curves can be used to determine an elastic
constant or stiffness of the biofilm. The derivation involves the analysis of the
relative compression due to the contact between two effective springs, the cantilever
and the biofilm. The biofilm stiffness, S, is related to the unitless slope, m (canti-
lever displacement/sample displacement), and the cantilever spring constant, k,
through the equation [17],
m
S=k
1 m
Therefore, when using the same cantilever for comparisons between different
plasma treatments, relative changes in the bacterial stiffness is a function of the
slope of the force-displacement curves only.
Figure 11.6 shows comparisons between the initial slope of the force-displacement
curve for the 0 min treatment (control) and 30 min plasma-treated samples on the
electropolished stainless-steel coupons. Curves at ten regions for each sample were
obtained. There is an overall increase in the slope of the displacement curves after
30 min of plasma treatment versus the control sample. The results differ by just
slightly more than one standard deviation. Specifically, the mean slope value of the
ten regions measured for the control sample is 0.627 with a standard deviation of
0.274. For the 30 min-treated sample, the mean slope value for the ten regions mea-
sured is 0.990 with a standard deviation of 0.040. Therefore, this increase in the
slope is consistent with an increase in the stiffness of the biofilm. These results
1.4
Adhesive Step (m) 1.2
1.0
0.8
0.6
0.4
0.2
0.0
1 3 5 7 9 11 13 15 17 19 21
Sample Region
Fig. 11.7 P. aeruginosa biofilm force-displacement curve adhesive step data for 0 min (control,
first ten bars) and 30 min plasma-treated samples (last ten bars). The height of each bar on the
graph corresponds to the mean adhesive step height of five curves obtained for each region. Error
bars represent plus and minus one standard deviation about this mean
differ from our earlier results on biofilms grown on glass coupons, where we found
a slight decrease in biofilm stiffness with 30 min plasma treatment [15].
From the same force-displacement curves obtained for the sample regions shown
in Fig. 11.6, adhesive step data were extracted and are displayed in Fig. 11.7.
It is apparent from this graph that there is a wide variability in adhesive step
values over the various regions of each sample. The mean adhesive step height of
the ten regions measured for the control sample is 0.642 mm with a standard devia-
tion of 0.350 mm. For the 30 min-treated sample, the mean adhesive step height for
the ten regions measured is 0.328 mm with a standard deviation of 0.249 mm.
Therefore, there is a reduction in adhesion with 30 min of plasma treatment. This
reduction with plasma treatment indicates that the biofilm would exhibit less adhe-
sion to surfaces, detering its retention. These results are consistent with our earlier
findings for samples grown on glass coupons [15]. Pristine polycarbonate coupons
had surface height variations on the scale of a few microns, precluding their use in
the AFM studies, which could not delineate between the features of the biofilm and
the substrate background.
We previously reported that Chromobacterium violaceum biofilm-forming cells
undergo sequential morphological changes after plasma treatment. Bacterial cells
may undergo modifications ranging from minimal changes to putative loss of cell
walls. In another contribution, we verified the relative roughness of cells by exam-
ining image cross sections and analyzing the standard deviation of the surface
height. These surface features are consistent with cells undergoing damage [9, 14].
The present study goes beyond those reports suggesting that the architecture and the
stability of the biofilm as a whole may be impacted by plasma treatment.
Figure 11.8 shows the results of the virulence tests on lettuce leaves. P. aerugi-
nosa is also a plant pathogen and it produces tissue damage. It is clear that the mid-
vein of leaves in panels a and d show no damage whereas both the leaf treated
Fig. 11.8 Lettuce leaves were injected with P. aeruginosa biofilm-forming cells treated with
plasma for 0, 5 or 30 min (panels b, c and d respectively) as indicated in Sect. 11.2.
A control injected with saline (panel a) was included as a negative control
with bacteria not subjected to plasma treatment (panel b positive control) or

treated with plasma for 1 min (panel c) show tissue damage. Although bacterial
cells treated with plasma for 5 min do not yield appreciable numbers of colonies on
a petri dish, it is obvious that those cells are still not only viable but also virulent.
11.4 Conclusions
Our results clearly show that bacterial biofilms can be inactivated by using gas dis-
charge plasma. The architecture and the stability, together with cell culturabilty, are
impacted by the plasma treatment. These results are evidence of the potential of
plasma as an alternative sterilization method against biofilms.
One of the issues of more concern regarding plasma-assisted cell inactiva-
tion is that, in most of the cases, the lethality of plasma is assessed solely based
on the number of colonies that can be counted after the treatment. However,
bacterial cells can respond to one or more environmental stresses by entering a
viable-but-non-culturable (VBNC) state [1921]. When cells are VBNC they
are unable to produce colonies on an agarized medium but they are still alive
and may retain pathogenicity. Therefore, not counting colonies after plasma
treatment cannot be considered as an obvious indication that the cells are dead.
We developed a virulence assay using lettuce plants for P. aeruginosa biofilms
and preliminary results suggest that cells retain viability after short exposures
to plasma. Therefore, and in agreement with previously reported results with
C. violaceum [3, 6, 7, 14], viability experiments should always be carried out
before drawing the conclusion that plasma is useful to kill cells based solely on
measurement of culturable cells.
Acknowledgments This work was supported by the U.S. National Institutes of Health under
Grant SCORE SC3 # 1SC3GM088070-01. Funding for the AFM was provided by the National
Science Foundation Nanotechnology Undergraduate Education Program, award # 0406533.
Anna J. Zelaya was supported by a fellowship from the U.S. National Institutes of Health RISE
Program 2R25GM061190-05A2. Sandra Sue Lwin was supported by the U.S. National Institutes
of Health under Grant SCORE SC3 # 1SC3GM088070-01. We thank Dr. Nina Abramzon for
allowing us to use her plasma reactor.
References
1. Brelles-Mario G (2010) Bacterial biofilm inactivation by gas-discharge plasmas. In: Brelles-

Mario G (ed) Biological and environmental applications of gas discharge plasmas. Nova
Science Publisher, New York, ISBN: 978-1-60741-945-7
2. Laroussi M (2010) Foreword. In: Brelles-Mario G (ed) Biological and environmental applica-
tions of gas discharge plasmas. Nova Science Publisher, New York, ISBN: 978-1-60741-945-7
3. Abramzon N, Joaquin JC, Bray J, Brelles-Mario G (2006) Biofilm destruction by RF high
pressure cold plasma jet. IEEE Trans Plasma Sci 34:13041309
4. Akishev YS, Grushin ME, Karalnik VB, Monich AE, Pankin MV, Trushkin NI, Kholodenko
VP, Chugunov VA, Zhirkova and other authors (2005) Sterilization/decontaminations of physi-
ological solution and dry surface by non-thermal plasma created in bubbles and jet. In:
Proceedings of the 2nd international workshop on cold atmospheric pressure plasmas,
pp 6972. ISBN:908086692X
5. Akishev Y, Grushin M, Karalkin V, Trushkin N, Kholodenko V, Chugunov V, Kobzev E,
Zhirkova N, Irkhina I, Kireev (2008) Atmospheric-pressure, nonthermal plasma sterilization
6. Becker K, Koutsospyros A, Yin SM, Christodoulatos C, Abramzon N, Joaquin JC, Brelles-
Mario G (2005) Environmental and biological applications of microplasmas. Plasma Phys
Control Fusion 47:B513B523
7. Brelles-Mario G, Joaquin JC, Bray JD, Abramzon N (2005) Gas discharge plasma as a novel
tool for biofilm destruction. In: Proceedings of the 2nd international workshop cold atmo-
spheric pressure plasmas, pp 6972. ISBN: 908086692X
8. Cooper M, Fridman G, Fridman A, Joshi S (2010) Biological responses of Bacillus strato-
sphericus to floating electrode-dielectric barrier discharge plasma treatment. J Appl Microbiol
109:20392048
9. Joaquin JC, Bray JD, Kwan C, Abramzon N, Vandervoort K, Brelles-Mario G (2009)
Understanding plasma assisted biofilm inactivation. Microbiology 175:724732
10. Kamgang JO, Briandet R, Herry JM, Brisset JL, Natali M (2007) Destruction of planktonic,
adherent and biofilm cells of Staphylococcus epidermidis using a gliding discharge in humid
air. J Appl Microbiol 103:621628
11. Morajev M, Yang X, Nowling GR, Chang JP, Hicks Rf (2004) Physics of high-pressure helium
and argon radio-frequency plasmas. J Appl Physics 96:70117017.
12. Sladek REJ, Fioloche SK, Sissons CH, Stoffels E (2007) Treatment of Streptococcus mutans
biofilms with a non-thermal atmospheric plasma. Lett Appl Microbiol 45:318323
13. van den Bedem LJM, Sladek REJ, Steinbuch RM, Stoffels Adamowicz E (2005) Plasma treat-
ment of S. mutans biofilms cultured in a simulated dental cavity model. In: Proceedings of the
XXVIIth ICPIG meeting, Eindhoven, The Netherlands, pp 1822
14. Vandervoort K, Abramzon N, Brelles-Mario G (2008) Plasma interactions with bacterial bio-
films as visualized through atomic force microscopy. IEEE Trans Plasma Sci 36:12961297
15. Zelaya A, Stough G, Rad N, Vandervoort K, Brelles-Mario G (2010) Pseudomonas aerugi-
nosa biofilm inactivation: decreased cell culturability, adhesiveness to surfaces, and biofilm
thickness upon high-pressure non-thermal plasma treatment. IEEE Trans Plasma Sci
38:33983403
16. Jones R, Pollock HM, Cleaver JAS, Hodges CS (2002) Adhesion forces between glass and
silicon surfaces in air studied by AFM: effects of relative humidity, particle size, roughness,
and surface treatment. Langmuir 18:80458055
17. Zhao L, Schaefer D, Marten MR (2005) Assessment of elasticity and topography of Aspergillus
nidulans spores via atomic force microscopy. Appl Environ Microbiol 71:955960
18. Oh YJ, Lee NR, Jo W, Jung WK, Lim JS (2009) Effects of substrates on biofilm observed by
atomic force microscopy. Ultramicroscopy 109:874880
19. Oliver JD (1993) Formation of viable but nonculturable cells. In: Kjelleberg S (ed) Starvation
in bacteria. Plenum Press, New York, pp 239272
20. Colwell RR, Huq A (1994) Vibrios in the environment: viable but nonculturable Vibrio chol-
era. In: Kaye T (ed) Vibrio cholerae and cholera: molecular global perspectives. Proceedings
of the American Society for Microbiology, ASM Press, Washington DC.
21. Rozak DB, Colwell RR (1987) Survival strategies of bacteria in the natural environment.
Microbiol Rev 51:365379
Chapter 12
Inactivation of Microorganisms in Model
Biofilms by an Atmospheric Pressure Pulsed
Non-thermal Plasma
Yuri Akishev, N. Trushkin, M. Grushin, A. Petryakov, V. Karalnik,

E. Kobzev, V. Kholodenko, V. Chugunov, G. Kireev, Yu. Rakitsky,
and I. Irkhina
Abstract Non-thermal plasma jet formed by self-running pulsed-periodical

high-current spark generator (PPSG) was used for atmospheric pressure inactiva-
tion of microorganisms including biofilms. A distinctive feature of the PPSG is a
formation of transient hot plasma clouds (plasma bullets) periodically flying out to
the target. We experimented with model biofilms of E. coli and Bacillus subtilis
monocultures which were grown on agar and surfaces of steel and polypropylene
coupons. High efficiency of plasma inactivation was demonstrated. This effect is
associated primarily with an interaction of transient hot plasma clouds with bio-
films. Besides complete or partial degradation of the cell membrane, weakening of
the cell wall of E.coli culture by active plasma was found.
12.1 Introduction
A distinguished feature of microorganisms is their ability to attach themselves to

different surfaces and form biofilms. Namely this ability is responsible for micro-
bial fouling of industrial and water supply systems. As a result, fouling can lead
to decreasing in the diameter of operating pipes [1]. Besides, biofilms promote
pipeline corrosion and damage of engineering structures [2]. Biodamage and
Y. Akishev (*) N. Trushkin M. Grushin A. Petryakov V. Karalnik

Low Temperature Plasma Department, SRC RF TRINITI,
Pushkovykh St-12, Troitsk, Moscow region 142190, Russia
e-mail: akishev@triniti.ru
E. Kobzev V. Kholodenko V. Chugunov G. Kireev Y. Rakitsky I. Irkhina
SRC RF for Applied Microbiology and Biotechnology,
Obolensk 142279, Russia
e-mail: kobzev_e@mail.ru
150 Y. Akishev et al.
biofouling of industrial materials are common phenomena which exhibit themselves

intensively if engineering systems are opened to direct exposure to moist and
warm environment [3].
Protection of industrial facilities, devices, materials, etc against the biofouling
and biodamage (including biocorrosion) is hard task to be solved by standard meth-
ods because, on the one hand, strong heating of the objects to be treated is not
always applicable and, on the other hand, biofilm-forming microorganisms are
highly resistant to biocides [1]. Due to this, bio-decontamination of water supply
systems requires higher biocidal doses and takes too long time (longer than 24 h).
At present, so-called non-thermal plasma (NTP) is one of the breakthrough and
innovative methods for cold (average gas temperature is close to room temperature)
bio-decontamination and sterilization of microorganisms including biofilms. NTP is
a gaseous mixture containing wide spectrum of bio-chemically active agents: charged
particles, free radicals and atoms like OH and O, excited nitrogen and oxygen
molecules, ozone, ultra-violet radiation, etc. NTP can be created with different
kinds of low and atmospheric pressure (AP) electric discharges such as DBD,
RF-, MW-discharges, and AC, pulsed and steady-state glow discharges [414]. There
are numerous publications demonstrating that NTP is promising approach in many
medical and microbiological applications including an inactivation of gram-positive
and gram-negative microorganisms, fungi and spores, etc [1420].
Here, we are dealing with AP pulsed NTP inactivation of microorganisms includ-
ing biofilms. Biofilms are spatially and metabolically structured microbial communi-
ties within extracellular polymeric matrix at the phase interface. A simple and
convenient research model of a biofilm is pure culture of colonies grown on agar or
surface of coupons. We used monocultures of Escherichia coli and Bacillus subtilis.
12.2 Atmospheric Pressure NTP Source Used

for Inactivation of Biofilms
Gas discharge generators forming axisymmetric plasma jets are used for remote
treatment of 2D or 3D objects. These generators can be divided into two groups
which fundamentally differ from each other by gas dynamic characteristics of
plasma jets formed. The fist group includes generators applying RF, DBD and DC
discharges and forming steady-state cold plasma jet. NTP created inside these gen-
erators is blown out continuously by flow of plasma forming gas at moderate veloc-
ity about of 30 m/s. The second group comprises the PPSG generators based on
use of pulsed-periodical high-current (around 300 A) spark excited in small cylin-
drical volume (typical sizes are 5 mm in a diameter and 5 mm in the length). In this
case, active plasma is produced with little portions of short duration. Plasma jet is
not formed due to blowing through the gap by plasma forming gas but because of
strong expansion of the gas rapidly heated by spark (due to that the operation of
PPSG is accompanied with a loud noise). Continuous blowing through is required
to recover the gap for the next pulse. Plasma jet (or plasma cloud) flying out the
generator is very hot and has high outlet velocity close to sound velocity. High gas
12 Inactivation of Microorganisms in Model Biofilms 151
Fig. 12.1 Sketches and electrical schemes of axisymmetric PPSGs for remote treatment at atmo-
spheric pressure developed by different authors: (a) [19]; (b) [20]; (c) [18]; SG is a compliment
spark gap (SG) in series to the main gas gap; (d) image of PPSG by [18], in the latter case diameter
of an activated area on target is 1 cm
temperature in hot plasma cloud (HPC) is transient. Because typical period-to-pulse

duration ratio is extremely high (104), an average gas temperature (Tg) at the treated
area is close to room temperature. The duration of plasma cloud formation, its outlet
temperature and composition of active species inside the cloud depend on electric
power and amount of energy loaded into the spark by an external circuit. Repetition
frequency of jet pulses is also determined by parameters of external circuit.
Figure 12.1 shows sketches of self-running PPSGs designed by Cybik et al. [19],
Dobrynin et al. [20] and authors of this paper [18]. Our investigations on spark
development [21] found out that the best parameters of spark plasma can be achieved
if the gap is stressed under strong overvoltage and the discharging time for the
capacitor C is the shortest. Taking this into account we designed the proper pin-to-ring
electrodes with a pin rounded hemisphericaly (but not acuate conically) and added
in the external circuit a compliment spark gap in series to the main gas gap
(Fig. 12.1c). It allowed us to increase an overvoltage up to 1214 kV and decrease
the discharging time to 4 ms due to diminishing the capacitance C up to 33 nF
(for comparison, the corresponding parameters in [20] are 4 kV, 330 nF and 40 ms
respectively). Pin-to-ring PPSG is able to operate steady for a long time in self-
running regime at repetition frequency 1030 Hz (for comparison, the PPSG by
[20] operates under frequency of about 1 Hz). Energy loaded in spark is around of
1 J per pulse. The working gases were N2 (mainly) and air.
In the area outside the spark gap an electric field is absent. It results in absence of the
excitation of molecular and atomic electron states by electron impact. That is why there
is no emission of 1+ and 2+ N2 systems from HPC flying to the target (the same situation
was observed in steady-state plasma jet in N2 [12]). However there is a strong emission
from the cloud in the N and Fe atomic lines. Atomic species pointed are produced due
to high temperature in spark that leads to partial dissociation of N2 and erosion of metal-
lic electrodes. These species have low energy electron levels which effectively are
Fig. 12.2 Variation of instant Tg and average Tg with a distance from the outlet nozzle
Fig. 12.3 Current and light characteristics of spark and HPC formed by PPSG. (a) Time behavior
of spark current and intensity (in a.u.) of total light emitted from HPC flying past narrow slit
located at 10 mm away from the PPSG outlet. (b) Optical spectrum of the light collected from the
area at the PPSG outlet. UV spectrum of the spark emitted in the axial direction through the hole
is similar to that observed from HPC (see b) but its intensity at least 1,000 times higher. Plasma
forming gas is air; electrodes are fabricated of steel. The recording devices are: oscilloscopes
Tektronix TDS 520C and TDS 2012; photomultiplier tube FEU-100; spectrometer AvaSpec-
2048FT-6-RM with a resolution of 0.100.15 nm in all spectral region
excited by collisions with vibration excited N2. In such a case we can assume that popu-
lation of low energy electron states is determined by vibration temperature (Tv) of nitro-
gen in the ground state. Because of high gas temperature in HPC, V-T relaxation goes
fast and Tv is close to Tg. So we can determine instant Tg in the HPC from Boltzmann
plot for Fe atomic lines. The results obtained are presented in Fig. 12.2a. Full circles
show instant Tg determined from registration of the 2+ N2 system to excite this system
in HPC we used an auxiliary microdischarge located in different points of the cloud
trajectory. Average Tg determined by K-type thermocouple is shown in Fig. 12.2b. One
can conclude the instant Tg in HPC is higher than average Tg at the target.
Figure 12.3 presents experimental data on characteristics of spark and HPC pro-
duced by PPSG. They are typical for N2 and air as working gases. Figure 12.3a
demonstrates two features of the PPSG: (1) amplitude of spark current pulse is
about of 500 A with typical width about of 4 ms; (2) the moving HPC has a steep
leading edge and a gentle tailing edge, i.e. gas dynamic impact is applied to a target
(the microorganisms) upon arrival the HPC; average velocity of the leading edge
over distance of 10 mm is about of 100 m/s. Figure 12.3b shows the most intense
emission of the PPSG corresponds to UV radiation in Fe atomic lines. One can
see that there is emission of O and N atoms, too.
12.3 Microbiology Materials and Methods
A biofilm is a spatially structured heterogenic microbial community presented by dif-

ferent microorganisms. The dynamics of the growth and substrate utilization is spe-
cific for any community and reflects generally small-scale cooperative and antagonistic
relationships among structured oxygen-poor ecosystems [22]. The simplest and most
suitable object for testing is model biofilm grown on agar by monoculture colonies.
We used two model biofilms generated by monocultures: bacterial strains in vegeta-
tive forms Bacillus subtilis MPV 7095 #1997 and Escherichia coli ATCC 25922
#2393. These biofilms were grown on agar and surface of mild steel and polypropyl-
ene coupons immersed in starvation (poor) medium (all provided by SRCAM&B).
Biofilms grown on agar are convenient to work with, but they are too simplified
models of natural biofilms. Model biofilms grown on the substrates of inert carriers
are much closer to natural biofilms [23] because in natural conditions agar is absent
on the surface. Therefore we inactivated the biofilms grown on agar and inert sur-
face as well.
A complete nutrient medium, fish meal hydrolysate (FMH), and minimal synthetic
media 8E and 27C were used to culture the bacteria. All nutrient media were produced
by SRCAM&B, Obolensk. Medium 8E consisted of: (0.8 g) MgSO47H2O; (0.5 g)
NaCl; (0.7 g) KH2PO4; (1.5 g) (NH4)2HPO4; (20 g) agar; water up to 1,000 ml; pH 7.2.
Medium 27C consisted of: (1 g) MgSO4; (0.5 g) K2HPO4; (20 g) agar; water up to
1,000 ml; pH 7.0. In culturing B. subtilis and E. coli glucose in final concentrations of
1% and 0.4%, respectively, was added as a sole source of carbon and energy.
For the first series of experiments on antimicrobial plasma treatment, the bio-
films were grown in Petri dishes filled with agar either 8E, 27C or FMH. The center
of each dish was inoculated with 500 ml of E. coli or B. subtilis overnight cell sus-
pension. The dishes were then installed in a thermostat and incubated for a time
required. The age of resultant biofilms varied from 2 h to 7 days (we call biofilms
young or old if they grown overnight or over several days respectively).
Typical characteristics of microorganisms in biofilms to be studied are presented
in Table 12.1 by example of agar-grown E.coli biofilms. The bacterial concentration
was found to reach 1011 CFU/biofilm in enriched medium already after 1-day
growth. As biofilms were getting old, total number of microorganisms did not
change (i.e. they have stabilized), but the biofilms increased in a diameter, and their
superficial density decreased (from 2.9 109 CFU/mm2 for a young biofilm to
3.6 108 CFU/mm2 for 3 days old biofilm grown on FMH-agar).
Biofilms were exposed to the plasma jet directed onto the dish center at a
distance of 78 mm from the outlet plasma source. After plasma treatment, the
Table 12.1 Characteristics of biofilms produced by monoculture Escherichia coli on agar

Age of the Diameter of the Number of microorganisms Surface density of the
Medium biofilm, days biofilm, mm per one biofilm, CFU biofilm, CFU/mm2
11
FMH 1 10.02 2.3 10 2.9 109
11
2 12.28 1.2 10 1.0 109
11
3 19.26 1.0 10 3.6 108
8E 2 6.14 1.7 109 5.6 107
5 4.26 1.6 1010 1.1 109
9
6 5.70 9.6 10 3.8 108
9
27 C 2 4.86 1.4 10 7.3 107
5 7.28 1.8 1010 4.4 108
6 6.72 2.5 1010 7.0 108
biofilms were washed off (using a sterile brush of boarish bristle) into tubes containing
a saline solution. Rinses were tenfold diluted and plated on agar to determine micro-
bial titers. The dishes were incubated in a thermostat overnight at 37C. The grown
colonies were counted.
For the second series of the experiments on antimicrobial plasma treatment, we
used biofilms that were grown on the surface of mild steel (20 7 1 mm) and poly-
propylene (10 10 0.1 mm) coupons. The coupons were provided by the Institute
Biopribor (Puschino, RAS). The coupons were placed into tubes filled with
medium 8E and inoculated with E. coli or B. subtilis overnight suspension.
Incubation was performed at 37C for 314 days, with shaking (200 rpm). In these
experiments we used the same nutrient media and PPSG operating parameters as for
the first series. After NTP treatment the biofilms were washed off (with use of a
sterile brush of boarish bristle) into tubes with a saline solution. Rinses were tenfold
diluted and plated on agar dishes to determine microbial titers. The dishes were
incubated overnight at 37C in a thermostat so that the cultures formed colonies.
The grown colonies were counted.
12.4 Results on Plasma Inactivation
12.4.1 The Viability of Model Biofilms Grown on Agar
Results on microorganism resistance against exposure time of plasma treatment are

shown in Fig. 12.4 by example of E.coli biofilms. Variable parameters in these
experiments were nutrient medium in agar and the age of biofilms.
As seen in Fig. 12.4, culture medium plays crucial role for bacteria in their sur-
vivability or resistance against active plasma: old E.coli biofilms grown on agar
with enriched medium FMH are less susceptible (or highly resistant) to active
plasma compared with those grown on agar with poor synthetic media 8E and 27C
Fig. 12.4 Number of E.coli cells grown on FMH agar (a) and 8E agar (b) survived in biofilms vs
plasma exposure time. Biofilm age is a variable parameter: (a) 1 2-h old culture, 2 overnight,
3 2 days old, 4 3 days old. (b) 1 2-h old culture, 2 2 days old, 3 5 days old, 4 6 days old. Statistical
scattering of our results does not exceed 30%
Fig. 12.5 Number of B. subtilis cells (grown on: (a) FMH agar, (b) 8 E agar) survived in biofilms
vs plasma exposure time. Biofilm age is a variable parameter: (a) and (b): 1 2-h old culture,
2 overnight, 3 2 days old, 4 7 days old. Statistical scattering of our results does not exceed 30%
(the results for biofilms grown on medium 27 C are practically the same as those for
medium 8E). In general, young E.coli biofilms were highly susceptible (or less
resistant) to plasma action. Nevertheless young biofilm grown over 2 h on the
enriched FMH-agar was incompletely inactivated by plasma after treatment over
5 min in spite of its rather low original titer.
Although absolute values of surviving bacteria in media FMH, 27C and 8E dif-
fer, there is a common tendency in their viability vs plasma exposure time: the total
number of microorganisms sharply decreases (by 25 orders of magnitude) in 1 min
of plasma exposure, and after that curves of microbial inactivation take a form close
to plateau. A similar tendency was observed for B.subtilis biofilms (Fig. 12.5).
B. subtilis biofilms were found to grow slower compared with E.coli biofilms.
For example, microbial titers of biofilms at the age of 1 and 2 days were similar,
whereas the titer of the biofilm grown on FMH medium for 7 days was higher by
Fig. 12.6 Number of E. coli cells (a), and B. subtilis cells (b) survived in biofilm grown on metal
and polymeric coupons vs plasma exposure time. Biofilm age is a variable parameter: (a) 1 and 2
metal coupon, 3 and 6 days old biofilm, 3 and 4 plastic coupon, 3 and 6 days old biofilm. (b) 1 and
2 metal coupon, biofilm grown for 1 and 2 weeks, 3 and 4 plastic coupon, biofilm grown for 1 and
2 weeks. Statistical scattering of our results does not exceed 30%
almost 3 orders of magnitude (Fig. 12.5a). Besides, B. subtilis biofilms are more
plasma resistant. Indeed, although total number of microorganisms in B. subtilis
biofilms grown on FMH medium was markedly lower than that of E. coli biofilms
under identical conditions, we failed to fully inactivate old B. subtilis biofilms.
While in contrast to the E. coli biofilms, young B. subtilis biofilms grown on
medium 8E for 2 h or overnight were fully inactivated when exposed to plasma for
3 min (Fig. 12.5b).
12.4.2 The Viability of Biofilms Grown on Metal

and Plastic Coupons
As mentioned above we suppose that properties of model biofilms grown on surface

of the inert carriers (e.g. on the surface of metal or polymeric coupons) are closer to
those of natural biofilms. According to our data, total number of microorganisms in
biofilms grown on the metal coupons was higher than that on the plastics ones
(Fig. 12.6). In our opinion, this is associated with poorer microbial adsorptive poten-
tialities of plastic coupons. Besides, the titer of the E.coli biofilm grown for 3 days
was found to be higher than that of the 6-day biofilm. Probably, a reason of that is
in-part degradation of the latter under starvation conditions.
The plasma treatment of E.coli biofilms grown on metal and plastic coupons led
to the death of microorganisms (Fig. 12.6a). Most of the biofilms became inacti-
vated already after the first minute of the treatment. A biofilms grown on the metal
coupon for 3 days appeared less responsive. B. subtilis biofilms grown on both types
of coupons appeared more resistant than similar E. coli biofilms (Fig. 12.6b). In
spite of the lower initial titer of B. subtilis biofilms compared to E. coli biofilms, it
took 13 min to kill them.
Fig. 12.7 (a) Dynamics of free nucleotides concentration in the supernatant vs NTP-exposure of
E.coli cell suspension at isotonic conditions. (b) Survivability of NTP-treated E.coli liquid culture
incubated in the solutions with different osmotic pressure: 1 isotonic solution, 2 hypotonic solu-
tion. Statistical scattering of our results does not exceed 30%
12.4.3 On Degradation of the Cell Membrane

and the Cell Wall by Active Plasma
To clarify the mechanism of NTP-cell inactivation, we have done the experiments

with microorganisms in liquids related to checking the integrity of some individual
cell structures (cell wall, cytoplasmic membrane and inta-cell components) after
plasma action. The method is based on measuring the number of free intracellular
nucleotides released by a cell in response to the NTP exposure (210 min). Cells
were precipitated by centrifugation (5810R Eppendorf; 12,000 rpm) for 20 min.
A concentration of supernatant nucleotides was determined from measurement of
optical density of a liquid at l = 260 nm by spectrophotometer (Shimadzu UV-1700).
The NTP was generated inside solution with the E.coli cell suspension. In that case,
UV radiation is not essential active agent in the inactivation process. Our setup for
generation of NTP inside a liquid is described in [24].
Free nucleotides (mainly low-molecular-weight ones) can occur in the supernatant
predominantly because of two reasons: (1) partial or complete degradation of the
cell cytoplasmic membrane (because of strong porosity, the cell wall is permeable
for free nucleotides; the damage of the cell wall does not contribute therefore so
much to the amount of nucleotides in the supernatant); (2) partial or complete break-
ing the integrity of intra-cell components due to penetration of plasma active spe-
cies into the cell.
The results obtained are presented in Fig. 12.7a. One can see in this figure the
concentration of nucleotides increases almost linearly with an increase in exposure
time of the E.coli cell suspension to the NTP. This experimental fact is serious evi-
dence that NTP treatment of microorganisms leads to partial or complete degrada-
tion of the cell cytoplasmic membrane and partial or complete breaking the integrity
of intra-cell components presumably due to penetration of plasma active species

into the cell.
To prove an existence of a degradation of the cell wall strength after plasma action
we varied a turgor (swelling) of the cells. It is well known that turgor pressure of the
cell contents against the cell wall is determined by osmotic pressure or hydrostatic
pressure produced by a solution in a space divided by a semipermeable membrane
due to a differential in the concentration of solute. If the solution is diluted enough
(hypotonic solution), the cell may burst because the degraded cell wall loses an abil-
ity to prevent the cell membrane from excessive stretching as the cell expands.
Hypotonic solution was obtained by tenfold dilution of the isotonic E.coli cell
suspension with distilled water. We have revealed the cells exposed to active plasma
species and incubated after that in hypotonic solution were inactivated in 2 min
(Fig. 12.7b) that is four times faster compared to the case with the cells in isotonic
solution. We associate a weak viability of the injured cells in hypotonic solution
(i.e. under increased turgor pressure on the cell wall) with a degradation of the cell
wall strength after plasma action.
12.5 Discussion
It was shown the 1-day (young or vegetative) biofilms are highly susceptible,
whereas the 3-day (old) biofilms are highly resistant to active plasma. The
decreased susceptibility of old biofilms is likely due to physiological ageing of
microorganisms, when the process of cell division is inhibited but both the amount
of accumulated cellular substances and strengthening of the cell wall increase.
Besides, the old biofilms may contain a significant amount of the dead cells
screening the living cells against plasma action.
It was revealed also that culture nutrient (enriched or poor medium) influences
drastically the viability of the cells injured by active plasma species. We associate
this fact with an existence of reparation processes inside the injured cell the cell
tries to repair the injuries delivered with active plasma species. The reparation goes
more effectively, if the injured cell is immersed in the enriched nutrient medium.
This process was discussed and taken into consideration in the mathematical model
of plasma-cell interaction published in [18].
The main feature of the pin-to-ring PPSG is that this plasma source activates an
object to be treated not continuously but periodically with intensive plasma clouds
(bullets). An important question needed to discuss is a spectrum of active agents
generated by PPSG developed. We distinguished three periodical stages in opera-
tion of the PPSG forming hot plasma bullets. These stages differ from each other in
types of active agents which can be responsible for remote inactivation of microor-
ganisms. In such an event, we can split each period in operation of the PPSG also
into three stages in bio-inactivation.
First stage has a short duration of about 5 ms and is associated with an appearance
of spark. At the first stage, only intensive spark UV radiation interacts with the
microorganisms to be treated. UV spectrum of the spark emitted in the axial direction

through the hole is similar to that observed from HPC at the outlet of the nozzle
(see Fig. 12.3b) but its intensity at least 1,000 times higher. Note that intensity and
spectrum of spark UV radiation depends strongly on sort of metal, of which the
electrodes are fabricated (our results show that UV radiation of Fe atoms is more
intensive compared to that produced by Al atoms), but in any case, spark UV radia-
tion exceeds drastically the intensity of UV radiation emitted by steady-state cold
plasma jets.
Second stage begins after arrival of HPC to the target to be treated. As a rule, the
time spent for traveling the HPC from the spark gap to the target is equal to several
tens or one hundred of microseconds. Duration of the second stage is determined by
cooling the HPC due to interaction with target and takes, according to estimations,
also several tens of microseconds. It turns out that the microorganisms activated by
strong UV radiation at the first stage are exposed additionally to high gas tempera-
ture exceeding 1,200 K at the second stage. Short time of the second stage is not
enough to heat essentially the bulk of the target but enough to create a thermal acti-
vation of thin upper layer of the treated area. We do not believe that transient high
temperature provides full thermal damage of the bacterial cells. We assume that
thermal activation leads to the burn of the microorganisms, and this burn increases
drastically the cell sensitivity to bio-chemically active species containing abun-
dantly in HPC. Note the second stage with transient high gas temperature and gas
dynamic impact applied to the microorganisms upon arrival the HPC on the target
is an exclusive property of the PPSG such peculiarity is absent in the case of inac-
tivation by plasma generators forming steady-state cold plasma jet.
Third stage. The second stage is followed by third stage when gas temperature
around treated area drops down to room temperature, and main active species are
atoms and radicals like O, N and OH, NO (their emission from HPC was registered)
and vibrationally excited molecules like N2(X,v) brought to the target by HPC.
Duration of the third stage is determined by life-time of radicals and is equal to
several tens of milliseconds. For the first glance, the third stage is similar to the situ-
ation existing on the target under remote NTP inactivation by plasma sources form-
ing steady-state cold plasma jet. In fact, there is strong difference. In the course of
third stage of PPSG treatment, cold active plasma species interact with the microor-
ganisms already pre-activated by intensive UV radiation at the first stage and tran-
sient high gas temperature at the second stage. Besides, there is a gas-dynamic
impact of HPC on the target. Taken together, it provides synergy effect and mark-
edly increases the total efficiency of plasma inactivation by the PPSG compared to
NTP inactivation by plasma sources forming steady-state cold plasma jet.
As it turned out, an inactivation efficiency of the PPSG is practically the same, if
the working gases are N2 or air. One of the explanations is the following. The HPC
flies out the PPSG in ambient air. In the case of N2 as plasma forming gas, nitrogen
active species (N, N*, N2*(e), N2*(v)) being presented in HPC mix up with air and
form active particles like O, NO, OH, etc. Due to that, a mixture of active species at
the area to be treated will be practically the same once plasma forming gas is air.
Another explanation assumes that main inactivation process is associated with a
transient interaction of hot gas with the microorganisms. In that case, practically
there is no difference what sort of gas (air or N2) will transfer a critical amount of
thermal energy to the cells.
12.6 Conclusion
The results obtained demonstrate that atmospheric pressure NTP produced by pin-
to-ring PPSG is an effective means to inactivate the microorganisms including bio-
films. Bio-inactivation by the PPSG is produced by pulsed periodical shooting the
targets to be treated by intensive UV flashes and hot and chemically active plasma
bullets of short duration. In such an event, the microorganisms are exposed not
only to strong UV radiation and chemically active species but to high transient gas
temperature as well. Such complex impact differs cardinally from that acting at the
inactivation by generators forming steady-state cold plasma jet. We found out
practically the same inactivation efficiency of the PPSG, if the working gases are
N2 or air.
It is shown that NTP treatment of microorganisms in liquid leads to partial or
complete degradation of the cell cytoplasmic membrane, the cell wall strength and
partial or complete breaking the integrity of intra-cell components. We assume that
main NTP active species in liquids are OH-radicals. In our opinion these species can
destroy not only the outside cell components these radicals can penetrate into the
cell due to their small sizes and interact with intra-cell components as well.
In total one can conclude that NTP PPSG antimicrobial treatment differs benefi-
cially from conventional methods to control biofilms: no chemically aggressive
reagents are required, and the plasma inactivation process takes short time compared
to long-lasting conventional procedures. Promising NTP method of bio-decontami-
nation based on pin-to-ring PPSG has much potential for practice and could compete
against traditional methods. There is one drawback of this method it is a loud
acoustic noise generated by pin-to-ring PPSG under its operation. However this
problem can be easily solved by using the proper sound isolation of the PPSG.
References
1. Lengeler J, Drews G, Schlegel HG (1999) Biology of the prokaryotes. Thieme, Stuttgart

2. Jigletsova SK, Rodin VB, Kobelev VS, Akimova NA, Alexandrova NV, Rasulova GE,
Mironova RI, Noskova VP, Kholodenko VP (2000) Increased environmental safety of biocides
used to control microbial corrosion. Appl Biochem Microbiol 36:694700
3. Videla HA (1996) Manual of biocorrosion. Lewis publishers, London
4. Ehlbeck J, Schnabel U, Polak M, Winter J, Woedtke T, Brandenburg R, Hagen T, Weltmann
K-D (2011) Low temperature atmospheric pressure plasma sources for microbial decontami-
nation. J Phys D: Appl Phys 44:013002 (18 pp)
of direct plasma interaction with living tissue. New J Phys 9:115020 (26 pp)
6. Sosnin EA, Lavrenteva LV, Masterova YaV (2004) Capacitive discharge sterilizing lamp in
iodine vapor. Lett JTF 30:8994
7. Anpilov A, Barkhudarov E, Bark Yu (2001) Electric discharge in water as a source of UV
radiation, ozone and hydrogen peroxide. J Phys D: Appl Phys 34:993999
8. Mesyats GA, Korolev YuD (1986) High-pressure volume discharges in gas-lasers. Uspekhi
Fizicheskikh Nauk 148:101122
9. Klimenko KA, Korolev YuD (1990) Pulse volume discharge in short interelectrode intervals as
a source of accelerated electrons. Zhurnal Tekhnicheskoi Fiziki 60:138142
10. Montie TC, Kelly-Wintenberg K, Roth JR (2000) An overview of research using the one atmo-
sphere uniform glow discharge plasma (OAUGDP) for sterilization of surfaces and materials.
11. Laroussi M (2002) Nonthermal decontamination of biological media by atmospheric-pressure
plasmas: review, analysis, and prospects. IEEE Trans Plasma Sci 30:14091415
12. Akishev Yu, Grushin M, Karalnik V, Petryakov A, Trushkin N (2010) Non-equilibrium con-
stricted DC glow discharge in N2 flow at atmospheric pressure: stable and unstable regimes.
J Phys D: Appl Phys 43:075202 (11 pp)
13. Akishev Yu, Grushin M, Karalnik V, Petryakov A, Trushkin N (2010) On basic processes
sustaining constricted glow discharge in longitudinal N2 flow at atmospheric pressure. J Phys
D: Appl Phys 43:215202 (18 pp)
14. Machala Z, Chladekova L, Pelach M (2010) Plasma agents in bio-decontamination by dc
discharges in atmospheric air. J Phys D: Appl Phys 43:222001 (7 pp)
15. Yamabe C, Miichi T, Ihara S (2000) Proceedings of 13th international conference GD-2000,
2.Glasgow, p 684
16. Moreau S, Moisan M, Tabrizian M, Barbeau J, Pelletier J, Ricard A (2000) Using the flowing
afterglow of a plasma to inactivate Bacillus subtilis spores: influence of the operating condi-
tions. J Appl Phys 88:11661174
18. Akishev Yu, Grushin M, Karalnik V, Petryakov A, Trushkin N, Kholodenko V, Chugunov V,
Irkhina I, Kobzev E, Zhirkova N, Kireev G (2008) Atmospheric pressure non-thermal plasma
sterilization of microorganisms in liquids and on the surfaces. Pure Appl Chem 80:1953
19. Cybik BZ, Wilkerson JT, Grossman KR (2004) Performance characteristics of the spark-jet
flow control actuator. AIAA meeting paper, Portland, USA, June 2004, pp 20042131
20. Dobrynin D, Arjunan K, Fridman A, Friedman G, Morss Clyne A (2011) Direct and control-
lable nitric oxide delivery into biological media and living cells by a pin-to-hole spark dis-
charge (PHD) plasma. J Phys D: Appl Phys 44:075201 (10 pp)
21. Akishev Yu, Aponin G, Karalnik V, Monich A, Trushkin N (2004) Spatiotemporal evolution of
the current and the integral and spectral emission characteristics of a negative corona in nitro-
gen during its transformation into a spark. Plasma Phys Rep 30:971982
22. Nikolaev YuA, Plakunov VK (2007) Biofilm City of Microbes or an analogue of multicel-
lular organisms? Microbiology (Translated from Mikrobiologiya) 76:149163
23. Kholodenko VP, Chugunov VA, Kobzev EN, Zhirkova NA., Irkhina IA., Tedikov VM,
Martovetskaya II, Kireev GV, Dyatlov IA (2008) Study of plasma-chemical method to inacti-
vate microorganisms of different groups. In: Book of abstracts, 11th annual biological confer-
ence of the European biosafety association, Florence, Italy, 34 April 2008, p 71
24. Akishev Yu, Grushin M, Karalnik V, Monich A, Pankin M, Trushkin N, Kholodenko V,
Chugunov V, Zhirkova N, Irkhina I, Kobzev E (2006) Generation of non-equilibrium plasma
in heterophase atmospheric pressure gas-liquid media and demonstration of its sterilization
ability. Plasma Phys Rep 32:10521061
Chapter 13
Low Temperature Atmospheric Argon Plasma:
Diagnostics and Medical Applications
Svetlana Ermolaeva, Oleg Petrov, Nailya Zigangirova, Mikhail Vasiliev,

Elena Sysolyatina, Sergei Antipov, Maxim Alyapyshev, Natalia Kolkova,
Andrei Mukhachev, Boris Naroditsky, Tetsuji Shimizu, Anatoly Grigoriev,
Gregor Morfill, Vladimir Fortov, and Alexander Gintsburg
Abstract This study was devoted to diagnostic of low temperature plasma produced
by microwave generator and investigation of its bactericidal effect against bacte-
ria in biofilms and within eukaryotic cells. The profile of gas temperature near
the torch outlet was measured. The spectrum in a wide range of wavelengths was
derived by the method of optical emission spec-troscopy. Probe measurements of
the floating potential of plasma were car-ried out. The estimation and adaptation
of parameters of plasma flow (tem-perature, velocity, ion number density) accord-
ing to medico-technical requirements were produced. The model of immersed
surface-associated biofilms formed by Gram-negative bacteria, Pseudomonas
aeruginosa and Burkholderia cenocepacia, and Gram-positive bacteria,
Staphylococcus aureus, was used to assess bactericidal effects of plasma treat-
ment. Reduction in the concentration of live bacteria in biofilms treated with
plasma for 5 min was demonstrated by measuring Live/Dead fluorescent labeling
and using direct plating. The intracellular infection model with the pathogenic
bacterium, Chlamydia trachomatis, was used to study the efficacy of microwave
argon plasma against intracellular parasites. A 2 min plasma treatment of mouse
S. Ermolaeva (*) N. Zigangirova E. Sysolyatina N. Kolkova A. Mukhachev

B. Naroditsky A. Gintsburg
Gamaleya Institute of Epidemiology and Microbiology,
Gamaleya st. 18, 123098 Moscow, Russia
e-mail: sveta@ermolaeva.msk.su
O. Petrov M. Vasiliev S. Antipov M. Alyapyshev V. Fortov
Joint Institute of High Temperatures RAS, Moscow, Russia
T. Shimizu G. Morfill
Max Planck Institute for Extraterrestrial Physics, Munich, Germany
A. Grigoriev
Institute of Biomedical Problems RAS, Moscow, Russia
164 S. Ermolaeva et al.
cells infected with C. trachomatis reduced infectious bacteria by a factor of

2106. Plasma treatment diminished the number of viable host cells by about
20%. When the samples were covered with MgF2 glass to obstruct active particles
and UV alone was applied, the bactericidal effect was re-duced by 5104 fold
compared to the whole plasma.
13.1 Introduction
Despite the large societal impact and economic burden, the problem of antimicro-
bial therapy of chronic infections remains largely unsolved [1]. Causative agents of
chronic infection are often resistant to the standard antimicrobial treatments. Besides
wide spreading of bacterial strains with multiple resistance to antibiotics, changes
in bacterial physiology in the course of prolonged infection affects sensitivity to
antimicrobial chemotherapy. One possible mechanism of the resistance of chronic
infections to antibiotics is the involvement of bacterial biofilms, a multilayer struc-
ture that includes bacteria and exopolysacharide matrix, and makes bacteria resis-
tant to conventional treatment strategies [2, 3]. Another evasive mechanism adopted
by some pathogenic microorganisms is through occupying intracellular niches,
which can be classified as intracellular parasitism [4, 5].
Physical methods of antibacterial treatment might be useful when traditional anti-
biotic therapy fails due to natural pathogen resistance or forming of specific niches
such as biofilms or intracellular vacuoles. Low temperature plasma is attractive for
such applications because (1) it possesses bactericidal activity; (2) the mechanism of
plasma antibacterial action seems to be independent of metabolic processes in
bacterial cells, and therefore it should be bactericidal for metabolically inert bacteria
in biofilms; (3) it has relatively low toxicity for eukaryotic cells and tissues [6, 7].
One of the purposes of this work was a development of methods for optical and
probe diagnostics of low temperature plasma. The plasma was produced by micro-
wave generator with frequency of 2.45 GHz at low power (~ 150 W) and with argon
as the feeding gas. Another aim was to evaluate the potential of low temperature
plasma as an antibacterial agent in therapy of chronic infections. Bactericidal activ-
ity of low temperature microwave argon was studied against bacteria in biofilms and
bacteria within eukaryotic cells, i.e. in conditions that modeled physiological status
of bacteria in the course of the chronic infection.
Biofilms are communities formed by surface attached bacteria [8, 9]. Biofilms
starts with bacterial adhesion, implemented by interaction of bacterial pili with a
surface. Attached bacteria multiply to form microcolonies. Further growth results in
a multilayer structure covered by exopolysacharide. Most biofilms exhibit some
level of heterogeneity in that patches of cell aggregates are interspersed throughout
an exopolysaccharide matrix that varies in density, creating open areas where water
channels are formed. Lower layers of the mature biofilm are not easily accessible to
chemical agents. Moreover, a part of the bacterial population is metabolically inert
that makes it resistant to antibiotics [8].
13 Low Temperature Atmospheric Argon Plasma 165
The role of biofilms in the pathogenesis of chronic wound infections was

proved by both clinical and laboratory studies [2, 8]. Wound biofilms include
established pathogens such as Staphylococcus aureus, Pseudomonas aerugi-
nosa, b-haemolytic Streptococcus, Enterococcus spp as well as bacteria where
their input in pathogenesis remains to be elucidated, e.g. Corynebacterium,
Bacteroides, and Peptostreptoccocus spp. [3]. Chronic lung infections associ-
ated with Pseudomonas aeruginosa and Burkholderia cenocepacia are depen-
dent on the ability of these pathogens to form biofilms [10]. Pathogenesis of
cystitis is associated with bacterial biofilms formed on tissues and stones in the
bladder [11].
Experimental studies in vivo of natural biofilms in both humans and animals are
severely restricted by problems of access, sampling and ethical issues. To reduce
complexity and facilitate investigations in the laboratory under controlled and repro-
ducible conditions, a number of simple biofilm model systems have been estab-
lished. These include flow-cell-grown biofilms, colony biofilms, pellicle biofilms
formed at the air-liquid interface, and immersed surface-associated biofilms [12].
The first model is of particular importance in the study of dental plaque [13]. The
last model is closer to conditions found when bacteria interact with tissues that are
covered with a body fluid [12]. Because some steps of biofilm formation (such as
adhesion to the support) are specific to immersed surface-associated models, bio-
film development under these conditions may involve regulatory pathways that dif-
fer from those identified in pellicle and macrocolony models, leading to particular
features and specific bacterial sensitivity to stresses. In this work, we used the
immersed surface-associated biofilm model [14] to evaluate bactericidal effects of
low temperature argon plasma.
Some bacteria developed an alternative way of survival within a host, and instead
of consolidation in a biofilm community, these bacteria enter host cells and multiply
within this niche [4, 5]. These intracellular parasites are common causative agents
of chronic infections, particularly infections of mucous surfaces. Mycobacterium
tuberculosis is an intracellular parasite that has been a major concern in therapy for
centuries, but it is still unbeaten [15]. Obligate intracellular bacteria of the genus
Chlamydia are widespread causative agents of chronic lung and urogenital infections,
but are also known wound contaminators [5]. Wound and skin disease-associated bac-
terial species that are normally extracellular, e.g. Staphylococcus and Streptococcus
spp, can penetrate eukaryotic cells to prolong their survival [16]. Intracellular local-
ization protects the pathogens against both humoral immune response and antibiotics,
and provides them with nutrients [4, 15]. These intracellular parasites manipulate host
cell responses, and often control intracellular signaling events to preserve the host cell
from apoptosis, thereby supporting their own survival [17].
Gram-negative bacteria Chlamydia trachomatis are obligate intracellular
parasites. C. trachomatis is a causative agent of chronic infections of urogenital
tract, eyes and lung [5]. Recent findings show that these bacteria are opportunistic
pathogens in chronic skin ulcers and other inflammatory skin conditions [14]. Being
obligate intracellular parasites, C. trachomatis can multiply only within the host
cells using the benefits supplied by the intracellular environment [5]. The C. trachomatis
Fig. 13.1 Main stages of the C. trachomatis life-cycle. Infection starts when extracellular elemen-
tary bodies (EBs) enter the mammalian cell. Upon entry, the EBs turns into reticulate bodies (RBs)
and transform the phagosome into a replicative vacuole, called an inclusion. RBs multiply within
the inclusion until back transformation occurs into EBs. The infectious cycle is repeated upon host
cell destruction
life-cycle includes two forms, infectious extracellular elementary bodies (EBs) and
vegetative intracellular reticulate bodies (RBs) (Fig. 13.1). Intracellular RBs multi-
plying within intracellular vacuoles called inclusions are susceptible to antibiotics
that cross host cell membranes. In contrast, EBs are non-vegetating and metaboli-
cally inactive, being highly resistant to antibiotics and other medications.
Chlamydiae actively interacts with host signaling and metabolic pathways to
induce its own uptake by the host cell, obtain access to intracellular nutrients, and
maintain the integrity of the host cell until EBs become mature [6]. Chlamydiae
manipulate signaling pathways to hinder the activation of innate immune responses
detrimental to bacterial or host survival. In particularly, Chlamydiae have been shown
to inhibit host cell apoptosis and manipulate intracellular signaling systems driven by
transcriptional regulators, p53 and NF-kB [17]. Intracellular survival of Chlamydiae
is strictly dependent on manipulation by the host resources and any interruption of
the host signaling pathways can be vitally important for the pathogen. To study effec-
tiveness of low temperature argon plasma against intracellular bacteria, the model of
C. trachomatis infection of the murine fibroblasts McCoy was used.
13.2 Plasma Source. Techniques and Results

of Plasma Torch Diagnostics
All experiments were performed with the MicroPlaSter b device (Fig. 13.2) that
produces microwave plasma [18]. The MicroPlaSter b device allowed the research-
ers to use two regimes an argon plasma and a placebo regime with a flow of
Fig. 13.2 The MicroPlaSter

b device operates infected
cells grown in a Petri dish
Fig. 13.3 The scheme of

diagnostics measurements
non-ionized argon gas. The plasma source consists of a 2.45 GHz microwave
power supply, a torch, and a gas supporting system. The torch is a concentric tube
device, which is oriented vertically. The torch design causes a pattern required in
the plasma gas flowing through the torch tubes and entering the plasma. The micro-
wave power is coupled into the plasma torch via a coaxial cable. The torch should
generate a highly stable plasma jet operated at low powers (60150 W) with low
gas flow rates (48 l/min). Diameter of a plasma flow in such conditions is not less
than 30 mm. Inert gas consumption controls is carried out by system of monitored
inflow. Speed of argon flow defines a length of plasma torch which was about
3545 mm in our experiments. To measure such plasma parameters as temperature,
floating potential, densities of plasma components, we chose characteristic regime
of plasma torch for biological experiments with microorganisms and mammal tis-
sue (power of the discharge 120 W, argon flow velocity 5 l/min, orifice gas
argon with purity of 99.998%). The plasma torch was fastened on an optical table
with possibility of its exact positioning in space by means of micrometric screws
(Fig. 13.3).
The room temperature was about 19C. We measured gas temperature in plasma
behind torch outlet by the shielded thermocouple to exclude distortions of measurement
Fig. 13.4 Gas temperature distribution in plasma behind the torch outlet
results owing to plasma influence on a thermocouple current (Fig. 13.4). We used a

chromel-alumel type of the thermocouple that is suitable for temperature measure-
ments in a range up to 1,100C.
Spectrometer Avesta ASP 150TF was taken for realization of a method of opti-
cal emission spectroscopy in plasma torch to obtain spectrum in a wide range of
wave lengths. The width of the spectrometer entrance slit was 100 ms. The infor-
mation about emission was gathered by series of CCD-detectors connected with
the computer. Monochromator resolution (for grating with 2,400 dashes per 1 mm)
was 0.016 nm, exposure time of CCD 0.02 s, signal was amassed during ten
expositions. The spectrometer spectral range was 1851,105 nm. Plasma light
emission was collected on the end of the optical fiber that was connected with
spectrometer.
Not all plasma components are safe and can be used for contact with biological
objects. For example, ozone is carcinogenic; UV radiation can damage DNA, etc.
Therefore, in addition to the development of new plasma sources, it is important to
investigate the role of plasma parameters, such as density of chemical compound,
intensity of UV-radiation, etc., and to develop special filters for plasma sources.
Various filters can be taken for definition of the contribution to bactericidal effect
of the various components generated in a plasma torch (Fig. 13.5). In the present
work we used a filter with the MgF2 glass allowing to block the plasma flow feeding
to biological object and to pass UV radiation. It allowed estimating the contribution
of an UV component for bactericidal plasma effects on intracellular bacteria.
The spectrum of absorption of MgF2 glass shows a value of absorbed light on
each wavelength. The transmittance of used MgF2 glass was almost constant, ~85%,
in the whole spectral region we measured. The standard optical scheme for mea-
surement of the spectrum of absorption involved heavy hydrogen and halogen lamps
that radiation was passed through MgF2 glass. Light passed through glass was trans-
ferred by optical fiber into a spectrometer connected to the computer.
Fig. 13.5 Filters for control various components generated in a plasma torch
Fig. 13.6 The results of spectrum measurements in the plasma torch
To obtain the quantitative data on plasma source composition the spectral diag-
nostic was used (Fig. 13.6). However, response of a spectrometer was too small for
definition of other active species in a plasma flow. It was offered to use for diagnos-
tics a special configuration of a torch with vertical windows for carrying out mea-
surements in the field of plasma generation and obtaining the intensity space
distribution in spectral lines of elements in vertical section of a plasma torch.
The results of spectrum measurements of a glow intensity distribution of an
active species in the plasma torch in vertical section are presented in Fig. 13.7.
As one can see in Fig. 13.8, the intensity and density of active species signifi-
cantly decreases out of generation area. However, the tests obtained by means of gas
analyzer MX2100 on distance of 20 mm for edge of the torch have shown concen-
tration O3 ~ 1,2 ppm, NO < 1 ppm, NO2 ~ 2,1 ppm that are substantial for sterilization
process of biological objects.
Fig. 13.7 Results of spectral measurements of a glow intensity distribution for active species (Ar)
in the plasma torch in vertical section
Fig. 13.8 Results of spectral measurements of a glow intensity distribution for active species (OH
and N2) in the plasma torch in vertical section
We designed special probe to measure floating potential of plasma. With the help
of it we obtained profiles of plasma potential along the torch. The probe was kept by
molybdenum holder. When measuring we regulated a position of torch to probe
from 0 to 45 mm. The profile of floating potential in plasma along an axis z is
Fig. 13.9 Profile of floating potential in plasma along axis z
presented on Fig. 13.9. Plasma (electron and ion) density in this area is estimated in
a range of 105106 cm3 that is sufficient for an effective charging of small biological
objects. Further, measurements had shown that in interval of 535 mm behind torch
outlet the potential of an electrode decreased to 5 V.
Based on the results described previously, we chose the optimal regime of plasma
treatment. The samples were treated with the plasma source at a distance of
2 0.2 cm to ensure that the gas temperature was 36 2C throughout the
experiments.
13.3 Bactericidal Activity of Low Temperature Plasma

Against Bacteria in Biofilms
The pathogenic bacteria, which are the most common causative agents of nosoco-
mial and wound infections, Pseudomonas aeruginosa (the strain PA103),
Staphylococcus aureus (the strain Sa78), and Burkholderia cenocepacia (the strain
Bc46) were used as model microorganisms. The different species were used to
check sensitivity to plasma in species differed in cell wall structure (Gram-negative
vs Gram-positive). The B. cenocepacia strain Bc46 was used to perform the quanti-
tative assessment as this strain high biofilm biomass due to a specific mutation [19].
The formation of surface-associated biofilms was performed on coverglass slices placed
into microtiter plates as previously described, but with slight modifications [20]. In
brief, an overnight culture was diluted 1000-fold in Luria-Bertani broth (LB broth,
Difco/BD, Franklin Lakes, NJ, USA) and cultured without agitation at 28C for
up to 72 h. Coverglasses 1.5 cm2 in area placed in the well to allow a biofilm to

form on its surface were removed after 16, 48 and 72 h, carefully washed with
Phosphate buffered saline (PBS, Amresco, Solon, Ohio, USA) and treated as
described below.
The coverglasses were placed on wet agar with the biofilms upwards and treated
with low temperature plasma or non-ionized argon gas for 5 min at a distance of
2 0.2 cm using the plasma source described above. Thereafter, the biofilms were
labeled by the Live/Dead cell viability assay (Invitrogen, Carlsbad, CA, USA)
according to the manufacturers instructions. Digital images were acquired with a
confocal laser scanning microscope (Zeiss Axiovert 200M LSM 510 META) as
described by [20]. The Live/Dead cell viability kit provided a two-color fluores-
cence assay of bacterial cells based of membrane integrity (Fig. 13.2). Dead bacte-
ria were labeled red and viable bacteria were labeled green.
In dependence on time point, newly forming (young) or mature biofilms were
observed. Young biofilms that formed after 16 h represented an early stage of
biofilm formation, i.e. when attached bacteria multiply to form microcolonies
(Fig. 13.10a, e). After 72 h, the mature biofilms formed as multilayer structures
covered by exopolysacharide (Fig. 13.10c, g). Plasma treatment of both young and
mature biofilms caused noticeable mortality among bacteria, with a prevalence of
dead (red labeled) bacteria in the plasma-treated biofilms, whereas the plasma
source. The other sample was left untreated as a control. Both samples non-treated
biofilms included more living (green labeled) than dead bacteria (Fig. 13.10a, c, e,
and g). The experiments were repeated three times.
To perform a quantitative assessment of the plasma bactericidal effect, the bio-
films formed by B. cenocepacia strain Bc46 on glass surfaces were used. This strain
produced high biofilm biomass due to a specific mutation [20]. The biofilms were
scraped with a sterile blade, transferred into PBS, and partly disrupted by pipetting.
The suspension was divided into two portions; one was applied to the surface of a
sterile Petri dish to form a layer of ~1 mm given low temperature plasma or non-
ionized argon gas treatment for 5 min at a distance of 2 0.2 cm with the were
totally disaggregated by intensive mixing with an ultrasound disintegrator at 5 W
for 7 s and serial dilutions were plated on blood-agar plates. The biofilm disruption
was followed by visualizing it with a Gram-staining method. Plasma reduction of
colony-forming bacteria in biofilms was found in three independent experiments.
Survival ranged from 0.005% to 0.5% of the initial bacterial load after 5 min treat-
ment. There were no survivals after 10 min treatment (Fig. 13.11). Non-ionized
argon gas produced no statistically significant change in bacterial viability.
Obtained results suggested that bacteria in biofilms are susceptible to low tempera-
ture plasma treatment. Similar results were obtained with other plasma sources applied
to model biofilms formed by Gram-negative and Gram-positive bacteria. Gas-
discharge plasma inactivated up to 99.6% of Gram-negative bacteria Chromobacteria
violaceum in biofilms after a single 5 min treatment [21]. Atmospheric pressure glow
discharge plasma was demonstrated against biofilms formed by Pantoea agglomerans
on the surface of white pepper [22]. A 6 log reduction was achieved after exposition
Fig. 13.10 Effect of low temperature argon plasma on viability of bacteria in biofilms. (a, b)
newly formed P. aeruginosa biofilms, (e, f) newly formed S.aureus biofilms. (c, d) mature
P.aeruginosa biofilms, (g, h) mature S. aureus biofilms. A, C, E and G control bacterial biofilms,
B, D, F and H biofilms treated with plasma for 5 min. Differential live/Dead dyes labeled alive
bacteria green and dead bacteria red
Fig. 13.11 Bacterial survival after biofilm treatment with plasma for 5 or 10 min. Bacterial con-
centrations are shown as denary logarithm of colony forming units (CFU) in 1 ml of suspension.
The mean values and SD are shown for each of three independent experiments. Decimal dilutions
of samples were plated in triplicates
of the Gram-positive bacterium, S. epidermis, to gliding discharge plasma for 70 min

[23]. The data demonstrated the considerable potential of low temperature plasma in
killing pathogenic bacteria in biofilms.
13.4 Bactericidal Activity of Low Temperature Microwave

Argon Plasma Against Intracellular Bacteria
The model of C. trachomatis infection of the murine fibroblasts McCoy was used to
study effectiveness of low temperature argon plasma against intracellular bacteria.
Cells were grown in Dulbeccos modified Eagles medium (DMEM, Invitrogen, US)
supplemented with 10% Fetal calf serum (FCS, HyClone, Australia), 2 mM Glutamine,
4.0 mg/ml Gentamycin and 5.0 mg/ml Amphotericin B in the 5% CO2 atmosphere. The
C. trachomatis Bu 434/L2 strain was routinely propagated in McCoy cells and elemen-
tary bodies (EBs) were purified by Renografin gradient centrifugation. Purified EBs
were resuspended in the sucrose-phosphate-glutamic acid buffer (SPG) and store fro-
zen at 70C. Titers were determined by infecting a cell monolayer with decimal dilu-
tions of the stock suspension. All experiments were performed with the multiplicity of
infection (MOI) 2. Infected McCoy cells were cultivated at 37C in the 5% CO2 atmo-
sphere up to 48 h post infection (hpi). Intracellular C. trachomatis were visualized with
luminescent microscopy as follows: infected McCoy cells were fixed with 50% metha-
nol, permeabilized with 1% Triton X-100 and stained with the monoclonal antibodies
specific for the C. trachomatis Major outer membrane protein (MOMP), which were
conjugated with the fluorescent dye fluorescein isothiocyanate (FITC,NearMedic Plus,
Moscow, Russia). Fluorescence of inclusion-containing cells were examined and
photographed under Nikon Eclipse 50i fluorescent microscope at x1350 magnification.
Fig. 13.12 C. trachomatis intracellular infection of McCoy cells at 48 hpi. Bacteria were visualized
by staining with FITC-labeled monoclonal antibodies (green). The cells were stained red.
(a) Control infection. (b) Infection was interrupted by plasma treatment of infected cells at 24 hpi
To perform a quantitative assay, serial lysate dilutions were used for infection of intact
McCoy cells. Infection was calculated at 48 hpi for all dilutions.
Treatment with low temperature argon plasma was performed as follows: just
before treatment, the culture medium was removed and about a ~1 mm layer of the
medium was left to preserve cells from desiccation during the treatment (Fig. 13.12a).
Cells were treated with plasma for 2 min and fresh DMEM medium was added
immediately afterwards. The treated cells were left for the next 24 h at 37C in the
5% CO2 in air atmosphere to complete the infection cycle. The cells were studied
microscopically as detailed above or cell viability was determined as follows. The
cells were stained with the 0.5% methylene blue dye for 2 h, lysed with 0.5% sodium
dodecyl sulfate (SDS, Sigma, USA) and the optical density was measured at OD540.
In parallel, the same experiments were carried out with non-ionized argon gas. As
control, non-treated cells were used. All experiments were repeated at least three
times in duplicate.
The control untreated cells contained big bacterium-containing inclusions that
occupy a considerable proportion of the cell volume (Fig. 13.12a). In contrast,
discontinued aggregates of bacterial cells without distinct borders were observed in
24 hpi plasma treated samples instead of regular inclusions (Fig. 13.12b).
To measure the concentration of live infectious bacteria in treated samples, the
cells were disrupted by ultrasound and the intact McCoy cells were infected with
the lysates (Table 13.1). Lysates obtained from untreated cells were used as a con-
trol. Secondary infection demonstrated reduction in the concentration of viable
infectious bacteria after treatment by a 5 logarithms; therefore, plasma treatment
had a considerable impact on viability of intracellular bacteria.
The important question arose about viability of the host cells upon plasma treat-
ment. Methylene blue staining demonstrated that the concentration of viable McCoy
cells only slightly reduced at 24 h post treatment with plasma. The overall reduction
in the concentration of viable McCoy cells was 20 2% (the mean from three times
made in duplicate). Thus, the plasma effect on viability of intracellular bacteria
considerably exceeded the toxic effect on the host cells.
Table 13.1 Concentration of infecting particles after treatment at different stages of the
Chlamydia trachomatis life-cycle
Treated
Chlamydia Non-ionized
forms Control Plasma treated argon gas UV treated
RBs at 24 hpi 2,0 107 IFU ml1 1,1 101 IFU ml1 1,2 107 IFU ml1 3,5 105 IFU ml1
The spectrum of argon plasma includes UV of A, B and C types proven to have

a bactericidal effect [24]. To evaluate the input of UV on killing intracellular bacte-
ria, the well with infected cells was covered with the MgF2 glass. The plasma emis-
sion spectra with and without the MgF2 glass were measured with the spectrometer
Ocean Optics USB4000 using a TCD1304AP detector. The detector operates in the
range 2001,100 nm and has an optical resolution of 0.3 nm. The integration time
was 10 s. The MgF2 let through ~ 85% of the UV with wavelengths 200 nm or higher
(see above). Measuring UV at lower wavelengths was not possible owing to the
limitations of the spectrophotometer. However, transmission of MgF2 glass is uni-
form within the range 1307,500 nm. Therefore, we extrapolated the data to lower
wavelengths and suggested that the MgF2 glass detained particles, but was essen-
tially transparent to UV.
When the active particles were detained with the MgF2 glass, the mortality among
intracellular bacteria was <0.1% relatively to whole plasma (Table 13.1). Therefore,
the role of UV in terms of the plasma bactericidal effect on intracellular bacteria
was important, but not predominant. Other known bactericidal activity against bac-
teria is due to surface lesions by provoking bombardment with plasma charged par-
ticles and active species ([25, 26]). However, direct bombardment is not possible for
intracellular bacteria, which are partitioned off by host membranes and cytoplasm.
Thus, the results suggested that killing of intracellular Chlamydia by microwave
argon plasma might be due to a plasma effect on the host cell rather than on bacteria
themselves. Interruption of signaling and/or metabolic host pathways that supported
bacterial intracellular survival seemed to be one of the possible reasons for the kill-
ing of intracellular bacteria. Details of the processes and signaling events in the host
cell that are affected by low temperature plasma have still to be elucidated. In the
meantime, the data suggest a substantial potential of low temperature plasma in the
inactivation of intracellular bacteria.
13.5 Conclusion
Biofilm formation and intracellular parasitism provide prolonged pathogen survival

within the host, defend bacteria from antibiotics and humoral immune response, and
are a frequent course of chronic infections. Traditional antimicrobial therapy often
fails to combat chronic infections as the physiological status of microorganisms in
the course of a chronic infectious process differs from the actively multiplying
microorganism model, which is typical of acute infections. In vitro models of bacterial
biofilms and intracellular infection were used to study the efficacy of low tempera-
ture argon plasma to eradicate bacteria in conditions, which are model conditions
typical of chronic infections. Five min plasma treatment reduced concentration of
viable bacteria in biofilms by a factor of a 100. Both Gram-negative and Gram-
positive bacteria in biofilms were sensitive to argon plasma. Mature biofilms did not
prevent bacterial killing. Argon plasma treatment of McCoy murine cells infected
with the obligate intracellular pathogen, C. trachomatis, interrupted intracellular
infection and caused noticeable mortality among bacteria. The loss of viability
among host eukaryotic cells was several logarithms less than mortality among bac-
teria. The results suggest that low temperature plasma can be effective in therapy of
chronic infections.
Acknowledgements We thank ADTEC Plasma Technology Co. Ltd., and in particular Mr.
Urayama for their part in the development of the MicroPlaSter device. We highly appreciate the
help of BioMedes Ltd. in manuscript preparation. The work was supported by Russian Ministry of
Education and Science (grants 02.740.11.0310 and 14.740.11.0118) and the Russian Foundation
for Basic Research (grant 10-02-01428).
References
1. World Health Organization (2008) The world health report 2008. World Health Organization,
Geneva
2. Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: a common cause of persis-
tent infections. Science 284:13181322
3. James GA, Swogger E, Wolcott R, Pulcini ED, Secor P, Sestrich J, Costerton JW, Stewart PS
(2008) Biofilms in chronic wounds. Wound Repair Regen 16:3744
4. Eswarappa SM (2009) Location of pathogenic bacteria during persistent infections: insights
from an analysis using game theory. PLoS One 4:e5383
5. Hammerschlag MR (2002) The intracellular life of Chlamydiae. Semin Pediatr Infect Dis
13:239248
plasma medicine. Plasma Processes Polym 5:503533
7. Kong M, Kroesen G, Morfill G, Nosenko T, Shimizu T, van Dijk J, Zimmermann J (2009)
8. Donlan RM, Costerton JW (2002) Biofilms: survival mechanisms of clinically relevant micro-
organisms. Clin Microbiol Rev 15:167193
9. Davey ME, OToole GA (2000) Microbial biofilms: from ecology to molecular genetics.
Microbiol Mol Biol Rev 64:847867
10. Ramsey DM, Wozniak DJ (2005) Understanding the control of Pseudomonas aeruginosa
alginate synthesis and the prospects for management of chronic infections in cystic fibrosis.
Mol Microbiol 56:309322
11. Hatt JK, Rather PN (2008) Role of bacterial biofilms in urinary tract infections. Curr Top
Microbiol Immunol 322:163192
12. Denef VJ, Mueller RS, Banfield JF (2010) AMD biofilms: using model communities to study
microbial evolution and ecological complexity in nature. ISME J 4:599610
13. Sissons CH (1997) Artificial dental plaque biofilm model systems. Adv Dent Res
11:110126
14. King LEJ, Stratton CW, Mitchell WM (2001) Chlamydia pneumoniae and chronic skin
wounds: a focused review. J Investig Dermatol Symp Proc 6:233237
15. Stewart GR, Robertson BD, Young DB (2003) Tuberculosis: a problem with persistence. Nat
Rev Microbiol 1:97105
16. Garzoni C, Kelley WL (2009) Staphylococcus aureus: new evidence for intracellular persis-
tence. Trends Microbiol 17:5965
17. Byrne GI, Ojcius DM (2004) Chlamydia and apoptosis: life and death decisions of an intracel-
lular pathogen. Nat Rev Microbiol 2:802808
18. Shimizu T, Steffes B, Pompl R, Jamitzky F, Bunk W, Ramrath K, Georgi M, Stolz W, Schmidt
H, Urayama T, Fuji S, Morfill G (2008) Characterization of microwave plasma torch for
decontamination. Plasma Processes Polym 5:577582
19. Romanova IM, Stepanova TV, Nesterenko LN, Balunets DV, Andreev AL, Shevliagina NV,
Borovaia TG, Gintsburg AL (2009) Persistence of Burkholderia cenocepacia bacteria
in vivo in dependence of their ability to form biofilms. Zh Mikrobiol Epidemiol Immunobiol.
4:2933
20. Kaminskaya A, Pushkareva V, Moisenovich M, Stepanova T, Avramenko N, Romanova J,
Litvin V, Gintsburg A, Ermolaeva S (2007) Insertion of Tetrahymena pyriformis cells within
Burkholderia cenocepacia biofilms stimulates biofilm formation. Mol Genet Microbiol Virol
22:186194
21. Joaquin JC, Kwan C, Abramzon N, Vandervoort K, Brelles-Mario G (2009) Is gas-discharge
plasma a new solution to the old problem of biofilm inactivation? Microbiology
155:724732
22. Vleugels M, Shama G, Deng X, Greenacre E, Brocklehurst T, Kong M (2005) Atmospheric
plasma inactivation of biofilm-forming bacteria for food safety control. IEEE Trans Plasma Sci
33:824828
23. Kamgang JO, Briandet R, Herry JM, Brisset JL, Natali M (2007) Destruction of planktonic,
adherent and biofilm cells of Staphylococcus epidermidis using a gliding discharge in humid
air. J Appl Microbiol 103:621628
24. Rentschler HC, Nagy R, Mouromseff G (1941) Bactericidal effect of ultraviolet radiation.
J Bacteriol 41:745774
26. Fricke K, Steffen H, von Woedtke T, Schrder K, Weltmann K (2011) High rate etching of
polymers by means of an atmospheric pressure plasma jet. Plasma Processes Polym
8:5158
Chapter 14
A Sub-microsecond Pulsed Plasma Jet
for Endodontic Biofilm Disinfection
Chunqi Jiang, Christoph Schaudinn, David E. Jaramillo,

Martin A. Gundersen, and J. William Costerton
Abstract A pulsed, tapered cylindrical plasma jet, several centimeter long and
<2 mm in diameter, has been generated by a concentric tubular device for root canal
disinfection. This plasma dental probe is typically powered with ~100 ns, 1 kHz,
multi-kilovolt electric pulses and filled with 5 SLPM (standard liter per minute) He/
(1%)O2 flow. We report here an in vitro study of the antimicrobial effect of the room
temperature plasma jet against monolayer Enterococcus faecalis biofilms on bovine
dentins. Resultant colony-forming unit counts were associated with changes in bac-
terial cell morphology observed using scanning electron microscopy (SEM) follow-
ing the treatment and control. Treatment of dentin discs cultivated with E. faecalis
monolayer biofilms with the plasma (average power 1 W) for 5 min resulted in
92.4% kill (P < 0.0001). Severe disruption of the cell membranes was observed
for the plasma treatment group, while the morphology of the cells remained intact
for the negative control group. In addition, a pilot ex vivo test was conducted to
examine the bactericidal effect of the plasma against saliva-derived biofilms culti-
vated in human root canals. Conspicuous biofilm disruption and cleared dentinal
surfaces were observed in the canal after the plasma treatment for 5 min. We
C. Jiang (*) M.A. Gundersen

Department of Electrical Engineering Electrophysics, Viterbi School of Engineering,
University of Southern California, 920 Bloom Walk, Los Angeles, CA 90089-0271, USA
e-mail: chunqi@usc.edu
C. Schaudinn
Electron Microscopy and Advanced Imaging Center, House Ear Institute,
Los Angeles, CA 90057, USA
D.E. Jaramillo
Endodontic Department, School of Dentistry, Loma Linda University,
Loma Linda, CA 92354, USA
J.W. Costerton
Center for Genomic Sciences, Allegheny-Singer Research Institute,
Pittsburgh, PA 15212, USA
180 C. Jiang et al.
conclude that this non-thermal pulsed plasma-based technology is a potential

alternative or supplement to existing protocols for root canal disinfection.
Non-thermal, atmospheric-pressure plasmas enhance the generation of reactive

chemical species and the interaction of these species with the materials under treat-
ment, while the bulk gas remains near room temperature. These properties have
made these plasmas highly attractive in a variety of biomedical and dental applica-
tions, including clinical instrument sterilization [1, 2], tissue engineering [3], wound
disinfection or stimulus for wound healing [4, 5], dental disinfection [6, 7], and
teeth whitening [8].
A room temperature plasma dental probe [8, 9] was recently developed for root
canal disinfection. Conventional root canal disinfection relies on adequate working
length control, mechanical instrumentation, antimicrobial irrigation and removal of
the smear layer in order to eliminate intracanal bacteria and debris [1012]. However,
these conventional methods result in rates of post-procedure infection exceeding
10% [13, 14]. The ecological organization of the endodontic pathogen cells into
protected sessile biofilms is one of the greatest challenges for root canal treatment
and a common cause of persistent bacterial infections [15, 16]. Therefore, new anti-
microbial strategies or adjunct procedures that work effectively against endodontic
biofilms would significantly benefit clinicians in endodontic therapy.
In this study, we evaluate the plasma-mediated antimicrobial effect against
Enterococcus faecalis biofilms inoculated on bovine dentin discs in vitro and saliva-
derived multi-species biofilms inoculated in human root canals ex vivo. E. faecalis,
a facultative gram-positive bacterium, has relatively low susceptibility to irrigant
solutions and to intracanal medicaments compared to many other endodontic patho-
gens, and considered as one possible cause of post-treatment disease after root canal
treatment [11, 17, 18]. Treatment of E. faecalis biofilm-colonized dentine will pro-
vide one of the representative studies to evaluate the efficacy of the non-thermal
plasma technique for endodontic biofilm control. In addition, the plasma treatment
of multi-species biofilms in root canals will provide a necessary first-step study for
potential clinical applications.
14.1.1 Plasma Source
A 3 cm long, 12 mm diameter plasma plume was generated by the plasma dental

probe, powered with 6 kV, ~100 ns voltage pulses at a rate of 1 kHz. The plasma
dental probe is based on a concentric tubular structure [8], as shown in Fig. 14.1a.
The center high voltage tubular electrode (6.35 mm OD, 3 mm ID) is separated by
a ceramic cylindrical structure from the ground chamber (34 mm OD, 12.7 mm ID).
The inner diameter of the gas flow channel is 3 mm. The high-voltage electrode is
recessed 5 mm from the outer surface of the ceramic. A custom-designed high-voltage
14 A Sub-microsecond Pulsed Plasma Jet for Endodontic Biofilm Disinfection 181
Fig. 14.1 (a) Schematic of the plasma dental probe, and (b) an image of the cold plasma jet
impinging into a human root canal
pulse generator was used to drive the plasma device. The inductive adder-based
pulse generator is able to generate up to 10 kV, 100 ns pulses at a rate from single-
shot to 5 kHz. Current and voltage were measured with a current monitor (Pearson
2877) and a high-voltage probe (Tektronix 6015A) in conjunction with a digital
oscilloscope. The average power of the plasma dental probe was obtained with inte-
gration of the product of the voltage and current waveforms. During treatment, the
average power was measured to about 1 W. A flow meter (Omega, FL-3839ST)
delivers He/(1%)O2 gas mixture (Prepared by Airgas) to the plasma dental probe at
a flow rate of 5 SLPM (Standard Liter per Minute).
The plasma jet impinging into a root canal is shown in Fig. 14.1b. The plasma
plume can be touched with bare hand without causing pain or burning. In an indepen-
dent study, the surface temperature of an instrumented human tooth under plasma
exposure for 15 min was recorded by a thin-film platinum resistance temperature
detector (Omega, TFD), fixed on the outside wall of the tooth. The temperature
increased from room temperature to 28C in 10 min, then stayed almost constant
between 28C and 28.5C for longer plasma exposure time. This indicated a steady
state of heat exchange was reached for the plasma-tooth system after 10 min. In addi-
tion, the gas temperature of the plasma plume was measured to be about 300 K or
27C with optical emission spectroscopy by comparing the measured emission spec-
tra of the 2nd positive system of N2 with simulated spectra [19].
Optical emission spectroscopy and ultraviolet (UV) absorption spectroscopy
were conducted to identify reactive chemical species contributing to the plasma
sterilization process [7, 19]. Emission spectra in the 200800 nm wavelength range
were obtained for the He/(1%)O2 plasma plume [7]. Emission lines from excited
nitrogen molecules, excited atomic oxygen, excited helium, and nitrogen ions were
observed. Among them, atomic oxygen, the most reactive species, is able to inacti-
vate cells and cause cell lysis by oxidation [20]. OH emission was not observed. No
significant UV emission was observed between 200 and 300 nm for the atmospheric
pressure plasma. In addition, UV absorption spectroscopy measured ozone density
in the order of 1015 cm3 [19].
182 C. Jiang et al.
14.1.2 E. faecalis Biofilm Cultivation for the in vitro Study
Freshly extracted, intact bovine molars were used for the in vitro tests. 6 mm
diameter, 1 mm thick dentin slices from the teeth roots were prepared, following the
protocol by Haapasalo and Orstavik [21]. The dentin discs were kept in tap water
during procedures to avoid dehydration. The smear layer was removed and the sur-
faces of the dentin discs were polished to ensure smooth dentin surfaces with Garnet
fine sandpaper disc (0.75 OD, Moore Company, Inc. Dearborn. MI). For steriliza-
tion, the dentin discs were treated with 6% NaOCl for 10 min followed by autoclav-
ing (121C, 20 min). After preparation of the specimens, the dentin discs were
immersed in sterile PBS solution until use for biofilm colonization.
An in vitro bacterial biofilm model was followed for the biofilm cultivation [22].
Prior to inoculation, ten bovine dentin slices were individually placed in lids of
500 ml Eppendorf tubes, which were then glued into six-well plates (Greiner bio-
one, Monroe, NC). All wells and the samples were disinfected with 6% NaOCl
solution and rinsed with sterile ddH2O. 7 ml of Luria Bertani (LB) broth (BD
Diagnostic System, Sparks, MD, USA) were added to each well and incubated
overnight at 37C to verify complete sterility. Then all wells were inoculated with
1 ml overnight culture of E. faecalis (ATCC 29212), obtained from the American
Type Culture Collection, and incubated for 6 days at 37C without shaking, with
daily change of 5 ml media.
14.1.3 Tooth Specimen Preparation and Saliva-Derived

Biofilm Cultivation
Appropriate Institutional Review Board (IRB) approval was obtained for human
teeth and saliva collection. Four freshly extracted human teeth were subjected to
standard endodontic instrumentation. The crowns were cut off at the cementum-
enamel junction with a diamond disc (Hyperflex double-sided, No. 911. Brasseler
USA. Savannah, GA, USA). The root canals were then cleaned and shaped with
ProTaper rotary files (F2 and F3) (Dentsply, USA). During the root canal shaping,
the irrigation protocol was as follows: 1 ml of 6% NaOCl after each instrumentation
procedure. After instrumentation was completed, the teeth were immersed in 17%
EDTA for 5 min and then transferred to 70% ethanol (to prevent contamination)
until use for biofilm colonization.
A protocol adapted from the in vitro biofilm model of Guggenheim B et al. [21] was
followed. Prior to use, the teeth were thoroughly rinsed with sterile ddH2O and subject
to autoclaving at 121C for 30 min. Saliva was collected and incubated in Todd-Hewitt
broth (BD Diagnostic System, Sparks, MD, USA) without shaking at 37C for 24 h. The
sterilized teeth were placed in a six-well plate and filled with 4 ml Todd-Hewitt broth.
The six-well plate containing the teeth was inoculated with 1 ml of pre-cultured saliva
biofilm and incubated for 4 days at 37C without shaking with daily change of media.
14.1.4 Microbiological Analysis and Scanning Electron

Microscopy for the In Vitro Study of the
Plasma-Mediated Bactericidal Effect
Total ten E. faecalis biofilm-inoculated bovine dentin discs, contained in individual

holders (lids of 500 ml Eppendorf tubes), were randomly divided into two groups
(n = 5 in each group): the negative control group and the plasma treatment group.
Prior to treatment, disc holders were removed from the six-well reactor. Any excess
liquid was carefully removed with filter paper. The dentin discs were individually
contained in tightly fitting holders for all the treatments.
The tooth slices of the negative control group were placed 1 cm below the device
nozzle and subject to the gas flow (at a flow rate of 5 SLPM) for 5 min. For plasma
treatment, the tooth slices were placed 1 cm below the plasma device nozzle and
exposed to plasma for 5 min.
After treatment, three randomly selected dentin discs from each group were pre-
pared for plating to determine the number of colony-forming units (CFUs). The
biofilm on the treated side of each dentin disc was removed to an Eppendorf tube by
pipetting up and down 4 25 ml LB broth, and subsequently plated on LB agar after
appropriate dilutions. Average CFU values and standard deviations from triplicate
experiments were obtained. The parametric test of one-way Analysis of Variance
(ANOVA) was used to analyze statistical significance.
The other four dentin slices (n = 2 for each group) were immediately fixed in 4%
paraformaldehyde (PFA) for 24 h at 4C. The specimens were then dehydrated in a
graded ethanol series and critical point dried. At this point, the dentin slices were
then removed from their holders, mounted on a stub, sputter coated with a 25 nm
layer of platinum and documented with scanning electron microscopy (SEM) (XL
30 S, FEG, FEI Company, Hillsboro, OR, USA).
14.1.5 Scanning Electron Microscopy and Confocal

Laser Scanning Microscopy for the Ex Vivo
Study of the Plasma Removal of Saliva Biofilms
For treatment, teeth were placed 5 mm below the plasma dental probe nozzle. Two
of the teeth were subject to the plasma exposure for 5 min. The other teeth, serving
as the control, were subject to the gas flow at the same flow rate for the same amount
of time, but with the plasma switched off.
One of the control tooth specimens was stained immediately with LIVE/DEAD
BacLight (6 mM for SYTO nine stain and 30 mM for propidium iodide) for 10 min
in the dark and washed with PBS thoroughly. The tooth was fixed with 4% formal-
dehyde for 1 h at room temperature in the dark and split crosswise with the dental
burr and examined in the confocal laser scanning microscope (CLSM 5 Pa inverted,
Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA) by immersing the sample in
184 C. Jiang et al.
sterile water in a slice chamber (Lab-Tek, Electron Microscopy Sciences, Hatfield,

PA, USA).
The other teeth were split longitudinally and transversely (in cross-section) with
the Hyperflex diamond disc. The teeth slices were dehydrated in a graded ethanol
line, critical point dried with carbon-dioxide and mounted on a stub. The samples
were sputter coated with 25 nm platinum and examined with SEM.
14.2 Results
14.2.1 Study of the Plasma-Mediated Bactericidal Effect In Vitro
(a) Microbiological analysis

A mean (SD) CFU count of 3.9 107 (3.2 106) (P < 0.0001) was obtained for
the negative control group (the gas flow-treated group). Plasma treatment
resulted in a significant reduction of the bacterial load to a mean (SD) CFU
count of 2.9 106 (5.8 105) (P < 0.0001), indicating a 92.4% kill. Treatment of
5.25% NaOCl resulted in the same order of magnitude reduction of the bacterial
load compared to the plasma treatment. The results of the CFU counts for all
groups after log transformation are given in Fig. 14.2.
(b) Scanning electron microscopy
The SEM images of the specimens of the negative control group show suc-
cessful growth of the E. faecalis biofilm monolayer on the dentinal surfaces
and in the dentinal tubules. Exposing biofilm-inoculated dentin discs to 5
SLPM He/(1%)O2 gas flow may have caused re-distribution of the bacterial
biofilms on the surface, resulted in bacterial-free micro-voids, as shown in
Fig. 14.3a. The bacterial morphology appeared intact (Fig. 14.3b) after the
treatment of the gas flow. In the above microbiological analysis study, the
total CFU numbers of the gas-treated group is significantly higher than those
of the plasma-treated group. Both of the results suggest that the gas flow
treatment can serve as a valid negative control. Severe disruption of the bacte-
rial integrity was observed for the regions directly under the plasma exposure,
as shown in Fig. 14.3c and d. SEM images of the plasma-treated specimens
with various magnifications consistently show morphologically damaged
cells (e.g. arrow 1 in Fig. 14.3d) on the dentinal surfaces after plasma expo-
sure. Many of the remaining morphologically complete cells appear deflated
and dying (e.g. arrow 2 in Fig. 14.3d). However, morphologically intact cells
were also observed in the areas near the edge of the discs for the plasma treat-
ment group. This indicates that the plasma treatment did not cover the entire
disc surface (6 mm in diameter) and explains the observed incomplete steril-
ization results by the 2 mm-in-width plasma jet.
Fig. 14.2 Microbiological

analysis: the colony-forming
unit counts of E. faecalis
biofilms for all treatment
protocols after log
transformation. (ANOVA,
P < 0.0001)
Fig. 14.3 SEM: (a) monolayer E. faecalis biofilms cultivated on bovine dentin discs without
treatment (the negative control); (b) the same monolayer biofilms at higher magnification
(8,000): E. faecalis cells appear morphologically intact (arrow 1); (c) after the plasma treat-
ment (He/(1%)O2 plasma, 5 SLPM, 5 min); (d) the same plasma treatment group at higher
magnification (10,000): membrane integrity severely compromised or damaged cells were
mostly observed (arrow 1). Morphologically complete but deflated or dying cells were also
observed (arrow 2)
186 C. Jiang et al.
14.2.2 Study of the Plasma Removal of Saliva Biofilms Ex Vivo
SEM images of the root canals for each case were taken at different locations and
with different magnifications (ranging from 36 to 20,000). The typical SEM
images of the gas flow-treated root canal (the negative control) are shown in
Fig. 14.4a and b. For the control teeth, biofilms covered the entire root canal surface,
as shown in Fig. 14.4a. Saliva biofilms in different development stages were
observed on the dentinal surfaces. Biofilm thickly embedded in exopolymeric matrix
is shown in Fig. 14.4b. Microcolonies with predominant morphotypes of filamen-
tous rods and cocci were observed. CLSM (at several regions with different magni-
fications) of the LIVE/DEAD BacLight-staining of the root after the control
experiment shows that the biofilm inside the root canal consists of cells with pre-
dominantly intact membrane integrity and hence viable bacteria. A representative
CLSM image of the biofilms from the apical region of the canal is shown in
Fig. 14.4c. In one of the plasma treated root canals (Specimen #3), the plasma plume
reached a depth of 1 mm in the root canal revealing a distinct zone of biofilm clear-
ance as shown in Fig. 14.4d. In the same canal, biofilms occupied the regions where
the plasma failed to reach. This produced a visible contrast line and clearance zone
for plasma-treated and non-plasma treated surfaces, as clearly evident in Fig. 14.4e.
A predominantly clean surface revealing histomorphologically intact and open den-
tinal tubules was observed, as shown in Fig. 14.4f. The SEM images of the other
plasma-treated root canal (Specimen #4) show less effective biofilm disinfection
compared to Specimen #3. Bacterial-free patches on the dentinal surfaces were
observed but no complete biofilm removal. Bacteria with disrupted cell walls coex-
ist among morphologically intact cells, as shown in Fig. 14.4g and h.
14.3 Discussion
The quantitative microbiological and the visual morphological effects of the non-
thermal plasma jet on monolayer E. faecalis biofilms were assessed in the in vitro
study. We used E. faecalis as our first-step and representative study of the anti-
biofilm effects of the plasma because E. faecalis has been used as a biological
marker in a great deal of endodontic studies [17, 23, 24] and persists even in hostile
environments where nutrients are reduced and in the presence of antimicrobial
agents [25, 26]. In the experiment, viable monolayer E. faecalis biofilms formed on
dentinal surfaces. The microbiological analysis showed that the plasma approach
achieved 92.4% kill. The SEM images imply that better results could be achieved if
the plasma jet scans through the entire surface to ensure complete coverage by direct
plasma exposure. Nevertheless, the SEM results clearly show plasma-mediated
E. faecalis biofilm disruption.
The pilot ex vivo study demonstrated the plasma-based technique for bacterial
biofilm disinfection in root canal systems. The test system we have chosen repre-
sents an exemplar, in that copious salivary biofilms were grown in the root canals,
so that we could extrapolate to a smear layer in a real root canal. For one of the
Fig. 14.4 SEM and CLSM: (a) a salivary biofilm-formed root canal after treatment of He/(1%)O2
flow at 5 SLPM for 5 min (the negative control); (b) detail of the boxed area in the previous image
(magnification, 565): the matrix-embedded salivary biofilm covers almost the entire length of the
root canal; (c) CLSM image of biofilms in the same control root canal: viable bacterial biofilms
were observed for the control specimen; (d) after plasma treatment for 5 min (specimen #3). Note
that the right half (the entrance) of the canal has a distinct zone of clearance with a visible vertical
line left to which biofilms are still present where the plasma failed to reach; (e) detail of the boxed
area in the previous image; (f) a typical plasma treated root canal surface (specimen #3), revealing
open dentinal tubules; (g) a plasma partially treated root canal surface (specimen #4). Integrity
severely compromised morphotypes of fusiform bacteria (arrows) were observed among morpho-
logically intact cells; (h) a region of the plasma treated surface (specimen #4) where the
membrane-disrupted cocci (arrows) were observed
188 C. Jiang et al.
tested samples, this biofilm was removed by plasma treatment so that a predominantly
clean surface, revealing intact and open dentinal tubules in the root canal, was
observed in the plasma treated region. The test results for the other sample, how-
ever, showed disrupted bacterial biofilms and partially cleaned dentinal surfaces,
caused by plasma treatment, but no complete biofilm removal. As the two tooth
samples were randomly selected, the canal parameters may vary, which resulted in
different canal diameters and complexities that favors the plasma treatment or vice
versa. The incomplete biofilm removal may be due to failed or partial entrance of
the plasma jet into both root canals. One canal may have smaller upper (near the
corona region) diameter compared to the other, which resulted in higher level of
difficulty of the plasma entrance into the canal. We are in the process of developing
and optimizing the plasma sources for generation of plasma plumes into the entire
root canal. It has been recently reported that such plasma jets consist of fast propa-
gating ionization fronts with better repeatability and directivity compared to corona
discharges [2729]. The presence of a tooth with a narrow aperture under the plasma
nozzle would perturb the gas flow and affect the ionization channel of the plasma
bullets. Consequently, the diameter of the plasma plume may appear to affect the
entrance of the plasma plume into the canal whose diameter is typical 2 mm. A
1 mm-diameter plasma needle has been recently developed to enter into and com-
pletely fill submillimeter Tygon tubes [30] as well as root canals. The biofilm disin-
fection results by the improved plasma source will be reported in the near future.
No emission peaks were observed for the spectral range of 200315 nm, indi-
cating UV radiation-induced direct DNA damage on bacterial cells was negligible
for this plasma jet, which agreed with the observations by others [31, 32] on atmo-
spheric pressure plasmas. In addition, both the measured gas temperature of the
plasma plume and the temperature of the dentinal surface under plasma exposure
showed that heat did not play a significant role during the bactericidal process.
The previously reported optical spectroscopic results [7, 19] showed that the
plasma generated reactive chemical species including atomic oxygen and ozone.
These reactive plasma species may cause enhanced oxidation and membrane
disruption of microbial biofilms, and thereby may contribute importantly to the
antimicrobial effects.
14.4 Conclusion
In this study, a sub-microsecond pulsed atmospheric pressure plasma jet was gener-
ated for endodontic biofilm disinfection. The plasma-mediated antimicrobial effects
against E. faecalis and salivary biofilms were conspicuously observed. The antimi-
crobial effects of the non-thermal plasma have shown a novel, plasma-based tech-
nique that can be a potential alternative or a supplement to existing protocols for
root canal disinfection.
Acknowledgements The authors thank Dr. Shawn Anderson for the donation of the tooth
specimens for the experiments. This work is supported by the National Institute of Dental and
Craniofacial Research (NIDCR), one of the National Institutes of Health (NIH) in the U.S.
Department of Health and Human Services.
References
1. Lee K, Paek KH, Ju WT, Lee Y (2006) Sterilization of bacteria, yeast, and bacterial endospores
by atmospheric-pressure cold plasma using helium and oxygen. J Microbiol 44:269275
2. Deng XT, Shi JJ, Chen HL, Kong MG (2007) Protein destruction by atmospheric pressure
glow discharges. Appl Phys Lett 90:13903
3. Brown IG, Bjornstad KA, Blakely EA, Galvin JE, Monteiro OR, Sangyuenyongpipat S (2003)
Growth of large patterned arrays of neurons using plasma methods. Plasma Phys Control
Fusion 45:547554
4. Fridman G, Peddinghaus M, Ayan H, Fridman A, Balasubramanian M, Gutsol A, Brooks A,
Friedman G (2006) Blood coagulation and living tissue sterilization by floating-electrode
dielectric barrier discharge in air. Plasma Chem Plasma Process 26:425442
5. Weltmann KD, Kindel E, von Woedtke T, Hahnel M, Stieber M, Brandenburg R (2010)
Atmospheric-pressure plasma sources: prospective tools for plasma medicine. Pure Appl
Chem 82:12231237
7. Jiang CQ, Chen MT, Gorur A, Schaudinn C, Jaramillo DE, Costerton JW, Sedghizadeh PP,
Vernier PT, Gundersen MA (2009) Nanosecond pulsed plasma dental probe. Plasma Processes
Polym 6:479483
8. Lee HW, Nam SH, Mohamed AAH, Kim GC, Lee JK (2010) Atmospheric pressure plasma jet
composed of three electrodes: application to tooth bleaching. Plasma Processes Polym 7:274280
9. Jiang CQ, Chen MT, Schaudinn C, Gorur A, Vernier PT, Costerton JW, Jaramillo DE,
Sedghizadeh PP, Gundersen MA (2009) Pulsed atmospheric-pressure cold plasma for endo-
dontic disinfection. IEEE Trans Plasma Sci 37:11901195
10. Heling I, Chandler NP (1998) Antimicrobial effect of irrigant combinations within dentinal
tubules. Int Endod J 31:814
11. Menezes MM, Valera MC, Jorge AO, Koga-Ito CY, Camargo CH, Mancini MN (2004) In vitro
evaluation of the effectiveness of irrigants and intracanal medicaments on microorganisms
within root canals. Int Endod J 37:311319
12. Colak M, Evcil S, Bayindir YZ, Yigit N (2005) The effectiveness of three instrumentation
techniques on the elimination of enterococcus faecalis from a root canal: an in vitro study.
J Contemp Dent Pract 6:94106
13. Nair PN, Sjogren U, Krey G, Kahnberg KE, Sundqvist G (1990) Intraradicular bacteria and
fungi in root-filled, asymptomatic human teeth with therapy-resistant periapical lesions: a
long-term light and electron microscopic follow-up study. J Endod 16:580588
14. Sjogren U, Figdor D, Persson S, Sundqvist G (1997) Influence of infection at the time of root
filling on the outcome of endodontic treatment of teeth with apical periodontitis. Int Endod J
30:297306
15. Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: a common cause of persis-
tent infections. Science 284:13181322
16. Estrela C, Sydney GB, Figueiredo JA, Estrela CR (2009) Antibacterial efficacy of intracanal
medicaments on bacterial biofilm: a critical review. J Appl Oral Sci 17:17
17. Gomes BP, Souza SF, Ferraz CC, Teixeira FB, Zaia AA, Valdrighi L, Souza-Filho FJ (2003)
Effectiveness of 2% chlorhexidine gel and calcium hydroxide against enterococcus faecalis in
bovine root dentine in vitro. Int Endod J 36:267275
190 C. Jiang et al.
18. Vianna ME, Gomes BP, Berber VB, Zaia AA, Ferraz CC, de Souza-Filho FJ (2004) In vitro
evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite. Oral Surg
Oral Med Oral Pathol Oral Radiol Endod 97:7984
19. Jiang C, Puech V, Magne L, Jeanney P (2009) Absolute ozone measurements for a low-energy
pulsed plasma needle. In: The 62nd Gaseous Electronics conference, Saratoga Springs, NY,
USA
20. Laroussi M, Alexeff I, Richardson JP, Dyer FF (2002) The resistive barrier discharge. IEEE
21. Haapasalo M, Orstavik D (1987) In vitro infection and disinfection of dentinal tubules. J Dent
Res 66:13751379
22. Guggenheim B, Giertsen E, Schupbach P, Shapiro S (2001) Validation of an in vitro biofilm
model of supragingival plaque. J Dent Res 80:363370
23. Murray BE (1990) The life and times of the enterococcus. Clin Microbiol Rev 3:4665
24. Jett BD, Huycke MM, Gilmore MS (1994) Virulence of enterococci. Clin Microbiol Rev
7:462478
25. Estrela C, Sydney GB, Figueiredo JA, Estrela CR (2009) A model system to study antimicrobial
strategies in endodontic biofilms. J Appl Oral Sci 17:8791
26. Sundqvist G, Figdor D (2003) Life as an endodontic pathogen. Ecological differences between
the untreated and root-filled root canals. Endod Top 6:328
27. Jiang C, Chen MT, Gundersen MA (2009) Polarity-induced asymmetric effects of nanosecond
pulsed plasma jets. J Phys D Appl Phys 42:232002
28. Lu X, Laroussi M (2006) Dynamics of an atmospheric pressure plasma plume generated by
submicrosecond voltage pulses. J Appl Phys 100:063302
29. Sands BL, Ganguly BN, Tachibana K (2008) A streamer-like atmospheric pressure plasma jet.
Appl Phys Lett 92:151503
30. Jiang C, Schaudinn C (2011) A curving bactericidal plasma needle. IEEE Trans Plasma Sci
39:29662967
31. Laroussi M, Leipold F (2004) Evaluation of the roles of reactive species, heat, and uv radiation
in the inactivation of bacterial cells by air plasmas at atmospheric pressure. Int J Mass Spectrom
233:8186
32. Lu X, Ye T, Cao YG, Sun ZY, Xiong Q, Tang ZY, Xiong ZL, Hu J, Jiang ZH, Pan Y (2008)
The roles of the various plasma agents in the inactivation of bacteria. J Appl Phys
104:053309
Chapter 15
Medical Plasma in Dentistry: A Future Therapy
for Peri-implantitis
Ina Koban, Lukasz Jablonowski, Axel Kramer, Klaus-Dieter Weltmann,

and Thomas Kocher
Abstract Biofilm formation plays a major role in the pathogenesis of many oral
diseases especially in peri-implantits. To evaluate the anti-biofilm effect of different
plasma devices and processes we used different dental biofilm models: Candida
albicans, Streptococcus mutans, Streptococcus sanguinis, aerobe multispecies
human saliva and anaerobe plaque biofilms. After 10 min treatment we reduced the
biofilms by 5 log10 steps using dielectric barrier discharge (DBD) plasma.
Chlorhexidine is the gold standard antiseptic which achieved in the same time only
a 1.5 log10 reduction. All plasma devices (DBD or plasma jets) damaged the mem-
brane of the microorganisms but only etching plasma sources can remove the bio-
film as shown in CLSM micrographs. It is possible to improve the plasma process
using antiseptics like octenidine. This combination significantly reduced CFU val-
ues after 1 min plasma treatment compared to the plasma control. Beside the anti-
biofilm effect an additional effect of plasma is the contact angle reduction of different
I. Koban (*)
Unit of Periodontology, Policlinics for Restorative Dentistry, Periodontology
and Endodontology, Ernst-Moritz-Anrdt University, Walther-Rathenau-Str. 49a,
17489 Greifswald, Germany
e-mail: ina.koban@uni-greifswald.de
A. Kramer
Institute for Hygiene and Environmental Medicine, Ernst-Moritz-Arndt University Greifswald,
Walther-Rathenau-Strae 49a, 17489 Greifswald, Germany
e-mail: kramer@uni-greifswald.de
K.-D. Weltmann
Leibniz Institute for Plasma Science and Technology e. V. (INP Greifswald), Felix-Hausdorff-Str.2,
Walther-Rathenau-Strae 49a, 17489 Greifswald, Germany
e-mail: weltmann@inp-greifswald.de
L. Jablonowski T. Kocher
Unit of Periodontology, Dental School, Ernst-Moritz-Arndt University Greifswald,
Rotgerber Str. 8, 17475 Greifswald, Germany
e-mail: lukasz.jablonowski@uni-greifswald.de; kocher@uni-greifswald.de
192 I. Koban et al.
titanium implant surfaces from 90 to super-hydrophilic (<5). This can improve the
implant healing process. Thus in the future, plasma could be an interesting treat-
ment option in dentistry, especially in treatment of peri-implantits.
15.1 Introduction
15.1.1 Problems in Peri-implantitis Therapy
Many diseases in the oral cavity are caused by bacteria. The most common infectious
diseases in humans include marginal periodontitis. It is caused by bacterial plaque that
can result in inflammation and bone shrinkage which eventually leads to tooth loss
(periodontitis) (Fig. 15.1) [1]. Even on dental implants, similar inflammatory reactions
can be observed. At present peri-implantitis is a well known problem in the focus of
dentistry. The inflammation of the peri-implant tissue is caused by a biofilm on the
implant surface, leading to bone loss and jeopardize the longevity of an implant
(Fig. 15.2). Thus, within 10 years after implant insertion the prevalence of peri-implan-
titis is about 20% [2]. The biofilm removal is of great importance and a prerequisite for
the re-osseointegration. There does not exist a generally accepted and successful
method for biofilm removal of titanium surfaces [3]. To promote the primary osseointe-
gration, implants are very rough. Mechanical methods for biofilm removal, as they are
available on teeth, should not be used because they damage the rough and elaborate
surface topography of implants and they do not reach the deep cavities and recessions.
The disinfection with irrigating solutions does not remove the adherent biofilm. The
problem is magnified by the complex geometry of the screw implant.
So far, no general treatment recommendations exist for the removal of biofilms
on titanium surfaces in peri-implantitis [3]. Possible methods of biofilm removal
with rotating diamond burs, laser or air abrasion devices are associated with a
change in the surface structure and topography. Further, the implant surface is con-
taminated by the decontamination method. The resulting smear layer and the modi-
fied surface topography do not permit re-osseointegration [4]. Therefore, the
regeneration of peri-implant bone after an intra-operative implant processing within
the therapy is not yet possible to predict.
The dentist is currently lacking facilities
which remove biofilms in vivo without an adverse change of the implant surface
in combination with
to promote osseointegration of affected implants via concomitant surface
modification.
Opportunities: The implant manufacturers are aware that a chemically active and
hydrophilic surface modification can promote the early healing process by a cellular
interaction in the first phase of wound healing. This type of surface modification has
been used for some time in the industrial production of implants [5] and leads to an
improved and more rapid tissue integration [6, 7]. If it would be possible, to achieve
15 Medical Plasma in Dentistry: A Future Therapy for Peri-implantitis 193
Fig. 15.1 X-ray image of a patient with periodontitis and tooth loss due to periodontal disease
Fig. 15.2 Radiograph

of two implants with
a peri-implantitis and a tooth
with periodontitis
similar modifications intraorally during peri-implantitis therapy, this could open

new avenues for treatment.
New insights from the use of low-temperature plasmas in the removal of biofilms
and the treatment of titanium surfaces and wounds suggest that its use in the treat-
ment of peri-implantitis opens up new treatment options.
15.1.2 Plasma Has Antimicrobial Effects
Early on, the antimicrobial effect of plasma on planktonic pathogens was detected
experimentally [8]. For this purpose, test organisms were streaked on agar plates
and treated with plasma. After a short treatment time inhibition zones could be
detected (Fig. 15.3).
194 I. Koban et al.
Fig. 15.3 Zone of inhibition

on Staphylococcus aureus
overgrown agar plate after
plasma treatment with
kINPen09 (Photo: Rutger
Matthes)
Fig. 15.4 Used plasma sources for laboratory tests: (a) hairline-pen, (b) kINPen in endodontic
model, (c) volume dielectric barrier discharge, (d) hollow electrode dielectric barrier discharge in
wells, (e) etching plasma jet on titanium plates
In the mouth, bacteria live in biofilms. The pathogens are covered in an extracel-
lular polymeric matrix (composed of polysaccharides, proteins, lipids and nucleic
acids) which protects them from environmental and physical influences as well as
antimicrobial substances. Hence, commercial antiseptics are insufficient in biofilm
treatment because they do not penetrate into the biofilm deeply enough. Biofilm
etching processes using plasma could solve these problems.
It was shown that a significant reduction of microorganisms, their metabolic
activity and inhibition of growth is possible with different plasma sources depend-
ing on the application time [911]. In order to test various plasmas in dentistry,
several sources are available (Fig. 15.4).
15.2 Methods and Results
We investigated the antimicrobial efficacy in monospecies (Streptococcus mutans/

Candida albicans) and multispecies biofilms (saliva) using a plasma jet (kINPen) and
two DBD (dielectric barrier discharge) processes [1214]. The experimental setup has
already been published and is briefly described below [15]. We used 48 h old biofilms
cultured on machined titanium discs with a diameter of 5 and 1 mm thickness (8 discs/
biofilm/test group). The kINPen (Fig. 15.4b) consists of a hand-held unit for the
generation of a plasma jet at atmospheric pressure, a DC power supply (system power:
8 W at 220 V, 50/60 Hz), and a gas supply unit (5 slm argon). In the centre of a
ceramics capillary (inner diameter 1.6 mm) a pin-type electrode (1 mm diameter) is
mounted. In the continuous working mode, a high frequency (HF) voltage (1.82 MHz,
26 kVpp) is coupled to the pin-type electrode. The plasma is generated from the top
of the centred electrode and expands to the surrounding air outside the nozzle. For the
first DBD (Fig. 15.4d, hollow electrodes DBD), microtitre plates were placed on
the grounded electrode, which was cooled by a Peltier-element in order to control the
temperature of the objects to be treated. Six hollow and thin metal tubes served as high
voltage electrodes and gas injection pipes in one function. The gas flew through these
electrodes, while a high rf-voltage (37.6 kHz, 8.4 kV) was coupled. Argon flow was
set to 1 slm per well, which is equivalent to a total flow rate of 6 slm. The second DBD
(Fig. 15.4c, volume DBD) consisted of two flat round metal electrodes, with one of
them being electrically grounded. The high voltage electrode is perforated to get a
better insight into the discharge gap. A Petri dish with the titanium discs was located
between these electrodes. The bottom of the Petri dish acted as dielectric for the DBD.
For cooling a Peltier-element was used, too. The gap in this system was 15 mm and
sealed air-tight. Argon gas (0.05 slm) flew into the system. The high sinusoidal volt-
age (40 kHz, 10 kV) applied between both electrodes generated the plasma. These
DBD sources act more extensively by simultaneous micro-scale lightning. After
plasma treatment the biofilms on titanium discs were placed into wells with 200 ml
0.9% NaCl solution and finally removed by ultrasonic scaling (20 min). Serial dilu-
tions of this resuspended biofilm solution were made and an aliquot portion of 0.1 ml
from each dilution was plated on Sabouraud glucose (Candida albicans), Brain Heart
Infusion (Streptococcus mutans) or Columbia sheep blood (saliva) agar plates and
incubated at 37C for 48 h. The colonies were counted and expressed as colony form-
ing units (CFU). The highest antimicrobial reduction rates were observed using
volume DBD [11]. Here, in 10 min the biofilm has been reduced by 5 log10 steps.
Chlorhexidine (0.1%) is the gold standard antiseptic which achieved in the same time
(10 min incubation, than inactivation of antiseptic effect) only a 1.5 log10 reduction. In
the microscopic image the effects of plasma sources were equal. In electron micro-
scopes, the destruction of the cell membrane was visible (Fig. 15.5). In the confocal
laser scanning microscope (CLSM) membrane damage and biofilm removal (using an
etching plasma source, Fig. 15.4e) could be detected by fluorescence staining
(Fig. 15.6). Dead bodies and debris after plasma treatment could prevent further treat-
ment of surviving bacteria. Therefore, killing of bacteria in biofilms is not enough.
196 I. Koban et al.
Fig. 15.5 Scanning electron

micrograph (2,000),
destroyed cells of Candida
albicans by plasma treatment
(kINPen09 plasma jet,
10 min, argon plasma)
Fig. 15.6 CLSM micrograph of a Streptococcus mutans biofilm before (left, 100) and after
(right, 100) plasma treatment (etching plasma jet 1 min, Ar+1%O2). Dark grey areas are live
cells, bright grey areas are membrane damaged cells. Important are the black areas in the right
micrograph after plasma treatment. Here the biofilm was removed
Etching plasma sources that can remove biofilms are needed. This jet (Fig. 15.4e) is
characterized by a grounded ring electrode and a centre rod electrode inside a quartz
capillary. The rod electrode is connected to the power source via a matching network.
The applied overall electric power of 65 W was held constant [16]. For biofilm removal
we used 5 slm argon and 1% oxygen.
Because of the antimicrobial effect plasma could be an interesting option for
endodontic application. The recent development of a new plasma device enables the
treatment of narrow cavities such as root canals (Fig. 15.4a) [17].
15.2.1 Synergistic Effects Between Plasma and Antiseptics
The antimicrobial efficacy of plasma could be increased by increasing the electrical

input power [18], but the plasma should be also tissue tolerable and applicable to
humans. As with any active substance the balance between efficacy and tolerability
has to be found. To potentiate the plasma effects without an increased input power we
investigated possible synergistic effects between atmospheric pressure plasma and
4
Reduction factor in log10 (CFU/ml) No
Ar gas
Ar plasma
*
3
0
control chlorhexidine octenidine control chlorhexidine octenidine
Streptococcus sanguinis biofilm saliva biofilm
Fig. 15.7 Reduction factors of Streptococcus sanguinis and saliva multispecies biofilm after treat-
ment with chlorhexidine and octenidine in combination with a following argon gas and plasma
treatment (kINPen 09, 1 min) * P < 0.05 versus Ar plasma control
the antiseptics chlorhexidine and octenidine. Here we used the model organism
Streptococcus sanguinis, which is one of the first colonizers adhering to saliva-coated
human tooth surfaces or dental materials [19]. In this monospecies biofilm model,
the highest log CFU reduction factor was found for argon (Ar) plasma without any
admixture (negative control). As a more realistic model concerning peri-mucositis
we used a multispecies saliva biofilm. Our results for this multispecies biofilm
showed that we found synergistic effects between plasma and agents. The combina-
tion octenidine and plasma significantly reduced CFU values compared to the plasma
control. Here, we achieved a similar reduction factor as with the monospecies biofilm
using plasma without admixture (Fig. 15.7). Therefore, it is effective to combine
plasma with special antiseptics. Further investigations are necessary to clarify the
mechanism of this synergism. Additionally other antiseptics should be tested.
15.2.2 Plasma Could Promote the Healing of Dental Implants
There is a reasonable chance to promote implant healing by surface modifications

using plasma. After short plasma treatment the contact angle of different titanium
implant surfaces has been reduced from 90 to super-hydrophilic (<5) (Fig. 15.8).
The degree of hydrophilicity affects cell attachment. Therefore, the healing of
implants could be improved [20]. The treatment of tooth discs with plasma showed
similar results. If applied in future, this could significantly assist periodontal wound
healing.
198 I. Koban et al.
Ar Plasma
Ar + 0.2% O2 Plasma
Ar + 1% O2 Plasma
Contact angle in
Contact angle in
Treatment time in s Treatment time in s
Fig. 15.8 Effect of plasma treatment (kINPen09) on the contact angle of titanium
In addition, a promotion of wound healing by plasma due to the following effects

may be possible [21]:
effective debridement of the wound surface [22]
antimicrobial activity against adherent bacteria and biofilms [9, 11]
tissue heating [23], improved circulation and promoted angiogenesis [24]
promotion of cell proliferation and differentiation in wound depth [25]
increasing the penetration of drugs into the wound [26] and
stimulation of immune cells by local generation of endogenous radicals [26].
15.3 Summary
Non-thermal plasma is applicable in narrow cavities, has an antimicrobial effect

against biofilms, reduces the contact angle and hydrophilizes the surface and could
improve the implant healing.
Thus in the future, plasma could be an interesting treatment option in dentistry,
especially in treatment of peri-implantits.
Acknowledgements This work was realized within the framework of the multi-disciplinary
research cooperation Campus PlasmaMed, particularly within the project PlasmaDent. The
authors acknowledge that this work was supported by a grant funded by the German Ministry of
Education and Research (BMBF, grant no, 13N9779). All titanium discs were kindly provided by
Straumann (Institut Straumann AG, Basel, Switzerland).
We thank Tina Dornquast, Claudia Lehnert and Hartmut Fischer for their excellent technical
assistance, Rdiger Titze for his skilful support in operating the plasma equipment and Karsten
Schrder for critical discussions as well as Christoph Schmidt and Sander Bekeschus for critical
reading of the manuscript.
References
1. Meisel P, Kocher T (2005) Photodynamic therapy for periodontal diseases: state of the art.
J Photochem Photobiol B Biol 79:159170
2. Roos-Jansaker AM, Lindahl C, Renvert H, Renvert S (2006) Nine- to fourteen-year follow-up
of implant treatment. Part 1: implant loss and associations to various factors. J Clin Periodontol
33:283289
3. Roos-Jansaker AM, Renvert S, Egelberg J (2003) Treatment of peri-implant infections: a lit-
erature review. J Clin Periodontol 30:467485
4. Klinge B, Hultin M, Berglundh T (2005) Peri-implantitis. Dent Clin North Am 49:661676,
viiviii
5. Rupp F, Scheideler L, Olshanska N, de Wild M, Wieland M, Geis-Gerstorfer J (2006)
Enhancing surface free energy and hydrophilicity through chemical modification of micro-
structured titanium implant surfaces. J Biomed Mater Res A 76:323334
6. Schwarz F, Sculean A, Wieland M, Horn N, Nuesry E, Bube C, Becker J (2007) Effects of
hydrophilicity and microtopography of titanium implant surfaces on initial supragingival
plaque biofilm formation. A pilot study. Mund Kiefer Gesichtschir 11:333338
7. Zhao G, Schwartz Z, Wieland M, Rupp F, Geis-Gerstorfer J, Cochran DL, Boyan BD (2005)
High surface energy enhances cell response to titanium substrate microstructure. J Biomed
Mater Res A 74A:4958
8. Dschlein G, von Woedtke T, Kindel E, Brandenburg R, Weltmann KD, Jnger M (2010)
Antibacterial activity of an atmospheric pressure plasma jet against relevant wound pathogens
in vitro on a simulated wound environment. Plasma Processes Polym 7:224230
9. Koban I, Hbner NO, Matthes R, Welk A, Kindel E, Weltmann KD, Kramer A, Kocher T
(2009) Antiseptic efficacy of selected agents and tissue tolerable plasma (TTP) on C. albicans
biofilms has the biofilm maturity influence on it? GMS Krankenhaushyg Interdiszip
4:Doc09
10. Matthes R, Bender C, Hbner NO, Mller G, Kohl K, Mentz J, Koban I, Kindel E, Kocher T,
Weltmann KD, Lademann J, Kramer A (2009) Influencing of the penetration of Pseudomonas
aeruginosa into a 3-D epidermis model and the inactivation with tissue tolerable plasma. GMS
Krankenhaushyg Interdiszip 4:Doc08
11. Koban I, Matthes R, Hbner NO, Welk A, Meisel P, Holtfreter B, Sietmann R, Kindel E,
Weltmann KD, Kramer A, Kocher T (2010) Treatment of Candida albicans biofilms with low
temperature plasma induced by dielectric barrier discharge and atmospheric pressure plasma
jet. New J Phys 12:073039
12. Foest R, Kindel E, Ohl A, Stieber M, Weltmann KD (2005) Non-thermal atmospheric pressure
discharges for surface modification. Plasma Phys Control Fusion 47:B525B536
13. Weltmann KD, Brandenburg R, von Woedtke T, Ehlbeck J, Foest R, Stieber M, Kindel E
sure plasma jets (appjs). J Phys D Appl Phys 41:194008
14. Weltmann KD, Kindel E, von Woedtke T, Hhnel M, Stieber M, Brandenburg R (2010)
Chem 82:12231237
15. Koban I, Holtfreter B, Huebner NO, Matthes R, Sietmann R, Kindel E, Weltmann KD, Welk
A, Kramer A, Kocher T (2011) Antimicrobial efficacy of nonthermal plasma in comparison to
chlorhexidine against dental biofilms in titanium discs in vitro proof of principle experiment.
J Clin Periodontol 38:956965
16. Fricke K, Steffen H, von Woedtke T, Schrder K, Weltmann KD (2011) High rate etching of
polymers by means of an atmospheric pressure plasma jet. Plasma Processes Polym
8:5158
17. Bussiahn R, Brandenburg R, Gerling T, Kindel E, Lange H, Lembke N, Weltmann KD, von
Woedtke T, Kocher T (2010) The hairline plasma: an intermittent negative dc-corona discharge
at atmospheric pressure for plasma medical applications. Appl Phys Lett 96:143701
200 I. Koban et al.
18. Halfmann H, Denis B, Bibinov N, Wunderlich J, Awakowicz P (2007) Identification of the

most efficient VUV/UV radiation for plasma based inactivation of Bacillus atrophaeus spores.
J Phys D Appl Phys 40:59075911
19. Hauser-Gerspach I, de Freitas PS, Dan Daniels AU, Meyer J (2008) Adhesion of Streptococcus
sanguinis to glass surfaces measured by isothermal microcalorimetry (IMC). J Biomed Mater
Res B Appl Biomater 85:4249
20. Schwarz F, Sager M, Kadelka I, Ferrari D, Becker J (2010) Influence of titanium implant sur-
face characteristics on bone regeneration in dehiscence-type defects: an experimental study in
dogs. J Clin Periodontol 37:466473
21. Kramer A, Assadian O, Below H, Bender C, Hammann A, Hartmann B, Hbner NO, Koban I,
Kocher T, Lademann J, Matthes R, Weltmann KD (2010) Perspektiven der Plasmamedizin.
Vakuum in Forschung und Praxis 22:3338
22. Hammann A, Hbner NO, Bender C, Ekkernkamp A, Hartmann B, Hinz P, Kindel E, Koban I,
Koch S, Kohlmann T, Lademann J, Matthes R, Mller G, Titze R, Weltmann KD, Kramer A
(2010) Antiseptic efficacy and tolerance of tissue-tolerable plasma compared with two wound
antiseptics on artificially bacterially contaminated eyes from commercially slaughtered pigs.
Skin Pharmacol Physiol 23:328332
23. Lademann J, Richter H, Alborova A, Humme D, Patzelt A, Kramer A, Weltmann KD,
Hartmann B, Ottomann C, Fluhr JW, Hinz P, Hbner G, Lademann O (2009) Risk assessment
of the application of a plasma jet in dermatology. J Biomed Opt 14(5)
24. Bender C, Matthes R, Kindel E, Kramer A, Lademann J, Weltmann KD, Eisenbei W, Hbner
NO (2010) The irritation potential of nonthermal atmospheric pressure plasma in the het-cam.
25. Stoffels E (2007) Tissue processing with atmospheric plasmas. Contrib Plasma Phys
47:4048
26. Kramer A, Hbner N, Assadian O, Below H, Bender C, Benkhai H, Brker B, Ekkernkamp A,
Eisenbei W, Hammann A, Hartmann B, Heidecke CD, Hinz P, Koban I, Koch S, Kocher T,
Lademann J, Lademann O, Lerch M, Maier S, Matthes R, Mller G, Partecke I, Rndler C,
Weltmann KD, Zygmunt M (2009) Chancen und Perspektiven der Plasmamedizin durch
Anwendung von gewebekompatiblen Atmosphrendruckplasmen (tissue tolerable plasmas, ttp).
GMS Krankenhaushyg Interdiszip 4:Doc10
Chapter 16
Inactivation of Candida Strains in Planktonic
and Biofilm Forms Using a Direct Current,
Atmospheric-Pressure Cold Plasma Micro-Jet
Wei-Dong Zhu, Peng Sun, Yi Sun, Shuang Yu, Haiyan Wu, Wei Liu,
Jue Zhang, and Jing Fang
Abstract A direct-current, atmospheric-pressure, He/O2 (2%) cold plasma microjet

is applied to Candida species (C. glabrata, C. albicans and C. krusei). Effective inac-
tivation is achieved both in air and in water within 5 min of plasma treatment. Same
plasma treatment also successfully inactivated candida biofilms on Petri dish. The
inactivation was verified by cell viability test (XTT assay). Severe deformation of
Candida biofilms after the plasma treatment was observed through scanning electron
microscope (SEM). Optical emission spectroscopy shows strong atomic oxygen emis-
sion at 777 nm. Hydroxyl radical (OH), superoxide anion radical (O2-) and singlet
molecular oxygen (1O2) are detected by electron spin resonance (ESR) spectroscopy.
The sessile minimal inhibitory concentrations (SMICs) of fluconazole, amphotericin
B, and caspofungin against the Candida spp. biofilms were decreased to 2-6 fold dilu-
tions in plasma microjet treated group in comparison with the controls. This novel
approach may become a new tool for the treatment of clinical dermatosis
Candidiasis, commonly referred to as yeast infection, is caused by Candida species

(spp) [1, 2]. It typically encompasses mucocutaneous disorders such as oral
candidiasis, vaginal and vulvovaginal candidiasis, as well as systemic infections
and potentially life-threatening diseases (often referred to as candidemia or
fungemia). Although Candida species are the microorganism exhibiting planktonic
W.-D. Zhu (*)

Department of Applied Science and Technology, Saint Peters College,
2641 Kennedy Boulevard, Jersey City, NJ 07306, USA
e-mail: wzhu@spc.edu
P. Sun S. Yu H. Wu J. Zhang J. Fang
Academy for Advanced Interdisciplinary Studies, Peking University,
Beijing, China
Y. Sun W. Liu
Department of Dermatology and Venereology, Peking University 1st Hospital,
Beijing, China
202 W. Zhu et al.
unicellular form, filamentous growth or complex multicellular structure is observed

mainly in the infected tissues [3]. Candida biofilms are structured microbial
communities and are much more resistant than the free-living planktonic cells. The
fact that they can attach to surfaces or encase a matrix of exopolymeric materials
[4, 5], has raised serious problems for various implanted medical devices such as
vascular and urinary catheters, joint prostheses, cardiac valves, artificial vascular
bypass devices, or more commonly used contact lens and dentures [6, 7]. The
complex structure of the biofilms makes them resistant to both host defense and
commonly used antifungal drugs [5, 8, 9]. The cells of Candida species can con-
stantly split from the structured microbial communities, spread and further cause
antifungal treatment failure, devices failure or persistent infections [10]. Therapeutic
methods for candida biofilm infections are very limited. On the other hand, although
Candida albicans infection is most commonly seen, the incidence of Candida non-
albicans is increasing in recent years due to (among other reasons) the frequent
prophylactic use of antifungal chemicals. Several non-albican species, such as
Candida glabrata (C. glabrata) and Candida krusei (C. krusei), may be resistant to
azole antifungal therapy.
In recent years, atmospheric pressure non-thermal plasmas (NTP) have drawn
much attention for their potential applications in medicine and biomedicine. They
have been reported to be effective in bacterial inactivation [11], blood coagulation
[12], tooth whitening [13], tumor treatment [14] and wound healing [15]. Early
work on the effectiveness of NPT on inactivating bacterial biofilms was performed
by a few groups [16, 17]. Compared with traditional therapeutic methods, non-
thermal plasmas as a physical method could be more economic and effective [18].
In addition, the gaseous form of plasmas provide the possibility to treat inhomoge-
neous surfaces, cavities and fissures down to the micrometer scale, and allow the
combination with minimally invasive surgery or with antimicrobial chemotherapy.
Several papers have reported using non-thermal plasmas against Candida albicans
[19, 20], but few have analyze their effect on the fluconazole-resistant Candida
strains. In this work, a direct current atmospheric pressure non-thermal plasma
microjet (PMJ), with helium and oxygen gas mixture as working gas, is applied to
several strains of Candida species including fluconazole-resistant C. albicans,
C. glabrata and C. krusei. Further investigation has been done on the fungicidal
capability of PMJ on Candida biofilms, and its effect on antifungal susceptibility of
candidal biofilms to common antifungal drug. Cell viability test (XTT assay) and
scanning electron microscope (SEM) were used after the plasma treatment to verify
the inactivation. Electron Spin Resonance (ESR) spectroscopy and optical emission
spectroscopy (OES) are employed to evaluate the reactive species generated.
16.1 Plasma Device
The plasma device comprises two copper tubes as electrodes separated by a

ceramic tube with the offset between the surfaces of the electrodes approximately
0.5 mm. The inner electrode is powered by a DC negative-polarity high-voltage
16 Inactivation of Candida Strains in Planktonic and Biofilm Forms 203
Flow
Gas
100
Flow - HV
Gas
5k 9.0 cm
2.0 cm
1.0 cm 2.0 cm
Petri Dish
Fig. 16.1 Schematic diagrams of a DC atmospheric pressure non-thermal plasma microjet work-
ing in air and in water, and their corresponding pictures
power supply through a 5 kW ballast resistor and the outer electrode is grounded
for safety considerations. The nozzle opening of the plasma device is 0.8 mm in
diameter and approximately 1 mm deep. Details about this device and electrical
circuit can be found in references [21, 22]. Premixed He/O2 (2% in volume) was
used as the working gas at a flow rate of 2.5 slm (standard liters per minute).
Figure 16.1 shows a schematic diagram of the plasma device in air, in water and
their respective pictures. When sustained in air, the visible jet is ~25 mm at a
sustaining voltage about 400 V and a discharge current of 35 mA. When immersed
in water, the PMJ was sustained in a quasi-steady gas cavity with approximately
the same sustaining voltage.
16.2 Candida Strains (Planktonic Phase)
Three types of Candida strains (planktonic phase), namely C. albicans (BMU

02971), C. krusei (ATCC6258, or BMU 00279) and C. glabrata (BMU 00271)
being either resistant or dose-dependent susceptible to fluconazole, were used in the
experiments. Fluconazole susceptible C. albicans (SC5314) was used as the control.
204 W. Zhu et al.
Each Candida strain was grown for 2 days on sabouraud dextrose agar (SDA) at
35C to ensure the viability and purity.
When treated in air, 100 ml diluted suspension (1 104 CFU/ml) were spread
evenly onto sabouraud dextrose agar (SDA) via a sterile plastic loop. This initial
concentration was chosen on purpose so that all results from various treatment times
can be fit into the CFU count detection limit in a later stage. The plasma treatment
(visible portion of the plasma touches agar) was limited to a 2 2 cm2 square area
(referred to as treated area) at the center of a 9 cm diameter Petri dish (as shown
in Fig. 16.1), with a treatment time ranging from 0 to 10 min. CFU counts from
controls with only gas flow are within error range of that from samples without any
treatment. The exit nozzle of the PMJ is maintained at about 1 cm away from the
treated media, where temperature was evaluated to be below 40C. This is not a
temperature that can affect the survival of the fungi.
For the treatment in water, conidial suspension was diluted to a concentration of
(13) 106 CFU/ml. Twenty milliliter suspension was treated with He/O2 PMJ from 0
to 4 min. After the plasma treatment, 200 ml suspensions was aspirated out and further
diluted 1,000 and 100-fold to perform antifungal susceptibility test and colony count-
ing, respectively. All experiments were repeated 3 times for statistical analysis.
The percentage of inactivation (PI, defined as 100% (1 CFU treated /CFU control ) )
of the Candida strains treated in air and in water are plotted in Fig. 16.2. In air, in
the treated area on Petri dish (Fig. 16.2a), there is a fast increase of the PI for all four
Candida species within the first minute, followed by a much slower change of PI. It
appears that different strains respond to plasma treatment slightly differently, with
C. glabrata reaching 100% inactivation in 2 min, while C. krusei only reaching 91%
after a 10-min plasma treatment. We also evaluated the CFUs in the untreated area
on Petri dish. Interestingly enough, the Candida strains in the untreated area were
also inactivated. Although the initial change of the PI with respect to time in the
untreated area was not as fast as it was in the treated area, significant PI (~90%)
was observed after a 10 min plasma treatment (Fig. 16.2b). It is possible that long
lived reactive species (such as ozone) can laterally transport to the untreated area,
where critical dosage is reached with the increase of treatment time.
While these Candida strains were treated in water, a fast increase of PI to above
90% was observed within the first 30 s. A 100% inactivation was achieved around
1 min treatment, as shown in Fig. 16.2c. This fast inactivation in water is related to
free radical production and will be discussed in Sect. 16.5.
XTT colorimetric-assay (Sigma, St. Louis, MO) was used to evaluate the cellular
viability of Candida strains through measuring the activity of enzymes that reduce
XTT to Formazan dyes. After PMJ treatment, 20 ml of the sample was added into
100 ml XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-car-
boxanilide) solution in a well of a 96-well microtiter plate. Each sample runs in
duplicate. The plates were then covered by aluminum foil and incubated at 37C for
2 h. The resulting supernatant was transferred via a multichannel pipette into the
wells of new microtiter plates, which were then read in a microtiter plate reader
(Bio-rad680). The optical density (OD) of the supernatant in each well was deter-
mined by measuring the absorbance at a wavelength of 490 nm. The OD of the
a 120 b
100
Percentage of Inactivation
100
80
80
60
60 In Air
40
In Air (Treated Area) (Untreated Area)
40
C. glabrata 00271 20 C. glabrata 00271
20 C. krusei 00279 C. krusei 00279
C. albicans 002971 0 C. albicans 002971
0 C. albicans SC5314 C. albicans SC5314
20
0 2 4 6 8 10 0 2 4 6 8 10
Time (min) Time (min)
c 120 d 120
C. glabrata 00271
100 100 C. krusei 00279

80 C. albicans 002971
80 XTT(%)
C. albicans SC5314
60
60
In Water 40
40
C. glabrata 00271 20
20 C. krusei 00279
0
C. albicans 002971
0 C. albicans SC5314 20
0 1 2 3 4 0 1 2 3 4
Time (min) Time(min)
Fig. 16.2 Percentage of inactivation of C. glabrata (BMU 00271), C. krusei (BMU 00279),
C. albicans (BMU 02971) and C. albicans (SC5314) treated with He/O2 PMJ (a) in air in the
treated area, (b) in air in the untreated area, (c) in water and (d) XTT assay
treated samples was then compared to that of the control to generate a percentage.
The XTT results of planktonic Candida strains are plotted in Fig. 16.2d. It shows
fairly good agreement with the inactivation in water. Zero metabolic activity was
achieved within 1 min except for C. albicans (SC5314) where 4 min is needed.
Nevertheless, this indicates that these Candica strains were essentially not viable
after the plasma treatment.
16.3 Candida Biofilms
Ten strains of Candida species isolated from different sources were used in the bio-
film study, as listed in Table 16.1.
After each strain was grown on potato dextrose agar (PDA) at 35C for 3 days to
ensure the viability and purity, a loopful of Candida cells was inoculated in 20 ml
yeast peptone dextrose liquid medium and incubated overnight in a shaker (150 rpm)
at 30C. Cells were then harvested from the liquid cultures by centrifugation
(3,000 g 5 min at 4C), and washed by ice-cold sterile PBS. RPMI-1640 medium
206 W. Zhu et al.
Table 16.1 Strains of Candida species used for biofilm study

Isolates Source Species
BMU00279 Sputum (ATCC6258) C. krusei
BMU05102 Oral mucosa C. krusei
BMU05137 Oral mucosa C. krusei
BMU00271 Blood C. glabrata
BMU01689 Knee C. glabrata
BMU04388 Intraperitoneal fluid C. glabrata
BMU02971 Pharynx C. albicans
BMU03213 Oral mucosa C. albicans
BMU04801 Oral mucosa C. albicans
SC5314 Blood (Sequencing strain) C. albicans
Fig. 16.3 A picture of the

He/O2 PMJ above a 96-well
microtiter plate
was used to re-suspend the pellet and adjust the final density to 1.0 106 cells/ml for
all strains. Hundred microliters of prepared suspension was pipetted into selected
wells of 96-well microtiter plates and every replication was separated by an empty
well. Biofilm was formed after incubation at 37C for 24 h. The medium was aspi-
rated in the wells which were then washed with sterile water three times to remove
planktonic cells. The biofilms were treated by PMJ for 10 s, 20 s, 30 s and 1 min,
respectively. A photo of the PMJ above the microtiter plate is shown in Fig. 16.3.
After PMJ treatment, 100 ml of sterile water was added into the well and washed
vigorously in order to re-suspend the biofilm cells. The suspension was then diluted
1,000 times with sterile water from which 100 ml was pipetted out and spread
evenly by a sterile plastic transferring loop onto SDA. The CFU counting was per-
formed after 24 h incubation at 35C. One hundred microliters RPMI1640 medium
followed by 100 ml of 2,3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-5-[(Phenyl-
Amino)Carbonyl]-2H-Tetrazolium Hydroxide (XTT, 1 mg/ml, Sigma-Aldrich Co.,
St. Louis, USA) and menadione (10 mM, Sigma-Aldrich Co., St. Louis, USA)
mixture were added into the wells treated by PMJ. The plates were then covered by
aluminum foil and incubated at 37C for 2 h. Eighty microliters of the resulting
b 100 100 a 100 100
Percentage Inactivation
80 80 80 80
XTT (%)
XTT (%)
60 C. krusei
60 60
C. glabrata 60
40 BMU 05137 40 40 BMU 00271 40

BMU 05102 BMU 01689
20 BMU 00279 20 20 BMU 04388 20
0 0 0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time (s) Time (s)
c 100
100
80 80
XTT (%)
60 C. albicans 60
BMU 04801
40 BMU 02971 40
SC5314
20 BMU 03213 20
0 0
0 10 20 30 40 50 60
Time (s)
Fig. 16.4 Percentage of inactivation and cell viability test (XTT assay) of C. glabrata, C. krusei
and C. albicans biofilms (iosolates from different sources, see Table 16.1) by He/O2 PMJ
supernatant was transferred via a multichannel pipette into the wells of new micro-
titer plates, which were then read in a microtiter plate reader (Bio-rad680). The
optical density (OD) of the supernatant in each well was determined by measuring
the absorbance at a wavelength of 490 nm. Each group contained negative and
positive controls.
Figure 16.4 shows the inactivation and XTT results of C. krusei, C. glabraca
and C. albicans biofilms versus treatment time. Three important observations: (1)
different Candida biofilms (even the same strain but isolated from different sources)
respond to plasma treatment slightly differently; (2) all Candida biofilms are inac-
tivated by PMJ rather quickly, all reaching approximately 100% within 30 s. (3)
XTT assay corresponds well with CFU counts, indicating the loss of viability of
the cells.
C. albicans (SC5314) biofilms (with and without PMJ treatment) were fixed
overnight with 2.5% glutaraldehyde and then dehydrated in ethanol. The samples
were gold-paladium coated and evaluated with SEM (Quanta 200FEG and NOVA
NANOSEM 430). Figure 16.5 shows five SEM images of C. albicans (SC5314)
biofilms treated with He/O2 gas flow and with He/O2 PMJ for different time periods.
Magnifications of these images are at or around 10,000 times. Figure 16.5a shows
that healthy yeast cell and pseudohyphae are with smooth surfaces. With the increase
of the plasma treatment time, cell rupture, distortion and shrinking were observed
208 W. Zhu et al.
Fig. 16.5 SEM images of Candida albicans (SC5314) treated with (a) He/O2 gas flow, He/O2 PMJ
for (b) 10 s, (c) 20 s, (d) 30 s and (e) 60 s
(Fig. 16.5be). The biofilm lost its original morphological characteristics and
degraded to clusters of cell fragments.
16.4 Antifungal Susceptibility Test
Antifungal susceptibility test was performed in samples treated with PMJ following
The Clinical and Laboratory Standards Institute recommended reference standard
M27-A3 with minor modification [23]. Two major concerns that motivated this test are:
(1) some fungi may survive a certain dosage of plasma treatment. They are possibly,
however, modified by the plasma and are more susceptible to traditional antifungal
treatment. (2) Clinical trials often combine traditional treatment methods and the
developing technique. As the toxicity and safety study of the plasma treatment is still
lacking, one might want to reduce the exposure to plasma to a minimum dosage while
achieving a considerable reduction of the dosage of traditional antifungal therapy.
Three commonly used antifungal drugs, namely fluconazole (Fuyang Genebest
Chemical Industry Co. Ltd, China), caspofungin (Merck, NJ, USA), and amphotericin
B (Sigma-Aldrich Co., St. Louis, USA) were used in the antifungal susceptibility
tests. From the stock solution of each antifungal agent, final working concentrations
were prepared in RPMI1640 medium to be 2561, 20.015 and 40.125 mg/ml,
respectively. Hundred microliters of diluted the respective solutions were added into
wells containing biofilms. After incubation at 37C for 24 h, their metabolic activi-
ties were evaluated with XTT assay as described above. Candida parapsilosis ATCC
22019 was included as a control. The sessile minimum inhibitory concentration
(SMIC50) is defined as the antifungal concentration at which a 50% decrease in
absorbance was detected in comparison with the control sample. All experiment pro-
cedures were performed three times for statistical analysis.
The SMIC50 results for C. albicans, C. krusei and C. glabarata treated with PMJ
for 10, 20 and 30 s are plotted in Fig. 16.6. Significant reductions of SMIC50 are
observed for all plasma treated Candida biofilms. SMIC50 ranges from 1 to 16 mg/
ml for fluconazole, 0.1250.25 mg/ml for amphotericin B and 0.0150.5 mg/ml for
caspofungin after a 10 s plasma treatment. These values are reduced to 1, 0.125
and 0.015 mg/ml, respectively, after 20 and 30 s treatments.
a 250 C. albicans b C. krusei c C. glabrata

Fluconazole (g/ml)
200
BMU 04801 BMU 05137 BMU 00271
SC5314 BMU 00279 BMU 04388
BMU 02971 BMU 05102 BMU 01689
150 BMU 03213
100
50
0 10 20 30 0 10 20 30 0 10 20 30
Time (s)
d 4
C. albicans e f
C. krusei C. glabrata
Amphotericin B (g/ml)
BMU 04801 BMU 05137 BMU 00271

3 SC5314 BMU 00279 BMU 04388
BMU 02971 BMU 05102 BMU 01689
BMU 03213
2
0 10 20 30 0 10 20 30 0 10 20 30
Time (s)
g 2.0
C. albicans h C. krusei i C. glabrata
1.6 BMU 04801 BMU 05137 BMU 00271
Caspofungin (g/ml)
SC5314 BMU 00279 BMU 04388

BMU 02971 BMU 05102 BMU 01689
1.2 BMU 03213
0.8
0.4
0.0
0 10 20 30 0 10 20 30 0 10 20 30
Time (s)
Fig. 16.6 SMIC50 of (a)(c) fluconazole, (d)(f) amphotericin B and (g)(i) caspofungin for the
biofilms treated for 10, 20 and 30 s
16.5 Reactive Species
Reactive species, charged particles and UV radiation are commonly considered to

(more or less) participate in the inactivation process. To study the reactive species
generated in the plasma, end-on light emission from the He/O2 PMJ was collected in
air through a quartz fiber optics cable to the entrance slit of a 0.75 m spectrometer
210 W. Zhu et al.
350 350
OH (A-X)
O 844
O 777
x50
300 300
He 706
Emission Intensity (A.U.)
He 667
Emission Intensity (A.U.)
He 587
x250
He 501
250 250
Ha 656
200 200
He 728
150 150
300 305 310 315
100
He 447
100
He 492
7 5
32 32
50 640 650 660

u
He 471
C
50
u
C
0
0
300 350 400 450 500 550 600 600 650 700 750 800 850
Wavelength (nm) Wavelength (nm)
Fig. 16.7 End-on optical emission spectrum of He/O2 PMJ in air (flow rate: 2.5 slm; current:
35 mA)
(Acton 2750) equipped with a 1,800 groove/mm grating. The dispersed emission
spectra were recorded by an intensified CCD camera (Roper Scientific I-MAX-1024)
in the exit plane of the spectrometer. The light was focused into one end of the fiber
optics cable via a quartz convex lens. Figure 16.7 shows the end-on spectra of He/O2
PMJ operated in air in the UV to near IR region. Major peaks are mostly from helium
emissions, while strong atomic oxygen emissions at 777 and 844 nm are also
observed. Aside from direct electron impact excitation and dissociative excitation,
helium metastables must have participated in the excitation of oxygen from the car-
rying gas as well as from the air. Water from the surrounding air were disassociated
and excited. Emissions from both OH (A-X) band in 306309 nm and Ha at 656 nm,
although very weak, were observed (insets in Fig. 16.7). Copper emissions were also
observed in the UV region due to the choice of the electrode material.
Free radicals are atoms, molecules or ions with unpaired electrons. They are
highly chemically reactive and can therefore react with biological material. They
may be produced directly in the plasma or at the plasma-liquid interface of the sys-
tem. We believe hydroxyl radical (OH) and singlet molecular oxygen (1O2), are of
particular importance in our system. Both OH (oxidation potential: 2.8 eV) and 1O2
(oxidation potential: 0.98 eV) tend to attack chitin and polysaccharides on cell wall
as well as unsaturated fatty acids on cell membrane. Their presence can compro-
mise the function of cell wall and membrane lipids and cause the transportation of
ions and polar compounds into the cell [24].
Electron spin resonance (ESR) spectroscopy is a direct method for detecting species
which possess an unpaired electron, whose spin states are split in an external magnetic
field. Upon application of a magnetic field of the resonance frequency between the two
states, a transition is induced, which is signaled by an absorption peak on the spectrum
of the magnetic field. As both radicals mentioned above (in particular OH) are of rather
short life-span and are therefore difficult to be detected directly in ESR spectrum, spin
trapping was applied to facilitate the detection by reacting short-lived radicals with a
spin trap reagents. As a result, persistent aminoxyl spin adduct radicals are produced,
which are of longer life-span and are easier to be detected.
a 1500 b 2000
DMPO-OH TEMPO
Signal Strength (A.U.)

Signal Strength (A.U.)
750 1000
0 0
3350 3400 3450 3500 3350 3400 3450 3500

Magnetic Field (Gauss) Magnetic Field (Gauss)
Fig. 16.8 Electron spin resonance spectra of (a) DMPO-OH (spin adduct of OH) and (b) TEMPO
(spin adduct of 1O2)
Twenty microliters (0.8 mol/L) 5,5-dimethyl-1-pyrroline-N-oxide (DMPO,

Sigma Aldrich Co., Ltd.) was added to 1 ml distilled water prior to the plasma treat-
ment to spin trap OH. Similarly, 20 ml (99.9%) 2,2,6,6-Tetramethylpiperidine
(TEMP, Sigma Aldrich Co., Ltd.) was added into 1 ml distilled water to spin trap
1
O2. Both media were treated with PMJ for 20 s. After each PMJ treatment, about
40 ml sample was imbibed by a capillary tube and sent to ESR resonance chamber.
Measurements of the ESR signals were carried out on an ER-200D-SRC ESR
spectrometer (Bruker Ltd, Germany) operated at room temperature at the following
conditions: central magnetic field, 3,420.00 G; sweep width, 200.0 G; frequency,
9.54 GHz; modulation frequency, 100 kHz and power 20 mW. The ESR spectra of
DMPO-OH (the spin adduct of OH) and of TEMPO (the spin adduct of 1O2) show
quartet and triplet patterns with peak ratios of 1:2:2:1 and 1:1:1, respectively
(as shown in Fig. 16.8).
DMPO can also spin trap superoxide anion radical (O2), or more specifically, its
conjugate acid (HOO), but at a much smaller reaction rate. The resulting ESR spec-
trum of the spin adduct DMPO-OOH should in principle show a 12 peak pattern.
However, no DMPO-OOH signal was detected. It is interesting to note that when
superoxide dismutant (SOD) was added into the system, DMPO-OH signal decreases
quickly, indicating that O2 likely the source of OH in water.
16.6 Discussions
Non-thermal plasma can effectively inactivate the plantonic cells of Candida spe-
cies (whether antifungal resistant of not) on Petri dish. We believe that in the treated
area, both charged particles and reactive species (short lived and long lived) partici-
pate in the inactivation process. In the untreated area, however, only longer lived
reactive species (such as O3) can contribute to the inactivation. Helium metastable
(~20 eV) has a longer life time, can essentially transport laterally to the untreated
area and interact with oxygen in air via stepwise excitation to create antifungal
212 W. Zhu et al.
species on-site. Nevertheless, Candida species in the untreated area can be inactivated
to the extent as in the treated area, but a bigger dosage (longer treatment time) seems
to be necessary. When the treatment was done in water, the Candida species were
not bound to agar as they were on Petri dishes but can move rather freely. They can
be brought to a close vicinity of the plasma (which was sustained in a quasi-steady
gas cavity in water). Furthermore, radicals with strong oxidative capability (such as
OH and 1O2) were generated in water. Both of these facilitate a fast inactivation of
Candida species.
Biofilms are structured microbial communities that are found in many places
(such as in catheters and on implanted artificial joints). They are resistant to both
host defense and commonly used antifungal drugs. The results here on ten Candida
strains (isolated from different sources) only show first evidence of the fast and
effective treatment of Candida biofilms and the change of their antifungal suscepti-
bility. The results were confirmed by cell viability test (XTT assay) as well as SEM.
The fact that biofilms were completely inactivated within 60 s in air while the
inactivation of planktonic fungi in the untreated area on Petri dish took 4 min may
seem that biofilms are easier to inactivate. However, one has to note that the treat-
ment of the planktonic fungi in air was performed on Petri dish in a 2 2 cm2 area.
The percentage of inactivation in the treated area is similar to that in the well of a
microtiter plate, which has a diameter of 6.86 mm. We believe that charged particles
and reactive species generated in the plasma are responsible for the inactivation.
Some of the radicals (such as OH) may not be produced directly in the plasma but
rather at the plasma surface interface (given surface water accumulation from sur-
rounding air). O2 must have been produced in the system. Evidence [25] has shown
that their conjugate acid (HOO) can peroxidize the fatty acid on cell membrane, at
pH lower than a critical value of 4.7. PH of our liquid system was found to change
from 7.3 only to approximately 7. O2 converts to OH in the system via a process
called Haber-Weiss reaction (O2 + H2O2 OH + OH + O2). This reaction is
accelerated with transition metal copper (electrode used in the device) as the cata-
lyst. Cu+/Cu2+ were detected in water by HPLC [26]. 1O2 is believed to be directly
produced in the plasma and subsequently transferred to the surface. Its lifetime in
air is in tens of minutes but is reduced to seconds in liquid. Nevertheless, oxidative
damages can be caused by these reactive oxygen species directly or indirectly, lead-
ing to impediment of ion transition. Furthermore, the oxidative molecules can com-
bine with DNA, causing additional cell damage [27].
In conclusion, atmospheric pressure He/O2 cold plasma microjet presents a new
approach to effectively inactivate C. albicans, C. glabara and C. krusei in plantonic
phase in air and in water as well as the biofilms induced by these strains (isolated
from different sources). However, issues concerning safety in operation, the function
of other killing agents in the plasma or at the plasma-surface interface, toxicity
study, their effect on healthy cells need to be further investigated in the future.
Acknowledgements Work supported in part by Bioelectrics Inc. (U.S.A.), the Peking University
Biomed-X Foundation and China International Science and Technology Cooperation (2008KR1330
Cold Plasma induced biological effect and its clinical application studies)
References
1. Concia E, Azzini AM, Conti M (2009) Epidemiology, incidence and risk factors for invasive
candidiasis in high-risk patients. Drugs 69:514
2. Arendrup MC (2010) Epidemiology of invasive candidiasis. Curr Opin Crit Care 16:445452
3. Rowen JL (2003) Mucocutaneous candidiasis. Semin Perinatol 27:406413
4. Hasan F, Xess I, Wang X, Jain N, Fries BC (2009) Biofilm formation in clinical Candida
isolates and its association with virulence. Microbes Infect 11:753761
5. DEnfert C (2006) Biofilms and their role in the resistance of pathogenic Candida to antifungal
agents. Curr Drug Targets 7:465470
6. Ramage G, Tomsett K, Wickes BL, Lopez-Ribot JL, Redding SW (2004) Denture stomatitis:
a role for Candida biofilms. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 98:5359
7. Bryers JD, Ratner BD (2004) Bioinspired implant materials befuddle bacteria. ASM News
5:232237
8. Ramage G, Wickes BL, Lopez-Ribot JL (2001) Biofilms of Candida albicans and their associ-
ated resistance to antifungal agents. Am Clin Lab 20:4244
9. Mukherjee PK, Chandra J (2004) Candida biofilm resistance. Drug Resist Updat 7:301309
10. Cauda R (2009) Candidaemia in patients with an inserted medical device. Drugs 69:3338
12. Fridman G, Peddinghaus M, Ayan H et al (2006) Blood coagulation and living tissue steriliza-
tion by floating-electrode dielectric barrier discharge in air. Plasma Chem Plasma Process
26:425442
13. Pan J, Sun P, Tian Y, Bai N, Zhou H, Wu H, Zhu W, Zhang J, Becker KH, Fang J (2010) A
novel method of tooth whitening using cold plasma micro-jet driven by direct current and
atmospheric-pressure air. IEEE Trans Plasma Sci 38:31433151
14. Vandamme M, Robert E, Pesnel S, Barbosa E, Dozias S, Sobilo J, Lerondel ALP, Pouvesle
J-M (2010) Antitumor effect of plasma treatment on U87 glioma xenografts: preliminary
results. Plasma Processes Polym 7:34
New J Phys 11:50135032
16. Abramzon N, Joaquin JC, Bray J, Brelles-Mario G (2006) Biofilm destruction by RF high-
pressure cold plasma jet. IEEE Trans Plasma Sci 34:13041309
17. Vleugels M, Shama G, Deng XT, Greenacre E, Brocklehurst T, Kong MG (2005) Atmospheric
33:824828
18. Heinlin J, Isbary G, Stolz W, Morfill GE, Landthaler M, Shimizu T, Steffes B, Nosenko T,
Zimmermann JL, Karrer S (2011) Plasma applications in medicine with a special focus on
dermatology. J Eur Acad Dermatol Venereol 25:111
19. Rupf S, Lehmann A, Hannig M, Schfer B, Schubert A, Feldmann U, Schindler A (2010)
Killing of adherent oral microbes by a non-thermal atmospheric plasma jet. J Med Microbiol
59:206212
20. Morfill GE, Shimizu T, Steffes B, Schmidt H (2009) Nosocomial infections a new approach
towards preventive medicine using plasmas. New J Phys 11:115019
21. Feng H, Sun P, Chai Y, Tong G, Zhang J, Zhu W, Fang J (2009) The interaction of a direct-
current cold atmospheric-pressure air plasma with bacteria. IEEE Trans Plasma Sci
37:121127
22. Liu F, Sun P, Bai N, Tian Y, Zhou H, Wei S, Zhou Y, Zhang J, Zhu W, Becker K, Fang J (2010)
Inactivation of bacteria in an aqueous environment by a direct-current, cold-atmospheric-
pressure air plasma microjet. Plasma Processes Polym 7:231236
23. Pierce CG, Uppuluri P, Tristan AR, Wormley FL Jr, Mowat E, Ramage G, Lopez-Ribot JL
(2008) A simple and reproducible 96-well plate-based method for the formation of fungal
biofilms and its application to antifungal susceptibility testing. Nat Protoc 3:14941500
214 W. Zhu et al.
24. Fridovich I (1995) Superoxide radical and superoxide dismutases. Annu Rev Biochem
64:97112
25. Ikawa S, Kitano K, Hamaguchi S (2009) Effects of pH on bacterial inactivation in aqueous
Polym 7:3342
26. Sun P, Wu H, Bai N, Zhou H, Wang R, Feng H, Zhu W, Zhang J, Fang J (2011) Inactivation of
Bacillus subtilis spores in water by a direct-current, cold atmospheric-pressure air plasma
microjet. Plasma Processes Polym, Online: DOI: 10.1002/ppap.201100041
27. Davies MJ (2003) Singlet oxygen-mediated damage to proteins and its consequences. Biochem
Biophys Res Commun 305:761770
Chapter 17
Non-thermal Atmospheric Plasma Treatment
for Deactivation of Oral Bacteria and
Improvement of Dental Composite Restoration
Qing Song Yu, H. Li, A.C. Ritts, B. Yang, M. Chen,

L. Hong, C. Xu, X. Yao, and Y. Wang
Abstract This paper reviews our recent research results of using non-thermal
atmospheric plasmas for oral bacterial deactivation and for composite restoration
improvement. Oral bacteria of Streptococcus mutans (S. mutans) and Lactobacillus
acidophilus (L. acidophilus) with an initial bacterial population density between
1.0 108 and 5.0 108 cfu/ml were seeded on various media and their survivability
with plasma exposure was examined. The plasma exposure time for a 99.9999%
cell reduction was less than 15 s for S. mutans and within 5 min for L. acidophilus.
To evaluate the dentin/composite interfacial bonding, extracted unerupted human
third molars were used by removing the crowns and etching the exposed dentin
surfaces with 35% phosphoric acid gel. After dental composite application and
light curing, the teeth were then sectioned into micro-bars as the specimens for
microtensile test. Student Newman Keuls (SNK) tests showed that the bonding
strength of the composite restoration to peripheral dentin was significantly
increased (by 64%) after 30 s plasma treatment of the dentin surfaces. These find-
ings indicated that non-thermal atmospheric plasma technology is very promising
for dental clinical applications.
Q.S. Yu (*) H. Li A.C. Ritts B. Yang

Center for Surface Science and Plasma Technology,
Department of Mechanical and Aerospace Engineering,
University of Missouri, E2403D Lafferre Hall, Columbia, MO 65211, USA
e-mail: yuq@missouri.edu
M. Chen
Nanova Inc., Columbia, MO 65203, USA
L. Hong
College of Dentistry, University of Tennessee, Memphis, TN 38136, USA
C. Xu X. Yao Y. Wang
School of Dentistry, University of MissouriKansas City, Kansas City, MO 64108, USA
216 Q.S. Yu et al.
Polymethacrylate-based dental composites have received widespread clinical

acceptance as alternative restorative materials to dental amalgam amid concern
regarding the potential health risks associated with mercury released from dental
amalgam. Polymer-based dental composites can be easily adapted to a wide variety
of direct placement applications, and are aesthetically appealing. Dental composite
restorations, however, have much shorter longevity when compared to amalgam
restorations [14]. An 8-year clinical study found that the failure rate for posterior
composite restorations was two to three times higher than amalgam restorations [5].
Many other studies reported very similar results [68]. The reduced longevity of
dental composite restorations is troubling as the removal of these restorations can
lead to extensive loss of sound tooth structure and eventual loss of the teeth after
two or three restoration replacements [9].
Extensive clinical and laboratory studies indicate that the reduced longevity of
dental composite restoration most often resulted from the interface failure (i.e.,
adhesive bonding failure of the composite restoration to the surrounding tooth
structure) [1015]. The interface failure results in the separation of the composite
restoration from dentin. The resulting gaps lead to marginal staining, sensitivity,
and recurrent caries [12, 13, 16, 17], which cause a significant portion of composite
restoration removal and replacements [2, 17], and therefore are significant in terms
of reducing the longevity of the composite restoration.
The interface failures in composite restoration are commonly found at the
adhesive/dentin interface [10, 18, 19]. Adhesion between enamel and composite is
generally adequate for clinical applications, while adhesive/dentin interface is weak
and the interfacial bonding strength deteriorates significantly over time [1821]. For
example, an in vivo study showed that the mean tensile bond strength values dropped
from 28.3 MPa at 24 h to 9.1 MPa at 23 years [19]. Unsuccessful dentin bonding
also means that there are sites at the tooth restoration interface that are vulnerable to
hydrolytic breakdown and susceptible to attack by bacterial enzymes [9]. The
improvement in adhesive/dentin interfacial bonding is essential to achieve robust
and durable composite restoration with increased longevity.
As an effective surface/interface engineering tool, non-thermal gas plasma tech-
nique provides a unique opportunity in dentin surface preparation to disinfect oral
bacteria and modifying dentin surfaces to significantly strengthen the interface
bonding, and thus to increase the longevity of composite restorations. In this paper,
we summarized our recent research results on plasma deactivation of oral bacteria
and surface treatment of dentin surfaces for interfacial bonding improvement with
dental adhesive and composite application.
17.1 Experimental
17.1.1 The Non-thermal Plasma Source
An atmospheric cold plasma brush was used in this study. The detail of this
plasma source was reported previously [22, 23]. A MKS mass flow controller
17 Non-thermal Atmospheric Plasma Treatment for Deactivation of Oral Bacteria 217
Fig. 17.1 Optical emission spectrum of Ar atmospheric plasmas. Plasma conditions were 1,500
sccm Ar flow and 15 W DC power input
(MKS Instruments, Andover, MA, USA) was used to control argon gas flow rate.
The discharge was ignited and sustained by a DC power supply (Pd 1556c, Power
Design Inc. New York, NY, USA). With a relatively high gas flow rate, the plasma
discharge formed inside the chamber could be blown out of the chamber to form a
brush-shaped low temperature plasma jet, i.e., a cold atmospheric plasma brush.
This plasma source can be operated under very low electrical power (as low as a few
watts), and as a result very low plasma temperature can be achieved. The gas phase
temperatures of such an argon atmospheric plasma, measured using an infrared
camera combined with a thermocouple thermometer range from 30C to 65C, with
the corresponding argon gas flow rate varying from 500 sccm (standard cubic
centimeter per minute, a volumetric flow rate at 273 K under 1 atm) to 3,500 sccm
and input power from 5 to 15 W.
The optical emission spectrum (OES) of the Ar atmospheric plasmas is shown in
Fig. 17.1. Besides Ar emission lines, N2 emission lines and one O emission line
(777.2 nm) were observed due to the interaction of the Ar plasmas with ambient air.
17.1.2 Plasma Deactivation of Oral Bacteria
Streptococcus mutans (S. mutans) ATCC 25175 and Lactobacillus acidophilus

(L. acidophilus) ATCC 4356 (American Type Culture Collection, ATCC, Manassas,
VA, USA) were used in this study. As Gram-positive, facultatively anaerobic bacte-
ria, S. mutans is one of the most implicated bacteria in smooth surface caries and
considered to be a major pathogen in dental caries [2426]. L. acidophilus has been
associated with dental caries, and the quantity of L. acidophilus in saliva is still used
as a direct measure of caries risk, which is known as Lactobacillus counts (Caries
Test) [27, 28]. P5 filter papers and glass slides and PTFE (Polytetrafluoroethylene)
films (0.1 mm in thickness) were used as the bacterial supporting media. Filter
papers have porous structures, which simulate the fissures of the tooth [29].
218 Q.S. Yu et al.
Glass slide was used as smooth solid surface similar to the intact tooth surface and
PTFE film was a polymer surface carrier. Tryptic Soy Agar and Tryptic Soy Broth
(Difco Bacto, Detroit, MI, USA), Standard Methods Agar (Becton Dickson,
Cockeysville, MD), and Lactobacillus MRS Agar (MRSA) and MRS Broth (MRSB)
(Difco Bacto, Detroit, MI, USA) were used. The detailed experimental procedures
were reported elsewhere [30].
17.1.3 Dentin Surface Preparation and Dental Materials
Extracted un-erupted human third molars stored at 4C in phosphate buffered saline

(PBS), containing 0.002% sodium azide, were used. These molars were collected
after patients informed consent under a protocol approved by the UMKC adult
health sciences institutional review board (IRB) [31]. Molars were prepared using
an Isomet 5,000 diamond saw purchased from Buehler (Lake Bluff, IL, USA).
Specimens were polished and etched for 15 s before rinsing with de-ionized (DI)
water [31]. The surface was dried by blotting the surface with a Kimwipes dust-
free tissue prior to cold plasma treatment. 600-grit silicon carbide abrasive paper
(Buehler, Lake Bluff, IL, USA) was utilized for polishing the dentin surface prior to
demineralization to mimic the clinical use of dental burs [32]. ScotchbondTM phos-
phoric acid gel by 3M ESPE (Dental Products, St. Paul, MN, USA) was utilized for
demineralizing the dentin surface, AdaperTM Single bond plus from 3M ESPE was
utilized as adhesive, and FiltekTM Z250 from 3M ESPE was utilized as the dental
composite for the restoration. Composite and adhesive were light cured by visible
light via Spectrum 800 from Dentsply (Milford, DE, USA). Compressed argon gas
(4.7 grade and 99.997% purity) purchased from Praxair (Columbia, MO, USA) was
used as plasma gas. The preparation details of test specimen for mechanical testing
were reported previously [33].
17.2 Plasma Deactivation of Oral Bacteria
Prior to plasma treatment, S. mutans and L. acidophilus bacteria with population

density in the range between 1.0 108 and 5.0 108 cfu cm1 were dispensed onto
the supporting media and then dried in a moderate vacuum incubator at 37C for
20 min. Figure 17.2 shows the cell surviving curve of S. mutans and L. acidophilus
with different plasma exposure times on the three different supporting media. It can
be seen that the Ar plasma brush is very effective in deactivation of S. mutans and a
very short plasma exposure time of less than 15 s gave a complete kill of the bacte-
ria. On the other hand, a longer plasma treatment time was required for deactivating
L. acidophilus than killing S. mutans.
There are two possible reasons for such a difference in bacterial deactivation
between these two kinds of bacteria. The first is that plasma etching, oxidation and
Fig. 17.2 Plasma sterilization effect on (a) S. mutans and (b) L. acidophilus seeded on different
support media. Plasma conditions were 2,000 sccm Ar flow rate and 10 W DC power input [30]
UV-radiation have been known to be the main plasma sterilization mechanisms.

Such sterilization mechanisms indicate that bacterial sizes and structure would
affect the plasma effectiveness and efficiency in bacterial deactivation. The bigger
cell size of L. acidophilus (3 mm in diameter) would have higher plasma tolerance
and are harder to kill than the smaller S. mutans (~1 mm in diameter). In order to
achieve the same intensity of plasma injection to a single cell, under the same plasma
conditions, several minutes of exposure time were needed to kill L. acidophilus,
while only tens of seconds of exposure time were needed for S. mutans. The second
reason is due to the limited spreading area (4 mm in diameter) of the seeded cells on
the supporting media. With similar cell population densities used in this study, such
a limited area leads to more overlapped cell layers for L. acidophilus than that of
S. mutans. Consequently, less L. acidophilus cells resided on the top layer were
directly exposed to plasma than that for S. mutans under the similar cell population
densities and plasma conditions [34]. Plasma species were blocked by the outer
layer cells from reaching the underneath layer of the seeded cells.
It was also noted that more efficient deactivation for both types of bacteria was
observed with filter papers as the supporting media. Based on our experimental
observation, the S. mutans cell droplets had the biggest spreading area on the filter
paper (4 mm in diameter) than those on the other two media. This large spreading
area would provide the most amount of cells resided on the top cell layers to be
exposed to plasma at the same time. Because of the highest water contact angle of
PTFE surface, the smallest droplet spreading area (2 mm in diameter) was observed
on the PTFE surface. From Fig. 17.2, it was observed that the plasma deactivation
rate for S. mutans became slower in the period from 3 to 9 s and re-accelerated after
10 s plasma exposure. This duration enabled plasma to decompose the remaining
debris of the lethal cells before the underneath layer cells were directly exposed to
the plasma.
Scanning Electron Microscopy (SEM) was used to examine the cell structural
changes of both S. mutans and L. acidophilus bacteria after plasma exposure. As
shown in Fig. 17.3, plasma treated S. mutans on glass slide resulted in a significant
alteration in size and transformation in morphology when compared with the
220 Q.S. Yu et al.
Fig. 17.3 SEM images of S. mutans and L. acidophilus cells without plasma treatment and after
plasma exposure for various time periods. The support media were glass slides and plasma condi-
tions were 2,000 sccm argon flow rate and 10 W DC power input [30]
untreated controls. After 5 s of Ar plasma exposure, majority of the remaining viable

S. mutans colonies showed distinct cell structural damage. Being consistent with the
cell survival curve shown in Fig. 17.2, 15 s Ar plasma exposure of S. mutans resulted
in a much more amount of cell debris than that after 5 s Argon plasma exposure. In
case of L. acidophilus as shown in Fig. 17.3, damages on cell wall were found with
60 s plasma treatment. More cell damage was observed with the cell colonies after
longer plasma exposure of 180 s (SEM image not shown). With further increase in
the plasma exposure time to 300 s, fragmentation of L. acidophilus colonies was also
found. Because of the cell structural damage observed in the SEM images, the cell
survival curves shown in Fig. 17.2 indicate that the bacteria were completely killed.
In comparison with L. acidophilus, the pronounced cell damages of S. mutans shown
in Fig. 17.3 explained the faster plasma deactivation rate of S. mutans.
To investigate plasma deactivation mechanisms of the bacteria, a UV-visible
spectrometer was used to monitor the absorbance peak intensities at wavelengths of
260 (DNA absorbance) and 280 nm (protein absorbance). The peak intensity of the
absorbance is related to the leakage amount of the intracellular proteins and DNAs.
Figure 17.4 show the intensity changes of such absorbance peaks with plasma expo-
sure time for S. mutans and L. acidophilus, respectively. It can be seen that a very
short plasma exposure (1 s) could significantly increase the peak intensity for both
protein and DNA absorbance, indicating dramatic leakage of the intracellular
Fig. 17.4 The change in absorbance (Abs.) intensity of intracellular protein and DNA leakage
from (a) S. mutans and (b) L. acidophilus with plasma treatment time. Plasma conditions were
2,000 sccm argon flow rate and 10 W DC power input [30]
proteins and DNAs. For both bacterial samples, a longer plasma exposure showed
continuous leakage of intracellular proteins and DNAs, in spite of leveling off of the
peak intensity of absorbency at wavelengths of 260 and 280 nm. These data suggest
that there were a large amount of protein and/or nucleic acids that were released into
the supernatant immediately after plasma exposure, which usually occurs when the
cell membranes are damaged as observed by SEM images shown in Fig. 17.3.
17.3 Plasma Treatment of Dentin Surface

for Composite Restoration
The most common adhesive/dentin interface failure in dental composite restoration

indicates the inadequate interface bonding. Currently, the surface energy values of
dentin (which is mainly collagen fibrils after acid etch step) and infiltrated adhesive
resin are mismatched. The underlying hypothesis is that compatible surface energy
levels will enhance penetration of adhesive resins into collagen fibrils on dentin
surfaces and thus improving interfacial bonding through micromechanical inter-
locking mechanism. Our experimental results have shown that dentin surface energy
levels can be easily/effectively modified by non-thermal plasma treatment.
Figure 17.5 shows the water contact angle change of dentin surface with plasma
exposure times. It can be seen a few seconds of plasma treatment could significantly
reduce the water contact angle of the dentin surfaces. This preliminary data
demonstrates that non-thermal plasmas are capable to rapidly alter dentin surface
characteristics and are effective in dentin surface modification.
Plasma treatment could also introduce new functionalities on collagen fibrils and
thus change the surface characteristics of dentin surfaces. The Fourier transform
infrared (FTIR) spectra of plasma treated dentin surfaces and the untreated controls
are shown in Fig. 17.6. FTIR surface analysis showed structural changes in the sur-
face of the demineralized dentin after plasma treatments. It was shown that there
222 Q.S. Yu et al.
50
Water Contact Angle (degree)

40
30
20
10
Completely
Penetrated
0
0 5 10 15 20
Plasma Treatment Time (second)
Fig. 17.5 Water contact angle change of dentin surfaces with plasma treatment time
Fig. 17.6 FTIR spectra of demineralized dentin before and after plasma treatment [33]
were two major changes in the representative spectrum of dentin surface after
plasma treatment. First, a new shoulder peak around 1,760 cm1 (in the oval)
associated with carbonyl stretch was found. This new peak has been confirmed by
subtraction line between the FTIR spectra of plasma treated and untreated dentin.
Second, an amide II shift of ~10 cm1 was observed (1,543 cm1 before to 1,533 cm1
after), which might indicate the secondary structural changes of dentin collagen
after plasma treatment. These chemical changes of the collagen fibrils may allow
more interactions with the adhesive resins applied subsequently.
After acid etching, the polished dentin surfaces were exposed to non-thermal
argon plasmas for predetermined time period as pictorially shown in Fig. 17.7. The
dental adhesive and dental composite resins were subsequently applied and light
cured to the dentin surfaces as instructed. Microbar test specimens were then pre-
pared with a cross-section of ~1.0 1.0 mm for tensile testing [33, 35]. The mean
Fig. 17.7 A pictorial view of

the non-thermal atmospheric
plasma brush applied to an
extracted tooth with crown
removed [33]
cross sectional areas of each test specimen between treatments were from 0.87 to
0.95 mm2 as measured using a digital caliper and no difference was detected among
the treatments of all groups by one-way ANOVA (p = 0.38). Typically, 2025 micro-
bar test specimens were prepared from one extacted tooth. Among these 2025
microbars, about 46 microbars obtained from the tooth center position were desig-
nated as inner dentin (I) and the rest 1520 microbars were designated as peripheral
dentin (P). The bars were adhered to a universal testing system TAHD Plus (Stable
Micro System Ltd, Golalming, Surrey GU7 1YL, UK) via cyanoacrylate resin (Zapit,
Corona, CA, USA) and subjected to tensile testing with strain rate of 0.5 mm/min.
No specimens from the peripheral dentin failed prematurely before testing.
Figures 17.8 and 17.9 show the statistical comparison of ultimate tensile strength
and modulus data obtained with test specimens prepared from plasma treated dentin
and the untreated controls. Statistically significant differences in tensile strength
between all specimens using Student-Newman-Keuls (SNK) method were observed.
A significant difference was found between the peripheral dentin that was plasma
treated for 30 s and all the other treatments. As the plasma treatment time was
increased beyond 30 s the tensile strength decreased. After100 s plasma treatment,
the tensile strength of the microbar specimens was increased as compared to the
controls, but not significantly. After 300 s plasma treatment, tensile strength of the
specimens was similar to the control specimens, with a larger variance.
The modulus for the 300 s plasma treatment specimens was also reduced
(Fig. 17.9). Modulus data showed a trend of lower modulus for inner dentin as com-
pared to peripheral dentin. The 30 s plasma treatment increased the modulus of the
interface bonding when compared to the controls; however increasing treatment
time did not further increase the modulus. Inner dentin specimens for 100 and 300 s
plasma trials were not successfully tested. Plasma treatment of inner dentin for 30 s
does not affect the mechanical properties as seen in Figs. 17.8 and 17.9.
The stronger interface bonding obtained with plasma treated dentin can be also
attributed to the new surface functionalities and thus modified surface characteristics
introduced through plasma treatment. Plasma treatment would introduce residue free
224 Q.S. Yu et al.
80.00
Ultimate Tensile Strength (MPa)

70.00 a
60.00
b
50.00
b,c b,c
40.00
30.00 c,d
d
20.00
10.00
0.00
0s P 30s P 100s P 300s P 0s I 30s I
Plasma Treatment Time/Dentin Location
Fig. 17.8 Statistical comparison of ultimate tensile strength obtained with test specimens
prepared from plasma treated dentin and the untreated controls (0s P and 0s I). P Peripheral
dentin, I Inner dentin. Different letters in the plot indicate statistically significant differences
(a = 0.05) [33]
1000 a
Interface Modulus (GPa)
800
b
b,c
600 c,d
d c,d
400
200
0
0s P 30s P 100s P 300s P 0s I 30s I
Plasma Treatment Time/Dentin Location
Fig. 17.9 Comparison of tensile modulus obtained with test specimens prepared from plasma
treated dentin and the untreated controls (0s P and 0s I). P Peripheral dentin, I Inner dentin.
Different letters in the plot indicate statistically significant differences (a = 0.05) [33]
radicals and peroxide functional groups on collagen fibrils surfaces. In chemistry,

these residue free radicals and peroxides could then initiate the polymerization, i.e.,
curing of the monomers in adhesive resins, and form covalent chemical bonds between
the resin and the collagen fibrils. In our study, hydroxyethyl methacrylate (HEMA)
monomer, a primary component in many commercial dentin adhesives, was applied to
both the plasma pretreated dentin collagens and untreated dentin collagens. 10 mins
Plasma pretreated collagen+HEMA, wash

Amide I 1638 C-O
Amide II 1158
1537
Amide III
C=O C-O-C
C-H
1240
1716 1078
1448
C-C
1030
Untreated collagen+HEMA, wash
Untreated collagen
Plasma pretreated collagen

1800 1700 1600 1500 1400 1300 1200 1100 1000
cm-1
Fig. 17.10 FTIR spectra of plasma treated collagen and the untreated controls with and without
applying HEMA
after, the dentin collagens were washed thoroughly with water for three times. FTIR
results (Fig. 17.10) show that HEMA was totally washed away from the untreated
dentin collagens. In contrast, in the plasma pretreated dentin collagens, the spectrum
clearly indicates the presence of HEMA as evident from the observed bands at 1,716
(C=O stretching), 1,158 (OC), 1,078 (COC), 1,030 (CC), respectively. It is also
noticed that HEMA monomers in the plasma pretreated collagens have polymerized.
This is based on the wavenumber shift of the OC band (at 1,158 cm1) in the pre-
treated dentin collagens. The position of this band in the HEMA monomer is located
at 1,162 cm1, shifted to lower wave-numbers in poly (HEMA). The results indicate
that the plasma introduced functionalities on dentin surfaces could initiate polymer-
ization of adhesive resin monomers and consequently form robust chemical bonding
at the adhesive/dentin interface. Compared with micromechanical bonding, chemical
bonding is much stronger, more stable, and durable. As a result, well improved inter-
face bonding strength was obtained (Fig. 17.8).
SEM images of the fracture surfaces generally showed that more composite
remained on plasma treated dentin surfaces when compared with the untreated con-
trols [33]. More specimens cohesively failed in the composite for plasma treated
specimens compared to the controls, except for the specimens prepared from 300 s
plasma treated dentin specimens. The control specimens had adhesive or mixed
failures more frequently than the plasma treated specimens. The large amount of
composite/adhesive observed on plasma treated dentin surfaces implies the dentin
adhesive interface is stronger than the bulk composite. These trends were also
observed with the test specimens that gave higher tensile strength. Plasma treated
226 Q.S. Yu et al.
specimens cohesively failed within the composite more frequently than the control
specimens which also implies a stronger interface. These experimental results
demonstrated that a proper plasma treatment of dentin could increase the bonding
strength of dental adhesives to dentin surfaces. It was also found that regional
variability and plasma modification duration affected the interface bonding strength
as measured using tensile test. An effective plasma treatment time was around 30 s
as observed from the mechanical testing data. This short plasma treatment is desired
in dental clinical applications because it will allow a quick dental composite restora-
tion. On the other hands, a prolonged plasma treatment could cause collagen fibers
to degrade and as a result lead to a weak interface.
17.4 Conclusions
Our experimental results showed that atmospheric plasma treatment was very
effective in rapid cleaning/disinfecting caries-causing oral bacteria. The results
obtained from this study showed the prospect of utilizing non-thermal plasma tech-
nology for dentin surface treatment to increase the chemical interactions of dentin
surfaces with dental adhesives, to introduce chemical bonding at the interface, and
thus to improve the interface bonding of composite restorations. It was demon-
strated that, under appropriate plasma conditions, plasma treatment of dentin could
increase the bonding strength of dental adhesives to dentin surfaces. The findings
from this research indicated that non-thermal atmospheric plasmas could be a
promising technique in dentistry for many clinical applications, e.g., oral bacterial
disinfection, caries early prevention, and improved composite restoration.
Acknowledgements The research work was supported in part by US National Science Foundation
(NSF) under contract of NSF-CBET-0730505 and US National Institute of Health (NIH) with
grant numbers of 1R43DE019041-01 and 2R44DE019041-02.
References
1. York AK, Arthur JS (1993) Reasons for placement and replacement of dental restorations in
the United States Navy Dental Corps. Oper Dent 18:203208
2. Mjor IA (1997) The reasons for replacement and the age of failed restorations in general dental
practice. Acta Odontol Scand 55:5863
3. Forss H, Widstrom E (2001) From amalgamto composite: selection of restorative materials
and restoration longevity in Finland. Acta Odontol Scand 59:5762
4. Lutz FU, Krejci I, Oddera M (1996) Advanced adhesive restorations: the post-amalgam age.
Pract Periodontics Aesthet Dent 8:385394
5. Collins CJ, Bryant RW, Hodge KLV (1998) A clinical evaluation of posterior composite resin
restorations: 8-year findings. J Dent 26:311317
6. Letzel H (1989) Survival rates and reasons for failure of posterior composite restorations in
multicentre clinical trial. J Dent 17:S10S17
7. Nordbo H, Leirskar J, von der Fehr FR (1993) Saucer-shaped cavity preparation for composite
resin restorations in class II carious lesions: three-year results. J Prosthet Dent 69:155159
8. Nordbo H, Leirskar J, von der Fehr FR (1998) Saucer-shaped cavity preparations for posterior
approximal resin composite restorations: observations up to 10 years. Quintessence Int
29:511
9. Wang Y, Spencer P (2005) Interfacial chemistry of class II composite restoration: structure
analysis. J Biomed Mater Res 75A:580587
10. Hannig M, Friedrichs C (2001) Comparative in vivo and in vitro investigation of interfacial
bond variability. Oper Dent 26:311
11. Davidson CL, DeGee AJ, Feilzer A (1984) The competition between the composite-dentin
bond strength and the polymerization contraction stress. J Dent Res 63:13961399
12. Wilson NHF, Burke FJT, Mjor IA (1997) Reasons for placement and replacement of restora-
tions of direct restorative materials by a selected group of practitioners in the United Kingdom.
Quintessence Int 28:245248
13. Friedl KH, Hiller KA, Schalz G (1995) Placement and replacement of composite restorations
in Germany. Oper Dent 20:3438
14. Griffiths BM, Naasan M, Sherriff M, Watson TF (1999) Variable polymerisation shrinkage and
the interfacial micropermeability of a dentin bonding system. J Adhes Dent 1:119131
15. Hilton TJ (2002) Can modern restorative procedures and materials reliably seal cavities? In
vitro investigations. Part 1. Am J Dent 15:198210
16. Brannstrom M (1984) Communication between the oral cavity and the dental pulp associated
with restorative treatment. Oper Dent 9:5768
17. Amaral CM, de Castro AKBB, Pimenta LAF, Ambrosano GMB (2002) Influence of resin
composite polymerization techniques on microleakage and microhardness. Quintessence Int
33:685689
18. Tam LE, Pilliar RM (1994) Effects of dentin surface treatments on the fracture toughness and
tensile bond strength of a dentin-composite adhesive interface. J Dent Res 73:15301538
19. Hashimoto M, Ohno H, Kaga M, Endo K, Sano H, Oguchi H (2000) In vivo degradation of
resin-dentin bonds in humans over 1 to 3 years. J Dent Res 79:13851391
20. Swift EJ Jr (2003) Dentin bonding. J Esthet Restor Dent 15:71
21. Burrow MF, Satoh M, Tagami J (1996) Dentin durability after three years using a dentin
bonding agent with and without priming. Dent Mater 12:302307
22. Duan YX, Huang C, Yu QS (2005) Low-temperature direct current glow discharges at
atmospheric pressure. IEEE Trans Plasma Sci 33:328329
23. Yu QS, Huang C, Hsieh F, Huff HE, Duan YX (2007) Bacterial inactivation using a low-
temperature atmospheric plasma brush sustained with argon gas. J Biomed Mater Res B Appl
Biomater 80:211219
24. Loesche WJ (1982) Dental caries, a treatable infection. Charles C. Thomas, Springfield. ISBN
0-3980-4767-7
25. Schaeken MJM, Van Der Hoeven JS, Franken HCM (1986) Comparative recovery of
Streptococcus mutans on five isolation media, including a new simple selective medium.
J Dent Res 65:906908
26. Keene HJ (1986) Sampling of cariogenic microorganism in human populations. Oral Microbiol
Immunol 1:712
27. Jay P (1938) Lactobacillus acidophilus and dental caries. Am J Public Health 28:759
28. Ljungh A, Wadstrom T (eds) (2009) Lactobacillus molecular biology: from genomics to
probiotics. Caister Academic Press, Norfolk. ISBN 978-1-904455-41-7
29. Goree J, Bin L, David D, Stoffels E (2006) Killing of S.mutans bacteria using a plasma needle
at atmospheric pressure. IEEE Trans Plasma Sci 34:13171324
30. Yang B, Chen JR, Yu QS, Li H, Lin MS, Mustapha A, Hong L, Wang Y (2011) Oral bacterial
deactivation using a low-temperature atmospheric argon plasma brush. J Dent 39:4856
31. Wang Y, Spencer P, Yao X (2006) Micro-Raman imaging analysis of monomer/mineral
distribution in intertubular region of adhesive/dentin interfaces. J Biomed Opt 11:024005-1
024005-7
228 Q.S. Yu et al.
32. Pashley DH, Sanoz H, Ciucchis B, Yoshiyamad M, Carvalhos RM (1995) Adhesion testing of
dentin bonding agents: a review. Dent Mater 11:117125
33. Ritts AC, Li H, Yu QS, Xu C, Yao X, Hong L, Wang Y (2010) Dentin surface treatment using
a non-thermal argon plasma brush for interfacial bonding improvement in composite restora-
tion. Eur J Oral Sci 118:510516
34. Brock TD (1998) Biology of microorganisms, 5th edn. Prentice Hall, Englewood Cliffs. ISBN
0-1307-6829-4
35. Sano H, Shono T, Sonoda H (1994) Relationship between surface area for adhesion and tensile
bond strength evaluation of a micro-tensile bond test. Dent Mater 10:236240
Part III
Plasma-Based UV Sterilization
Chapter 18
Features of the Sterilization by VUV/UV
Irradiation of Low-Pressure Discharge Plasma
Vyacheslav V. Tsiolko
Abstract The review is devoted to peculiarities of sterilization of items by VUV/UV

radiation of the discharge plasma both in case of the items immersed into the discharge
plasma (direct plasma treatment), and in case of flowing afterglow plasma (remote
plasma treatment). The issues of influence of such factors as UV irradiation
spectrum, substrate temperature on the UV sterilization efficiency are also considered.
For the first time, plasma use for inactivation of microorganisms was proposed by
Menashi in 1968 in patent [1]. Sterilization of internal surfaces of glass or plastic
containers was performed by means of pulsed corona discharge in argon at atmo-
spheric pressure. It has been shown that the sterilization of spores with surface
density up to 6 105 spores/cm2 is achieved in a time of less than 0.1 s. Subsequently,
this author together with Ashman proposed usage of RF low pressure discharge on
chlorine, bromine and iodine containing gases for the surface sterilization [2]. In
both patents, sterilized items were placed immediately in the region of the discharge
plasma generation. In patent [3], another approach to design of the device for
sterilization was used: items to be processed and location of the plasma generation
were spatially separated along dielectric working chamber, and the sterilization was
performed by flowing afterglow argon plasma from low pressure capacitive RF
discharge. Subsequent patents proposed usage of different device designs, discharge
types and plasma generating media for the plasma sterilization. In patent [4] it was
proposed to use the plasma excited by ultra-short laser beam for sterilization of
internal surfaces of containers. It has been also shown that the use of low-pressure
capacitive RF discharge plasma in oxygen provides sterilizations of B. subtilis
spores on the surface of packed instruments for about 60 min [5]. Plasma of
V.V. Tsiolko (*)

Department of Gas Electronics, Institute of Physics NAS of Ukraine,
Av. Nauki, 46, 03028 Kiev, Ukraine
e-mail: tsiolko@iop.kiev.ua
232 V.V. Tsiolko
low-pressure inductive RF discharge in a working mixture, which included aromatic,

heterocyclic and saturated or unsaturated acyclic aldehydes, was used for the
sterilization in patent [6]. The same discharge type on hydrogen peroxide vapor was
used for the sterilization in patent [7]. In patent [8], metal vacuum chamber of the
sterilizer contained perforated electrode along the chamber wall, and low-pressure
RF discharge on mixtures of O2 and CF4 was ignited between the chamber and the
electrode. Processed items were located inside perforated electrode, and thus
the sterilization was performed by flowing plasma from the discharge gap between
the chamber wall and perforated electrode. Low-pressure microwave discharge on
mixtures of nitrogen, oxygen, argon and helium was used in patents [9, 10]. The
plasma generator consisted of either several MW generator tubes located inside
sterilizing chamber [9], or a single MW plasma generator attached immediately to
top wall of the chamber. Sterilization of the items located in the chamber either
between generator tubes, or below single plasma generator, was performed by
flowing afterglow plasma. In mid-1990s, subsequent development of ideas stated in
patents [710] resulted in creating the sterilization systems based on RF (Advanced
Sterilization Products) and MW (AbTox Inc). plasma generators with use of plasma
generating medium represented by hydrogen peroxide vapor in the first system, and
mixture of hydrogen peroxide and peracetic acid vapors in the second one.
Analysis shows that till the middle of 1990s, all low-pressure plasma sterilization
systems were conditionally subdivided into several groups. By the design (indepen-
dently on types of plasma generating media and discharges), they are: (1) direct
plasma sterilizer, in which processed items are placed immediately in the discharge
plasma; (2) flowing afterglow plasma one, in which locations of placement of
processed items and discharge plasma generation are spatially separated. By the
type of plasma generating medium, they are: (1) true plasma sterilizer with the
medium represented by gases/gas mixtures which do not exhibit bactericidal features
under normal conditions (oxygen, nitrogen, argon, etc.); (2) plasma based steril-
izer with plasma generating medium represented by substances which exhibit
bactericidal features themselves (aldehydes, hydrogen peroxide, acids and so on).
In the present work, role of UV radiation of the plasma only in case of true plasma
sterilization systems will be considered for both direct plasma, and flowing after-
glow plasma treatments. It is motivated by fact that in case of true sterilizers, UV
radiation of the plasma can perform a determining (or, at least, an essential) role in the
sterilization process. And in case of plasma based sterilizers, the main effect in the
sterilization is contributed by both active species themselves, and the species formed in
processes of dissociation, excitation and ionization (see, for example [11]).
18.1 Survival Curves of Spores and Their VUV/UV

Sensitivity Short View and Comments
Prior to immediate discussion of peculiarities of sterilization by VUV/UV irradiation

of the plasma, let us mention briefly about the main tool which is most widely
used for investigation of the sterilization process. It is represented by survival
18 Features of the Sterilization by VUV/UV Irradiation of Low-Pressure 233
8
10
7 7 -2
10 10 cm
6 -2
Number of survivors
10
6 10 cm
5 -2
5 10 cm
10 4 -2
10 cm
4
10
3
10
2
10
1
10
0
10
0 200 400 600 800 1000
Sterilization time, s
Fig. 18.1 Survival curves for B. subtilis spores at sterilization by the hollow cathode discharge
plasma in air for test objects with different mean surface spore density values. Each point at the
graphs represents averaged results of counting for 612 test objects (24 individual sets). Power
density 0.01 W/cm3, P = 10.6 Pa (in part according to [16])
curves or dependencies of a number of survived microorganisms on either treatment

time, or fluence in cases of the use of flows of particles, ionizing and VUV/UV
irradiation for the sterilization (almost always presented on semi-log graphs). In
case of use of conventional sterilization methods (dry/wet heat, low molecular
weight reactive chemical sporicides), the shapes/types of experimentally obtained
survival curves are essentially different and may be conditionally designated as:
(1) shoulder, sigmoid, logarithmic, upward concavity, biphasic with tail
(Fig. 18.1 in [12]). The most typical ones are sigmoid, upward concavity and bipha-
sic with tail survival curves, at that the first two of those in first approximation may
be treated as variations of biphasic with tail one. In principle, tailing of the survival
curves up to now is a subject of discussions, and is explained by inactivation mecha-
nisms and resistance, as well as by the reasons independent of those mechanisms.
Particularly, in [13] it was shown that the spores obtained by isolating cells from
colonies of the tail section were more heat resistant than the original ones (D104c = 0.15
and 1.9 min for spores from the original population and from the tail, respectively).
That is, the shape of survival curves (particularly, tailing) may be also due to the
presence of subpopulations of spores with different resistances. In case of plasma
sterilization, interpretation of behavior of the survival curves is even more compli-
cated due to fact that in this case the microorganisms are affected by several steril-
izing factors at once: VUV/UV irradiation, electrically neutral active species and
ions produced by the discharge plasma. Besides, the plasma method is principally
surface one, and in many cases, when the spores at the surface of processed items
are stacked and mixed with biological residues, their sterilization will occur in
layer-wise manner due to small penetrability of above plasma sterilizing factors.
While the main mechanism of spore destruction or deactivation by VUV/UV irra-
diation is considered to be DNA damage, the action of active species is associated,
first of all, with spore etching and erosion. The inactivation rates of the two
mechanisms are markedly different, which results, by opinion of the authors of [14],
234 V.V. Tsiolko
in three-phase survival curves. While at the first phase inactivation rate is determined
by DNA damage of the upper spore layer by VUV/UV irradiation, kinetics of the
second phase is determined by slower erosion processes (due to both etching by the
active plasma species, and VUV/UV photodesorption). The third phase happens
only when VUV/UV irradiation gets an access to the lower spore layers due to
removal of the upper ones. It is obvious that the situation, which occurs in actual
plasma sterilization, is essentially more complicated. Particularly, as it was shown
in [15], ion flows from the plasma may essentially accelerate spore erosion caused
by electrically neutral active plasma species. Thus, in general case, following from
common survival curves it is very difficult to separate influence of particular
plasma factors on the sterilization efficiency. Influence of the surface density of
B. subtilis spores at the test objects on a behavior of survival curves is demonstrated
in Fig. 18.1 (in part after [16]).
In these experiments, metal Petri dishes with bottom surface of 10 cm2 were
used as test objects. During the preparation of the test objects, Petri dishes were
at first treated in low pressure argon discharge plasma for surface wettability
improvement, and then the spore water suspension was deposited homogeneously
onto the dishes bottom (Initial population of the spores was varied in a range
105109). Quality of deposited spore layers at the surface of Petri dishes was mon-
itored by means of scanning electron microscope (SEM). One can see from
Fig. 18.1 that in the range of initial mean surface spores concentration 104106
spores/cm2, survival curves posses practically the same behavior (especially in the
second phase). As it was shown in [1719], in this case the main role is performed
by VUV/UV plasma irradiation. But increase of the spore concentration up to 107
spores/cm2 leads to drastic alteration number of survived spores at the same
sterilization times grows by more than one order of magnitude. Results of SEM
investigation of the test objects, in turn, have shown that, whereas at variation of
initial spore surface density from 104 to 106 spores/cm2, the spores are located in
a single layer practically everywhere at the test objects, then subsequent increase
of the spore surface density up to 107 spores/cm2 leads to clumping and occlusion
of the spores at the test objects surface. Thus, it is obvious that growth of the
number of survived spores at the increase of their surface density up to 107 spores/
cm2 is due to diminishing of VUV/UV irradiation action caused by its absorption
by upper layers of stacked spores. Due to that, in this case the spore inactivation
efficacy will be determined not only by VUV/UV irradiation action, but by slower
erosion processes as well.
Behavior of the spore survival curves at small initial surface density of the spores
(104106 spores/cm2) at the test objects also enables making a conclusion about the
origin of tailing of the spore survival curves. Since the experiments described above
were accomplished with the same strain of B. subtilis spores, and main role in the
inactivation is performed only by VUV/UV irradiation (that is, only one inactiva-
tion mechanism works in this case), then the most probable reason of biphase
character of the survival curves may be due to presence of two subpopulations of
spores with different resistances (as in case described in [13]).
Fig. 18.2 Dependencies of

inactivation rate constant 80 UVP
R (1020 m2) for B. subtilis UVR
spores strains UVP, UVR RCE
DNA
K*100, cm1; R, a.u.

and RCE (After [20]) and 60
extinction coefficient K of
DNA (After [21]) vs VUV/
UV wavelength
40
20
0
40 80 120 160 200 240 280
Wavelength, nm
As to separation of VUV/UV irradiation contribution to overall inactivation

ability of the plasma, the most typical approach is the use of optical filters which
prevent coming of active species and ions onto used test objects. Use of the filters
with different cut-off wavelengths also allows estimating the most efficient wave-
length ranges of VUV/UV irradiation. At conducting such experiments we must be
also sure that the method of preparation of the test objects should provide controlled
single-layer deposition of the spores onto their surface. Otherwise, absorption of
UV radiation by the upper spore layers introduces difficulties in correct comparison
of the results obtained with different plasma parameters (type of gas mixture and its
pressure, etc.) and, consequently with different spectrum shape of VUV/UV
irradiation.
At determining the most efficient wavelength ranges of VUV/UV irradiation,
one should pay attention to one more important aspect. As it is known, inactiva-
tion efficacy of VUV/UV irradiation action on the microorganisms essentially
depends on wavelength of this radiation, at that behavior of this dependence for
different types/strains of the microorganisms may be considerably different.
Particularly, in [20] with the use of quasi-monochromatic synchrotron radiation it
has been shown that inactivation action spectra in wavelengths range 50300 nm
for two types of spores B. subtilis (UVR, UVS and UVP; RCE and RCF), differ-
ing in repair and/or recombination capabilities, essentially distinguish from each
others. Inactivation action spectra for typical representatives of these two groups
of B. subtilis spore strains UVR, UVP (deficient in removal mechanism of spore
photoproducts) and RCE (recombination deficient) are presented on Fig. 18.2.
(The figure also exhibits, for comparison, spectrum dependence of DNA extinc-
tion coefficient K).
One can see from the figure that in VUV range about 125175 nm recombination
deficient B. subtilis spores RCE were much more sensitive than the spore strains
deficient in removal mechanism of spore photoproduction. Another peculiarity is
insensitivity of all spore strains at 190 nm and in VUV range 50120 nm, in spite
236 V.V. Tsiolko
of fact that DNA extinction coefficient reaches its maximum values. In general, the
authors state that in each particular case the behavior of inactivation action spectrum
is determined by three factors: absorption of the VUV/UV radiation by outer layers
of the spores, the efficiency of DNA mortal damage production, and peculiarities of
repair mechanisms for each strain of the spores.
In turn, shape of VUV/UV irradiation spectrum of plasma in wavelength range
responsible for inactivation (that is, below 300 nm) is also a complicated func-
tion of several parameters: type of gas or gas mixture, the pressure, and the type
of plasma used for inactivation direct or flowing afterglow one. Usage of
gas with known electron transitions gives us principal possibility to obtain
required radiation spectrum shape. Particularly, when using pure gases/vapors
one can expect that: (1) in oxygen or water vapor, main contribution to VUV/UV
radiation of the plasma occurs due to emission of bands of the second negative
system (SNS) O2+ (A2Pu - X2Pg) (194300 nm) and Schumann-Runge system
(SRS) O2 (B3Su - X3Sg), ((244300 nm), (2) in nitrogen the radiation is repre-
sented by bands of Lyman-Birge-Hopfield system N2, (a1Pg - X1Sg+) (140
260 nm, with radiation intensity maximum in VUV branch) and
Lyman-Birge-Hopfield system N2, a1Pg - X1Sg+) (170200 nm), (3) in hydrogen
or NH3 radiation of H2 continuum in wavelength range 170300 nm (with max-
imum at about 200 nm) occurs. However, the situation is complicated at the use
of gas mixtures. Particularly, in case of N2 and O2 mixture, the main contribution
to UV radiation is provided by the bands of g (A2+ X2) and/or b (B2P X2P )
systems NO, at that their relative contribution to overall UV irradiation depends
both on concentration ratio of gas mixture components, as well as on nature of
the plasma which generates the species (direct or flowing afterglow). (It
should be noted that any pure gas actually contains admixture of ambient air
coming from the walls of gas feeding system and chamber. As well see below,
this admixture can also influence VUV/UV radiation spectrum). Such difference
of VUV/UV radiation spectrum of gas mixture plasma from spectra produced by
plasma of the mixture components is explained by fact that in case of gas mixture
essential changes are possessed by component content of the plasma species,
electron energy distribution function (which determines excitation efficacy of
molecule electron levels), and in addition to that, new channels of generation of
VUV/UV quanta occur.
In any case, one can state that spectrum distribution of VUV/UV plasma radia-
tion in a range below 300 nm is very uneven. Since there is a difference in inactiva-
tion action provided by the plasma irradiation at each particular wavelength, in
comparison of inactivation efficacy for different VUV/UV spectrum shapes one
should take into consideration germicidal portion of the emission [22, 23]. In other
words, one should weigh inactivation action at each wavelength. Due to that,
actual, that is weighed inactivation fluence rate F for each type of the spectrum is
presented by the equation
I (l )G (l )dl ,
300
F=
l min
where I(l) is spectrum distribution of VUV/UV radiation, G(l) is inactivation rate

for particular microorganism type, usually normalized by its value at 254 nm. One
can find here certain difficulty due to a lack of data on inactivation action spectrum
for each particular microorganism. That is why, as an alternative to action spectra,
in many cases the absorbance spectrum of DNA is used as a surrogate. In spite of
drawbacks of such approach, the DNA spectrum approximates the action spectra of
many microorganisms in the range 200300 nm with good enough precision, and
may be considered as acceptable alternative.
It should be also noted that, due to non-linear nature of dependencies of the num-
ber of survived spore on time/fluence of UV irradiation, correct determination of the
sterilization efficiency dependence on the discharge plasma parameters is possible
only by comparison of complete survival curves. The efficiency determination
with the use of separate points from these curves may lead to wrong conclusions
18.2 The Most Efficient VUV/UV Radiation: Flowing

Afterglow and Direct Plasma Cases
Prior to proceeding with particular results of the researches, let us consider briefly
principal difference of features of flowing afterglow and direct plasma from the
viewpoint of generating VUV/UV radiation.
Generation of radiation in the plasma may occur by two ways: either due to a
direct excitation of certain electron level of plasma particle by the electron hit, or
at interaction of species formed in result of processes of dissociation and excitation
of gas medium particles. Since both in the first and the second case, the primary
role is performed by plasma electrons, efficiency of generation of VUV/UV radia-
tion is in any case determined by an appearance of plasma electron energy distribu-
tion function, and is mainly defined by the quantity of fast electrons with energies
exceeding thresholds of dissociation, excitation and ionization. In case of flowing
afterglow plasma, with moving away from the generation zone plasma, electrons
are cooled by collisions with ambient gas molecules (that is, quantity of fast
electrons decreases), which results in the decrease of generation efficacy of UV
(first of all, VUV) quanta. Besides, a component contents of the plasma species
also changes, which influences the appearance of UV radiation spectrum. Obviously,
the actual behavior is somewhat more complicated due to a big quantity of elemen-
tary processes occurring in the plasma. On the other side, when the plasma moves
away from the location of its generation, the temperature of electrically neutral
plasma species (which defines the temperature of processed items) also decreases
due to collisions. Thus one can see that the use of direct plasma for inactivation
enables more efficient VUV/UV irradiation, but at the same time, probability of
overheating processed items exists. And in flowing afterglow plasma, the tempera-
ture of the items is lower, but the irradiation efficacy decreases both due to the
lower intensity of UV radiation as a whole, and the depletion of VUV radiation
spectrum range.
238 V.V. Tsiolko
Although pioneer patents devoted to the plasma sterilization appeared a long

ago, the initial detailed studies of its mechanisms started only at late 1990s. It was
due, first of all, to two reasons: increase of quantity of expensive multiple-use items
made of thermo-labile materials used in medical practice, and approval of laws
limiting the use of EtO (12/88) sterilizers. In [1719], hollow cathode glow DC
discharge was used for direct plasma sterilization. Choice of that particular dis-
charge type as the plasma generator was due to several reasons: first, such discharge
plasma was relatively weakly perturbed by sterilized items placed into it; second,
use of such discharge enabled creation of uniform plasma with the density 1010 cm3
in a volume of several tens liters just with specific power in the discharge 30
40 mW/cm3; third, the presence of a large quantity of fast plasma electrons enabled
efficient generation of UV radiation. In the mentioned proceedings it has been
shown that the most efficient plasma generating gas with respect to the sterilization
of B. subtilis spores is oxygen, subsequently followed by air, carbon dioxide, hydro-
gen, argon, nitrogen. Experiments with the use of optical filters have shown that the
main role in sterilization of open surfaces is performed by UV irradiation from the
plasma. It has been also determined that charged plasma species (including ions
with energy of 500600 eV) do not play essential role in the sterilization process.
All those investigations were carried out with the use of test objects with surface
spore density of 106 spores/cm2 and less.
In [14, 2428], flowing afterglow plasma from MW discharge on Ar, Ar - O2 and
N2 - O2 mixtures were used for the sterilization. By indirect experiments it was
determined that in case of N2 - O2 mixture, the main role in the sterilization is per-
formed by UV radiation of NOb system in 250320 nm range originated in the
afterglow plasma. Essence of these experiments consisted in the measurements of
dependencies of concentration of oxygen atoms in the plasma and radiation inten-
sity of NOb bands on O2 content in the mixture. Since the dependencies of inactiva-
tion rate of B. subtilis spores and UV radiation intensity on oxygen content in the
mixture were in a good correlation with each other, the conclusion was made about
dominating role of UV radiation in the sterilization process. Confirmation of this
conclusion is also given by fact that SEM photographs of the treated spores have
shown that spore erosion at O2 concentration of 0.7% (total sterilization) was practi-
cally absent. At the same time, at 10% O2 concentration (maximum concentration
of O atoms), the sterilization was not achieved, although the spores were heavily
damaged.
The reason of such behavior of NO(B) concentration, which is responsible for
generation of UV radiation of NOb system in afterglow flowing MW discharge on
the mixture of oxygen and nitrogen, was explained in [29]. It has been shown that
the main mechanism of NO(B) state generation is the reaction N + O + M
NO(B) + M, (M = N2, O2), whereas its loss is defined by radiative decay and quench-
ing by N2 and NO(X) (at O2 concentration values above 5% first of all by NO(X)).
In result of competition of these processes of generation and loss, NO(B) state
concentration reaches maximum at oxygen content in the mixture 0.72%.
A state of investigations on plasma inactivation of microorganisms for early 2000
is presented in more details in review [27].
Although a number of proceedings devoted to studies of the plasma sterilization

during past decade is big enough, results of a majority of them either simply dem-
onstrate a fact of inactivation action of the plasma, or repeat already known facts.
Due to that, let us consider only the most interesting works dealing with the role of
VUV/UV irradiation in the inactivation of microorganisms.
In [30], a comparison of spectrum sensitivity of B. subtilis and Aspergillus niger
spores at their inactivation by VUV/UV NH3 microwave direct plasma radiation
was performed. (As it was already noted above, in such plasma, radiation of H2
broad continuum is generated in a range 170300 nm with maximum at about
200 nm). PET foil with deposited 106 spores was used as test object. The spores
mean surface density was about 4 104 spores/cm2, which enabled a monolayer
distribution of the spores on the foil surface. For separation of separate ranges from
broad VUV/UV radiation spectrum, optical filters with different lcut-off 307, 275,
233, 164 and 125 nm were used. With the use of these filters survival curves for each
spore type were obtained with the same discharge parameters. Comparison of sur-
vival curves for the B. subtilis spores have shown that inactivation process starts
with the use of filter with lcut-off = 275 nm, and is abruptly enhanced on adding light
at wavelengths down to 233 nm. Under filter with lcut-off = 164 and 125 nm plasma
action effect is about the same and corresponds to inactivation efficacy provided by
the plasma without use of any filter. From such behavior of survival curves conclu-
sion was made that VUV/UV NH3 plasma radiation in the range from 307 to 164 nm
is the dominating mechanism for the inactivation of B. subtilis spores. In case of
Aspergillus niger spores, the situation was somewhat different an analysis of
obtained survival curves have shown that the main role in the inactivation is
performed by irradiation in the range between 125 and 233 nm. Thus we can see
that the sensitivity curve for Aspergillus niger spores is shifted toward shorter
wavelength values, as compared with B. subtilis spores. Accordingly to the opinion
of the authors, such difference in the spectrum sensitivity of the spores is probably
due to pigmented cell wall of A. niger spores. In other words, in case of Aspergillus
niger spores, UV irradiation is absorbed more efficiently by the outer layers of the
spore shell, and does not reach DNA.
The works [31, 32] were devoted to identification of the most efficient UV
plasma irradiation for B. atrophaeus spores (ATCC 51189) inactivation with the
use of double ICP on Ar, N2 and O2 mixture. (This spore strain is one of pigmented
variants of B. subtilis). Spores on the test samples with surface density of about
106 spores/cm2 formed a monolayer structure. For separation of particular spectrum
regions, MgF2, M235 and SiO2 optical filters with cut-off wavelengths 112, 235
and 300 nm, respectively, were used. For optical diagnostics in the most interesting
for us wavelength range from 110 to 300 nm, absolutely calibrated spectrometer
with spectral resolution 0.35 nm was used. Radiation spectrum of the plasma for
Ar:N2:O2 mixture (100:4:1 sccm) is presented in Fig. 18.3. For l < 210 nm UV
radiation originated from Lyman-Birge-Hopfield system N2, (a1Pg X1Sg+), and
for l = 200275 nm from NO (A2+ X2 ). Results of the experiments on ster-
ilization of the spores without filters (that is, by joint action of both active species,
and UV irradiation), as well as those with the use of mentioned above filters, are
240 V.V. Tsiolko
Fig. 18.3 Spectral density

of VUV/UV emission from
MW discharge plasma for
Ar:N2:O2 (100:4:1 sccm),
P = 10 Pa (After [32])
shown in Fig. 18.4. (R is a logarithm of ratio of initial number of the spores N0 = 106
to that of survived ones Ns).
As one can see from Fig. 18.4, the main role in the sterilization of B. atrophaeus
spores is performed by UV radiation in the wavelength range l 235300 nm,
whereas the influence of VUV radiation is less important. Although B. atrophaeus
is one of subspecies of B. subtilis, the obtained results are in certain contradiction
with the data of [30] where VUV irradiation performed dominating role in the inac-
tivation. Possible reasons for such discrepancy may be due to both the difference in
spectrum sensitivity of these spores, and different spectrum shapes of VUV/UV
irradiation broad continuum of H2 radiation in [30] and band-shaped radiation
spectrum of N2 and NO molecules in [31, 32], and probably to combination of these
factors.
The high frequency (200400 MHz) discharge on argon was used in [33]
for study of UV radiation role in the process of direct plasma sterilization of
B. atrophaeus (ATCC 9372) spores. (As well as in the works discussed above,
spores at the used test objects possessed monolayer arrangement). In that work, fil-
ters made of MgF2, SiO2 and Pyrex (lcut-off = 112, 190 and 330 nm respectively) were
used. Results of the experiments are given in Fig. 18.5.
One can see from the figure that sterilization of the spores is mainly performed
by VUV radiation in wavelength range 112190 nm. Spectrum identification for
VUV radiation in the range of 112180 nm was not accomplished, and its integral
intensity was measured by means of photomultiplier. Since cut-off wavelength of
filter is defined as the wavelength with 50% transmission, radiation intensity in
VUV range may be due to emission of argon resonance lines located near 105 and
107 nm. However, due to fact that experimental conditions did not allow direct
measurements of intensities of these lines, another approach was used. By the
method of absorption spectroscopy, the dependence of relative population density
of the metastable level 3P2 on argon pressure was determined. Since the authors had
to determine relative variations of the population density, rather than absolute ones,
Fig. 18.4 Sterilization

efficiency R for B. atrophaeus 6
without filter
spores depending on cut-off
wavelength at different 5
wavelengths for Ar:N2:O2
(100:4:1 sccm), P = 10 Pa. 4
Treatment time 60 s MgF2=112 nm M235=235 nm
R
(After [32]) 3
2 SiO2=300 nm
1
0
0 100 200 300
cut-off / nm
Fig. 18.5 Number of

survivors spores after 180 s
Ar plasma treatment with no
filter and with three high-pass
filters. P = 750 mTorr
(After [33])
and respective populations of resonant and metastable levels of the 3p54s orbital
configuration are of the same order and vary similarly to the functions of the opera-
tion conditions, such substitution was justified. It has been also shown that, if the
sterilization efficiency increases along with argon pressure growth, then relative
population density of the metastable level 3P2 (and, respectively, radiation intensity
of lines at 105 and 107 nm) decreases. At the same time, integral radiation intensity
in the range of 112180 nm increases with the pressure growth. Thus, it is clear that
radiation of argon resonance lines at 105 and 107 nm does not contribute to spore
inactivation. Simultaneous measurements of radiation spectrum in the range of
200400 nm have indicated presence of radiation of NOg system, the 306.4 nm sys-
tem of OH and the 2nd N2 positive system, which gives evidence to presence of
admixtures (residual air, water vapor) in argon. Due to that, the authors have assumed
that the main contribution to VUV radiation in the range of 112190 nm is done by
242 V.V. Tsiolko
radiation of these admixtures. Indirect confirmation of this assumption follows from

the fact that VUV radiation intensity increases with argon pressure growth. (This
growth results in the increase of absolute values of concentrations of the admixtures
and, consequently, in the increase of the radiation intensity provided by these admix-
tures). By opinion of the authors, the fact that sterility is reached in 180 s without
filter, while about 30 spores still remain alive in the case of MgF2 filter, may be due
to the change of experimental conditions. In absence of the filter, the plasma is
generated in an immediate proximity of the spores, which may result in the increase
of VUV fluence coming to them.
Although spectrum identification for VUV radiation was not done in that work,
following from the presence of radiation of NOg and the 2nd N2 positive systems in
the range of 200400 nm, one can assume that in the range of 112180 nm, as well
as in [31, 32], UV radiation is originated from Lyman-Birge-Hopfield system N2,
(a1Pg X1Sg+). And the difference in the most efficient ranges of UV irradiation
(although the same kind of bacteria was used) may be due to low intensity of plasma
radiation in the range of 200300 nm in [33]. Unfortunately, it is difficult to deter-
mine more exactly the reason for such mismatch because it is not possible to make
correct comparison of distributions of VUV/UV irradiation intensities obtained in
[3133].
Experiments on the sterilization by UV radiation of the plasma described in [34,
35] were accomplished at setup somewhat different from conventional ones. While
in conventional low-pressure plasma setups used for the sterilization the discharge
plasma and processed BIs are located in the same vacuum chamber, in mentioned
case BIs (quartz Petri dishes with E. coli suspension) were located in ambient air,
and were treated by UV radiation from the plasma generated by low pressure hollow
cathode discharge through quartz window with lcut-off 175 nm. However, this dif-
ference is not principal since the main purpose of the work consisted in researching
the influence of UV radiation spectrum shape on its sterilizing features. Due to fact
that experimental studies were performed with the use of UV radiation with essen-
tially different spectrum shape, in those papers the method of determining effective
irradiation dose for the studied sample was used, which enabled correct comparison
of the results obtained with the use of the mentioned UV sources. Essence of the
method consisted in weighing spectrum distributions of intensity absolute values
for each used type of UV radiation. The weighing curve was obtained by multi-
plying DNA absorption spectra and transmission curve of BaF2 filter with lcut-
off
= 215 nm, normalized by 1 at l = 254 nm. (As it was found in preliminary
experiments, UV radiation with wavelength l 215 nm did not provide essential
inactivation effect). The experiments were performed with the use of UV radiation
of hollow cathode discharge plasma on air, deuterium, mixtures of deuterium with
oxygen, oxygen and water vapor. Simultaneously, the experiments with the use of
monochromatic (l = 254 nm) UV radiation of DB-30 low-pressure (LP) mercury
lamp and broadband UV radiation of PRK-400 middle-pressure (MP) lamp were
performed. The survival curves for all these cases are presented in Fig. 18.6. One
can see from the figure that bacteria survival curves obtained at the use of plasma
UV radiation from discharges on oxygen, water vapor, and mixtures of deuterium
Fig. 18.6 Survival curves

obtained at treatment of
E. coli water suspension
density by UV radiation of
LP and MP mercury lamps
and radiation of hollow
cathode discharge (HCD) on
air, oxygen, water vapor and
mixtures of deuterium with
oxygen (After [34, 35])
with oxygen practically coincide with each other. Curves obtained at the use of UV
radiation of mercury lamps and that of the discharge plasma on air are also close to
each other, however, they are located above the first set of the curves. Such essential
difference in behavior of the curves gives an undoubted evidence to the fact that
bactericidal features of UV radiation in 215300 nm wavelength range depend not
only on radiation dose absorbed by DNA (as it was observed in the case of mono-
chromatic radiation), but on shape of the radiation spectrum as well. Comparison of
spectra of radiation absorbed by DNA for the discharges on different gases shows
that maximum inactivation efficiency is provided by the discharges plasma with UV
radiation having maximum flux rate in 215230 nm range.
To determine the fact whether high inactivation efficacy by UV radiation with
maximum fluence rate in 215230 nm range is specific only for vegetative form of
microorganisms, or has more general character, authors of [30, 31] have done veri-
fication of this effect for the case of spores B. subtilis received from Scientific
Research Institute of Standardization and Control of Medical Biological Preparations
(Moscow, Russia). The experiments were performed at the setup, which was
described in details in [17]. Measurements of spectrum dependencies of the plasma
UV radiation in wavelength range of 200300 nm on the discharge glow time tg
were performed by means of spectrometer SL40-2-2048USB (SOLAR TII, Ltd)
with the spectral resolution 0.2 nm. At the measurements, the end of quartz wave-
guide of the spectrometer was located in a plane, which corresponded to placement
of Petri dishes with B. subtilis spores during medical-biological researches. The
discharge parameters were the same as in the case of inactivation of these spores in
[1719]: pressure of oxygen and ambient air was varied in a range of 416 Pa,
specific power in the discharge Wd in a range of 0.0025-0.0125 W/cm3. For correct
comparison of inactivation results with the use of UV radiation with different
spectrum shape, weighing of spectrum distributions of the intensity of UV radia-
tion from oxygen and air plasma was performed. Inactivation action spectra for
B. subtilis spores type RCF from [21] was used as weighing function. (It should
244 V.V. Tsiolko
a b
60 240
"Weighed" UV fluence rate Ew, a.u.
"Weighed" UV fluence rate, a.u.

50 200
40 160
30 120
20 80
10 40
0 0
200 220 240 260 280 300 200 220 240 260 280 300
Wavelength, nm Wavelength, nm
Fig. 18.7 Spectral distributions of weighed fluence rate Ew of UV radiation from oxygen (a) and
air (b) discharge plasma. Pressure P = 15 Pa, Wd = 0.08 W/cm3
be noted that the use of the inactivation spectra, obtained in [21] for other spore
types, as weighing functions results in values of weighed UV fluence rate Ew
and fluence Fw of the radiation, which differ by no more than 2025%, as compared
to the values in case of RCF weighing function use). Figure 18.7a and b shows the
weighed spectrum fluence rate Ew distributions of UV radiation from oxygen and
air plasma.
As one can see from the analysis of these spectra, at oxygen use the main
contribution to UV radiation of the plasma occurs due to the emission of the second
negative system O2+ (A2Pu X2Pg) and Schumann-Runge system O2 (B3Su X3Sg),
and at the use of ambient air and nitrogen due to the emission of g system NO (A2S+
X2P). For both cases fluence Fw of UV radiation practically linearly grows with tg
and Fw value for the discharge plasma in ambient air considerably exceeds UV
fluence for the discharges in oxygen in the whole range of tg variation. The experi-
ments have also shown that at all gas pressures, the values UV fluence value grows
up linearly with the increase of discharge power Wd. In Fig. 18.8 the numbers of
survivors B. subtilis spores vs UV weighed fluence Fw from discharge on oxygen
and ambient air plasma are presented.
These survival curves were obtained by converting dependencies of the number
of survivors spores on the sterilization time ts (see insert in Fig. 18.8) with the use
of respective dependencies of fluence Fw on tg. One can see from Fig. 18.8 that
B. subtilis spores sterilization in case of use of UV irradiation from oxygen plasma
is reached at fluence Fw approximately five times less than in case of air plasma.
The observed large difference in the behavior of survival curves for inactivation
of the spores by UV from oxygen plasma on one side, and from air one on another
side, unambiguously shows that bactericidal features of UV radiation in 200300 nm
range depend not only on the UV fluence, but also on the shape of radiation spec-
trum. And this effect is possibly inherent not only to the types of the microorgan-
isms described above. High efficiency of UV radiation having maximum in
wavelength range 200240 nm may be presumably due to: (1) difference in nature
of DNA damage caused by UV radiation in the mentioned wavelength range, as
7
10 10
7
6
6 10
10
Number of survivors
5
10
4
5 10
10
Number of survivors
3
10
2
4 10
10 1
10
0
3 10
10 a b
-1
10
2 0 100 200 300 400
10 Inactivation time ti, s
1
10
0
10
a b
-1
10
0,0 0,1 0,2 0,3 0,4 0,5
"Weighed" UV fluence Fw, a.u.
Fig. 18.8 Dependencies of the number of B. subtilis survivors on weighed UV fluence Fw at

treatment of the spores by UV radiation of discharge in oxygen (a) and air (b). Each point at the
graphs represents averaged results of counting for 36 test objects (12 individual sets)
compared to that occurring at the use of radiation with other wavelengths;

(2) stronger damage caused by radiation in the mentioned wavelength range to other
biological molecules, particularly enzymes which are responsible for reparation of
the damaged DNA; (3) synergetic effect of simultaneous action of UV quanta having
broad energy spectrum in this wavelength range on the DNA.
18.3 Heat and UV Radiation in Plasma: Synergy Effect

on Spore Inactivation
Plasma sterilization efficiency enhancement with the increase of temperature of

processed items was already noted in [24, 36]. In [36], it has been determined that
inactivation rate of B. atrophaeus spores, directly exposed to CO2 discharge plasma,
is strongly non-monotonically depending on the substrate temperature. But there
are no mentions about the possible mechanisms of this effect. In [26], the observed
increase efficiency of B. atrophaeus spore inactivation by oxygen plasma at the
increase of the substrate temperature from 15C to 50C was attributed to a higher
etching rate of the spores at higher temperature. After that, for more than almost
10 years practically none of researchers paid attention to this effect, and it was
unknown which of possible inactivation mechanisms (UV or active species) is
responsible for the discovered inactivation growth with temperature increase.
246 V.V. Tsiolko
And only in [37], the effect of temperature of Petri dish with B. atrophaeus spores
on the efficiency of their inactivation by UV radiation (NOb system) from flowing
afterglow MW N2-O2 discharge plasma was studied in detail. During the experi-
ments, the Petri dish was tightly closed by CaF2 optical filter (lcut-off = 112 nm) to
prevent plasma active species entering. The temperature of the Petri dish could be
changed from 4C to 80C. It has been shown that inactivation rate increases mono-
tonically with the temperature of Petri dish only in the case when heat and UV
radiation are applied simultaneously. In the cases when Petri dishes were heated
before or after their exposure to plasma afterglow at low temperature, synergetic
effect was not observed. Authors explain the growth of UV spores inactivation rate
with the temperature increase by fact that the heat provides the energy required to
surmount the potential barrier(s) encountered as the chemical reaction, initiated by
photon excitation. The energy barrier corresponds to molecular (conformation)
rearrangements occurring after photoexcitation, as the reaction develops to reach
the final state creating the lethal damage to the spore DNA strands.
In a majority of accomplished researches on VUV/UV plasma irradiation inacti-
vation, the temperature of the test objects was not stabilized and grew up with expo-
sure time. Let us consider qualitatively a character of the discovered effect influence
in case of investigating VUV/UV inactivation of the spores by radiation with differ-
ent spectrum distribution of the irradiation fluence rate. From insert in Fig. 18.8 one
can see that the sterilization by UV irradiation of air plasma is achieved about 3 min
later than that by UV irradiation of O2 plasma. Temperature of exposed to plasma
Petri dishes grows up practically linearly in time with a rate of about 1.5C/min.
That is, at reaching sterilization, temperature values of the test objects differed very
slightly no more than by 45C, whereas in [37] considerable difference in a slope
of survival curves was observed at temperature difference of 2030C. Certain
increase of the inactivation efficacy in case of air plasma use could result just in
moving air survival curve toward oxygen one. That is, it would just somewhat
decrease influence of the difference in spectrum distributions of UV fluence rate of
oxygen and air plasmas on the inactivation efficacy, without influence on the
effect as a whole.
18.4 Conclusions
Although during the past decade intense researches of low-pressure plasma UVU/
UV microbial inactivation were performed, their results cause contradictory feeling.
On one side, many researches were accomplished on the generation of UVU/UV
radiation of different discharge plasmas (DC, RF, HF and MW types) in various gas
mixtures at the different pressure, and dependencies of VUV/UV inactivation effi-
cacy on radiation spectrum shape and other parameters were determined for differ-
ent types of microorganisms (vegetative, fungi, spores). On the other side, conditions
of particular researches differ considerably, which makes correct quantitative com-
parison of the obtained results practically impossible.
For solving the problem, one should standardize the conditions of conducting the
researches. Particularly, definite protocol is required for determining VUV/UV
spectral distribution of fluence rate and VUV/UV fluence given to the microorgan-
isms. (At present, published articles present only spectrum distributions of UVU/
UV radiation intensity in arbitrary units, usually measured at random points of the
space). Obviously, this task is more difficult than that for the case of determination
of the fluence in an apparatus containing monochromatic or broadband UV lamps
[23], but the efforts on development of such protocol will definitely justify them-
selves in the future.
Used test objects with the microorganisms should be also standardized. Variety of
used test objects (polymer foils, glass plates, glass and polymer Petri dishes, etc.)
may also cause difficulties in comparison of the results of different works. Particularly,
at the same discharge plasma parameters, VUV/UV fluence value will be different
for plane test objects and Petri dishes due to the difference in acceptance angle of the
irradiation. Use of test objects with different mass, made of materials with different
heat capacity may also have an effect on the results of the researches due to influence
of the object temperature on VUV/UV inactivation efficacy [37].
Errors in determining efficacy of the plasma VUV/UV inactivation may occur
when separate points of the survival curves are used instead of the complete curves,
due to nonlinearity of dependencies of the number of survived microorganisms on
VUV/UV irradiation fluence.
As a whole, one can say that taking these wishes into consideration at accom-
plishing the researches may not only clarify understanding the processes during
VUV/UV inactivation, but as well promote the use of this method in medical
practice.
Acknowledgments The author would like to offer thanks to Dr. V. Yu. Bazhenov for helpful
discussions and assistance, Dr. Z. Machala for the encouragement and critical reading of the
manuscript.
References
1. Menashi WP (1968) Treatment of surfaces. US Patent 3,383,163

2. Ashman LE, Menashi WP (1972) Treatment of surfaces with low-pressure plasmas. US Patent
3,701,628
3. Fraser SJ, Giletter RB, Olson RL (1974) Sterilization and packaging process utilizing gas
plasma. US Patent 3,851,436
4. Tensmeyer LG (1976) Method of killing microorganisms in the inside of container utilizing a
laser beam induced plasma. US Patent 3,955,921
5. Bithell RM (1982) Package and sterilizing process for same. US Patent 4,321,232
6. Gut Boucher RM (1982) Seeded gas plasma sterilization method. US Patent 4,207,286
7. Jacobs PT, Lin S-M (1987) Hydrogen peroxide plasma sterilization system. US Patent
4,643,876
8. Jacobs A (1989) Process and apparatus for dry sterilization of medical devices and materials.
US Patent 4,801,427
248 V.V. Tsiolko
9. Campbell BA, Moulton A (1992) Plasma sterilizer and method. US Patent 5,115,166
10. Campbell BA (1993) Circular waveguide plasma microwave sterilizer apparatus. US Patent
5,184,046
11. Soloshenko IA, Tsiolko VV, Khomich VA, Bazhenov VYu, Ryabtsev AV, Schedrin AI, Mikhno
IL (2002) Features of the sterilization using low-pressure DC discharge hydrogen peroxide
plasma. IEEE Trans Plasma Sci 30:14401444
12. Cerf O (1977) Tailing of survival curves of bacterial spores. J Appl Bacteriol 42:119
13. Ruiz P, Ocio MJ, Cardona F, Fernndez A, Rodrigo M, Martnez A (2002) Nature of the
inactivation curves of Bacillus Pumilus spores heated using non-isothermal and isothermal
treatments. J Food Sci 67:776779
14. Moisan M, Barbeau J, Crevier M-C, Pelletier J, Philip N, Saoudi B (2002) Plasma sterilization.
Methods and mechanisms. Pure Appl Chem 74:349358
15. Raballand V, Benedikt J, Wunderlich J, von Keudell A (2008) Inactivation of Bacillus atropha-
eus and of Aspergillus niger using beams of argon ions, of oxygen molecules and of oxygen
atoms. J Phys D Appl Phys 41:115207 (8pp)
16. Khomich AV, Soloshenko IA, Tsiolko VV, Mikhno IL (1997) Cold sterilization of medical
devices and materials by plasma DC glow discharge. In: Proceedings of the 12th international
conference on gas discharges and their applications, vol 2, Greifswald, pp 740744
17. Khomich VA, Soloshenko IA, Tsiolko VV, Mikhno IL (1998) Investigation of principal factors
of the sterilization by plasma DC glow discharge. In: Proceedings of the international congress
on plasma physics, Prague, pp 27452748
18. Soloshenko IA, Khomich VA, Tsiolko VV, Mikhno IL, Shchedrin AI, Ryabtsev AV, Bazhenov
V Yu (1999) Experimental and theoretical investigation of cold sterilization of medical instru-
ments by plasma DC glow discharge. In: Proceedings of the 14th international symposium on
plasma chemistry, vol 5, Prague, pp 25512556
19. Soloshenko IA, Tsiolko VV, Khomich VA, Shchedrin AI, Ryabtsev AV, Bazhenov VYu,
Mikhno IL (2000) Sterilization of medical products in low-pressure glow discharge. Plasma
Phys Rep 26:792800
20. Inagaki T, Hamm RN, Arakawa ET, Painter LR (1974) Optical and dielectric properties of
DNA in extreme ultraviolet. J Chem Phys 61:42464250
21. Munakata N, Saito M, Hiera K (1991) Inactivation action spectra of Bacillus subtilis spores in
extended ultraviolet wavelengths (50300 nm) obtained with synchrotron radiation. Photochem
Photobiol 54:761768
22. Giese N, Darby J (2000) Sensitivity of microorganisms to different wavelengths of UV light:
implications on modeling of medium pressure UV systems. Water Res 34:40074013
23. Bolton JR, Linden KG (2003) Standardization of methods for fluence (UV dose) determina-
tion in bench-scale UV experiments. J Environ Eng 129:209215
24. Moreau S, Tabrizian M, Barbeau J, Moisan M, Leduc A, Pelletier J, Lagarde T, Rohr M, Desor
F, Vidal D, Ricard A, Yahia LH (1999) Optimum operating conditions leading to complete
sterilization at low substrate temperature in a plasma flowing afterglow. In: Proceedings of the
12 international colloquium plasma processes, Antibes, France
25. Moisan M, Barbeau J, Pelletier J, Philip N, Saudi B (2001) Plasma sterilization: mechanism,
potential and shortcomings. In: Proceedings of the 13th international colloquium plasma
processes, Antibes, France
26. Moreau S, Moisan M, Tabrizian M, Barbeau J, Pelletier J, Ricard A, LH Y (2000) Using the
flowing afterglow of a plasma to inactivate Bacillus subtilis spores: influence of the operating
conditions. J Appl Phys 88:11661174
27. Moisan M, Barbeau J, Moreau S, Pelletier J, Tabrizian M, LH Y (2001) Low-temperature
28. Philip N, Saoudi B, Crevier M-C, Moisan M, Barbeau J, Pelletier J (2002) The respective roles
of UV photons and oxygen atoms in plasma sterilization at reduced gas pressure: the case of
N2O2 mixtures. IEEE Trans Plasma Sci 30:14291436
29. Pintassilgo CD, Kutasi K, Loureiro J (2007) Modeling of a low-pressure N2O2 discharge and
post-discharge reactor for plasma sterilization. Plasma Sources Sci Technol 16:S115S122
30. Feichtinger J, Schulz A, Walker M, Schumacher U (2003) Sterilization with low-pressure
microwave plasmas. Surf CoatTechnol 174175:564569
31. Halfmann H, Denis B, Bibinov N, Wunderlich J, Awakowicz P (2007) A double inductively
coupled plasma for sterilization of medical devices. J Phys D Appl Phys 40:41454154
J Phys D Appl Phys 40:59075911.25
33. Pollak J, Moisan M, Keroack D, Boudam MK (2008) Low-temperature low-damage steriliza-
tion based on UV radiation through plasma immersion. J Phys D Appl Phys 41:135212
(14pp)
34. Soloshenko IA, Bazhenov VY, Khomich VA, Tsiolko VV, Potapchenko NG (2006) Comparative
research of efficiency of water decontamination by UV radiation of cold hollow cathode dis-
charge plasma versus that of low- and medium-pressure mercury lamps. IEEE Trans Plasma
Sci 34:13651369
35. Soloshenko IA, Bazhenov VY, Khomich VA, Tsiolko VV, Potapchenko NG, Goncharuk VV
(2006) Utilization of ultraviolet radiation of cold hollow cathode discharge plasma for water
disinfection. Ukr J Phys 51:10631070
36. Hury S, Vidal DR, Desor F, Pelletier J, Lagarde T (1998) A parametric study of the destruction
efficiency of Bacillus spores in low pressure oxygen-based plasmas. Lett Appl Microbiol
26:417421
37. Boudam MK, Moisan M (2010) Synergy effect of heat and UV photons on bacterial-spore
inactivation in an N2O2 plasma-afterglow sterilizer. J Phys D Appl Phys 43:295202 (17pp)
Chapter 19
Applications of Excilamps in Microbiological
and Medical Investigations
Victor F. Tarasenko, E.A. Sosnin, O.S. Zhdanova,

and E.P. Krasnozhenov
Abstract In a course long-term and comparative studies it has been shown, that the
DBD XeBr-excilamps looks as a good choice for various microorganisms inactiva-
tion. The first data about bacteriophage inactivation by XeBr-excilamp has been
obtained. Radiant modules for industrial treatment on contaminated water have
been developed. The XeCl-excilamp for treatment of skin diseases has been created
and tested.
19.1 Introduction
Spontaneous radiation sources, such as the excimer and exciplex lamps (excilamps)
find wide applications for science and engineering, in particular in biology and
medicine [17]. The main reasons are the following:
The great part of excilamps light energy concentrates in UV or VUV spectral
range which depends on gas mixture in a bulb. Photons with energies 510 eV
(UV and vacuum UV spectral ranges) can initiate and support different chemi-
cal, physical, and biological processes. Other advantages of excilamp are their
simplicity in comparison with UV and VUV lasers, and have long lifetime.
In the current review the excilamps are described briefly, and the most part of text
is dedicated to applications of excilamps in biology and medicine.
V.F. Tarasenko (*) E.A. Sosnin

Laboratory of Optical Radiation, High Current Electronics Institute,
Akademicheskii ave. 2/3, 634055 Tomsk, Russian Federation
e-mail: vft@loi.hcei.tsc.ru
O.S. Zhdanova E.P. Krasnozhenov
Microbiology and Virology, Sub-department of Siberian State Medical University,
Uchebnaya ave. 39, 634050 Tomsk, Russian Federation
252 V.F. Tarasenko et al.
19.2 Study and Development of Excilamps
Since 1992 the researches and development of spontaneous radiation sources

(in the first place excilamps) were carried out in the High Current Electronics
Institute. The excilamps filled in pure inert gases or inert gas-halogens mixtures
and excites by pulse-periodical or d.c. discharges. Efficient radiation of Ar2,
Kr2, Xe2, KrBr*, KrCl*, XeI*, XeBr*, XeCl*, Cl2* molecules and I atoms was
obtained in rare gas or in rare gas Br2 (Cl2, I2) mixtures. As a rule we use the
electrodeless discharge for increasing of excilamp lifetime. Our electrodeless
excilamps could be separated into two categories: capacitive discharge (CD)
and dielectric barrier discharge (DBD) driven excilamps. In both categories the
electrodes in a gas discharge device are covered with dielectric (it is fused
quartz in our case) that led to the high lifetime of the working mixture. The
dielectric layers have thickness up to several mm and dielectric constant of
3.54.
The difference between working pressure values and discharge gap values in
typical conditions causes the difference of discharge form and the spectral band
width of CD and DBD driven excilamps (Fig. 19.1). For CD driven excilamp is typi-
cal the big gap and low pressure values of gas mixtures (up to 10 Torr). As a result
the CD excilamp spectra are relatively broad and represent a few optical transitions.
For DBD devices the discharge gap is small (up to 10 mm) but a pressure of gas
mixtures is relatively higher (up to several hundred Torr). Therefore the spectra
become narrowband as we see at Fig. 19.1.
The spectral, temporal, energy and lifetime characteristics of CD and DBD
excilamps have been performed. In optimal conditions the radiant power for the just
sealed off KrCl (l ~ 222 nm) excilamp exceeded 100 W has been obtained. Maximal
radiation power density was of 50 mW/cm2. UV output power of ~75 W and
efficiency up to 10%, respectively, at l ~ 308 nm (XeCl* excilamp) were obtained
under excitation by pulses with frequency of 100 kHz. Application of water cooling
allows increasing the radiant power of DBD coaxial excilamps. VUV output power
of ~120 W at l ~ 172 nm was obtained under excitation by pulses with frequency of
~100 kHz.
The excilamp radiation efficiency was determined as h = Prad/Pin, where Pin is the
total power deposited to the lamp (input power), Prad radiation power.
The lifetime of gas mixtures in small XeCl and KrCl barrier discharge excilamps
over 12,000 and 8,000 h was demonstrated (some our results concerning excilamps
lifetime see in [5, 8, 9]).
The extraordinary characteristics of excilamps led to a lot of applications, which
had been demonstrated in a number of recent studies [17]. The example of devel-
oped excilamps (BD_X model) is illustrated in Fig. 19.2. There are DBD KrCl- and
XeBr-excilamps photo with output window size 13 80 cm. The DBD KrCl-exilamp
with the radiation maximum at l = 222 nm, and XeBr-exilamp with the radiation
maximum at l = 282 nm have radiant power up to 25 and 30 W, accordingly. Other
details were described in [10].
19 Applications of Excilamps in Microbiological and Medical Investigations 253
Fig. 19.1 Emission spectrum and general view of lighting of XeBr-excilamp excited by CD (left)
and DBD (right). The operating pressure values were 3 and 97 Torr, accordingly
Fig. 19.2 General view of KrCl- and XeBr-excilamps (BD_X model)

19.3 Applications
19.3.1 UV Inactivation of Biological Systems by Excilamps
This chapter is devoted to our experimental study of UV inactivation of biological

systems. We start these studies in 2001 [11] and now clearly see the advantages of
excilamp applications in this field.
UV irradiation has been shown to be a powerful tool in inactivating of both micro-
organisms and cells such as bacteria, viruses, protozoan parasites, some spores, liv-
ing cells and subsystems such as enzymes, aminoacids, and lipids (see refs. in [4]).
VUV or UV excilamps appear as an interesting option to conventional light
sources for UV disinfection. Thus, one should distinguish between two different
disinfection methods: the inactivation of microorganisms by UV irradiation (e.g. by
KrCl-, XeBr-, and KrBr-excilamps) or their total VUV-induced photomineralization
(by Xe2-excilamp).
For the first time, the bactericide action of incoherent VUV- and UV-radiation
using Xe2- and KrCl-excilamp flow-through photoreactors (electric input power Pel
of 150 W) was already manifested by Oppenlnder and Baum in 1996 [12].
The comparative analysis of inactivation by excilamps and other means (plasma
processing, laser irradiation, LP Hg lamps) has demonstrated that excilamps are the
competitive technical systems [13, 14]. In the scientific literature have been pre-
vailed the studies, where the specific excilamp gives inactivation effect on specific
microorganisms. So, the inactivation effect of excilamps was demonstrated for a
number of microbiological objects (yeasts, heterotrophic bacteria, spores, strains,
unicellular organisms, cultures of living cells (see refs in [4])). Therefore we have
started to do the comparative studies between inactivation of different microorgan-
isms by different excilamps.
In our first study the E. coli microbial samples were exposed by CD KrCl-, XeCl-
and XeBr-excilamps. It had been demonstrated that CD XeBr-excilamp is the most
efficient light source for inactivation [11]. How we have explained this fact? It is
known that the effect of radiation within the bactericidal range is associated primar-
ily with the dimerization processes in the bases of DNA molecules and that the
DNA absorption spectrum (Fig. 19.3) has two pronounced peaks near a wavelength
of 200 nm and in the band of 250270 nm. Therefore the action of XeBr-excilamp
is determined by its spectrum having a long short-wavelength tail of 260282 nm
that covers half of the first DNA absorption peak.
Our later studies had been shown that DBD driven XeBr excilamp is also attrac-
tive for inactivation. Let us to give a several results that prove this fact.
In our study [15] the XeBr-excilamp (282 nm) and low-pressure Hg-lamp
(253.7 nm) radiation impact on Escherichia coli bacterial strain have been presented.
The mercury lamps are in wide use owing to their simple power-supply systems and
easy maintenance. However, the radiant power of LP Hg lamps is very sensitive to
ambient thermal changes, which should be considered in LP Hg disinfection reactors
engineering.
Fig. 19.3 Action spectrum of UV inactivation on E. coli (1), DNA absorption spectrum (2) and
maxima of radiation bands of various excilamps and low pressure mercury lamp (LP Hg-lamp)
The peak intensity of B-X band of XeBr* molecule (282 nm) is seen to be at
about the same distance from the action spectrum maximum, the same as resonance
line of a LP Hg-lamp (Fig. 19.4). That is Dl1 ~ Dl2. This suggests that the both
lamps have comparable bactericidal effect. Note that we do not know any other
papers reporting on such direct comparison made. The model of XeBr-excilamp
(model XeBr_BD_P, High Current Electronics Institute SB RAS) with the follow-
ing parameters: discharge gap 0.8 cm, tube diameter 4.2 cm, UV radiant exitance of
3 mW/cm2, and a spectrum shown in Fig. 19.4, was used in the experiments.
Another lamp was the conventional germicidal LP Hg-lamp (TUV-15). In the
experiments, the lamp was specially furnished with a diaphragm in order to provide
the radiation doses comparable in values with the radiation doses of the DBD-driven
XeBr-excilamp. As a result, the Hg lamp provided radiant exitance of 2.5 mW/cm2.
The lamps radiant power was defined in absolute units by using a C8026 (Hamamatsu
Photonics KK) photodetector with the H8025-222 head. Just after the initiation, the
XeBr-excilamp achieved its mode, and the LP Hg-lamp needed 2.5 min for its initial
heating to provide stable luminous flux.
The object of study was the pure culture of Escherichia coli (strain K12 ATCC
25922) provided by the Scientific Research Institute of Balneology (Tomsk, Russia). Our
selection of colibacillus for study was determined by its belonging to the main species of
the enterobacterium group taken for sanitation of desinfection efficiency by UV-radiation.
Besides that, the E. coli has one of the most high resistance coefficients among enterobac-
teria group. The bacterial cultures were supported on beef-extract agar (BEA) and kept at
the temperature of 4C. In preliminary study, based on the method of multiple dilutions,
the optimal concentration of microbial dredge was found for experiments.
Fig. 19.4 Different spectral characteristics, essential to this investigation: 1 UV action spectra of
DNA, 2 the absorption spectrum of DNA, 3 emission spectrum of DBD-driven XeBr-excilamp,
4 resonance line of LP Hg-lamp [15]
Primary and secondary irradiation of E. coli via various exposure values were
carried out. Usually, the irradiation was made 5 cm distant from the lamp to 10 cm
Petri dish with contaminated surface. Thereby the heating effect of substrate was
minimized and values of irradiation were kept high. Thus, uniform illumination of
contaminated surface was additionally provided. The microorganisms, which
survived after irradiation by a dose leading to inactivation of 99.9% of E. coli, were
allocated in pure culture and subjected to reirradiation. The qualitative and quantita-
tive analysis and estimation of bacterial colonies morphology (size, form, consis-
tence, character of edge) were made. The results of the experiments are shown on
Fig. 19.5.
Thus, according to our hypothesis, both types of the light sources provide the
similar bactericidal effect due to their radiation in the spectral range where UV
action spectra of DNA have comparable values. As compared with traditional mer-
cury bactericidal lamps, the advantages of the modern excilamps, accentuating to
their bactericidal application are: (1) high photon flux qp, extracted from plasma
without self-absorption; (2) no elementary mercury in bulbs, which conforms with
ecology; (3) extraordinary geometric freedom of bulbs; (4) momentary launching
and full radiant power after ignition; (5) variable tuning of photon flux qp.
As we noted above for the fist time we have established bactericidal effect of
CD XeBr-excilamps due to their wideband spectra in 2002. Such a spectrum has
a short-wave tail from 230 to 282 nm (B-X band), which covers a half of the first
maximum of DNA absorption. Besides, the spectrum has a D-X band with a
Fig. 19.5 Inactivation of surface inoculated E. coli, performed with various UV-doses of XeBr-
excilamp () and LP Hg-lamp (o) irradiation. The first bacteria generation results are on the left,
and the second bacteria generation results are on the right [15]
maximum at 221 nm, which has a short-wave tail from 210 to 221 nm. It has been
shown that the microorganisms, which survived after the first irradiation by that
excilamp, kept their radiation susceptibility unchanged [15]. UV resistance pro-
tection of microorganisms under the action of wideband UVB irradiation is
explained by the fact that biological structure of bacteria bears very many induced
failures of biological structure, making it improbable to appear stable mutants to
UVB irradiation (the viewpoint validity is being considered in the reviews [16]).
At LP Hg-lamp ruled irradiation one might expect appearance of the greater
number of UV resistant mutants than at irradiation by a wideband radiation source.
Just in this respect maybe be interpreted the fact obtained in the present investiga-
tion. At irradiation of bacteria, survived after the first irradiation (their average
share was 0.5%), the survival curve obtained for LP Hg-lamp activated bacteria
has moves aside from the curve obtained for XeBr-irradiated bacteria. In other
words, resistance of E. coli, activated by the LP Hg-lamp, is higher in XeBr-
excilamp irradiation case.
In [17] the comparison of XeBr-, KrCl- and KrCl + KrBr-excilamps radiation
impact on 5 microbiological cultures (Escherichia coli (ATCC 25923),
Staphylococcus aureus (25923) and extracted from human skin representatives
p. Sarcina, p. Pseudomonas and p. Bacillus) have been presented.
Emission spectrums of DBD-driven excilamp under using are presented on
Fig. 19.6. Data about bactericidal efficiency is assembled to Table 19.1.
Fig. 19.6 Emission spectrums of DBD-driven KrCl- and KrBr + KrCl-excilamps
Table 19.1 Experimental data of surface irradiation dose which gives

99.9% of bactericidal efficiency for different excilamp [17]
HS, J/m2
Microorganism XeBr* KrCl* KrCl* + KrBr*
Escherichia coli (ATCC 25923) 60 85 65
Staphylococcus aureus (25923) 150 370 320
p. Sarcina 90 120
p. Pseudomonas 110 160
p. Bacillus 100 190 130
It is shown that surface irradiation dose which gives 99.9% of bactericidal effi-
ciency for different excilamp and for LP Hg-lamp have a comparable values (if we
take into the mind the data from [18]). In the second place the bactericide efficiency
are decrease in a row of lamps XeBr (282 nm) > KrCl_KrBr (222 and 206 nm) > KrCl
(222 nm).
In 20092010 we have studied the sensitivity of hospital infectious agents to UV
radiation of excilamp and LP Hg-lamp using the abovementioned cultivation and
irradiation methods [19].
The object of study was the pure culture of Escherichia coli (strain ATCC 501),
Klebsiella pneumonia (strain ATCC 2482), S. aureus (strain ATCC 209) and two
cultures selected from patients of Tomsk Savinich Hospital C. albicans and
P. aeruginosa. Suspension of daily cultures (with concentration 105 CFU/ml in vol-
ume 0,1 ml) was inoculated into a meat infusion agar. As the control a suspension
of daily cultures in concentration 103 CFU/ml was used.
Fig. 19.7 Sensitivity of test cultures to UV radiation of excilamp and LP Hg-lamp after 15-s time
exposure (and at the same UV exposure)
The results of our research are illustrated in Fig. 19.7. From this figure we notice
that: (1) XeBr-excilamp irradiation gives better germicidal effect for E. coli,
P. aeruginosa cultures; (2) S. aureus and C. albicans cultures have the same
UV-resistance for both light sources.
The low UV-sensitivity of K. pneumoniae could be explained by presence of
sheath (capsule) in their biological structure. This capsule absorbs a part of radia-
tion flux and decrease the DNA damage frequency.
Our recent results (2010) are dedicated to the sensitivity of MS2 bacteriophage
under UV radiation of LP Hg-lamp and XeBr-excilamp. The model XeBr_BD_P
and LP Hg-lamp (TUV-15) were used in the experiments. The object of study was
the MS2 bacteriophage (strain PH-1505), which cultivated on E. coli K 12 F + (strain
B-3254) after irradiation. Both cultures are from Russian industrial bank of micro-
organisms. UV sensitivity of bacteriophage has been determined by well-known
Gratia method [20]. The exposure value was equal to 45 J/m2. Virocide action of
radiation has been estimated by quantity of blank places on Petri dishes (see
Fig. 19.8).
The greater viroicidal influence on tested culture has been achieved by means of
XeBr-excilamp (Fig. 19.9). We think that it is related to damaging genetic block of
bacteriophage as well as protein shell too. Our results testifiers that the excilamps
could be use in antiviral applications.
Concluding this chapter, let us note that some microorganisms and cells possess
UVA/VIS repair mechanisms (photoreactivation) that substitute or dissociate thy-
mine dimers. Under these circumstances, excilamps as narrow-band emission
Fig. 19.8 Photo of Petri dish

after the experiment. Each dark
place corresponds to the locus
where a survived MS2
bacteriophage has killed E. coli
Fig. 19.9 Sensitivity of MS2 bacteriophage to UV radiation of XeBr excilamp and LP Hg-lamp
at the same UV doses (45 J/m2) compared with control
sources should be more efficient than wide-band MP Hg lamps. Sure, this problem
needs further studies to be done.
19.3.2 UV Phototherapy of Skin Diseases
One of the most effective methods of psoriasis curing is UVB phototherapy.

Radiation is absorbed by endogenous chromophores, especially by DNA nucle-
otides, which lead to suppression of DNA synthesis in epidermal cells, for example
in psoriatic plaques. Photochemical reactions of these molecules result in altera-
tions of skin and then lead to the curing effect. Apparently, the DNA damage is the
general mechanism at UV curing of skin diseases. Particularly, UV radiation affects
Fig. 19.10 Example of psoriasis curing by the XeCl excilamp (model BD_P, see previous chapter)
in Siberian State Medical University (BD_compact model, Optical Radiation Laboratory, Russia,
output window square 30 cm2, UV photon exitance of 40 mWcm2): before curing (left) and after
10 days at suberythermogenic doses treatment (right) [25]
the production of soluble mediators, the expression of cell-surface receptors, to

induce apoptosis in pathogenetic relevant cells (cf. [21, 22]).
In 1980, a spectrum of UV radiation effect on psoriasis was obtained and showed
that the effective UV radiation spectrum for psoriasis curing lay in the area 296
313 nm [23]. More than 90% of the DBD XeCl-excilamp radiant energy is within
the anti-psoriasis action spectrum. Thus, this excilamp is also a good variant for
psoriasis curing, which was proposed for the first time in 1994 [24]. For this aim in
2003 we have developed a compact XeCl excilamp for French start-up company
DermOptics SAS. It was the first prototype of compact device [25, 26]. Now a
key idea of this device are used in commertional Quantel derma 308 Excimer
System. Since 2004 we have tested XeCl-excilamp (Fig. 19.10). The merits of such
a therapy method are a good tolerance by patients and the use of suberythermogenic
doses. In comparison with a XeCl-laser, the excilamp is cheaper and simpler in use.
There are no principal restrictions for XeCl-excilamp radiant area increase, which
allows to develope large-scale set-ups both for local and total-body irradiation.
19.4 Conclusion
A study of excilamps applications for researches in photomedicine and photobiol-

ogy has been carried out. In a course long-term and comparative studies it has been
shown, that the CD and DBD XeBr-excilamps are the best choice for various micro-
organisms inactivation. The first data about bacteriophage inactivation by excilamp
has been obtained. We suppose that the further efforts should be concentrated on
microorganisms photoreactivation (after excilamps irradiation) studies and
reproducibility of results (because of specific character of microbiological tests).

The XeCl-excilamp for treatment of skin diseases has been created and tested.
Acknowledgements This work was supported in part by the Federal Target Program The scien-
tific and scientific-pedagogical personnel of Innovative Russia, State contract No. 02.740.11.0562.
Discussions with L.V. Lavrenteva, U. Kogelschatz, T. Oppenlender and technical assistance of
S.M. Avdeev, A.V. Gritzyta, M.V. Erofeev, D.V. Schitz, V.S. Skakun are gratefully acknowledged.
References
1. Kogelschatz U (2004) Excimer lamps: history, discharge physics and industrial applications.
Proc SPIE 5483:272286
2. Baum G, Oppenlnder T (1995) VUV-oxidation of chloroorganic compounds in an excimer
flow through photoreactor. Chemosphere 30:17811790
3. Lomaev MI, Skakun VS, Sosnin EA, Tarasenko VF, Shitts DV, Erofeev MV (2003) Excilamps:
efficient sources of spontaneous UV and VUV radiation. Phys Usp 46:193210
4. Sosnin EA, Oppenlnder T, Tarasenko VF (2006) Applications of capacitive and barrier
discharge excilamps in photoscience. J Photochem Photobiol C Photochem Rev 7:145163
5. Lomaev MI, Sosnin EA, Tarasenko VF, Shits DV, Skakun VS, Erofeev MV, Lisenko AA
(2006) Capacitive and barrier discharge excilamps and their applications. Instrum Exp Tech
49:595616
6. Sosnin EA, Sokolova IV, Tarasenko VF (2008) Development and applications of novel UV and
VUV excimer and exciplex lamps for the experiments in photochemistry. In: Sanchez A,
Gutierrez SJ (eds) Photochemistry research progress. Nova, New York, pp 225269
7. Kogelschatz U (2003) Dielectric-barrier discharges: their history, discharge physics, and
industrial applications. Plasma Chem Plasma Process 23:146
8. Sosnin EA, Erofeev MV, Lisenko AA, Tarasenko VF, Shits DV (2002) Study of the service
characteristics of a capacitive-discharge excilamp. J Opt Technol 69:509511
9. Avdeev SM, Sosnin A, Tarasenko VF (2010) Factors that limit the service life of sealed
chlorine-containing barrier-discharge exciplex lamps. J Opt Technol 77:4244
10. Shits DV, Avdeev SM, Skakun VS, Sosnin EA, Tarasenko VF (2011) Powerful portable mod-
ule fpr UV irradiation based on inert gas-halogen mixtures. Russ Phys J 53:109112 (in
print)
11. Sosnin EA, Lavrenteva LV, Yusupov MR, Masterova YV, Tarasenko VF (2002) Inactivation of
Escherichia coli using capacitive discharge excilamps. In: Proceedings of the 2nd international
workshop on biological effects of electromagnetic fields, Rhodes, Greece, pp 953957, 711 Oct
12. Oppenlnder T, Baum G (1996) Wasseraufbereitung mit Vakuum-UV/UV-Excimer-
Durchflussphotoreaktoren. Wasser-Abwasser 137:321325
13. Lavreneva LV, Sosnin EA, Masterova YaV (2003) UV inactivation of microorganisms: com-
parative analysis of methods. Bull Tomsk State Univ Biol Sci 30:163176
14. Laroussi M (2002) 2002. Non-thermal decontamination of biological media by atmospheric
pressure plasmas: review, analysis and prospects. IEEE Trans Plasma Sci 30:14091415
15. Avdeev SM, Sosnin EA, Velichevskaya KYu, Lavrenteva LV (2008) Comparative study of
UV radiation action of XeBr-excilamp and conventional low-pressure mercury lamp on bacte-
ria. Proc SPIE 6938:693813
16. Kalisvaart BF (2004) Re-use of wastewater: preventing the recovery of pathogens by using
medium-pressure UV lamp technology. Water Sci Technol 50:337344
17. Avdeev SM, Velichevskaya KYu, Sosnin EA, Tarsenko VF, Lavreteva LV (2008) Analysis of
germicidal action of UV radiation of excimer and exciplex lamps. Light Eng 16:3238
18. Guidance P (2004) Using of bactericidal UV radiation for air decontamination in a housing,
Ministry of Public Health of Russian Federation. 28 p, 3.5.190404
19. Zhdanova OS, Sosnin EA, Krasnoszhenov EP, Tarasenko VF, Avdeev SM, Gritsuta AV (2010)
Hospital infections agents sensitivity to XeBr excilamp irradiation. J Infect Pathol 17:6264
20. Gratia A (1936) Des relations numericues entre bacteries lysogenes at particules de bacterio-
phage. Ann Inst Pasteur 57:652694
21. Krutmann JJ (1998) Therapeutic photoimmunology: photoimmunological mechanisms in
photo(chemo)therapy. Photochem Photobiol B. 44:159164
22. Hnigsmann H (2001) Phototherapy for psoriasis. Clin Dermatol 26:343350
23. Parrish JA, Jaencke KF (1981) Action spectrum for phototherapy of psoriasis. J Invest Dermatol
76:359362
24. Oppenlnder T (1994) Novel incoherent excimer UV irradiation units for the application in
photochemistry, photobiology, photomedicine and for waste water treatment. Eur Photochem
Assoc Newslett 50:28
25. Dmitruck VS, Sosnin EA, Obgoltz IA (2006) The first attempt of XeCl-excilamp application
in complex psoriasis curing. Proc SPIE 6263:316321
26. Sosnin EA, Erofeev MV, Tarasenko VF, Skakun VS, Shitz DV, Mersey T, Meilhac L (2006)
Radiation source. Patent RU2 271590. Priority date 15 Mar 2004
Chapter 20
Xenon Iodide Exciplex Lamp as an Efficient
Source for the UV Surface Cleaning
and Water Decontamination
Mykola Guivan, H. Motomura, and M. Jinno
Abstract Discharges in mixtures containing xenon and iodine are interesting

because the exciplex XeI* emits in germicidal spectral region (lmax = 253 nm).
In this paper the optimization of a dielectric barrier discharge (DBD) excited lamp
operated with a Xe/I2 mixture is reported. Approximately 76% of the excilamp out-
put was due to the BX transition of XeI* exciplex at 253 nm. Short voltage rising
and falling time under pulse excitation is more important for the efficiency enhance-
ment than the pulse duration. The average radiation power of 10.3 mW/cm2 was
measured under pulsed excitation at a frequency of 80 kHz. An efficiency improve-
ment from about 3% for AC to 59% for square pulse excitation was obtained.
A good cleaning effect of glass surface and sterilization action was achieved with
the DBD-driven XeI* exciplex lamp. The contribution of an atomic iodine emission
in the range of 178207 nm has been confirmed. Germ reduction experiments with
the XeI* excilamp have been carried out in a water flow reactor.
20.1 Introduction
Conventional technology for disinfection by UV irradiation is based on low-pressure

mercury lamps. The development of a new mercury-free UV lamp is very important
due to the environmentally unfriendly nature of mercury. The excilamps (excimer or
exciplex lamps) based on mixtures containing xenon and iodine vapours emitting
M. Guivan (*)
Department of Quantum Electronics, Uzhgorod National University,
Pidgirna 46, 88000 Uzhgorod, Ukraine
e-mail: m_guivan@rambler.ru
H. Motomura M. Jinno
Department of Electrical and Electronic Engineering, Ehime University,
Matsuyama, Japan
266 M. Guivan et al.
mainly due to XeI(B2s1/2X2s1/2) at l = 253 nm are considered to be the efficient sources

in the UVC (200290 nm) range. Recently, the sterilization action of a DBD-driven
XeI* excilamp operated with a Xe/I2 mixture has been reported for the inactivation of
Bacillus Subtilis spores in a steady state mode [1]. In this study, we report the investiga-
tion of XeI* excilamp operated with the Xe/I2 mixture and using it for the UV surface
cleaning and UV inactivation of Bacillus Subtilis spores in a water flow reactor.
From the available reports, XeI* excilamp efficiencies vary from 1% to 30%.
Gellert and Kogelschatz [2] reported that the XeI* emission in DBD was weaker
than the exciplex emissions with other halogens. Zhang and Boyd [3] claimed an
efficiency of 22% from an AC excited XeI* DBD lamp at a total pressure 50 kPa
(Xe/I2 mixture) measured by means of the actinometric method. In contrast to previ-
ous reports, a much lower value (3%) has been obtained experimentally by Voronov
et al. [4], when the UV output was measured in the range of 230280 nm. A coaxial
DBD lamp with a gas gap d = 3.5 mm, filled with p(Xe) = 50 kPa and p(I2) = 2 kPa
was driven by AC power supply at an input power level of 1.1 kW and f = 300 kHz.
In [5] the XeI* excilamp efficiency under pulsed excitation was not over 5%.
Recently, Carman et al. [6] reported a detailed experimental and modeling study of
the efficiency of a DBD coaxial XeI* lamp excited by pulsed bipolar square-wave
circuits with a risetime of about 200 ns. Maximum conversion efficiency of electri-
cal output to UV (253 nm) was in the range 12% at d = 2.8 mm, p(Xe) = 1025 kPa
and p(I2) = 40 Pa. The authors concluded that direct quenching of the XeI(B) by I2
limits the output efficiency.
20.2 Experimental
In these experiments we used a dielectric barrier discharge lamp in coaxial design

consisting of two quartz tubes and electrodes, which were applied to the inner sur-
face of the smaller tube and the outer surface of the outer tube [1]. The outer quartz
tube (UV grade, transmittance of 70% at l = 200 nm) was 30 mm in diameter and
the discharge gap d was 7.4 mm. The active length of the lamp (l = 10 cm) was
defined by the outer electrode. The inner electrode was made of a copper plate cov-
ered by a thin smooth aluminium foil. For the outer electrode we used a 0.1 mm
nickel wire wrapped tightly on the outer tube with a 2 mm pitch providing transpar-
ency of 95%. Experiments were carried out with the sealed off lamp, filled with a
Xe/I2 = 13.3/0.04 kPa mixture, which has been determined as optimum from the
viewpoint of maximal UV output [1]. The lamp was air-cooled by means of a fan,
placed above the lamp bulb at a 3 cm distance.
The lamps were excited in Xe/I2 mixtures using different excitation circuits:
(a) AC power supply As-114B (NF Electronic Corp.), U = 03.3 kV rms,
I = 020 mA rms, f = 25159.9 kHz;
(b) custom built power supply (PS1), providing bipolar pulses with U = 04.4 kV
peak-to-peak, rising and falling times 0.9 and 0.6 ms, f = 21.5115 kHz;
20 Xenon Iodide Exciplex Lamp as an Efficient Source 267
(c) home made power supply (PS2), based on fast high voltage transistor push-pull
switch HTS 31-01-GSM (BEHLKE) and providing unipolar pulses with
U = 03 kV, rising and falling times Dtrise = 60 ns and Dtfall = 40 ns respectively,
f = 10 kHz;
(d) home made power supply (PS3), based on HTS 81-06-GSM push-pull switch
(BEHLKE), providing unipolar pulses with U = 08 kV, controlled rising and
falling times down to 160 and 60 ns respectively, f = 1080 kHz.
The voltage U applied to the excilamp was measured using a high voltage
probe (Sony Tektronix P3000 or Tektronix P6015). The waveforms of current I
and charge Q were registered by means of 100 W resistor or low-inductance 3.3 nF
capacitor placed in series with the excilamp and monitored by digital oscilloscope
TDS 3034B (Tektronix). The input power Pin was determined from the Lissajous
UQ figures.
The radiation power of the excilamp in absolute units was measured by a UV
power meter C8026 (Hamamatsu Photonics K.K., Electron Tube Centre) equipped
with a calibrated H8025-254 sensor head. The correct determination of the total
radiant flux requires the measurement of the angular distribution of radiation
from the tested DBD excilamp, because it differs from a Lambert source or a thin
linear lamp in geometry in the effect of the outer electrode and the emission from
the ends of the lamp. The distance L between the middle of the lamp and the
sensor head must be about 10 times longer than the active length of the lamp l.
Since the emission from the lamp is isotropic around the axis, the total UV radi-
ant flux Pout from the lamp (in the range of the sensor head sensitivity) can be
represented by
Pout = 2pL2 Pmeas (q )sin q dq ,

p
(20.1)
0
where Pmeas(Q) is the specific UV power in mW/cm2 measured by the power meter
in a certain direction and Q is the angle between the lamp axis and the sensor head
position, L = 1 m when l = 10 cm. The lamp efficiency was determined as the ratio
between the total UV radiant flux Pout and the input Pin: h = (Pout / Pin ) 100%.
Emission spectra were measured by means of HR4000 (5 mm slit, 1,200 g/mm
grating, 200400 nm) and USB2000 (10 mm slit, 600 g/mm grating, 200850 nm)
spectrometers (Ocean Optics). The registration system was calibrated within the
200850 nm wavelength range by the standard xenon lamp L7810-02
(Hamamatsu). For measurements of the emission features in the VUV range
(<200 nm) the VM-502 0.2 m vacuum monochromator (Acton) equipped with an
ICCD camera (PI-MAX, Princeton Instruments) and a PMT R928 (Hamamatsu)
were used. Calibration was performed by means of a deuterium lamp L648M
(Hamamatsu).
Figure 20.1 shows the experimental arrangement for the UV treatment of B. sub-
tilis spores in the water flow reactor. A chamber with a water spread system was
made of stainless steel. The test water (1 l) was circulated by a tubing pump
(Masterflex, Cole Parmer Instrument Co.) at a constant speed of 5.4 l/min.
Fig. 20.1 Experimental

arrangement for the UV
sterilization of B. subtilis
spores in a water flow reactor
The spread system provided a uniform water layer with a width and thickness of 90
and ~0.5 mm, respectively. To evaluate the exponential decrease of survival rate (N/
N0), the UV dose (cumulative energy density) needed to reduce a culturable cell
number was measured by a CFU counting assay. The distance between the XeI*
excilamp and the water layer was 4 cm. The test water was irradiated from 1 to
30 min using this experimental setup.
The relative energy, i.e. the contribution of each emitting species i in the total
output, was calculated as the area ratio Si/S170-400 from the VUVUV spectrum,
corrected on the spectral sensitivity of the registration system. The effect of
atomic iodine lines in the total UV output was estimated taking into account the
measured line width of 0.120.14 nm (FWHM). About 85% of radiation was
concentrated in the germicidal region. We have revealed that the atomic iodine
emission rises faster with frequency in comparison to XeI*. For pulsed excitation
with short voltage rising and falling times, when the operating frequency changes
from 10 to 80 kHz, the XeI*(253 nm) intensity increases ~5-fold, whereas the
I*(206 nm) intensity 19 times, and the intensity ratio 206 nm/253 nm grows
almost to 4 times [7]. In other words, we propose that by operating at a higher
frequency it is possible to get the greatest intensity of atomic iodine emission
and, consequently, better sterilization action due to larger DNA absorption. Note
also that the VUV part can be increased by using a quartz tube with enhanced
transmittance for l < 200 nm, such as Suprasil 311, 312 or Heralux plus [ 8 ] .
It was important for us to compare how the irradiation spectra affect the B. subtilis
spores lethality.
20.3 Results
20.3.1 Electrical Characteristics
Representative examples of measured voltage U(t) and external total current I(t)
waveforms, as well as UQ loops for Xe/I2 = 13.3/0.04 kPa mixture under the differ-
ent excitation schemes are shown in Fig. 20.2. The temporal evolution of all the
internal electrical quantities (discharge current, voltage across the dielectric, volt-
age across the discharge gap, consumed energy per cycle, and instantaneous input
power) in the gap were calculated using the technique described in [9, 10] and will
be presented elsewhere.
When using AC excitation, in each half-cycle of the applied voltage U0, the cur-
rent waveform consists of a displacement current and sharp current peaks caused by
the discharge (Fig. 20.2a), with characteristics depending on the operating fre-
quency and applied voltage. The peak current did not exceed 55 mA and their dura-
tion was in the range 0.92 ms. As U0 increased, the first current pulse shifted ever
more to the left of the peak of the applied voltage and, at high amplitudes U0 ~ 3.7 kV,
it occurred at the phase of reverse applied voltage. Up to three current pulses (three
three mode) were observed in each half-period under the present experimental con-
ditions. Similar behavior for the AC driven DBDs has been reported before, for
example, for He [11], Kr/I2 [12], and CdBr2/(Ne, Ar, Kr, Xe, N2) [13] mixtures.
For pulsed excitation the peak current was in the range 0.40.8 A and strongly
depended on the Dtrise and Dtfall values. The duration of the current pulses (FWHM)
did not exceed 200 ns. The same peak current was observed when Dtrise Dtfall
(Fig. 20.2e), whereas the shorter voltage pulse edge provides the greater current
amplitude (Fig. 20.2c, g). Note that pulsed excitation provides peak input power
into the discharge about one order higher than AC.
The DBDs driven by unipolar pulses show different electrical behaviour from
bipolar (Fig. 20.2e, g). The power input proceeds only during the primary discharge.
One part of the injected energy from the external circuit directly supports the pri-
mary discharge; the rest is stored by memory charges, to be released later to energize
the secondary discharge shortly after the pulse falling edge. The number of current
pulses for pulsed bipolar operation is completely controlled by the Dtrise and Dtfall.
The UQ loops can be efficiently used not only for the determination of the con-
sumed energy by the discharge, but also for the calculation of the lamp capacitance
Clamp for periods corresponding to the different discharge phases [9, 10]. Besides
that, from comparison of the calculated Clamp for cylindrical capacitor with known
geometric parameters and the slope dQ/dU in measured UQ loops, it is possible to
determine the conductivity of the discharge gap in the given time period [9]. In our
case we calculated Clamp ~ 7.5 pF, whereas the slope in Fig. 20.2b gives 1825 pF for
Off periods when the discharge current is negligible. The shape of the UQ loop
shows that at a given pressure under AC excitation discharge current flows during
a Xe/I2=13.3/0.04 kPa, f = 30 kHz b 100 1750 V (rms), 30 kHz, Pinput=9.9 W
3 60
2 40
50
Charge (nC)
Current (mA)
1 20
Voltage (kV)
0 0 0
1 20
2 40 50
3 60
100
0 10 20 30 40 50 2 0 2
Time (ms) Voltage (kV)
c Xe/I2=13.3/0.04 kPa, f = 60 kHz d 3.8 kV pp, 60 kHz, Pinput=17.62 W
2 0.4 100
1 0.2 50
Charge (nC)
Voltage (kV)
Current (A)
0 0.0 0
1 0.2
50
2 0.4
100
0 5 10 15 20 2 1 0 1 2
e 1
Xe/I2=13.3/0.04 kPa, f = 10 kHz, Duty: 50% f U0 = -3 kV, f = 10 kHz, Pinput=2.75 W
100
0 0.4
50
Voltage (kV)
Current (A)
Charge (nC)
1 0.0
0
2 0.4
50
3 0.8 CDBD=7.8 pF
100
9.5 10.0 10.5 60.0 60.5 61.0 3 2 1 0 1
Xe/I2=13.3/0.04 kPa, f = 60 kHz, Duty: 50%
g h U0 = 3.07 kV, f = 60 kHz, Pinput=17.52 W
100
3 0.4
50
Voltage (kV)
Current (A)
Charge (nC)
2 0.0
0
1 0.4
50
0 0.8
5 10 15 100
0 1 2 3 4
Fig. 20.2 Measured voltage, current waveforms (a, c, e, g) and corresponding Volt-Coulomb
Lissajous figures (b, d, f, h) for a mixture Xe/I2 = 13.3/0.04 kPa under the different excitation modes
used in the experiments. Displacement current and switching noise are already subtracted for the
current waveform under pulsed excitation at f = 60 kHz (g)
Fig. 20.3 Angular Distance to the lamp: 1 m

distribution of the irradiance
90
at L = 1 m from the lamp axis. 8
Bipolar pulse, Upp = 4.2 kV, 120 60
f = 60 kHz, 50% duty cycle 6

150 30
4
Irradiance, mW/cm2
2
0 180 0
Lamp
2
4 210 330
6
240 300
8
270
almost the entire voltage cycle excluding short intervals in the vicinity of the peak
of the applied voltage (Fig. 20.2b).
The slope obtained for pulsed bipolar operation PS1 with Dtrise = 0.9 ms and
Dtfall = 0.6 ms is about 14 pF (Fig. 20.2d). This means that the discharge gap is already
slightly conductive during the ignition phase before the breakdown. When using
PS2, providing square pulses with short rising and falling times of ~50 ns, the UQ
Lissajous figure is a parallelogram with two vertical sections for On periods
(Fig. 20.2f). This is similar to the observed one for a plasma a display cell with
square wave voltage (f = 100 kHz) [14]. The slope dQ/dU = 7.8 pF coincides with the
calculated lamp capacitance. For unipolar pulsed operation with PS3 (Dtrise = 160 ns,
Dtfall = 60 ns), the dQ/dU for Off periods remains close to 8 pF, but at higher fre-
quency a breakdown occurs at lower voltage and the right On section in Fig. 20.2h
changes position from vertical to sloping.
20.3.2 An Angular Distribution of Radiation
Figure 20.3 shows the angular irradiance distribution of the DBD XeI* excilamp
measured at 1 m distance between the centre of the lamp and the sensor head. The
angular dependence shows that the total flux for the improved electrode system is
89% with respect to the isotropic light source which has Pmeas(90). The obtained
value also differs from a linear cylindrical light source (p/4 78.5%). We would like
to note an interesting feature from Fig. 20.3. The radiation from the ends of the
DBD XeI* excilamp does not fall to neglected values but has a significant effect into
the total radiant flux. Taking into account the lamp dimensions and emitting
10
Xe/I2 = 13.3/0.04 kPa
f = 60 kHz
8
XeI*(B - X)
)
3/2
o
I*( P5/2 - P
2
Intensity (a.u.)
) 1/2
6 o
I*( P3/2 - P
2
4
4
1/2
XeI*(B - A)
o
I2*(D ' - A')

I*( P3/2 - P
XeI*(C - A)
2
2
4
0
200 250 300 350
Wavelength (nm)
Fig. 20.4 VUV-UV emission spectrum of the excilamp with a Xe/I2 = 13.3/0.04 kPa mixture.
Bipolar pulses, Upp = 4.2 kV
surfaces (side Sside = 26.8 cm2 and end Send = 6 cm2), one can calculate that 1 cm2 area
provides, at L = 1 m, irradiance perpendicularly to the lamp axis Pmeas(90) = 0.28 mW/
cm2, while in the direction of the lamp axis Pmeas(0) = 0.5 mW/cm2. This should be
considered in the design of the reflecting fittings for the DBD-driven XeI*
excilamps.
20.3.3 Spectral Characteristics
In the experimental reports to date, the emission spectra of DBD-driven XeI* excil-
amps are presented mainly for wavelengths l > 200 nm. Recently Carman et al. [6],
based on computer modeling, predicted an efficient formation and intense radiation
of Xe2* excimer in the VUV range (l = 172 nm) from a Xe/I2 mixture. The overall
intrinsic conversion efficiency from electrical energy to UV output from the plasma
was calculated at 9.6% for 253 nm and 19.4% for 172 nm. We wanted to check this
prediction and investigate the VUV emission from a Xe/I2 mixture experimentally.
Figure 20.4 shows the spectra of the excilamp in the VUV and UV range operat-
ing with the optimized working mixture Xe/I2 = 13.3/0.04 kPa under pulsed excita-
tion (PS1). Measurements reveal the strong XeI*(BX) exciplex radiation at
lmax = 253 nm. Weaker emissions of XeI*(BA) (lmax = 320 nm), XeI*(CA)
(lmax = 265 nm), and I2*(DA) (lmax = 342 nm) were also observed. Atomic iodine
is represented in the spectrum by the emission lines in the range 178188 nm (178.3,
179.9, 183.0, 184.4, 187.6 nm) and at l = 206.2 nm (5p46s 5p5 transitions). It is
interesting that the observed 4P3/22P1/2 (184.4 nm) line intensity is negligible com-
pared to the most intensive 4P5/22P3/2 (183.0 nm) line contrary to the VUV spec-
trum of an AC excited DBD excilamp operated with a Kr/I2 = 40/1.33 kPa mixture,
where both lines were registered with similar intensity [12]. It also differs from
the emission spectrum of a DC glow discharge with a Xe/I2 mixture, where the
4
P5/22P3/2 (183.0 nm) line intensity counts only for 2% of the 2P3/22P1/2 intensity
at 206.2 nm [15].
We did not observe Xe2* excimer radiation from the XeI* excilamp (Fig. 20.4),
while a DBD on pure xenon at the same pressure and discharge conditions produced
intense broadband Xe2* emissions in the VUV region with a cut-off at l ~ 173 nm
due to the low transmittance of the quartz bulb. The XeI* and I2* emissions under
the present conditions do not fall to the background level but form a continuum in
the range 190360 nm. This might be important for practical applications of the
XeI* excilamp. Similar features have been reported before [16]. Recently, I2* emis-
sions produced by the DBD-driven excilamps were studied in detail [17, 18].
20.3.4 Effect of Excitation Mode on the UV Output

and Efficiency
For all the excitation modes, the UV radiation power rises almost linearly with
increasing the input electrical power under the present discharge conditions
(Fig. 20.5a). Figure 20.5b shows the scaling of the excilamp efficiency with the
input power. The maximum efficiency obtained using pulsed unipolar excitation
with short pulse edges (PS2, PS3) is significantly (1.73.7 times) greater than that
obtained using AC excitation. In order to get the highest efficiency possible, the
voltage rising and falling times should be as short as possible. The operating fre-
quency affects the discharge appearance, and, as a result, the efficiency of the excil-
amp. The average radiation power of 10.3 mW/cm2 at the lamp surface was achieved
at f = 80 kHz.
20.4 Discussion
The presented results show that the enhanced performance of the XeI* excilamp is
derived from a discharge which is more diffuse than that achieved at the same Xe/I2
mixture pressures using AC excitation. The spatial distribution, UV power and effi-
ciency of the DBD-driven XeI* excilamp can be improved significantly by utilizing
pulsed excitation. Moreover, even for pulsed excitation the conversion efficiency is
very sensitive to the duration of voltage rising and falling times or, in other words,
Fig. 20.5 (a) UV power and 1 - AC, f = 60 kHz

(b) efficiency of the XeI* 2 - Pulsed Bipolar, f = 60 kHz
excilamp as a function of 10 3 - Pulsed Unipolar, f = 10 kHz
input power under different 4 - Pulsed Unipolar, f = 60 kHz
UV Power (mW/cm2)
excitation modes: AC, 8
f = 60 kHz (1), pulsed bipolar
PS1, f = 60 kHz, 50% duty 6
(2), pulsed unipolar PS2, 2
f = 10 kHz, 50% duty (3), 4 3 4
and pulsed unipolar PS3, 1
f = 60 kHz, 50% duty (4). 2
Xe/I2 = 13.3/0.04 kPa mixture a
0
Efficiency (%) 12
8 3
4
4 2
1 b
0
0 4 8 12 16 20
Input power (W)
to the dU/dt. Small increases of Dtrise by about 100 ns (for pulsed excitation) cause
changes in the discharge pattern and UV output. Moreover, for pulsed operation the
current amplitude is determined by the pulse edge steepness. The application of
fast-rising square voltage pulses to the discharge gap allows the discharge to occur
more homogeneously throughout the entire active length of the excilamp.
Comparison of the electrical characteristics (UI and UQ) depicted in Fig. 20.2
and excilamp efficiency (Fig. 20.5) show that conditions for high efficiency are
obtained for the excitation modes corresponding to the near vertical lines for On
periods in the UQ Lissajous figures.
The mechanism of efficiency enhancement compared to that presented in [4, 6]
has been described in [19]. Excellent coincidence in the temporal behavior of the
XeI*(BX) emission and the Xe(1 s5) absorption has proved that in DBD under the
present conditions a harpoon reaction is the dominant source of XeI*(B) population.
We have diminished the effect of the unwanted formation of Xe2*(3S) and Xe2I* by
operating in lower xenon pressures p and with a larger discharge gap d in order to
sustain the optimal value of the pdparameter. Neither Xe2* nor Xe2I* emission was
registered in the spectrum of the developed XeI* excilamp with the optimized work-
ing mixture (Fig. 20.4). This is why we used a gap 2.12.6 times larger than in [6].
We think that it is one of the main sources of efficiency improvement compared to
the values presented in [6]. Another reason is the use of pulsed excitation with shorter
Fig. 20.6 Glass plates w/o

treatment (left) and after
1 min. UV irradiation by
means of XeI* exciplex
lamp (right)
Fig. 20.7 Normalized Xe/I2=13.3/0.04 kPa, f = 60 kHz

100
number of CFU (B. subtilis B. subtilis spores, 104 CFU/ml
spores) as a function of the XeI* 4 cm, flow reactor
cumulative UV dose from the 101
CFU (normalized)
XeI* excilamp. Distance to

the lamp: 4 cm and 1 l of
flowing water. Bipolar pulse, 102
Upp = 4.2 kV, f = 60 kHz, 50%
duty cycle
103
104
0.0 0.4 0.8 1.2 1.6

UV Dose (J/cm2)
rising and falling times. We can assume that under these excitation modes (PS2 and
PS3) the most appropriate discharge conditions (mean electron energy, electron tem-
perature, electron energy distribution function (EEDF)) are created for the conver-
sion of ground state Xe atoms into low-level Xe*(6 s) states and to prevent energy
losses associated with stepwise excitation, ionization and ion heating [20].
The developed XeI* DBD excilamp can be successfully used for practical appli-
cations, such as UV cleaning, surface treatment and UV sterilization. The glass
plates were chosen as the test objects for the UV surface cleaning. It can be seen
from the Fig. 20.6 that after 1 min irradiation the water drop on the glass surface has
a contact angle about three times less in comparison with the untreated surface.
It means, the UV radiation from the XeI* lamp destroyed the oil film on the surface
and can be used for the UV cleaning.
Figure 20.7 shows the changes in the normalized number of CFU in the water
flow reactor as a function of the cumulative UV dose of XeI* excilamp, emitted dur-
ing the UV treatment. One experiment was carried out when the volume of irradiated
water was covered with a plate preventing the penetration of foreign microflora from
outside, and another one without a cover. Almost the same CFU reduction was
observed in both measurements. In all experiments, an increase in the exposure time

and UV dose always resulted in CFU decrease. The D-value (one-order reduction)
was about 0.4 J/cm2. It can be seen from Fig. 20.7 that a reduction by more than
4 orders of magnitude of CFU was achieved at the cumulative UV dose from the
excilamp about 1.7 J/cm2. Note that in the steady-state mode, a reduction of CFU by
more than six orders of magnitude was achieved when the UV dose was about
4045 mJ/cm2 and the D-value for the XeI excilamp was about 59.5 mJ/cm2 [1].
An additional effect of I* emission in the range of 178188 nm and at 206.2 nm has
been confirmed. We can assume that such a strong effect of the short-wavelength part
of the XeI* excilamp emission spectrum and, consequently, better sterilization action
are observed because the absorption of UV radiation by both the DNA and spore coat
protein of B. subtilis is the highest for l < 230 nm [21]. The higher UV doses, needed
for inactivation of B. subtilis spores in the water flow reactor in comparison with the
steady state mode, can be explained by the short reaction time. This is also the pos-
sible reason for the presence of a threshold at about 0.1 J/cm2 in CFU reduction.
Taking into account the water flow circulating velocity, the cross section ratio of the
tubing and the water layer on the plate, and the plate length, we can estimate that the
total exposure time of the same portion of water is about 49 s for a 30 min irradiation,
since the processing time of water per single pass in the water flow reactor is 0.3 s
and the same portion is irradiated 162 times during 30 min irradiation. Then, taking
into account the irradiation of 0.97 mW/cm2 at the distance of 4 cm from the lamp,
we can calculate that the UV dose, which reaches the same portion of water, does not
exceed 4042 mJ/cm2. This is close to the energy range of the steady state mode [1].
Note that it is possible to improve the reactor design placing the lamp along the water
stream and using a set of the excilamps. Thus, the latter experiment has shown the
possibility to use the XeI* excilamp (or a set of the excilamps) for water decontami-
nation in flow systems.
The advantage of the developed XeI excilamp in comparison with the low-
pressure mercury lamps should be noted. Microorganisms can adapt to the irradia-
tion of the monochromatic mercury lamp at a definite wavelength and generate
resistant mutants. However, it is more difficult to adapt to the polychromatic emis-
sion of the excilamp [22].
20.5 Summary
A detailed experimental study of the characteristics of a XeI* DBD excilamp excited

by AC sinusoidal, pulsed bipolar and unipolar voltage waveforms was carried out.
Pulsed excitation enhances XeI* exciplex production. A preferred operating mode
for optimizing the atomic iodine output, in the VUV and deep UV ranges, was
determined requiring the operating frequency to be increased. For an air cooled
system, a UV radiation power density of 10.3 mW/cm2 was obtained under pulsed
excitation at f = 80 kHz. The conversion efficiency from electrical input to maxi-
mum UV output under pulsed unipolar excitation (PS2) with edge steepness of
~50 ns was 9%, which is about 2.7 times greater than the maximum efficiency under
AC excitation (3.3%). In order to reach the highest efficiency, the voltage rising and
falling times should be as short as possible, and plasma breakdown has to occur
close to the peak of the voltage pulse.
The sterilization action of the DBD-driven XeI* excilamp was tested on the inac-
tivation of B. subtilis spores. A reduction by more than 4 orders of magnitude of
CFU in B. subtilis spores was achieved in a water flow reactor and the D-value was
about 0.4 J/cm2. An additional effect of the I* radiation at 206 nm and in the VUV
range (178188 nm) was confirmed. This research demonstrates that the DBD-
driven XeI* excilamp can be used for the UV cleaning and inactivation of microor-
ganisms in movable systems (drinking water treatment or food package
sterilization).
References
1. Motomura H, Guivan MM, Jinno M (2009) Development of DBD-driven xenon iodide excil-
amp and attempt for its sterilization application. In: Proceedings of the international light
sources workshop LSW 8, Jhong-Li, Taiwan.
2. Gellert B, Kogelschatz U (1991) Generation of excimer emission in dielectric barrier dis-
charges. Appl Phys B 52:1421
3. Zhang JY, Boyd IW (1998) Efficient XeI* excimer ultraviolet sources from a dielectric barrier
discharge. J Appl Phys 84:11741178
4. Voronov A, Reber S, Schilling FJ (2006) High power XeI* excimer lamps: technology and
perspectives. In: Proceedings of the XVI international conference on gas discharges and their
applications, Xian, China, pp 601602
5. Sosnin EA, Oppenlnder T, Tarasenko VF (2006) Applications of capacitive and barrier dis-
charge excilamps in photoscience. J Photochem Photobiol C Photochem Rev 7:145163
6. Carman RJ, Ward BK, Mildren RP, Kane DM (2007) An experimental and modeling study of
efficiency for a 253 nm xenon iodide lamp excited by dielectric barrier discharge. In:
Proceedings of the 11th international symposium on the science and technology of light
sources LS-11, Shanghai, China, pp 271280
7. Guivan MM, Motomura H, Jinno M (2008) Xenon iodide formation in dielectric barrier dis-
charge excilamp. Extended Abstracts (the 69th Autumn Meeting), The Japan Society of
Applied Physics, Nagoya, 1, p 186
8. http://www.heraeus.de/
9. Lomaev MI, Sosnin EA, Tarasenko VF, Shitts DV, Skakun VS, Erofeev MV, Lisenko AA
(2006) Capacitive and barrier discharge excilamps and their applications (Review). Instrum
Exp Tech 49:595616
10. Manley TC (1943) The electric characteristics of the ozonator discharge. Trans Electrochem
Soc 84:8396
11. Akishev YuS, Demyanov AV, Karalnik VB, Pankin MV, Trushkin NI (2001) Pulsed regime
of the diffusive mode of a barrier discharge in helium. Plasma Phys Rep 27:164171
12. Volkova GA, Zvereva GN (2004) Analysis of the parameters of a barrier discharge in Kr-I2 and
Xe-I2 mixtures. Opt Spectrosc 96:373381
13. Guivan MM, Malinin AN (2007) Spectral characteristics of a broadband exciplex spontaneous
emission source upon mixtures of the cadmium dibromide vapor with gases. Opt Spectrosc
102:376381
14. Kogelschatz U (2003) Dielectric-barrier discharges: their history, discharge physics, and
industrial applications. Plasma Chem Plasma Proc 23:146
15. Shuaibov AK, Shimon LL, Hrabova IA (2004) Emissive characteristics of small density elec-
tro-discharge plasma on mixes of inert gases. J Phys Stud 8:338345
16. Frame JW, John PC, DeTemple TA, Eden JG (1998) Continuous wave emission in the ultravio-
let from diatomic excimers in a microdischarge. Appl Phys Lett 72:26342636
17. Avdeev SM, Sosnin EA, Tarasenko VF (2007) Optical characteristics of plasma of I*2, Cl*2,
Br*2 halogen dimer barrier-discharge excilamps. Opt Spectrosc 103:526532
18. Avdeev SM, Zvereva GN, Sosnin EA (2007) Investigation of the conditions of efficient I*2
(342 nm) luminescence in a barrier discharge in a Kr-I2 mixture. Opt Spectrosc 103:910919
19. Guivan MM, Motomura H, Jinno M (2008) Excitation of xenon iodide in DBD-driven excil-
amp. The second central European symposium on plasma chemistry II CESPC, Brno, Czech
Republic, pp 6566
20. Carman RJ, Mildren RP (2003) Computer modelling of a short-pulse excited dielectric barrier
discharge xenon excimer lamp (l 172 nm). J Phys D Appl Phys 36:1933
21. Harm W (1980) Biological effects of ultraviolet radiation. Cambridge University Press,
Cambridge
22. Sosnin EA, Lavrenteva LV, Yusupov MR, Masterova YV, Tarasenko VF (2002) Inactivation
of Escherichia coli using capacitive discharge excilamps. In: Proceedings of the 2nd interna-
tional workshop on biological effects of electromagnetic fields, Rhodes, Greece, pp 953957
Part IV
Plasma Tissue Treatment
and Wound Healing
Chapter 21
Antisepsis of the Skin by Treatment with
Tissue-Tolerable Plasma (TTP): Risk
Assessment and Perspectives
Jrgen Lademann, Heike Richter, Alexa Patzelt, Martina C. Meinke,

Joachim W. Fluhr, Axel Kramer, Klaus-Dieter Weltmann,
and Olaf Lademann
Abstract The application of tissue tolerable plasma (TTP) is well suited for
disinfection of living tissue. In particular, when treating chronic wounds, it has
several advantages in comparison to the classical application of antiseptics, which
do not penetrate sufficiently into the tissue or inhibit wound regeneration. The mode
of action of the plasma is mainly based on synergetic effects between temperature
increase and the formation of free radicals, which destroy the bacteria and fungi.
In the present paper a risk assessment of TTP in dermatology is given. The inves-
tigations have been carried out with an atmospheric pressure plasma-jet working
with Argon as a discharge medium. It was found that during the plasma treatment of
tissue, the antioxidative potential is reduced only in the upper part of the stratum
corneum, but not in deeper cell layers. Selecting the optimum parameters of the
plasma formation, the UV exposure of the skin is less than in the case of UV irradia-
tion of the sun on a summer day at noon.
If the duration of the plasma treatment of the skin is in the optimal range for
wound healing, no thermal damage has to be expected.
Additionally, it could be demonstrated that plasma is able to reach the follicular
reservoir for antisepsis where germs are located.
J. Lademann (*) H. Richter A. Patzelt M.C. Meinke J.W. Fluhr

Department of Dermatology and Allergology, Charit Universittsmedizin Berlin,
Charitplatz 1, 10117 Berlin, Germany
e-mail: juergen.lademann@charite.de
A. Kramer O. Lademann
Institute of Hygiene and Environmental Medicine, Ernst-Moritz-Arndt University,
Walther-Rathenau-Str. 49a, Greifswald, Germany
K.-D. Weltmann
Department of Internal Medicine, Helios Klinik, Bad Saarow, Germany
282 J. Lademann et al.
21.1 Introduction
The life expectancy of the population in industrial countries is continuously

increasing, as a result of improved living conditions. Older people frequently
suffer from chronic wounds caused by various diseases, for instance, venous
dysfunction, diabetes mellitus, pressure ulcers, arterial circulatory disorder
[1]. Regardless of the fact that several highly efficient antiseptics are
commercially available, the antiseptic treatment of chronic wounds still remains
a problem, because of deep infections. Antisepsis is often insufficient under
these conditions, due to the occurrence of scabbing and crusting of the
wound [2].
In the past, electrical plasma discharges have been frequently used in biometrical
science for disinfection and sterilization of material surfaces [3]. Plasma systems
usually have a temperature of several hundred degrees. Recently, it was reported
that cold plasma (Tissue Tolerable Plasma TTP) could be applied onto living
tissue. In in vitro studies on cell culture, it could also be demonstrated that this new
plasma possesses excellent antiseptic properties [4, 5].
In the present paper, a risk assessment of cold plasma in dermatology is
given.
In principle, there are three plasma properties, which should be evaluated under
the aspect of safety:
1. As the result of a gaseous discharge, radicals are produced on the skin surface.
The human skin is continuously exposed to free radicals, which are produced
by environmental factors such as UV sun irradiation [6]. The human skin has
developed a protection system against the destructive action of these highly
reactive molecules in form of the antioxidative network. Therefore, the influ-
ence of the plasma on this antioxidative network of the skin was
investigated.
2. During the plasma formation, also UV-irradiation is produced [7]. The emission
spectrum of the plasma on the skin surface and in different depths of model
tissue (pig ear skin) was investigated depending on different physical parameters
influencing the formation of the plasma, such as gas pressure, discharge
voltage.
3. In a series of experiments, the influence of the duration of the plasma treatment
on the temperature of the skin surface was analyzed relating to different physical
properties influencing the plasma parameters (e.g., gas pressure, discharge
voltage).
Additionally, the action mechanism of the plasma was investigated concerning

the high efficiency in skin antisepsis [810]. Lange-Asschenfeldt et al. [11] could
demonstrate that the germs are not only located on the skin surface, but also in the
hair follicles, from where they re-colonize the skin surface after antisepsis. For a
highly efficient disinfection of the skin, the destruction of the germs in the hair
follicles is essential.
21 Antisepsis of the Skin by Treatment with Tissue-Tolerable Plasma (TTP) 283
21.2.1 Tissue Samples and Volunteers
All investigations excluding the analysis of the antioxidants were performed on pig
ear skin, which is a suitable model for human tissue [12]. Fresh pig ears had been
obtained from a nearby butcher. Before being used in the experiments, the pig ears
were washed with cold water and dried with paper towels. Sliced skin samples, with
a thickness of 200 mm and a size of 3 3 cm, were obtained from the pig ears by
using the Dermatom GA 148 (AESCULAP AG and Co. KG, Tuttlingen,
Germany).
Approval for the experiments had been obtained from the Veterinary Board,
District of Berlin-Koepenick.
The analysis of the influence of the plasma treatment of the antioxidants was
carried out in vivo on six volunteers, because it was demonstrated previously that
the radical formation in necrotic tissue is strongly reduced in comparison to the
in vivo situation. The volunteers were aged between 28 and 47 years. Approval for
the study had been obtained from the Ethics Committee of the Charit -
Universittsmedizin Berlin, Germany.
21.2.2 Plasma-Jet
In the present study, the plasma-jet (kinpen 09) was utilized, which had been
developed at the Institute of Plasma Physics, Greifswald, Germany and manufac-
tured by Neoplas GmbH [13, 14]. The plasma-jet has been described in detail previ-
ously [4, 14]. Argon gas was used as a discharge medium in the plasma-jet. The gas
flow was 4 standard liters per minute (slm). The applied voltage was 170 V. The top
of the plasma stream acts in the mode of a brush during the treatment of the skin.
The plasma stream had a length of 17 mm, whereas the plasma-tissue interaction
zone was approximately 1 mm in diameter, depending on the nozzle-to-tissue dis-
tance. In Fig. 21.1, the nozzle of the plasma-jet and the plasma stream are shown.
The device kinpen 09 has CE-Certification for fulfilling the standard of
electrical safety in humans.
21.2.3 Tape Stripping
To evaluate the plasma emission in the tissue, the spectra of the plasma beam were
analyzed behind layers of corneocytes removed by tape stripping and behind sliced
skin. Tape stripping is a well suited method for removal of single cell layers of the
stratum corneum of the skin [15]. Adhesive films (Tesa Film No. 5529, Beiersdorf
Fig. 21.1 Plasma-jet in operation
AG, Hamburg, Germany) were applied and successively removed from the same
skin area. By repeating this procedure, the stratum corneum could be removed
sequentially, cell layer by cell layer.
21.2.4 Spectral Analysis of the Plasma
The spectrum of the plasma emission was analyzed in the spectral range between
200 and 600 nm, using a fiber-based spectrometer EPP2000 (SI Scientific Instruments
GmbH, Gliching, Germany). The optical quartz fiber (Laser- und Medizin-
Technologie GmbH, Berlin, Germany) contains a scattering body at the fiber tip,
which collects the surrounding light and transfers it to the fiber.
21.2.5 Temperature Measurements
The temperature of the plasma stream on the skin surface was determined using a
digital thermometer GTH 1200A (Greisinger electronic GmbH, Regenstauf,
Germany). The measuring spot was approx. 1 mm in diameter.
21.2.6 Determination of the Disinfection Efficiency

of the Skin Surface
The disinfection efficacy of TTP was compared to a standard liquid antiseptic

(octenidine). The bacterial colonization was investigated prior and subsequent to
skin treatment with octenidine and TTP. The investigations were carried out on skin
areas, which remained untreated, on skin areas treated with the antiseptic octenidine
and on TTP treated skin. Immediately after treatment, the procedure for the deter-
mination of the amount of bacteria on the skin surface started in accordance with the
Williamsons protocol [16].
21.2.7 Determination of the Disinfection Efficiency

in the Hair Follicles
For an efficient disinfection it is necessary that plasma is able to reach the follicular
reservoir to destroy the bacteria and fungi, which are localized in this area. The
investigations were performed on porcine skin by using a solution containing a
chlorophyll dye. The fluorescent properties of the dye changed during the plasma
tissue interaction, as described by Lademann [10].
Histological sections were obtained from biopsies removed from the non-treated
and plasma-treated tissue samples. The fluorescence signal of the dye in the hair
follicles was analyzed in both cases using laser scanning microscopy.
21.2.8 Laser Scanning Microscopy
The structure of the skin surface of the tissue samples, before and after plasma treat-
ment, was analyzed by laser scanning microscopy (Stratum, Optilas Ltd.,
Melbourne, Australia). The aim of these investigations was to investigate whether
there is a thermal damage to the skin surface after plasma treatment. The laser scan-
ning microscope consists of a base station containing the excitation laser (argon
laser, l = 488 nm) and the spectrometer in the control unit [17]. The base station is
connected by optical fibers to the hand piece, where the optical imaging system and
the focus control unit are positioned. The maximal penetration depth of the radia-
tion into the tissue was approximately 150 mm, which implies that the skin could be
analyzed up to the upper papilla structure [17]. A detailed description of the laser
scanning microscope is given by Kandarova et al. [18].
21.2.9 Raman Microscopic Analysis of the Radical Formation

During Plasma Treatment
The radical formation during plasma treatment was analyzed indirectly by deter-
mining the concentration of the carotenoids in the skin.
These investigations were undertaken in vivo on the forearms of healthy human
volunteers, because the radical formation in necrotic tissue is strongly reduced on
account of its low oxygen concentration. The radicals formed in the tissue react with
the antioxidants, including the carotenoids. These highly reactive molecules are
neutralized by the antioxidants, before they can damage cells or cell compartments.
If the concentration of the free radicals exceeds a critical value, the antioxidants are
destroyed and consequently their concentration is reduced. By means of this proce-
dure, the carotenoids can be used as marker substances for radical formation in the
skin.
The carotenoid concentration was determined in different depths of the stratum
corneum by means of the Raman laser scanning microscope (River Diagnostics,
Rotterdam, Netherlands)
21.3.1 Risk Assessment
Using the optimal operation parameters of the plasma-jet, the risk aspects were
evaluated regarding UV-radiation, temperature effects and radical formation.
The emission spectrum of the plasma consists of one intensive line at 310 nm and
several smaller bands between 325 nm and 450 nm (Fig. 21.2). The emission signal
at 310 nm results from the OH radicals. Using the described plasma-jet system with
Argon as a charge medium, no emission bands at wavelengths lower than 300 nm
could be detected. The UV light at 310 nm is efficiently absorbed by the stratum
corneum of the skin. A single corneocyte layer removed by tape stripping reduces
the transmittance to 25% of the plasma radiation at 310 nm. The stratum corneum
of the human skin consists of 1525 cell layers, depending on the body site. This
means that almost no UV-radiation reaches the living cells of the skin. This was also
confirmed by transmission measurements of sliced skin [19].
The same spectroscopic arrangement, which was applied in the plasma
experiment, was used to measure the intensity of the sun radiation, on a sunny day
in March at noon. Comparing the sun radiation and the plasma emission, it was
found that the radiation dose produced by the plasma-jet at 310 nm was one order
of magnitude below the minimal erythema dose, necessary for the formation of skin
damage detectable as sunburn.
The temperature determined during the plasma treatment on the skin surface was
between 35C and 45C depending on the moving velocity. Changes in the cellular
structure of the stratum corneum caused by thermal effects could be detected only
at low moving velocities in the first two cell layers of corneocytes. This demon-
strates that the thermal action of the plasma is highly superficial and is limited to the
stratum corneum without affecting the living tissue.
Analyzing the carotenoid concentration in different depths of the stratum
corneum, before and after plasma treatment, it was found that the carotenoids were
partly destroyed only in the upper cell layers of the stratum corneum. In the
corneocyte layers close to the living epidermis no changes in the carotenoid level
could be observed [20]. This means that the radical formation is a superficial effect.
200
180
160
Intensity [arb. units]
140
120
100
80
60
40
20
0
200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600
Wavelength [nm]
Fig. 21.2 Emission spectrum of the plasma-jet under optimal conditions
The radicals are produced in high concentrations on the skin surface where the bac-
teria and fungi are located. In Fig. 21.3, the typical change of the carotenoids con-
centration in the stratum corneum of one volunteer prior to and after plasma
treatment is demonstrated. Additionally, as shown by histological investigations the
plasma penetrates effectively into the hair follicles [21], where radical formation
processes can be expected as discussed below.
21.3.2 Efficacy of Disinfection
In the disinfection experiments, the plasma-tissue interaction zone had a diameter of

approximately 2 mm. The plasma beam was moved at an average velocity of
10 mm/s over the porcine tissue samples. This movement corresponds to the opti-
mal velocity of the plasma, as reported previously [7]. For better comparison of the
results, the absolute numbers of bacterial colonies determined on the untreated skin
areas of every tissue sample were standardized to 100%.
Both the disinfection of the skin surface with octenidine and the treatment with
plasma-jet led to a significant reduction in the bacterial colonization, which was on
average 52 colonies per cm2 (p < 0.05). The application of octenidine (99% disinfec-
tion efficacy) led to an increased reduction of bacteria in comparison to TTP (94%
disinfection efficacy). Figure 21.4 shows two agar plates (Merck AG, Darmstadt,
Germany) demonstrating the bacterial growth on samples before (Fig. 21.4a) and
after (Fig. 21.4b) plasma treatment. In Fig. 21.5 the efficiency of disinfection after
treatment with octenidine and after plasma treatment is presented. These differences
140
before plasma treatment
after plasma treatment
120
Carotenoid concentration [m]
100
80
60
40
20
0
0 2 4 6 8 10 12 14
Depth of the SC [m]
Fig. 21.3 Carotenoid concentration in the stratum corneum prior to and after plasma treatment
Fig. 21.4 Determination of the bacterial colonies on agar plates before (a) and after (b) plasma
treatment
can be explained by the small size of the plasma beam of 2 mm. On account of this
small diameter, a thorough and homogeneous disinfection of the complete skin sur-
face seems difficult. It cannot be excluded that small residues on the skin surface
remained unaffected by the plasma stream during treatment. Consequently, in all
probability, bacteria may have survived in these skin areas leading to an increased
number of bacterial colonies.
120
100
Efficancy of disinfection [%]
80
60
40
20
0
Octenidine Plasma jet
Fig. 21.5 Efficacy of disinfection after in vivo treatment of the skin with octenidine and plasma
Analyzing the fluorescence characteristics of the chlorophyll dye in the hair

follicles, it could be demonstrated that TTP penetrates deeply into the hair follicles,
whereupon the hairs act as a conductor for the plasma [21]. The plasma is dispersed
on the surface extending deeply into the hair follicle. Therefore, it can be concluded
that bacteria and fungi located in the follicular reservoir are destroyed more effi-
ciently by the plasma than by conventional liquid antiseptics, which do not penetrate
efficiently into the hair follicles.
21.4 Conclusions
The treatment of the skin with tissue tolerable plasma is a highly efficient method
for skin disinfection. The diameter of the plasma-tissue interaction zone must be
selected in such a manner that the tissue surface is homogeneous and completely
treated. In comparison to classical application of antiseptics, the TTP has the
advantage of disinfecting also the hair follicles, which are a reservoir for bacteria
and fungi.
The temperature increase on the skin surface during the plasma treatment is
moderate. The thermal damage was observed or was related only to the upper cell
layers of the stratum corneum. The UV exposure of the skin by the investigated
plasma treatment was one magnitude below the erythema level. Also, the radical
formation during the plasma treatment is located only on the skin surface, sufficient
enough to destroy the bacteria and fungi. However, the radicals do not damage the
living epidermis. Consequently, TTP has a high perspective in medicine for disin-
fection and wound healing. No risk potentials are to be expected for the patient
when the system investigated in this study is applied in vivo.
Acknowledgments This work was realized within the framework of the multi-disciplinary
research cooperation of Campus PlasmaMed, particularly within the project PlasmaWound.
The authors acknowledge that this work was supported by a grant funded by the German Ministry
of Education and Research (BMBF, Grant No, 13 N9779).
References
1. Phillips TJ (2001) Current approaches to venous ulcers and compression. Dermatol Surg
27(7):611621
2. Sekkat N, Kalia YN, Guy RH (2002) Biophysical study of porcine ear skin in vitro and its
comparison to human skin in vivo. J Pharm Sci 91(11):23762381
3. von Woedtke T, Kramer A, Weltmann KD (2008) Plasma sterilization: what are the conditions
to meet this claim? Plasma Process Polym 5(6):534539
4. Weltmann KD, Kindel E, Brandenburg R, Meyer C, Bussiahn R, Wilke C, von Woedtke T
(2009) Atmospheric pressure plasma jet for medical therapy: plasma parameters and risk esti-
mation. Contrib Plasma Phys 49(9):631640
Chem 82(6):12231237
6. Zastrow L, Groth N, Klein F, Kockott D, Lademann J, Renneberg R, Ferrero L (2009) The
missing linklight-induced (2801,600 nm) free radical formation in human skin. Skin
Pharmacol Physiol 22(1):3144
Hartmann B, Ottomann C, Fluhr JW, Hinz P, Hubner G, Lademann O (2009) Risk assessment
of the application of a plasma jet in dermatology. J Biomed Opt 14(5):054025
8. Lademann O, Richter H, Patzelt A, Alborova A, Humme D, Weltmann KD, Hartmann B,
Hinz P, Kramer A, Koch S (2010) Application of a plasma-jet for skin antisepsis: analysis of
the thermal action of the plasma by laser scanning microscopy. Laser Phys Lett
7(6):458462
9. Hammann A, Huebner NO, Bender C, Ekkernkamp A, Hartmann B, Hinz P, Kindel E, Koban I,
Koch S, Kohlmann T, Lademann J, Matthes R, Muller G, Titze R, Weltmann KD, Kramer A
Skin Pharmacol Physiol 23(6):328332
10. Lademann O (2011) Antisepsis of the follicular reservoir by treatment with tissue-tolerable
plasma (TTP). Laser Phys Lett 1-5. doi:10.1002/lapl.201010123
11. Lange-Asschenfeldt B, Alborova A, Kruger-Corcoran D, Patzelt A, Richter H, Sterry W,
Kramer A, Stockfleth E, Lademann J (2009) Effects of a topically applied wound ointment on
epidermal wound healing studied by in vivo fluorescence laser scanning microscopy analysis.
J Biomed Opt 14(5):054001
12. Meyer W, Schwarz R, Neurand K (1978) The skin of domestic mammals as a model for the
human skin, with special reference to the domestic pig. Curr Probl Dermatol 7:3952
13. Foest R, Kindel E, Ohl A, Stieber M, Weltmann KD (2005) Non-thermal atmospheric pressure
discharges for surface modification. Plasma Phys Control Fusion 47:B525B536
14. Weltmann KD, Brandenburg R, von Woedtke T, Ehlbeck J, Foest R, Stieber M, Kindel E
sure plasma jets (APPJs). J Phys D Appl Phys 41(19):194008
15. Lademann J, Jacobi U, Surber C, Weigmann HJ, Fluhr JW (2009) The tape stripping procedure
evaluation of some critical parameters. Eur J Pharm Biopharm 72(2):317323
16. Williamson PT, Roth JF, Haddingham T, Watts A (2000) Expression and purification of recom-
binant neurotensin in Escherichia coli. Protein Exp Purif 19(2):271275
17. Martschick A, Teichmann A, Richter H, Schanzer S, Antoniou C, Sterry W, Lademann J

(2007) Analysis of the penetration profiles of topically applied substances by laser scanning
microscopy. Laser Phys Lett 4(5):395398
18. Kandarova H, Richter H, Liebsch M, Lademann J (2007) Stratum corneum architecture of
reconstructed human skin models monitored by fluorescent confocal laser scanning micros-
copy. Laser Phys Lett 4(4):308311
19. Lademann J, Jacobi U, Richter H, Otberg N, Weigmann HJ, Meffert H, Schaefer H, Blume-
Peytavi U, Sterry W (2004) In vivo determination of UV-photons entering into human skin.
Laser Phys 14(2):234237
20. Fluhr JW, Sassning S, Lademann O, Darvin ME, Schanzer S, Kramer A, Richter H, Sterry W,
Lademann J (2011) The influence of skin treatment with tissue tolerable plasma on the antioxidant
concentration in human skin. Experimental Dermatology. DOI: 10.1111/j.1600-0625.2011.01411.x
21. Lademann O, Kramer A, Richter H, Patzelt A, Meinke MC, Rwert-Huber J, Czaika V,
Weltmann K-D, Hartmann B, Koch S (2011) Antisepsis of the follicular reservoir by treatment
with tissue-tolerable plasma (TTP). Laser Phys Lett 8(4):313317
Chapter 22
Cold Microsecond Spark Discharge
Plasma Production of Active Species
and Their Delivery into Tissue
Danil Dobrynin, Gregory Fridman, Gary Friedman,

and Alexander Fridman
Abstract Mechanisms of the Plasma Medicine techniques, first of all plasma

sterilization and healing of wounds, are immediately related to the effects of reactive
neutral and charged species produced by plasma and delivered to the treated object.
Here we report experimental results on measurement of production of reactive
oxygen species in liquid media and their delivery into tissue by microsecond spark
discharge plasma. We also show that a simple agarose gel model may closely mimic
physicochemical characteristics of tissue.
Keywords Spark discharge Reactive oxygen species Plasma medicine Tissue

model
In recent years, the range of atmospheric pressure applications for medical and
biological purposes is growing fast, and a new field of Plasma Medicine was formed
[15]. Various types of plasmas, both thermal and non-thermal, are now widely
studied for the purposes of blood coagulation [2, 6, 7], sterilization of living tissues
[1, 2, 6], treatment of various wounds and burns [2, 6, 8], gastrointestinal diseases [9],
and even cancer [10]. This opens up new horizons in both medical and physical
D. Dobrynin (*)
Electrical and Computer Engineering Department, Drexel University,
3141 Chestnut Street, Philadelphia, PA 19104, USA
e-mail: dvdobrynin34@gmail.com
G. Fridman G. Friedman A. Fridman
Drexel Plasma Institute, Drexel University, 3141 Chestnut Street,
Philadelphia, PA 19104, USA
e-mail: fridman@drexel.edu
294 D. Dobrynin et al.
sciences, as well as in biomedical and electrical engineering. The success of plasma

medicine depends on developing a deeper understanding of the physics, chemistry,
and biology of plasma-tissue interactions.
Cold atmospheric pressure plasma discharges have been shown to be effective
when applied for wound sterilization and decontamination purposes and wound
healing [2, 5, 8]. Action of specific charged or neutral active species or radiation is
frequently associated with the corresponding specific effect (e.g., anti-inflammatory
effect of nitric oxide (NO), and highly oxidative hydroxyl radical and other reactive
oxygen species (ROS)). Traditionally, production of these biologically extremely
important reactive species have been measured mostly in gas phase, i.e. at the pro-
duction point plasma itself or plasma afterglow. Although these measurements are
undoubtedly necessary and useful, the real effect of plasma treatment is almost
never direct (except for special cases of, for example, low pressure sterilization of
surfaces or polymer treatment), and is often associated with so-called water chem-
istry. Indeed, in the case of wounds, where a large amount of various biological
liquids (e.g. blood, pus) is present, plasma produced reactive species are first react
and diffuse in media, and then for successful activation of signaling pathways of
healing mechanism have to be delivered even further deep into tissues and cells.
Therefore, measurements of production and delivery of plasma produced ROS,
followed by numerical modeling of these processes linked to the plasma source,
seem to be extremely important for the development of fundamental understandings
of the plasma-tissue interaction.
Here we report the results on measurement of simultaneous production of
anti-oxidant NO together with oxidative ROS in liquid media by microsecond
atmospheric pressure spark discharge. We also show, that plasma produced active
species can be transferred several millimeters deep into tissues, and this process can
be successfully simulated by agarose gel tissue phantoms.
In our study we have used DC spark discharge plasma in a pin-to-hole electrode

configuration (PHD plasma, Fig. 22.1), previously described in [11] and [12].
The discharge was ignited by applying high positive potential with magnitude of
about 4 kV to the central electrode. This resulted in a formation of dense ener-
getic discharge which exists for about 35 ms with average energy of about 1.8 J
per pulse.
Measurements of hydrogen peroxide (H2O2), and nitric oxide (NO) produced by
plasma in phosphate buffered saline (PBS) were done using fluorescent dyes,
Amplex UltraRed reagent (Invitrogen), DAF-2 (Cayman Chemical) respectively
according to manufacturers protocols. Superoxide (O2) was measured indirectly
by adding superoxide dismutase (Fisher Scientific) into PBS solution containing
Amplex UltraRed reagent before the plasma treatment. Fluorescence was measured
22 Cold Microsecond Spark Discharge Plasma Production of Active Species 295
Fig. 22.1 General schematic of the Pin-to-Hole spark Discharge (PHD) plasma system and a
photograph of the discharge in operation
using an LS55 (Perkin Elmer) fluorescent spectrometer equipped with well plate
reader accessory.
Agarose gel has been traditionally used to mimic biological substrates like
tissues, skin, cell layers, etc. Although it does not represent real tissue we show
that one is able to alter the gels buffering ability, density and fluidity to closely
resemble tissue. Agarose gels of 1.5% wt are prepared using standard procedure
with pure agar powder (Fisher) in either distilled water or phosphate buffered
saline (PBS, Fisher). Measurements of H2O2 penetration into agarose gels and
tissues were done using Amplex UltraRed reagent (Invitrogen, ex/em:
530/590 nm) fluorescent dye. 75 mL PBS containing 100 mM Amplex UltraRed
with 200 U/mL horseradish peroxidase (MP Biomedicals) were placed in between
1 mm thick 4 4 cm agar slices and incubated for about 15 min before the treat-
ment in order to provide presence of the dye in the agar volume. In order to
measure the H2O2 in tissue, the dye was injected using a syringe into a 1 cm thick
4 4 cm skinless chicken breast tissue samples at various points to the depth of
up to 1 cm. Treated samples were sliced in a vertical direction with thickness of
1 mm, and fluorescence was measured using an LS55 (Perkin Elmer) fluorescent
spectrometer equipped with XY reader accessory (Fig. 22.2). To obtain calibra-
tion curves for hydrogen peroxide in the plasma treated samples a standard sta-
bilized 3% H2O2 (Fisher) water solution properly diluted to obtain various
concentrations was used.
In Fig. 22.3 we report the results of plasma production of H2O2 (with and without
presence of superoxide dismutase), and NO delivery into PBS solution is shown in
Fig. 22.4. In order to ensure that the H2O2 specific dye is not altered by UV radiation
produced by plasma, first measurement was done through a quartz glass. As shown
Fig. 22.2 Chicken breast after plasma treatment with H2O2 fluorescent dye: photograph and fluo-
rescent images from the top and side of the sample (intensity is in arbitrary units, position of the
sample was different during measurements)
Fig. 22.3 Production of hydrogen peroxide and superoxide in PBS (100 ml) by microsecond spark
discharge
Fig. 22.4 Delivery of nitric oxide as measured by fluorescent dye DAF-2 in PBS (100 ml) by
microsecond spark discharge
in Fig. 22.3, the microsecond spark discharge produces a relatively stable amount
of about 40 mM of hydrogen peroxide in the solution. Addition of superoxide
dismutase (SOD) which catalyzes superoxide dismutation reaction
2O2 + 2 H +
SOD
H 2O2 + O2 , allows indirect measurement of O2 (Fig. 22.3). In
Fig. 22.4 we show that together with ROS we measure a significant amount of NO
produced by plasma in the PBS (compared to 8,000 ppm NO balanced with N2 from
a tank, Air Products) Simultaneous production of both ROS and RNS, and specifi-
cally H2O2 and NO may result in a number of biologically important effects. For
example, it has been shown, that presence of both NO and H2O2 may induce
apoptosis in cancerous cells [13], inactivate bacteria [14], and form biologically
important singlet oxygen [15].
The results of H2O2 measurements in chicken breast tissue are shown in
Fig. 22.5: with longer treatment time depth of penetration as well as concentra-
tion of hydrogen peroxide increases. In general, several millimoles per liter of
H2O2 are produced in tissue after plasma treatment, while it diffuses 1.53.5 mm
deep. The measurement results for H2O2 produced by plasma treatment in agar
gels are shown on Fig. 22.5. Hydrogen peroxide concentration on the agar gel
surface was 1.9 mM after 1 min of plasma treatment. The difference of the H2O2
concentration profile may be related to actual structural differences between aga-
rose gel and tissue, in which macroscopic irregularities (e.g. fibers, pores, etc.)
are present.
7
tissue 30s
tissue 60s
6 tissue 120s
120s agar 30s
5 agar 60s
agar 120s
[H2O2], mM
60s
3
2
30s
0
0 1 2 3 4 5 6 7
Depth, mm
Fig. 22.5 Hydrogen peroxide penetration depth in tissue and agarose gel
22.3 Conclusion
The results show that PHD discharge effectively produces both ROS and RNS
species in the media, and active species may be delivered into the tissues to the
depths of several mm, therefore providing not only surface effects (inactivation of
pathogens, first of all), but also therapeutic effects inside of treated tissues. The
agarose tissue model shows that plasma effects may be transferred several millime-
ters deep inside a tissue, as measured in an ex vivo chicken tissue model. We have
detected a penetration behavior of a simplest active component, hydrogen peroxide,
but other species may be detected and measured using other fluorescent dyes readily
available. In addition, we showed that a simple agar gel model may express similar
physicochemical properties as a real tissue, resulting in comparable penetration
effects of active species.
References

plasma medicine. Plasma Process Polym 5(6):503533
3. Kong M, Morfill G, Nosenko T, Shimizu T, van Dijk J, Zimmermann JL (2009) Plasma medi-
cine: an introductory review. New J Phys 11:115012
4. Morfill GE, Kong MG, Zimmermann JL (2009) Focus on plasma medicine. New J Phys
11:115011
5. Laroussi M (2009) Low-temperature plasmas for medicine? IEEE Trans Plasma Sci
37(6):714725
6. Chen Cheng-Yen, Hsin-Wen Fan, Kuo SP, Jenghwa Chang, Pedersen T, Mills TJ, Cheng-Chiu
Huang (2009) Blood clotting by low-temperature air plasma. IEEE Trans Plasma Sci
37(6):993999
7. Kalghatgi SU, Fridman G, Cooper M, Nagaraj G, Peddinghaus M, Balasubramanian M,
Vasilets VN, Gutsol A, Fridman A, Friedman G (2007) Mechanism of blood coagulation by
nonthermal atmospheric pressure dielectric barrier discharge plasma. IEEE Trans Plasma Sci
35(5, Part 2):15591566
8. Kalghatgi S, Friedman G, Fridman A, Clyne A (2010) Endothelial cell proliferation is enhanced
by low dose non-thermal plasma through fibroblast growth factor-2 release. Ann Biomed Eng
38(3):748757
9. Chakravarthy K, Dobrynin D, Fridman G, Friedman G, Murthy S, Fridman A (2011) Cold
spark discharge plasma treatment of inflammatory bowel disease in an animal model of ulcer-
ative colitis. Plasma Med 1(1):319
10. Vandamme M, Robert E, Pesnel S, Barbosa E, Dozias S, Sobilo J, Lerondel S, Le Pape A,
Pouvesle J (2010) Antitumor effect of plasma treatment on U87 glioma xenografts: prelimi-
nary results. Plasma Process Polym 7(34):264273
11. Dobrynin D, Gostev V (2006) Medical microplasmatron. In: Proceedings of the 3rd interna-
tional workshop on microplasmas, Greifswald, Germany, p 108
12. Dobrynin D, Fridman A, Friedman G, Starikovskiy A (2010) Microsecond thermal plasma jet
generation for biomedical applications. In: XVIII international conference on gas discharges
and their applications (GD 2010), Greifswald, Germany, 510 Sept 2010
13. Filep JG, Lapierre C, Lachance S, Chan J (1997) Nitric oxide co-operates with hydrogen per-
oxide in inducing DNA fragmentation and cell lysis in murine lymphoma cells. Biochem J
321:897901
14. Pacelli R, Wink DA, Cook JA, Krishna MC, DeGraff W, Friedman N, Tsokos M, Samuni A,
Mitchell JB (1995) Nitric oxide potentiates hydrogen peroxide-induced killing of Escherichia
coli. J Exp Med 182:14691479
15. Di Mascio P, Bechara E, Medeiros M, Briviba K, Sies H (1994) Singlet molecular oxygen
production in the reaction of peroxynitrite with hydrogen peroxide. FEBS Lett 355:287289
Chapter 23
Surface Dielectric Barrier Discharge
Jet for Skin Disinfection
Yves Creyghton, Rogier Meijer, Paul Verweij,

Frank van der Zanden, and Paul Leenders
Abstract A consortium consisting of the research institute TNO, the medical

university and hospital St Radboud and two industrial enterprises is working on a
non-thermal plasma treatment method for hand disinfection. The group is seeking
for cooperation, in particular in the field of validation methods and potential
standardization for plasma based disinfection procedures. The present paper
describes technical progress in plasma source development together with initial
microbiological data. Particular properties of the sheet shaped plasma volume are
the possibility of treating large irregular surfaces in a short period of time, effective
plasma produced species transfer to the surface together with high controllability of
the nature of plasma species by means of temperature conditioning.
Hand hygiene is an important topic in hospitals and medical practices as it reduces

transmission of infectious diseases. Frequently applied disinfection of hands and
underarms by available alcohol based disinfectants is time demanding, results in a
too dry skin and often causes skin irritation. This research project is aimed at a
standardized plasma-based hand disinfection procedure in accordance with basic
Y. Creyghton (*) R. Meijer

TNO Thin Film Technology, De Rondom 1, POB 6235,
5600HE Eindhoven, The Netherlands
e-mail: yves.creyghton@tno.nl
P. Verweij
UMC St Radboud Nijmegen, Eindhoven, The Netherlands
F. van der Zanden
Bactimm BV, Eindhoven, The Netherlands
P. Leenders
Filtex Air Filtration BV, Eindhoven, The Netherlands
302 Y. Creyghton et al.
disinfection requirements, without drawbacks of current methods, to be safely

applicable and available at affordable cost. Plasma produced in ambient or nitrogen
enriched humid air produces a variety of reactive oxygen and nitrogen species (ROS,
RNS) such as O radicals, the superoxide anion O2 and nitric oxide NO [1]. Directed
to a wet surface, transferred plasma species create secondary reactive products in
solution such as particular acid compounds and peroxides [2].
One must be aware of the fact that those chemical reactive species are not only
known from other types of sources (UV, ionizing radiation) but that in the biomedi-
cal research field many of them are produced by cells themselves [3]. Enzymatic
production of reactive oxygen species naturally occurs in cells and cell membranes.
Selected ROS and RNS may be subject to transmission through cell membranes,
and may have functions in cellular signaling essential for the proper development
and proliferation of cells, wound response and healing [4].
Different types of plasma sources which are under investigation for medical
application can be classified according various criteria such as electric energy cou-
pling modes (microwave, dielectric barrier and arc discharges), gas composition
and flow as well as location of the ionizing high electric field region either in
vicinity of the skin or wound, at remote distance or a combination of both. Though
plasma generation at the skin surface is possible even at low voltage potential, the
remote generation of reactive plasma species is preferable for reasons of increased
safety and reduced dependence of skin conductivity and morphology. In plasma jets
fast transport of neutral and charged reactive species to the substrate is achieved by
means of forced flow. There is growing recognition that using remote plasma jets
with N2-O2 mixtures, transport of ROS and RNS is of critical importance while UV
radiation and electric field induced electroporation play a minor role in microbial
inactivation at the remote surface [5]. However, influence of composition, tempera-
ture and flow of the plasma produced gas on skin/wound disinfection still needs
further investigation in order to standardize the treatment.
A variety of atmospheric pressure plasma sources including surface dielectric
barrier discharges (SDBD) have been applied to surface disinfection in general
and medical skin or wound disinfection in particular [6, 7]. In this paper we pres-
ent the use of an innovative SDBD plasma source with distinctive geometrical
properties [8].
23.1 Plasma Source and Experimental Procedure
The plasma jet source has been constructed using an alumina ceramic tube with an
inner temperature conditioned high voltage conductor. Two parallel flattened sur-
faces of the initially circular tube form two series of a thin electrical gas discharge
spaces with the adjacent exterior electrode as shown in Fig. 23.1. The exterior elec-
trodes have protruding ribs touching the ceramic and serving as starting points for a
large number density of surface dielectric barrier discharges (SDBD) on the ceramic
surface. The surface discharges are created in volume spaces of 5 5 S mm3 where
23 Surface Dielectric Barrier Discharge Jet for Skin Disinfection 303
Fig. 23.1 Cross sectional view of the SDBD electrode structure. The gas flow path is oriented in
perpendicular direction through narrow discharge spaces (5.0 0.5 mm2)
different metal electrode structures allow a lit width S ranging from 50 to 500 mm.
The active length of the electrode is 300 mm. The source offers various options:
It can be operated under a wide variety of electrical conditions (AC + DC, repeti-
tively pulsed), gases (N2-O2, noble gases) and gas flow conditions. SDBD plasma
is stable as function of time and homogeneously distributed in space.
The elongated electrode structure enables fast treatment of a large surface area
using a single unidirectional displacement.
The high speed at moderate gas flow of the thin sheet of plasma activated gas
allows treatment at relatively large distances, up to 50 mm.
Temperature conditioning of the ceramic core allows high power and improved
control of the ratio of reactive nitrogen to reactive oxygen species [RNS]/[ROS].
The high voltage electrode is effectively screened from the earthed external elec-
trodes which can be safely touched under plasma operation.
Since the SDBD plasma jet electrode structure provides a gas flow over its entire
length of 300 mm, it is necessary to remove the surplus of gas from the treating
volume. For this purpose a gas withdrawal system has been provided. This system
consists of two gas sucking elements placed at both sides of the electrode configura-
tion, as shown in Fig. 23.2. Alternatively the sample holder plate is replaced by a
gas permeable screen and gas removal elements are placed below this screen.
Substrates are treated by the plasma jet after placing them on a sample holder
which is positioned on a movable sample holder support plate. The time period of
plasma treatment is controlled by means of the moving speed of the sample. Before
or after plasma treatment the sample can be humidified by passing the sample
through an aerosol spray cabin as shown in Fig. 23.2.
The liquid spray unit (Spray Systems Inc.) provides a 300 50 mm2 wide spray
pattern. The liquid and air pressure conditions of the flat spray type nozzle have
been optimized for homogeneous spraying of <100 mm size mist droplets. The minimum
sprayed liquid density is 0.05 mg/cm2. Liquid density fluctuations over the treating
Fig. 23.2 Experimental configuration showing the relative positions of the SDBD electrode struc-
ture, gas removal elements, the spray cabin and the movable sample holder plate. Parameters D and
d design the vertical and lateral distance of a treating point on a substrate with respect to the centre
of the source nozzle
area are determined at less than 23%. A special shape of the spray cabin allows for
the use of the central most homogeneous part of the spray cone by removal of spray
cabin wall deposited liquid.
The line shaped plasma jet nozzle with 300 mm length (Fig. 23.3) is positioned
at adjustable distance D from the surface to be treated. For microbial inactivation
tests, for instance five stainless steel sample holders (25 mm in diameter) can be
disposed in a row receiving similar treatment from the linear source. In addition, as
shown in Fig. 23.2, a temperature probe and a gas sample point for ozone and NOx
analysis have been included. The BMT930 ozone monitor has been calibrated up to
concentration levels of 20 ppm. It has a 15 s sample measurement time which is
acceptable for obtaining an indication of ozone at the location of the sample when
moving the sample holder plate at a low speed of 1 mm/s. The temperature of the
liquid inside the core of the high voltage electrode is measured with a Neoptix opti-
cal fiber system.
A Pillar CS6030 power supply has been used in combination with a HT3 trans-
former from ITW Surface Treatment to supply voltage and current to the plasma.
The required amplitude of the voltage is 6 kV. For the given capacitance of dis-
charge configuration (~200 pF) the power supply was controllable from 50 to
600 W (maximum frequency was 25 kHz). Initial tests have been performed using
Fig. 23.3 Photograph of the

SDBD plasma jet (without
gas removal elements)
relatively high electrical power conditions in order to demonstrate the feasibility

of treatment of a large surface area (such as the surface of two hands) within a
short period of time. The system has been operated under various conditions but
standard conditions used for results presented in this paper are unless otherwise
mentioned: Slit width S = 0.5 mm, electrode length L = 300 mm, sample holder
plate velocity v = 2 mm/s, plasma power per tube P = 500 W, gas is air with a flow
rate of 40 L/min (distributed at both sides of the ceramic core of the electrode),
the gas removal flow rate is 23 times the gas supply rate. Under those conditions
a temperature increase of the inlet gas flow from 25C to 40C has been deter-
mined while the electrode cooling fluid increased from 20C to 31C at 25 L/h
(~320 W).
23.2 Influence of Gas Dynamics and Its Further Optimization
Measurements of temperature and ozone concentration have been performed using

the Pt100 probe, the ozone sampling point and gas removal elements as shown in
Fig. 23.2. In this case the gas withdrawal rate has been set at 3 times the inlet flow.
The experiments have been done feeding pure nitrogen to the jet as ozone levels
appeared too high for the calibrated range of the available ozone analyzer. As shown,
even the plasma species of nitrogen, probably metastable excited nitrogen N2(A)
and atomic N radicals, provide a significant ozone level. Further, it is demonstrated
that gas removal is effectively performed since at a lateral distance (d in Fig. 23.2)
larger than 30 mm from the source the ozone concentration near the substrate is less
than 1 ppm and at a distance of 100 mm the ozone concentration is below 0.06 ppm
which is below the safety value (Tables 23.1 and 23.2).
Table 23.1 Temperatures (C) as function of vertical distance (D) and horizontal distance (d)
from the plasma jet nozzle
500 W
40 L/min d = 0 mm (C) d = 10 mm (C) d = 30 mm (C) d = 100 mm (C)
D = 0 mm 44.5
D = 20 mm 33.2 23 23 22.8
D = 40 mm 22.3 21 21 20.9
The substrate plate velocity is 1 mm/s
Table 23.2 Ozone concentration (ppmV) as function of vertical distance (D)

and horizontal distance (d) from the plasma jet nozzle
500 W
40 L/min d = 0 mm d = 10 mm d = 30 mm d = 100 mm
D = 0 mm >20
D = 20 mm >20 15.9 8 2.6
D = 40 mm 4.4 1.13 0.34 0.04
In order to design a gas removal system for the application of hand disinfection
in combination with the linear SDBD plasma jet source, the temperature and flow
fields have been calculated using the finite element software package Comsol. An
initial result of a flow field calculation is shown in Fig. 23.4, showing the flow pat-
tern through and around two fingers represented by cylindrical rods at 2 cm distance
of the source nozzle.
In the calculated flow pattern a vortex zone is observed with a gas recirculation
zone directly downstream the slit shaped nozzle. While the flow velocity in the two
parallel electrode slits of the plasma jet reaches initially 2 m/s, the two gas streams
leaving the slits (at 6 mm distance) confine to a single stream with a velocity in the
0.20.4 cm/s range. The flow velocity at the back side of fingers is far below 0.1 m/s
thus showing that movement of hands or various plasma jets are required to obtain
a sufficient treatment of the hand surface.
23.3 Inactivation of Microorganisms
The test surfaces are glass slides (contaminated area = 20 35 mm2) or stainless steel
discs with a diameter of 20 or 25 mm. Prior to use, the slides or discs are cleaned by
placing them in 5% decontaminating solution and sonicated for 15 min. The discs
are rinsed with purified water and sterilized by autoclaving using moist heat (15 min,
121C).
Small agar plates are inoculated with bacteria or spores thereof and treated with
a various number of plasma treatment conditions. From a serial dilutions series,
agar plates are inoculated to a concentration of 105, 104, 103 or 102 cfu per plate. The
Fig. 23.4 Calculated gas flow in between and around two fingers 6 mm apart from each other
and at 20 mm distance from the line shaped nozzle. The calculation assumes plane symmetry
across the plane at the left picture border. The numbers on the left and bottom are in units of meter.
The flow velocity (right hand side bar) is given in m/s. Applied flow condition: 40 L/min per tube
(20 L/min through each slit), 300 mm length. The whole bottom area is used for gas withdrawal
with a total flow rate twice the gas flow rate via the electrode
plates have been incubated and the number of surviving germs is counted. Agar
plates are incubated at 3537C for 2448 h. The data, compared to non treated
samples give an estimate of the bactericidal and sporicidal activity of the treatment.
The colony counts of the positive control are use as baseline to calculate the achieved
reduction. This procedure has applied for E. coli, S. aureus and spores of B. globigii
and B. subtilis.
E. coli and B. globigii have been treated in dry form on glass slides. First results as
shown in Fig. 23.5 indicate that ~20 s treatment (25 mm samples with 2 mm/s) at
10 mm distance from the source nozzle results in a significant reduction of colony
forming units from E. coli. A thin film of distilled water (~0.1 mg/cm2) causes a
dramatic further increase of the inactivation efficiency. Replacing water with a 5%
chloroxylenol solution (Dettol) is shown to cause a small but significant synergetic
effect. A one log decrease of B. globigii spores has been observed in this case as well.
Figures 23.6 and 23.7 show the influence of the percentage oxygen and the treat-
ment time respectively. With a decreasing amount of oxygen, inactivation decreases
for all types of microorganisms, however in pure nitrogen a single log inactivation
is maintained.
Fig. 23.5 Biocidal effects for E. coli treated by a 500 W plasma jet with 2 mm/s sample move-
ment. P, NP design plasma and no plasma treatment, S, NS design spray and no spray treatment.
The percentage oxygen concentration in nitrogen has been varied. The distance D from the source
nozzle is 10 mm. For each condition a minimum and maximum CFU count result are shown,
representing the statistic variation in CFU count results
Fig. 23.6 Microbiological inactivation as a function of O2% in N2-O2 gas mixture. The distance D
from the source nozzle is 15 mm. The log reduction values are average values of five samples
treated simultaneously by the plasma jet system
Fig. 23.7 Microbiological inactivation as a function of substrate velocity. Other conditions are the
same as given with Fig. 23.6
The decreased reduction of microorganisms as a result of lowering the oxygen

percentage in Fig. 23.6 seems contradictory with the results shown in Fig. 23.5
which includes an example of increased reduction while lowering the oxygen con-
centration. The cfu counts presented in Fig. 23.6 are the average counts using a
group of five samples receiving similar treatment. Figure 23.5 shows cfu counts per
sample and we have observed occasionally a larger than 1 log deviation in cfu counts
which may be attributed to a non-homogeneous distribution of gas flow. The inho-
mogeneity is mainly located at the edges of the linear plasma source. Increasing the
sample holder velocity from 2 to 10 mm/s decreases the inactivation effect. However,
no difference is observed between inactivation data obtained at relatively large sub-
strate speed of 10 and 20 mm/s. More dedicated experiments will be required to
understand the dependence of the inactivation degree on the residence time of the
sample in the treating zone. Moreover, there is a need for a better delineation of the
treating zone. Local ozone measurements indicate that not all plasma activated gas
is removed from the sample surface area when it moves away from the direct plasma
treating zone. In general there is a need for determining the nature and the effect of
short living reactive species such as radicals and ions, relative to the effect of more
stable reactive species whether they are present in the gas phase or liquid phase.
23.4 Outlook for Optimization and Application
This initial investigation of the SDBD plasma jet source shows that effective and fast
disinfection treatment of a large three dimensional object such as a human hand is
feasible at acceptable temperatures and in a reasonable short period of time (<2030 s).
One of the process parameters which need further optimization is the gas inflow rate
which can be strongly reduced by making the slit width between the ceramic tube and
the external electrodes of the source much smaller. The combination of mild liquid or
cream disinfectants with plasma dissociation which may cause the creation of addi-
tional reactive species is giving new perspectives. An important effort is still to be made
to control gas flow patterns in a treating device in order to minimize gas consumption,
gas removal and/or circulation without exposing the environment to unacceptable
levels of stable gaseous plasma products such as ozone and nitrogen oxides.
References
1. Nosenko T et al (2009) Designing plasmas for chronic wound disinfection. New J Phys 11.
http://iopscience.iop.org/1367-2630/11/11/115013
2. Kalghatgi S (2011) Effects of non-thermal plasma on mammalian cells. PLoS One 6:e16270
3. Bartosz G (2009) Reactive oxyfen species: destroyers or messagers? Biochem Pharmacol
77:13031315
4. Ross C et al (2006) Involvement of reactive oxygen species and reactive nitrogen species in the
wound response of Dasycladus vermicularis. Chem Biol 13:353364
5. Liebmann J et al (2011) Biological effects of nitric oxide generated by an atmospheric pressure
gas-plasma on human skin cells. Nitric Oxide 24:816
6. Lee HW et al (2011) Modelling of atmospheric pressure plasmas for biomedical applications. J
Phys D Appl Phys 44:127
7. Gadri RB et al (2000) Sterilization and plasma processing of room temperature surfaces with a
one atmosphere uniform glow discharge plasma. Surf Coat Technol 131:528542
8. Creyghton Y et al (2008) A surface dielectric barrier discharge plasma unit and a method of
generating a surface plasma. Patent application number WO2008082297
Chapter 24
Cold Atmospheric Plasma for Clinical
Purposes: Promising Results in Patients
and Future Applications
Georg Isbary
Abstract Infected chronic wounds are both socioeconomic and medical problem.
Cold atmospheric plasma (CAP) has already proven its efficacy in killing bacteria on
agar plates but also the first prospective randomized controlled trial in patients. As an
add-on therapy CAPs proved a highly significant decrease in bacterial load in 5 min
plasma-treated wounds (34%, p < 106, n = 291, 36 patients) in comparison with
wounds that received only standard wound care. This reduction is found in all kinds
of germs, even multiresistant ones. Two minutes of plasma treatment led to a signifi-
cant reduction in bacterial load as well (40%, p < 0.016, n = 70, 14 patients). The
treatment is very well tolerated and no side effects occurred until now (in total more
than 2,000 treatments in over 220 patients). The results of this study revealed the
potential of atmospheric argon plasma treatment as a new approach to kill bacteria in
terms of mutiresistancy. With the same CAP device other dermatologic diseases were
treated successfully, e.g. Hailey-Hailey disease. New plasma devices using surround-
ing ambient air have not only greater bactericidal but also virucidal properties. These
devices may herald a new era in public, personal, pet, and food hygiene, same as in
decontamination. Investigations of human compatibility are promising.
24.1 Introduction
After the historical finding of the antibacterial properties of penicillin by Alexander

Fleming in early 1928, the medical society celebrated a new era in the battle against
pathogenic bacteria. This enthusiasm grew after successful use in patients in 40-some
and the emergence of many other antibiotic substances in the so called modern era of
G. Isbary (*)
Department of Dermatology, Allergology and Environmental Medicine,
Hospital Munich, Koelner Platz 1, 80804 Munich, Germany
e-mail: dr.isbary@googlemail.com
312 G. Isbary
antibiotics. However, bacteria stroke quickly back and became rapidly resistant towards
the new panaceas. Nowadays, in twenty-first century, it is daily routine at health care
facilities to verify resistance levels of bacteria before antibiotic treatment and to find
isolated patients due to infections with multiresistant germs in hospitals all over the
world. To treat bacterial infections has become a challenging task. On the one hand we
face rapidly growing resistance towards antibiotics and on the other hand we have a
lack of new antimicrobial substances [1]. Global health care associations consider mul-
tiresistant germs as a global threat, with a special focus on methicillin-resistant
Staphylococcus aureus (MRSA). This germ has a very high prevalence in countries
regardless the medical standard [2]. Infections with MRSA kill nearly 19,000 hospital-
ized patients in the U.S. annually, which is similar to the number of deaths caused by
AIDS, tuberculosis and viral hepatitis combined [3]. Furthermore, infections with
drug-resistant pathogens do increase death, illness, and direct costs by 30100% [4].
Especially, on chronic wounds, pathogenic bacteria can contribute to impaired healing.
Even colonization can lead to non-healing ulcers. Chronic wounds of the lower leg
have prevalence higher than 1% of the population in developed countries. These
wounds do cost billions for Health Care Systems. In European countries they are esti-
mated to account for 12% of the annual health care budget [5]. Venous ulcers are in
general a tedious task to get healed. On average they need 24 weeks to heal, 15% never
heal, and recurrence is found once or in multiple times in 1571% of all cases [6, 7].
In 2003 there was a very interesting finding of plasma physicists who achieved to
generate cold atmospheric plasmas (CAPs) with antibacterial properties. This new
generation of cold atmospheric plasmas shares the same benefits as their hot brothers,
except the enormous heat production. High-temperature plasmas are generally used in
standardized procedures to sterilize equipment or to remove, cauterize and cut tissues
[810]. CAPs have the great advantage of the possibility to provide in-vivo applica-
tions without harming surrounding tissue by heating, since they operate usually below
40C. They have not only bactericidal, but also fungicidal and virucidal properties due
to a highly reactive mix of reactive species, charging reactions, ultraviolet radiation,
and optical and infrared emissions. All the plasma characteristics can be tuned due to
the mechanism plasma is generated (e.g. microwave frequency, radio frequency, high
voltages) and due to the surrounding gas mixtures and concentrations. The great
potential and the broad flexibility to generate CAPs resulted in a rapid, world-wide
ever-growing number of scientists exploring the field of CAPs. Ironically, this trend
shows some similarities to pandemics. The next decades will tell us if CAPs can
answer the high expectations to a broad range of medical indications and others like
in hygiene, bio-decontamination, food security, and other new emerging fields.
24.2 Cold Atmospheric Plasma and Wounds
In 2005 the Max Planck Institute for Extraterrestrial Physics in Garching (Germany)
developed an indirect argon CAP device, called MicroPlaSter alpha, driven by
microwave technology (2.46 GHz, 86 W, Ar 2.2 slm, distance to the wound 2 cm)
24 Cold Atmospheric Plasma for Clinical Purposes: Promising Results 313
Fig. 24.1 Highly significant higher reduction in bacterial count (~34%, P < 106) in lasma-treated
area (dark grey bar, bottom) compared with standard wound care alone (light grey bar, top)
in collaboration with ADTEC Plasma Technology Co. Ltd. (Hiroshima/Japan) for

medical applications. The details of the torch is well described in the publication of
Shimizu et al. [11]. The device was the first CAP-device to get ethics committee
approval for clinical trials as an add-on therapy besides standard wound care in
patients after proving its efficacy and safety in a preclinical trial. The study was
realized at our department of dermatology, allergology and environmental medi-
cine, Hospital Schwabing-Munich (Germany) with patients who suffered from
chronic infected wounds of different origin. Patients enrolled had at least one wound
large enough to cover a treatment and control leg or had two separated wounds.
Each patient received a daily 5-min plasma treatment on a randomized wound(s).
The same standard wound care was applied on both areas during the course of the
treatment. Once a week, two standard swabs were taken before and directly after
plasma application to detect the type of bacteria, similar to the control area. The
other days we used nitrocellulose filters (Sartorius Stedim Biotech GmbH, Aubagne,
France) to detect changes in bacterial load in both areas. These filters were semi
quantitatively assessed by a manual count after incubation on agar plates. Control
area was undressed during the plasma application. In this trial we proofed a highly
significant decrease in bacterial load in 5 min plasma treated wounds (34%, p < 106,
n = 291, 36 patients, see Fig. 24.1) in comparison with wounds that only received
standard wound care [12]. No side effects occurred until now and plasma treatment
was efficient in all germs. The distribution of bacteria treated in-vivo on wounds in
patients (see Fig. 24.2) demonstrates, that plasma is an efficient new tool against all
types of bacteria, including multiresistant ones, like MRSA. This was the first clini-
cal proof of the bactericidal effects of a CAP device in patients.
In another interim analysis (unpublished data) using the same device and an
identical setting we proved that also a daily 2 min plasma treatment (40%, p < 0.016,
n = 70, 14 patients) resulted in a significant higher reduction in bacterial load than
control. The bactericidal effects seem to be triggered by reactive oxygen, nitrogen
and hydrogen species, charging reactions, ultraviolet radiation and optical and
314 G. Isbary
Fig. 24.2 Distribution of relevant bacteria found on wounds
infrared emissions. The exact bactericidal mechanism is yet unknown and a lot of
effort is done to encode the mode of action. Nevertheless reactive species seem to
play a major role.
In total, we treated over 220 patients in more than 2,000 applications (together
with the department of dermatology, University of Regensburg) using this micro-
wave plasma device and its successor (MicroPlaSter , see Fig. 24.3, which changed
in design and has an additional placebo leg where inert argon gas with the same
temperature as used for plasma treatment can be applied) without having any adverse
effects like pain, yet. Until now, it is unclear whether plasma treatment enhances
wound healing itself. Since wound healing is a long lasting procedure special
designed and controlled clinical trials in patients are needed. Nevertheless there are
some in-vitro or animal studies which support a dosage dependant beneficial effect
of plasma on wound healing and there is data from a NO rich air plasma device from
Russia.
Landsberg and co-workers [13] reported about a positive influence of indirect
DBD on cell regeneration of human keratinocytes (HaCaT). In their study they used
an in vitro wound model (scratch assay). Scratch assay is an established model to
compare closure times. They measured a significantly enhanced wound closure
after plasma treatment compared to untreated control.
Another approach was used of Topala and co-workers [14]. They treated defined
burn wounds on Whistar rats with a daily treatment of a helium plasma jet for 40s.
Even if they detected elevated oxidative stress (markers used: malondialdehyde,
reduced glutathion, glutathione peroxidase, and superoxide dismutase and catalase) a
possible inducer of cell necrosis in plasma treated rats compared to controls.
Interestingly, wound healing in defined plasma dosages was enhanced as proven by
planimetric and histological findings.
Fig. 24.3 Plasma-treatment of chronic ulcer of the left leg using MicroPlaSter -device
But the applied dosage and plasma device play an important role. Keidar and
co-workers [15] reported about the importance of the applied plasma dosage. Mild
helium plasma jet treatments resulted in a reduction of migration rate by factor of 2.
Medium levels lead to cell detachment and intensive treatment to cell desiccation.
The changes in migration rate were explained by plasma-affected integrin expres-
sion. The same group investigated the correlation between tertiary mouse fibroblast
migration rate and the influence of direct plasma jet application [16]. Hundreds of
helium plasma jet application resulted in significant reduced migration rate, whereas
500 s resulted in an even lower migration rate. The authors concluded that as appli-
cation time increases, cells migrate slower. Total reduction of migration rate was
approximately 30%. But it is important that the zone of reduced cell migration cor-
related to the 5 mm direct treatment zone of the jet. This may have a direct influence
on migration rate if directly applied plasma dosage was too high for the cells.
The only data providing evidence of enhanced wound healing due to exogenic NO
rich air plasma comes from a Russian device called Plazon. Many patients were treated
in different clinical trials in Russia. Publications in western journals or online libraries
with this device are not available; same as information about levels of UV (especially
UV-C) or other safety parameter are not published but presented data are indeed
impressive. A clinical trial proved its benefit in 318 patients with venous and arterial
ulcers. Reduction in planimetric determined wound size was significantly decreased
in NO-treated wounds (1.7% per day compared to 0.7% in control group) [17, 18].
316 G. Isbary
Patients suffered from wounds with sizes varying from 6 to 200 cm2. Plasma was
applied from 10 to 30 days (NO concentration ranged from 300 to 500 ppm).
Appearance of granulation and boundary epithelisation, cleansing of ulcers from
exudates and necrosis were much better and faster in NO-treated wounds. Acceleration
factor of 2.5 was stated by authors. Complete healing was achieved 2.54 times
faster than in control leg without plasma. Generally large wounds responded better to
NO therapy. Another trial investigated the effect on diabetic foot ulcers who did not
respond under conventional therapy regimes for 2 months. Those hard-to-heal
wounds responded as well to Plazon therapy [19]. Complete epithelisation was
achieved in small wounds within 68 treatments. Hospitalization time was reduced
by a factor of 2.3 and amputation rate decreased 1.9 times.
24.3 Treatment of Hailey-Hailey Disease
An advanced type of CAP device (MicroPlaSter , frequency 2.45 GHz; power of

80 W; gas flow of 4 slm; treatment temperature 2234C, 5 min treatment time)
showed its benefit in a skin disease, called Hailey-Hailey disease (HHD) [20]. It is
an autosomal dominant condition as a result of mutations in the ATP2C1 gene,
which encodes the human secretory pathway Ca2+/Mn2+-adenosine triphosphatase
ATP2C1, found on chromosome 3q21.1 [21]. Histologically the disease is charac-
terized by suprabasal acantholysis resulting in oozing erosions in the flexures of
patients. It is particularly difficult to control during the hot summer months when
sweat and friction aggravate the eruption. In addition, the eroded lesions of HHD
commonly develop secondary infection. A patient suffered from erythematous, ooz-
ing skin lesions in the right axilla and right side of the groin. These lesions had
failed to heal using topical fusidic acid/betamethasone (Fucicort Cream; LEO
Pharma GmbH, Ballerup, Denmark) used for the previous 3 years. Addition of CAP
treatment resulted in a fast healing response in axilla, compared to randomized con-
trol lesions in the right groin, which continued with topical fusidic acid/betametha-
sone. After aggravation during holidays, because of using topical potassium
permanganate in too high concentrations, we started again CAP treatment. After
previous positive results of additional CAP therapy we decided to treat both regions
with plasma this time. After a total of 11 additional plasma treatments, both areas
healed, leaving only residual erythema (Fig. 24.4). All burning sensations stopped.
It is unclear which mechanisms of plasma led to quick healing response in HHD.
But there are two possible mode of actions: On the one hand we were able to treat
the secondary infection (swab specimen revealed secondary infection with Candida
albicans and Proteus mirabilis) and on the other hand the plasma derived reactive
oxygen species (ROS) could positivly influence the disturbance in this system.
Cialfi et al. reported the importance of signaling effects of oxidative stress in the
development of HHD [22]. The accumulation of ROS in keratinocytes derived from
cutaneous lesions suggested that disturbances in the oxidant-antioxidant system
could play a key role in the pathogenesis of this disease.
Fig. 24.4 Appearance of right groin after 4 additional plasma treatments (left) and after a total of
11 plasma treatments, leaving only residual erythema remaining (right)
24.4 Conclusions
Cold atmospheric plasma technology demonstrated its efficacy not only in in-
vitro but also in the treatment of infected wounds in patients. Our studies prove
the first time, that an additional plasma treatment beside standard wound care can
enhance the reduction of germs on chronic infected wounds significantly. This can
be achieved either by a 5 min plasma treatment, or by a 2 min application.
Furthermore, the study showed in vivo, that plasma reduces different types of
germs, even the multiresistant ones, like MRSA. Therefore, cold atmospheric
plasmas are promising tools for superficial antibacterial applications on infected
wounds in future. There is no evidence that bacteria can become resistant to this
highly reactive mixture of reactive oxygen, hydrogen and nitrogen species, charg-
ing reactions, ultraviolet radiation and optical and infrared emissions. Another
important finding is, that no side effects occurred until now after more than 2,000
plasma applications yet.
The technology is not confined to chronic infected wounds any more. We
demonstrated, that a patient with Hailey-Hailey disease profited by an addi-
tional 5 min plasma treatment, as well. The treated lesions failed to heal with
topical disinfectant and glucocorticoid therapy for many years. Within few
plasma treatments lesions healed, leaving only residual erythema. Burning sen-
sations stopped.
New generations of cold atmospheric plasmas evolve bit by bit. These devices
open the field to completely novel indications, especially for hygiene. One example
is a cold atmospheric plasma which uses a technology called surface micro dis-
charge. It has not only the advantage of a very flexible and freely scalable design
using ambient surrounding air but also the cheap construction costs. First unpub-
lished results are very promising regarding tolerance to the skin besides a highly
antimicrobial potential towards bacteria, spores and even viruses. This technique is
only one example of many others.
However, the exact bactericidal mechanisms of cold atmospheric plasmas are not
understood and a lot of effort has to be done to get this knowledge. This awareness
318 G. Isbary
can help us to enhance future cold atmospheric plasma technologies to have even
more potent devices in the never ending battle against bacterial infections.
Like in other technical developments we are still in the starting blocks of this
innovative scientific leg but the future is in our hands and well see if we can meet
the high expectations.
References
1. Payne DJ, Gwynn MN, Holmes DJ, Pompliano DL (2007) Drugs for bad bugs: confronting the
challenges of antibacterial discovery. Nat Rev Drug Discov 6:2940
2. Grundmann H, Aires-de-Sousa M, Boyce J, Tiemersma E (2006) Emergence and resurgence
of meticillin-resistant Staphylococcus aureus as a public-health threat. Lancet 368:874885
3. Klevens RM, Edwards JR, Richards CL, Horan TC, Gaynes RP, Pollock DA, Cardo DM
(2007) Estimating health care-associated infections and deaths in U.S. Hospitals, 2002. CDC
Public Health Report March-April 122:160166
4. Cosgrove SE, Carmeli Y (2003) The impact of antimicrobial resistance on health and economic
outcomes. Clin Infect Dis 36:14331437
5. Etufugh CN, Phillips TJ (2007) Venous ulcers. Clin Dermatol 25:121130
6. Kurz X, Kahn SR, Abenhaim L, Clement D, Norgren L, Baccaglini U, Berard A, Cooke JP,
Cornu-Thenard A, Depairon M, Dormandy JA, Durand-Zaleski I, Fowkes GR, Lamping DL,
Partsch H, Scurr JH, Zuccarelli F (1999) VEINES task force report. Int Angiol 18(2):83102
7. Heit JA (2002) Venous thromboembolism epidemiology. Semin Thromb Hemost 28(2):313
8. Bogle MA, Arndt KA, Dover JS (2007) Evaluation of plasma skin regeneration technology in
low-energy full-facial rejuvenation. Arch Dermatol 143:168174
9. Elsaie ML, Kammer JN (2008) Evaluation of plasma skin regeneration technology for cutane-
ous remodeling. J Cosmet Dermatol 7:309311
10. Kilmer S, Semchyshyn N, Shah G, Fitzpatrick R (2007) A pilot study on the use of a plasma
skin regeneration device (Portrait: PSR3) in full facial rejuvenation procedures. Lasers Med
Sci 22:101109
11. Shimizu T, Steffes B, Pompl R, Jamitzky F, Bunk W, Ramrath K, Georgi M, Stolz W, Schmidt
HU, Urayama T, Fuji S, Morfill G (2008) Characterization of microwave plasma torch for
decontamination. Plasma Process Polym 6:577582
12. Isbary G, Morfill G, Schmidt HU, Georgi M, Ramrath K, Heinlin J, Karrer S, Landthaler M,
Shimizu T, Steffes B, Bunk W, Monetti R, Zimmermann JL, Pompl R, Stolz W (2010) A first
prospective randomized controlled trial to decrease bacterial load using cold atmospheric
argon plasma on chronic wounds in patients. Br J Dermatol 163:7882
13. Landsberg K, Neumann M, Haehnel M, Scharf C, Weltmann KD, von Woedtke T (2010)
Atmospheric pressure plasma shows rapid regeneration of human cells. In: Proceedings of 3rd
international conference on plasma medicine, Greifswald, Germany, 1924 Sept
14. Topala I, Nastuta AV, Grigoras C, Dumitrascu N (2010) Study of oxidative stress markers dur-
ing epithelial regeneration induced by atmospheric pressure plasma treatment. In: Proceedings
of 3rd international conference on plasma medicine, Greifswald, Germany, 1924 Sept
15. Keidar M, Volotskova O, Shashurin A, Stepp MA (2010) Plasma control of cell migration.
In: Proceedings of 3rd international conference on plasma medicine, Greifswald, Germany,
1924 Sept
16. Volotskova O, Shashurin A, Stepp MA, Pal-Ghosh S, Keidar M (2010) Plasma-controlled cell
migration: localization of cold plasma-cell interaction region. Plasma Med 1:8592
17. Shekhter AB, Kabisov RK, Pekshee AV (1998) Byull Eksp Biol Med 8:210
18. Shekhter AB, Serezhenkov VA, Rudenko TG, Pekshev AV, Vanin AF (2005) Nitric Oxide Biol
Chem 12:210
19. Shulutko AM, Antropova NV, Kryuger YA (2008) Surgery 12:43

20. Isbary G, Morfill G, Zimmermann J, Shimizu T, Stolz W (2011) Cold atmospheric plasma a
successful treatment of lesions in Hailey-Hailey Disease. Arch Dermatol 4:388390
21. Szigeti R, Kellermayer R (2006) Autosomal-dominant calcium ATPase disorders. J Invest
Dermatol 11:237023765
22. Cialfi S, Oliviero C, Ceccarelli S, Marchese C, Barbieri L, Biolcati G, Uccelletti D, Palleschi
C, Barboni L, De Bernardo C, Grammatico P, Magrelli A, Salvatore M, Taruscio D, Frato L,
Gulino A, Screpanti I, Talora C (2010) Complex multipathways alterations and oxidative stress
are associated with HaileyHailey disease. Br J Dermatol 3:518526
Chapter 25
Tissue Tolerable Plasma and Polihexanide:
Are Synergistic Effects Possible to Promote
Healing of Chronic wounds? In Vivo
and In Vitro Results
Claudia P. Bender, Nils-Olaf Hbner, Klaus-Dieter Weltmann,

Christian Scharf, and Axel Kramer
Abstract The assumption is that tissue tolerable plasma works as promoter for
wound healing and can be beneficially combined with the antiseptic polihexanide to
avoid bacterial recolonization. The effects of a combined plasma polihexanide
(PHMB) application on cell integrity, cytotoxicity and its irritative and inflamma-
tive potential were tested in vitro and in two dogs in vivo.
25.1 Introduction
Two important characteristics of a chronic wound beside other reasons are chronic
inflammation and critical bacterial colonization. The latter is one of the main
reasons to impede the healing process. Only a few of planctonic bacteria are
C.P. Bender (*)

Institute of Hygiene and Environmental Medicine, University Medicine Greifswald,
Walther-Rathenau-Str. 49a, 17489 Greifswald, Germany
Private Practice, Tierarztpraxis Karrin, Karrin 15, 17440 Krslin, Germany
e-mail: claudia.bender@uni-greifswald.de
N.-O. Hbner A. Kramer
Institute of Hygiene and Environmental Medicine, University Medicine Greifswald,
Walther-Rathenau-Str. 49a, 17489 Greifswald, Germany
K.-D. Weltmann
Leibniz Institute for Plasma Science and Technology e.V. (INP Greifswald),
C. Scharf
Department of Otorhinolaryngology, Head and Neck Surgery, University Medicine Greifswald,
Walther-Rathenau-Str. 43 45, 17475 Greifswald, Germany
322 C.P. Bender et al.
sufficient to adhere to the wound surface; to multiply and to develop into

microcolonies over short time, which form larger aggregates known as biofilms
[1, 2]. In this context, two therapeutic problems have to be solved: cleaning of the
critically colonized or biofilm loaded wound surface and turning the persisting
chronic inflammation into an acute process to initiate the healing. In recent time,
tissue tolerable plasma (TTP) was distinguished to enhance wound healing [3].
We were able to show in the modified HET-CAM (hens egg test on the chorioal-
lantois membrane) that plasma application can induce aseptic inflammations,
which are suitable to modulate chronic inflammations [4, 5]. The antimicrobial
efficacy of TPP is well described elsewhere [6, 7]. Furthermore, application of
plasma on biofilms show a high effectiveness against biofilms [8, 9]. Still, the
disadvantage of TPP is that the antimicrobial effect is only immediate and without
remament effect to prevent bacterial recolonization. To prevent bacterial recoloni-
sation on wounds, plasma should be used in short intervals and application times
that are probably harmful to sensitive tissues. In this manner, wound healing
would be impaired, because to long treatment times lead to severe damage and
strong inflammation [5].
Therefore, it seems to be supportive to combine plasma therapy and antiseptic
treatment, for instance polihexanide, to prevent the bacterial recolonisation.
Polihexanide (PHMB, Polyhexamethylene Biguanide) is a first line agent for
treatment of chronic wounds due to its broad antimicrobial spectrum, well tissue
tolerability, its ability to bind to the organic matrix [10] and stimulating effect on
wound healing [11, 12]. The synergies of plasma and polihexanide are probably as
follows: as first treatment, plasma gives a stimulus to the wound that lead to an
inflammatory response as shown in Bender et al. [5]. This inflammatory response
with hyperamia, coagulation, and inflammation associated with angiogenesis and
contracture disruptes the healing stagnation and initiates the healing cascade of an
acute inflammation. After that, an antiseptic with well wound tolerability as poli-
hexanide could be applied as useful supplement to prevent bacterial recolonisation,
thus the antiseptic supports the plasma effect in a synergistic way. Beyond that the
positive side effects of both therapies (plasma: antiseptic effects, polihexanide:
stimulating effect) enhance each others desired effect.
We therefore combined plasma treatment with the antiseptic polihexanide
in vitro to examine if a combined treatment led to pronounced side effects as
inflammation or to higher cell toxicity. The positive results encouraged us to apply
the combined treatment in two dogs as case reports for curative therapeutic use.
25.2 Plasma
The plasma was generated with the atmospheric pressure plasma jet (Neoplas
GmbH, Greifswald, Germany, CE certification No. 609.003.1) (Fig. 25.2) as
described in Weltmann et al. 2009 [13], with argon being used as a carrier gas.
The gas flow was 5 slm per min. The plasma was used in continuous mode.
25 Tissue Tolerable Plasma and Polihexanide 323
The visible plasma tip was 11 mm from the nozzle; the thermal output was 160 mV,
the input power 3.9 W.
25.3 In Vitro Tests
Normal human epidermal keratinocytes were cultivated on cell-culture-inserts,

(Millicell cell culture inserts, catalog no PIHP01250, Millipore, Billerica MA,
USA) for 14 days after airlift, according to Poumay et al. [14], using
DermaLifeK Medium Complete Kit (CellSystems Biotechnologie Vertrieb
GmbH, Troisdorf, Germany) to become a stratified reconstructed human epider-
mis (RHE, Fig. 25.1).
The integrity of the reconstructed human epidermis was determined by means of
the Trans Epithelial Electrical Resistance (TEER). We also used the reconstructed
human epidermis to assess cytotoxicity effects of combined plasma polihexanide
treatment on keratinocytes in three-dimensional cell structure.
The reconstructed human epidermis was treated with plasma punctually for 10s
and 20s, respectively (Fig. 25.2). The visible plasma tip was just in contact with the
cornified side of the epidermis. As control of mechanical gas effects, a group was
treated with argon gas for 20s.
After plasma treatment, on the cornified side of the epidermis was dropped
300 ml polihexanide 0.02% (Lavanid 1, Serag Wiessner, Naila, Germany) and 0.04%
(Lavanid 2, Serag Wiessner, Neila, Germany), respectively. After a contact time of
30 min, the epidermis was rinsed three times with Balances Salt Solution (BSS),
300 ml.
Triton X-100 is a detergent and was used as positive control, because it
induces an increase of permeability of membrane structures and thereby the
reduction of the barrier functions of the epidermis. Fifty microliters of Triton
X-100, 1% was dropped on the cornified side of the epidermis for a contact time
of 2 h. In parallel, 50 ml of Balanced Salt Solution (BSS) was applied as nega-
tive control. After the contact time of 2 h, the epidermis was rinsed three times
with BSS, 300 ml.
Fig. 25.1 Cross-section of a reconstructed human epidermis, Haematoxylin/Eosin staining

(PCF-Membran: polycarbonate membrane)
Fig. 25.2 Left: Schematic of the atmospheric pressure plasma jet. Right: Treatment of reconstructed
human epidermis with the atmospheric pressure plasma jet
25.3.1 Trans Epithelial Electric Resistance (TEER)
To monitor the integrity and barrier function of the reconstructed human epidermis
the transepithelial electrical resistance was measured before and after treatment
with the EVOM/STX2 (WPI Germany, Berlin, Germany).
The 10s plasma treatment led to a very little decrease of the TEER, whereas the
20s plasma treatment led to approximately 50% decrease of the TEER. This gives
an advice of the affected integrity of the keratinocytes and that the barrier function
of the epidermis is reduced. But compared to the chemical corrosive standard Triton
X 1%, the reduction of the barrier function is moderate. A possible dose effect
relation of plasma impact is visible (Fig. 25.3).
25.3.2 Viability-MTT-Assay
The MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl

blue; CAS number 298-93-1] assay is a quantitative colorimetric assay for measuring
the activity of enzymes which reduce MTT as a yellow tetrazole to purple formazan in
living cells [15]. The assay detects only living cells and the signal generated is depen-
dent on the degree of activation of the cells. A main application allows assessing the
viability and the proliferation of cells. The assay can also be used to determine cytotox-
icity of medical agents and toxic materials, since those agents stimulate or inhibit cell
viability and growth as described in the OECD guideline 431 (in vitro skin corrosion:
human skin model test). According to this guideline, the MTT assay was used to appraise
the cytotoxic effects of TPP and TPP/polihexanide, respectively, on keratinocytes.
4.5
TEER before treatment
TEER 4
[kOhm] TEER after treatment
3.5
2.5
1.5
0.5
0
50l TXT1%, 2h 50l BSS, 2h Gas-control 20s Plasma 10s Plasma 20s
Fig. 25.3 Transepithelial Electrical Resistance (TEER) before and after (i) 2 h contact time of
50 ml Triton X 100, 1% (50 ml TTX 1%, 2 h) as positive corrosive control, (ii) 2 h contact time of
50 ml Balanced Salt Solution (50 ml BSS, 2 h) as negative control, (iii) 20s argon gas treatment
(Gas-control 20s), (iv) 10s and 20s plasma treatment; Error bars: confidence intervals, n = 6,
alpha = 0.05
120,00
100,00
80,00
viability [%]
60,00
40,00
20,00
0,00
50l BSS, 2h
300l, 30min
300l, 30min
TTX,1%,2h
Gas, 20s
Gas, 20s +
Gas, 20s +
Plasma, 10s
Plasma,10s +
Plasma, 10s +
Plasma, 20s
Plasma, 20s +
Plasma, 20s +
Lavanid 2
Lavanid 1
Lavanid 1,
Lavanid 2,
Lavanid 2
Lavanid 1
Lavanid 2
Lavanid 1
50l
Fig. 25.4 Viability (%) of the reconstructed human epidermis after treatment with (i) 50 ml Triton
x-100, 1%, (ii) 50 ml BSS, (iii) Lavanid (1 and 2), (iv) Argon gas 20s, (v) Argon gas+Lavanid
(1 and 2), (vi) plasma (10s and 20s) and (vii) combined treatment plasma/polihexanide
(10s+Lavanid1, 10s+Lavanid2, 20s+Lavanid1, 20s+Lavanid2). Error bars: confidence intervals;
n = 6, alpha = 0.05
After the combined treatment, any liquid remaining on the reconstructed human
epidermis was removed. The epidermis comprising cell-culture-inserts were then
placed into 24-well plates containing 300 ml MTT solutions per well and incubated for
3 h in a 5% CO2 incubator at 37C. Then they were immersed in 2 ml of acidified
Ethanol as extractant solution over night at 8C. The absorbances of 200 ml extractant
solutions were measured in 96-well microtiter plates at 560 nm. The test was evaluated
as% viability compared to the negative control (2 h contact time BSS) (Fig. 25.4).
Even if the sole plasma treatment led to an increase of permeability (measured as

decrease of TEER), the combined TPP/polihexanide treatment did not lead to a
relevant decrease in viability. This means the cytotoxicity of the combined treat-
ment is only marginal increased. It seems unclear, why the 20s plasma treatment
revealed a higher viability then the 10s treatment. Possibly, the plasma treatment
induces an inflammation associated activation of enzymes that appear only after
longer impact times. Further investigations are necessary.
25.3.3 Modified HET-CAM
In the modified HET-CAM, we assessed if the combined TPP/polihexanide treat-

ment led to pronounced irritation or inflammation.
The HET-CAM [16] is a replacement method for the Draize-Test on rabbit eyes
to test acute toxicity, irritation and corrosion in accordance to OECD-Guideline 405
and is in accordance with the Directive 86/609/EEC on the protection of animals
used for experimental and other scientific purposes.
Vakzine Lohmann specific pathogen free eggs (VALO SPF, Lohmann GmbH,
Cuxhaven, Germany) were incubated for 10 days at 37 1C and 65 7% relative
humidity in a small-motored breeder (KMB F/2, Ehret GmbH, Emmendingen,
Germany). The experiments were conducted on day 10 after opening the eggs at the
blunt pole and removing the inner shell membrane. After evaluation on day 10
(directly and 5 min after treatment) and on day 11 (24 h after treatment), the eggs
were frozen at 20C, killing the embryos.
In a previous study it was found that Plasma (kINPen09, Argon as feeder gas,
4 slm) induces inflammation in dependence of treatment time. It was shown that a
5s-treatment in the continuos mode led to moderate inflammation, with effects pre-
dominantly reversible, slight hemorrhage and contracture. These effects are almost
adequate to 20s argon-gas treatment. A 10s and 20s-treatment led to in evidence
granulomas, hamorrhage, contracture and coagulation, while these effects were pre-
dominantly not reversible. A dose-effect relation was clearly visible [5].
Notwithstanding the previous study, in this study the gas flow was 5 slm. To avoid
mechanical and thermal damage of the CAM, the distance from the nozzle to the
CAM was 15 mm. Both, the distance of the nozzle to the CAM as well as the gas
flow have influence on the effects, what led to different results compared to previous
studies [4, 5].
To evaluate the reactions, the response patterns granuloma development (with
associated spoke wheel-like angiogenesis), hemorrhages, coagulation and contracture
were blinded classified by two calibrated, evaluators according to a scoring
system, rating the response of each CAM depending on the degree of severity (from
degree 1 no response to degree 5 heavy response) as described in Bender et al.
[5]. The individual values were then added to form a score sum (SS), and the mean
value was calculated from the score sums of all CAM of one treatment mode
(score sum mean, SSM, n = 6) (Fig. 25.5). The score allocated to the plasma tolerance
10
6
directly
SSM 5 after 5min
after 24h
4
0
300l Lavanid1 plasma10s+300l Lavanid1 plasma 10s gas10s+300l Lavanid1
Fig. 25.5 Score sum mean (SSM) of the chorioallantoic membranes (each group n = 6),
immediately after treatment, after 5 min and after 24 h
Table 25.1 Assignment of score sum (SS) to the compatibility

SS Compatibility
57 Very good, without inflammation
7.19 Very good, with mild inflammation
9.111 Moderate, with medium inflammation
11.113 Poor, with severe inflammation, applicable to limited extent, only
>13 Insufficient, with high-grade inflammation, application is not recommended
towards sensitive tissues like wounds is described in Table 25.1. The allocation is
based on the reaction of the CAMs in line with Kramer and Behrens-Baumann
(1997) [17]. Five different and clearly visible tolerance categories can be distin-
guished. Allocating the SSM, we tried to take this classification into account.
Single treatment with polihexanide 0.02% (Lavanid 1) led to a SSM of 8.7 within
24 h, what is a well compatibility (Table 25.1). Also single plasma treatment had a
similar compatibility and led to a SSM of 8.8. The combined treatment led to a
slight worsening of the compatibility, but the SSM of 9.2 still implies a moderate
compatibility. There was no evident difference to the combined treatment of gas/
polihexanide (SSM 8.2). In comparison to the previous study [5] the plasma effects
in this study are weaker because of the increased distance from the nozzle to the
CAM, (10s plasma after 24 h: 8.8 [here] versus 12.89 [5]).
Following these results and the results of earlier studies [5, 6] it was shown that
the plasma compatibility is dependent on several different parameters as flow rate,
distance, power, treatment time and treatment mode. The combined treatment of
TTP and polihexanide in this study led to a very good compatibility in the HET-
CAM within 5 min. Because of the good predictivity of the HET-CAM for non-
irritants in vivo [18] it is probable that a combined treatment is tolerable for wounds
using the tested settings. The HET-CAM provides a good model to determine
compatible parameters for plasma treatments of eyes and wounds.
25.4 In Vivo Application: Two Case Reports
The positive results from the in vitro assays encouraged us to test the combined
treatment in two dogs. Both dogs suffered of chronic wounds for several years,
conventional therapies (surgical debridement and wound closure in case of the
chronic rhagade, self adhesive dressing and different ointments several times a
week, prescribed by a veterinarian and applied by the owner in both cases, protec-
tive bandages in the second case) failed. The treatment was well tolerated by both
animals. In one case, the treatment led to a complete healing after 11 weeks,
which was particularly remarkable because the treatment with TPP or polihexanide
alone did not led to healing. This supports the presumption, that TPP and poli-
hexanide have synergistic effects in promoting healing of chronic wounds. In the
second case, the treatment was complicated by constant licking of the wound by
the dog, but the healing progressed after applying a ruff and the wound has
improved since then.
25.4.1 First Patient
The first patient was a 9-year-old German shepherd dog with a chronic rhagade of
the nasal planum. It can be assumed that the nose was regularly exposed to bacteria
because the dog licked his nose several times a day and rummaged in the soil some-
times. Swabs were not taken. The wound had persisted for 4 years and was pre-
treated with 0.02% polihexanide for a period of 17 months leading to a wound
surface reduction by approximately 30% of the original wound surface. Subsequently,
the wound healing process stagnated.
The wound was experimentally treated by applying TTP combined with 0.02%
polihexanide solution for two weeks. A slight amelioration was visible. After an
interruption of 3 weeks (break through holidays), the therapy was continued apply-
ing exclusively TTP for a period of 11 weeks to find out, if a sole plasma treatment
would lead to healing. The dog was subjected to the plasma treatment in two ses-
sions of 15s, each, two or three times a week. During the treatment, the tip of the
visible plasma contacted the vital tissue (distance to the nozzle about 11 mm, 5 slm
argon gas, derived from [4]) (Fig. 25.6). The plasma jet was moved quickly over the
wound surface on a meandering course. Consequently, the complete wound surface
was in contact with the plasma several times. The sole plasma therapy was finished
because there was no visible progress in the healing process. After the plasma treat-
ment was finished, the wound was treated with polihexanide daily until the wound
finally closed after a further 11 weeks (Fig. 25.6).
Fig. 25.6 Plasma treatment (top left), state at the treatment start (top right) and healing progress
after 6.5 weeks (down left) and 11 weeks (down right)
25.4.2 Second Patient
The second patient was a 12-year-old male German shepherd dog with a chronic
wound on the left dorsal carpus (foreleg), occurred after a trauma, caused by a
barbed wire fence 3 years ago. The wound was treated several times by a veterinary
with different ointments and protective bandages without any success.
Before starting the treatment, a swab was taken from the wound and mass of
haemolytic staphylococci, greening Streptococci and sporformers were found. The
wound was experimentally treated by applying TTP exclusively for two weeks
(Fig. 25.7). Due to the bigger size of the wound the treatment time was prolonged
to 90s on a meandering course, two times a week. Because the dog did not stop lick-
ing, the plasma treatment was continued in combination with daily treatment of
0.02% polihexanide for another 2 weeks. After an interruption of two weeks to
observe if the wound healing occurs, the therapy was continued because the con-
tinuative licking impeded the healing progress and a beginning of exsudation
Fig. 25.7 Plasma treatment (top left), state at the treatment start (top right) and healing progress
after 3 weeks (down left) and 8 weeks (down right)
became visibly. The combined therapy was continued for another nine sessions, two
plasma applications per week. The healing progressed after applying a ruff and the
wound has improved since then (Fig. 25.7). After the plasma treatment had finished,
the wound was treated with polihexanide daily until the wound finally closed after
a further 8 weeks.
25.5 Discussion of the Case Reports
In the first case, when the plasma treatment was stopped and polihexanide was
applied again, the wound, which had persisted for 4 years, continuously healed and
closed completely within 11 weeks. Accordingly, the wound healing supposedly
progressed as follows: The initially sole polihexanide treatment stimulated the
wound healing simultaneously repelling the critical colonization so that the wound
grew smaller by about one-third. Thereafter, healing of the wound stagnated. The
combined application of polihexanide and TTP followed by sole treatment with
TTP revived the wound healing process. During this time, however, the process had
obviously changed significantly so that the wound after the discontinuance of the
TTP application clinically changed to become a wound progressively healing until
complete closure. This effect is probably attributable to the TTP application. When
plasma was applied in the modified HET-CAM point by point in the range of sec-
onds, the following responses could be observed: hyperemia, hemorrhage and, after
24 h, contraction, granuloma formation with spoke wheel-like angiogenesis and
extravasale coagulation. It can be assumed that similar processes occur in vital tis-
sue of mammals. Accordingly, the plasma treatment should induce a variety of
effects causing the wound to transform from the chronic into the acute phase, for
instance by possible stimulation of the fibroblasts and keratinocytes, angiogenesis,
contraction and coagulation. The TTPs capability of tissue alteration induced
inflammation and improved blood circulation produced further stimuli for wound
healing processes [19] most likely as a summation of the physico-chemical plasma
components [20]. The healing effect of TTP could be explained by generation of
reactive oxygen species (ROS) during TTP treatment, which have a significant
molecular effect on the stimulation of wound healing. ROS can directly stimulate
MMPs (matrix metalloproteinases) and allow them to cleave and release Hb-EGF
[heparin-binding EGF (epidermal growth factor)]. Additionally, ROS can induce
tyrosine phosphorylation of PDGF (Platelet Derived Growth Factor) alpha- and
beta-receptors. Binding of both growth factors to its affinity receptor elicits a variety
of cellular responses, e.g. MAPK (mitogen-activated protein kinase) activation fol-
lowed by an induction of transcription. It is released by platelets upon wounding
and plays an important role in stimulating the growth of adjacent cells and thereby
healing the wound [2124]. Nevertheless, all affecting impact factors are dose
dependent. Further investigations should, therefore, be aimed at elucidating the cel-
lular mechanisms of the plasma effects on wound healing.
Positive possible side effects are the antiseptic action of TTP [6, 7] and its capa-
bility of inactivating biofilms [8, 9]. This is particularly important for the nasal
wound to be treated as the regular licking in the area of the injured nasal planum
involves an increased entry of bacteria into the wound [2527]. The antiseptic treat-
ment with polihexanide, which had been continued after the plasma treatment, had
the effect that a new critical colonization and biofilm formation could be avoided.
Contrary to an experts recommendation [28] we applied polihexanide for an
extended period of time, so as to avoid the risk of biofilm formation in the case of
the dog licking its nose.
It can be derived from the wound healing process that the TTP induces an
inflammation, which is possibly associated with a cellular stimulation as probably
visible in the MTT-assay. The inflammatory response transforms the chronic inflam-
mation into an acute one, the further progress of which is stimulated by healing
effects including that of polihexanide. To date, the time sequences in which these
effects occur are still unknown. As a result, the secondary wound healing did not
progress any further when the wound was treated with TTP two or three times a
week, implying that inflammatory effects are probably predominating. After the
TTP treatment was terminated the second stage of clinical visible healing began and
the wound finally closed completely. The application of polihexanide at this stage
prevented the formation of new biofilms and a disturbance of the wound healing.
In the second case, the licking surely will be a major reason to retard the wound
healing. The dog licked his wound even before the plasma treatment. The reason
for the licking seems unclear - it could be itching and/or pain and/or a bad habit.
Nevertheless, the wound closure failured when protective bandages and ointments
were applied in previous therapies. Therefore, it can be assumed that other reasons
among the licking as critical bacterial colonization and chronic inflammation led to
a disturbance in the wound healing process. With the exclusion of licking, the
combined plasma therapie led to healing, what was not achieved in earlier
treatments.
It is notable that the TTP was well tolerated by the animals. Thus, painful surgery
or anesthesia for such intervention could be avoided, what is a huge advantage in
anaesthesia risk patients.
Chronic wounds in animal patients are not standardized, what makes comparing
difficult. In these two cases, it lacks of knowledge about the optimum treatment
times and frequencies. The in vitro tests give advices for the maximum single
treatment, but it is still unknown what are the optimal treatment intervals and
frequencies. Further investigations are still necessary.
25.6 Conclusion
To conclude, the applied in vitro methods (modified HET-CAM for irritation - and
inflammation potential; reconstructed human epidermis for the assessment of the
cytotoxicity and integrity) are useful for screening plasma sources for their suitabil-
ity for chronic wound treatment and to determine the parameters for medical appli-
cations alone and in combination with polihexanide.
The in vivo TPP applications have been tolerated by the dogs without pain.
On one hand, the different treatment regimes and the small number of cases
allow no strong conclusion with regard to synergistic TPP-polihexanide effects. On
the other hand, the final success of the combined treatment supports the hypothesis
that the combined TPP-polihexanide treatment has possible synergistic effects,
particular in regard to the fact that the initially sole polihexanid treatment in the first
case led not to healing. The combined TPP-polihexanide treatment could be a
promising option for the treatment of chronic wounds. But as only a very little
amount of experience in the plasma application on chronic wounds exists, further
investigations should, therefore, be aimed at elucidating the cellular mechanism of
plasma promoted wound healing, combined TPP-polihexanide application and
optimizing the course of treatment.
References
1. James GC, Swogger E, Wolcott R, de Lancey Pilcini E, Secor P, Sestrich J, Costerton JW,
Stewart PS (2008) Biofilms in chronic wounds. Wound Repair Regen 16:3744
2. Davis SC, Ricotti C, Cazzaniga A, Welsh E, Eaglstein WH, Mertz PM (2008) Microscopic and
physiologic evidence for biofilm-associated wound colonization in vivo. Wound Repair Regen
16:2329
3. Lloyd G, Friedman G, Jafri S, Schultz G, Fridman A, Harding K (2010) Gas plasma: medical
uses and developments in wound care. Plasma Process Polym 7:194211
4. Bender C, Matthes R, Kindel E, Kramer A, Lademann J, Weltmann K-D, Eisenbei W, Hber
N-O (2010) The irritation potential of nonthermal atmospheric pressure plasma in the HET-
CAM. Plasma Process Polym 7:318326
5. Bender C, Partecke L-I, Kindel E, Dring F, Lademann J, Heidecke C-D, Kramer A, Hbner
N-O (2011) The modified HET-CAM as a model for the assessment of the inflammatory
response to tissue tolerable plasma. Toxicol In Vitro 25:530537
6. Daeschlein G, von Woedtke T, Kindel E, Brandenburg R, Weltmann K-D, Juenger M (2010)
Antibacterial activity of an atmospheric pressure plasma jet against relevant wound pathogens
in vitro on a simulated wound environment. Plasma Process Polym 7:224
7. Hammann A, Huebner NO, Bender C, Ekkernkamp A, Hartmann B, Hinz P, Kindel E, Koban
I, Koch S, Kohlmann T, Lademann J, Matthes R, Mller G, Titze R, Weltmann KD, Kramer A
Skin Pharmacol Physiol 23:328332
8. Koban I, Matthes R, Hbner NO, Welk A, Meisel P, Holtfreter B, Sietmann R, Kindel E,
Weltmann KD, Kramer A, Kocher T (2010) Treatment of Candida albicans biofilms with low
temperature plasma induced by dielectric barrier discharge and atmospheric pressure plasma
jet. New J Phys 12:073039
9. Hbner NO, Matthes R, Koban I, Rndler C, Mller G, Bender C, Kindel E, Kocher T, Kramer
A (2010) Efficacy of chlorhexidine, polihexanide and tissue-tolerable plasma against
pseudomonas aeruginosa biofilms grown on polystyrene and silicone materials. Skin Pharmacol
Physiol 23(suppl1):2834
10. Hbner N-O, Kramer A (2010) Review on the efficacy, safety and clinical applications of
polihexanide, a modern wound antiseptic. Skin Pharmacol Physiol 23(suppl1):1727
11. Roth C, Beule AG, Kramer A, Hosemann W, Kohlmann T, Scharf C (2010) Response analysis
of stimulating efficacy of polihexanide in an in vitro wound model with respiratory ciliary
epithelial cells. Skin Pharmacol Physiol 23(suppl1):3540
12. Kramer A, Roth B, Mller G, Rudolph P, Klcker N (2004) Influence of the antiseptic agents
polihexanide and octenidine on FL cells and on healing of experimental superficial aseptic
wounds in piglets. A double-blind, randomised, stratified, controlled, parallelgroup study. Skin
Pharmacol Physiol 17:141146
mation. Contrib Plasma Phys 49:631640
14. Poumay Y, Dupont F, Marcoux S, Leclercq-Smekens M, Hrin M, Coquette A (2004) A simple

reconstructed human epidermis: preparation of the culture model and utilization in in vitro
studies. Arch Dermatol Res 296:203211
15. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J Immun Methods 65(12):5563
16. Luepke NP (1985) Hens egg chorion allantoic membrane test for irritation potential. Food
Chem Toxicol 23:287291
17. Kramer A, Behrens-Baumann W (1997) Prophylactic use of topical anti-infectives in ophthal-
mology. Ophthalmologica 211:6876
18. Scheel J, Kleber M, Kreutz J, Lehringer E, Mehling A, Reisinger K, Steiling W (2011) Eye
irritation potential: usefulness of the HET-CAM under the globally harmonized system of
classification and labeling of chemicals (GHS). Regul Toxicol Pharmacol 59(3):471492
19. Stoffels E (2007) Tissue processing with atmospheric plasmas. Contrib Plasma Phys
47:4048
Plasma Process Polym 2:391400
21. Wang Q, Turlington A, Heo S, Blanco A, Tian J, Xie Z, Yan B, Wan Y (2005) Extracellular
matrix activity and caveolae events contribute to cell surface receptor activation that leads to
MAP kinase activation in response to UV irradiation in cultured human keratinocytes. Int J
Mol Med 15:633640
22. Brenneisen P, Sies H, Scharffetter-Kochanek K (2002) Ultraviolet-B irradiation and matrix
metalloproteinases: from induction via signaling to initial events. Ann NY Acad Sci
973:3143
23. Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T (1995) Requirement for generation of
H2O2 for platelet-derived growth factor signal transduction. Science 270:296299
24. Gonzlez-Rubio M, Voit S, Rodrguez-Puyol D, Weber M, Marx M (1996) Oxidative stress
induces tyrosine phosphorylation of PDGF alpha-and beta-receptors and pp 60c-src in mesan-
gial cells. Kidney Int 50:164173
25. Rubin JE, Chirino-Trejo M (2010) Pharyngeal, rectal and nasal colonization of clinically
healthy dogs with Staphylococcus aureus. Vet Microbiol 143:440441
26. Saphir DA, Carter GR (1976) Gingival flora of the dog with special reference to bacteria
associated with bites. J Clin Microbiol 3:344349
27. Stepanovic S, Dimitrijevic V, Vukovic D, Dakoc I, Sacic B, Svabic-Vlahovic M (2001)
Staphylococcus sciuri as a part of skin, nasal and oral flora in healthy dogs. Vet Microbiol
82:177185
28. Dissemond J, Gerber V, Kramer A, Riepe G, Strohal R, Vasel-Biergans A, Eberlein T (2010)
A Practice-oriented recommendation for the treatment of critical colonised and local infected
wounds using polihexanide. J Tissue Viability 19(3):106115
Chapter 26
Helium Atmospheric Pressure
Plasma Jet: Diagnostics and Application
for Burned Wounds Healing
Ionut Topala and Andrei Nastuta
Abstract A new field of plasma applications developed in the last years, entitled
plasma medicine, has focused the attention of many peoples from plasma community
on biology and medicine. Subjects that involve plasma physics and technology
(e.g. living tissue treatment or wound healing, cancer cell apoptosis, blood coagula-
tion, sterilization and decontamination) are nowadays in study in many laboratories.
In this paper we present results on optical and electrical diagnosis of a helium
atmospheric pressure plasma jet designed for medical use. This type of plasma jet was
used for improvement of the wound healing process. We observed a more rapid mac-
roscopic healing of the plasma treated wounds in comparison with the control group.
Atmospheric pressure plasma sources are employed nowadays in various industrial

applications, using many configurations and experimental solutions to fulfill the
needs of modern technologies [1, 2]. We can easily identify some classes of
discharges based on operation principle and discharge mechanism: arc, jet and
torches, corona, dielectric barrier discharge and resistive discharge, hollow cathode
and microdischarges. They are driven by dc or ac/pulsed voltage sources, having
frequency values spread on a broad range: low frequency (Hz-kHz), RF (13.56 MHz)
and microwaves (2.45 GHz).
Continuing this long tradition of atmospheric pressure plasma applications, a
new research field shaped his profile in the plasma community: the plasma medicine
[1, 35]. Currently it exist a number of intensively studied phenomena involving the
use of atmospheric pressure plasmas in medicine, such as living tissue treatment
(e.g. wound healing), cancerous cell destruction (e.g., by apoptosis or ablation),
blood coagulation, modification of bone tissue (dentistry and orthopedics),
I. Topala (*) A. Nastuta

Plasma Physics Laboratory, Faculty of Physics, Alexandru Ioan Cuza University,
Bd. Carol I, No. 11, 700506 Iasi, Romania
e-mail: ionut.topala@uaic.ro
336 I. Topala and A. Nastuta
sterilization and decontamination. A large variety of plasma sources were employed,

they being labeled in different ways. Well known names are: plasma needle [69],
plasma pencil [1012], plasma gun [13, 14] or atmospheric pressure plasma jet (APPJ)
[1518].
Parameters of these plasma sources (e.g. temperature and concentration of
charged species, gas temperature, concentration and distribution of reactive species)
are spread over large domains of values. Having this in mind, standardization of
operational parameters of atmospheric pressure plasma sources represents a neces-
sity before any large medical use and remains an open challenge. Parameters like
maximum current value, charge, power density, repetition rate, applied voltage, gas
mixture can be used to achieve this goal.
26.1 Main Parameters of Atmospheric Pressure

Plasma Jet for Medical Use
The atmospheric pressure plasma jet (APPJ) designed in our laboratory is based on
a cylindrical barrier discharge with external electrodes. Various kinds of geometries
are reported in the literature to generate dielectric barrier discharge (DBD) plasma.
Arc regime transition is avoided due to the presence in the electrode gap of dielec-
tric layer. This also assures the current limitation and constraints the system to work
in pulsed mode. Two working regimes are known: filamentary discharge (random
streamers) and homogenous discharge (diffuse or glow) [19, 20].
We have used a quartz tube, 4 mm inner diameter and 6 mm outer diameter, to
generate an atmospheric pressure plasma jet. Aluminum tape electrodes (10 mm
width for the power electrode and 4 mm width for the ground electrode) are wrapped
on the external surface of the tube, being separated by a 10 mm gap. This solution
offers a high experimental flexibility. The set-up can be easily moved and adapted
to work in biological environment, or can be integrated in other experimental
arrangements to perform in-situ analyses of plasma or its effects on biological mate-
rial. Helium (spectral purity, 99.99999%) is flowing inside the quartz tube with
electronically controlled flow rate. For flow rate values smaller than 7 L/min a lami-
nar flow regime is assured. The residence time of helium atoms in the electrode gap
has values up to 15 ms. The speed of helium atoms increases linearly from 1.4 m/s
at 1 L/min to 14 m/s at 10 L/min. The use of helium as working gas can be consid-
ered expensive for many applications. However for medical applications this is not
necessarily true, since the typical treatment durations are explored in the seconds to
only few minutes range.
The analysis of the discharge current shape proves that our discharge works in a
homogenous regime. The current is characterized by a sharp peak during the increase
of the voltage pulse, when the ground electrode is acting as cathode and the powered
electrode as anode. This is called usually the primary discharge. Unique for the
DBD set-up is the appearance of a secondary discharge during the falling part of the
applied voltage pulse, the above mentioned roles of the electrodes being now
26 Helium Atmospheric Pressure Plasma Jet: Diagnostics and Application 337
Fig. 26.1 Influence of voltage pulse (a) amplitude on main parameters of the discharge: current
(b), power (c), charge and current pulse duration (d), measured as full width at the half maximum
(frequency 2 kHz, helium flow rate 3 L/min)
inverted (Fig. 26.1a). The necessary energy to develop this second discharge is
assured by the charges deposited onto the dielectrics surface during the primary
discharge [21].
The value of the discharge current peak for these two discharges separated in
time is controlled mainly by the applied voltage amplitude (Fig. 26.1b). These low
values, in the mA range, assure the safety use of this plasma jet in direct contact
with living animals without any perceptible sensory or motor response. This was
tested in our laboratory on Wistar rats back skin and on human fingers.
The power peak values, obtained as the product of the primary current peak and
the corresponding value of the instantaneous voltage, is increasing together with the
increasing of the voltage (Fig. 26.1c). The same remark is valid for the transported
charge between the electrodes. Due to the sharpening of the primary current peak
(Fig. 26.1d), the average value of the pulse power slowly increases in the range
12.5 W (Fig. 26.1c).
The values of the electron density, obtained from both experiments and simula-
tions, in helium atmospheric pressure glow discharge are situated in the range 1010
1012 cm3 [19, 22, 23].
Utilization of plasma in direct contact with living animals must take into
account not only the charge effects, but also the presence UV radiations and
chemical active species, that can be related to plasma toxicity. Last but not the
least, local temperature or pH modifications in biological supramolecular sys-
tems can induce denaturation or destruction of molecules or supramolecular
assemblies.
The emission spectrum of the discharge contains helium lines, centered at 388.1,
501.5, 587.5, 667.8, 706.5, 728.1 nm. Lines or bands of impurities from the air were
identified in the APPJ emission spectrum at the following wavelengths: O (777.4 nm),
N2+ (391.4, 427.8, 470 nm), N2 (315.9, 337.1, 357.6, 380.4 nm), OH (308.9 nm), H
(486.1, 656.3 nm). No emission was found in the 200300 nm range. The time aver-
aged emission intensity increases with the voltage and for a fixed value of the volt-
age the maximum value of the emission intensity was found between the electrodes.
For all mentioned excited species, excepting the molecular nitrogen, lower emission
intensity was found outside the quartz tube, i.e. in the jet region.
The concentration values of the oxygen and nitrogen reactive species generated
in the APPJ are presented in the Table 26.1.
Plasma generation of UV radiations is a major concern regarding the medical use
of plasma based devices. An exception is obviously the use of plasma for steriliza-
tion and decontamination, where UV radiations were found to be a key factor for an
efficient process [1, 2729]. Nevertheless, depending on the operating conditions,
the effect of UV photons in the sterilization process can be neglected [30]. Typical
dose values for the plasma jets or other plasma sources designed for living tissue
treatment were found to be less than the necessary dose to induce skin or cell dam-
age [26, 31, 32].
The temperature of our helium APPJ was estimated by various methods. First of
all the rotational temperature of nitrogen molecular ion was estimated using the
Boltzmann plot method [33]. Between the electrodes the temperature values are
around 300 K, while in the jets tip region these values reach at 700 K. This is
clearly an overestimation of the gas temperature value. If we focus the plasma jet on
a laboratory thermocouple (situated at 15 mm from the edge of the quartz tube) we
obtain values of 296.5 K at 4 kV, 299.5 K at 6 kV and 304.5 K at 8 kV. Moreover,
pictures obtained with laboratory infrared camera of the plasma jet prove that the
plasma jet has an overall temperature equal to the background gas in the room
(Fig. 26.2).
Table 26.1 Typical concen- Specie Density (cm3)

tration of oxygen and nitrogen
O 10131014
reactive species generated in
atmospheric pressure plasma OH 10151017
1
jets [2426] O2 10141016
O3 10151017
O2 10101012
H2O2 10141016
NO 10131014
Fig. 26.2 Visible and infrared picture of the helium APPJ in contact with a human finger (voltage
pulse amplitude 6 kV)
Recently, pH modification was reported as an effect of the plasma action on

aqueous solutions [34]. No data are available for the moment regarding the skin or
wound pH values after plasma treatments.
26.2 Time and Space Propagation of the Plasma Structures
Time and space propagation of the plasma jet from the production region to the liv-
ing tissues is of a special interest. Most of the plasma sources designed for medical
use are pulsed discharges, the frequency has values in the kHz range up to micro-
wave range. This leads to an exposure of living tissues to UV-visible photons pulses
and to pulsed free radical chemistry.
In order to study the appearance and propagation of the helium APPJ (voltage
pulse amplitude 6 kV), we have used the high speed photography technique.
Pictures of the APPJ were taken for the plasma jet focused on a quartz window,
placed at 15 mm from the tubes edge and for the plasma jet facing a human finger,
placed at the same 15 mm distance from the tubes edge (Fig. 26.2). The time t = 0
was considered the moment when the discharge current corresponding to the pri-
mary discharge starts to increase.
As already reported in the literature, the jet is not spatially homogenous during the
current peak, during both primary and secondary discharges. A closer look in the
nanoseconds range as exposure time reveals the existence of plasma structures, so-
called plasma bullets, which can be found in distinct regions as the time increases. The
plasma propagation mechanism in air can be explained using a streamer model based
on photo ionization [35, 36] or using an ionization wave model [37]; new hypotheses
are under investigation and even glow discharge like behavior can be obtained [38].
During the primary discharge, inside the quartz tube the discharge appears as
homogenous luminous structure, in the anodes region and heading towards the cath-
ode. A discharge channel is then formed in the center of the quartz tube. After the
expansion in the air, the plasma bullets are no longer homogenous, a ring-shape
Fig. 26.3 Typical pictures of the APPJ behavior in on the surface of a quartz substrate (side view
and on-axis view) and a human finger (side view); temporal behavior of the plasma light intensity
at the human finger surface, as obtained from the analysis of ICCD images
profile of the emission intensity being recorded due to the higher density of molec-
ular nitrogen in the plasma volume [3941]. If the helium APPJ is focused on a
substrate the plasma bullet spreads on the surface and then is extinguish. We found
a different behavior of plasma structures dynamics onto dielectric substrates (i.e.
the quartz window) and onto human tissues (Fig. 26.3). In the space between the
tubes edge and the substrate the plasma jet has a bullet like behavior. Then the
plasma is reaching the substrate and it starts to spread. For a specific duration a
plasma structure exists in the front of the substrate. This duration is very short for
the quartz substrate, of around 1 ms and is very long for the human finger, of about
20 ms (Fig. 26.3).
Upon our knowledge, these are the first ICCD investigations of the plasma jet
action on a living human tissue. It should be emphasized that a long living
plasma structure exists at the human finger surface, in comparison with plasma
behavior on a dielectric surface. This leads to longer exposure durations of human

tissues to plasma action. In this way using the frequency as a parameter we can
control very well the effective time of direct plasma action on a living tissue.
26.3 Application of Helium APPJ for Burned Wounds Healing
Many studies revealed the great potential of plasma in the treatment of skin wounds,
with an especial attention devoted to chronic infected wounds. Indirect or direct
action of plasma on wounds was found to induce the healing of the injured tissues
[4245].
Our study was focused on plasma treatment of fresh model wounds obtained on
the Wistar rats back skin by chemical burns. The exposure of the rats skin to a
sulphuric acid solution caused a second degree burn, both the superficial dermis
and the epidermis being damaged. Control wounds were selected to study the natu-
ral wound regeneration, while other wounds were used to study the influence of the
plasma treatment on the regeneration process. These wounds were exposed 40 s
daily to the helium plasma jet, without any preliminary local cleaning or wound
conditioning. Hematological, biochemical and histological data were monitored
over the observation period (21 days) in order to follow the evolution of systemic
and local effects. Experimental procedures are described in detail in our previous
publications [33, 46, 47]. The experiments were carried out together with our col-
leagues from the Gr.T. Popa University of Medicine and Pharmacy, Physiopathology
Department, Iasi, Romania, under the supervision of Prof. Dr. Magda Badescu.
The values of selected hematological and biochemical parameters at day 21 are
as follows: white blood cell, 5.8 109 cells/L for control group, 9 109 cells/L for
natural wounds recovery group and 5.6 109 cells/L for plasma treated wounds
group; fibrinogen, 175 mg/dL for control group, 260 mg/dL for natural wounds
recovery group and 185 mg/dL for plasma treated wounds group; C3 component,
100 mg/dL for control group, 210 mg/dL for natural wounds recovery group and
100 mg/dL for plasma treated wounds group.
After 21 days the natural wounds recovery process is not finished. The center of
the initial wounds is still visible. As against this behavior, the recovery process for
the plasma treated wounds appears to be accelerated. The wounds disappeared
almost completely (Fig. 26.4).
Further biochemical analyses of skin homogenates from the wound region,
revealed differences on oxidative stress markers as against control group [33, 46,
47]. Values of measured markers are as follows: malondialdehyde, 0.22 mmol/mL
homogenized skin for control group, 0.48 mmol/mL homogenized skin for natural
wounds recovery group and 0.82 mmol/mL homogenized skin for plasma treated
wounds group; reduced glutathione, 0.82 nmol/mg protein for control group,
0.48 nmol/mg protein for natural wounds recovery group and 0.38 nmol/mg pro-
tein for plasma treated wounds group; glutathione peroxidase, 79 mmol GSSG/
min/mg protein for control group, 64 mmol GSSG/min/mg protein for natural
Fig. 26.4 Typical clinical

appearance of (a) fresh
wounds and (b) after 21 days:
left side, daily plasma
treatment for 40s wounds and
right side, natural wounds
recovery
wounds recovery group and 31 mmol GSSG/min/mg protein for plasma treated
wounds group; catalase, 16,000 U/mg protein for control group, 10,000 U/mg
protein for natural wounds recovery group and 5,000 U/mg protein for plasma
treated wounds group.
Plasma action was found to affect biological function in cells and tissues, trough
peroxidation of lipids from cell membranes, oxidative modification of proteins
involved in signalling pathways, oxidative DNA damage [4, 48].
To conclude, helium atmospheric pressure plasma jet represents a good option
as a plasma source for medical applications. Its operation is reproducible and can
be easily standardized, while the economic aspects can be adjusted to obtain a
profitable system. The mechanism of plasma action and its effects in biology and
medicine are not yet fully understood [49]. For a better understanding of mecha-
nism and the reaction pathways that are responsible for the benefic effects of
plasma use in medical applications, experiments regarding plasma effects on
supramolecular biological systems like proteins are carried now our group. Plasma
effects on protein structure and the relations between structure and function are
investigated. The results will offer a much clear image of the plasma effects on
biological systems.
Acknowledgements The authors thank to Dr. Constantin Grigoras (Gr.T. Popa University of
Medicine and Pharmacy, Iasi, Romania) for its valuable help in wound healing experiments and for
the fruitfull discussions.
This work was supported by CNCSIS-UEFISCSU, grant PN II-RU 297/2010-2011 and ESF in
Romania, grant POSDRU/89/1.5/S/63663.
References
1. Fridman A (2008) Plasma chemistry. Cambridge University Press, New York

2. Tendero C, Tixier C, Tristant P, Desmaison J, Leprince P (2006) Atmospheric pressure
plasmas: a review. Spectrochim Acta B 61:230
plasma medicine. Plasma Process Polym 5:503533
4. Kong MG, Kroesen G, Morfill G, Nosenko T, Shimizu T, Dijk J, Zimmermann JL (2009)
Chem 82:12231237
6. Stoffels E, Kieft IE, Sladek REJ, van den Bedem LJM, van der Laan EP, Steinbuch M (2006)
Plasma needle for in vivo medical treatment: recent developments and perspectives. Plasma
Sources Sci Technol 15:S169S180
7. Goree J, Liu B, Drake D (2006) Gas flow dependence for plasma-needle disinfection of S.
mutans bacteria. J Phys D Appl Phys 39:3479
8. Zhang X, Li M, Zhou R, Feng K, Yang S (2008) Ablation of liver cancer cells in vitro by a
plasma needle. Appl Phys Lett 93:021502
9. Pena-Eguiluz R, Perez-Martnez JA, Solis-Pacheco J, Aguilar-Uscanga B, Lopez-Callejas R,
Mercado-Cabrera A, Valencia-Alvarado R, Munoz-Castro AE, Barocio SR, de la Piedad
Beneitez A (2010) Instrumentation for a plasma needle applied to E. coli bacteria elimination.
Eur Phys J Appl Phys 49:13109
10. Janca J, Zajickova L, Klima M, Slavicek P (2001) Diagnostics and application of the high
frequency plasma pencil. Plasma Chem Plasma Process 21:565579
11. Laroussi M, Lu Xu (2005) Room-temperature atmospheric pressure plasma plume for bio-
medical applications. Appl Phys Lett 87:113902
12. Laroussi M, Hynes W, Akan T, Lu X, Tendero C (2008) The plasma pencil: a source of hyper-
sonic cold plasma bullets for biomedical applications. IEEE Trans Plasma Sci 36:12981299
13. Shashurin A, Keidar M, Bronnikov S, Jurjus RA, Stepp MA (2008) Living tissue under treat-
ment of cold plasma atmospheric jet. Appl Phys Lett 93:181501
14. Robert E, Barbosa E, Dozias S, Vandamme M, Cachoncinlle C, Viladrosa R, Pouvesle JM
(2009) Experimental study of a compact nanosecond plasma gun. Plasma Process Polym
6:795802
mation. Contrib Plasma Phys 49:631640
16. Xiong Q, Lu X, Ostrikov K, Xiong Z, Xian Y, Zhou F, Zou C, Hu J, Gong W, Jiang Z (2009)
Length control of He atmospheric plasma jet plumes: effects of discharge parameters and
ambient air. Phys Plasmas 16:043505
17. Daeschlein G, Scholz S, Arnold A, von Woedtke T, Kindel E, Niggemeier M, Weltmann K-D,
Junger M (2010) In vitro activity of atmospheric pressure plasma jet (APPJ) plasma against
clinical isolates of demodex folliculorum. IEEE Trans Plasma Sci 38:29692973
18. Jiang N, Ji A, Cao Z (2010) Atmospheric pressure plasma jets beyond ground electrode as
charge overflow in a dielectric barrier discharge setup. J Appl Phys 108:033302
19. Massines F, Segur P, Gherardi N, Khamphan C, Ricard A (2003) Physics and chemistry in a
glow dielectric barrier discharge at atmospheric pressure: diagnostics and modeling. Surf Coat
Technol 174:814
20. Roth JR, Rahel J, Dai X, Sherman DM (2005) The physics and phenomenology of One
Atmosphere Uniform Glow Discharge Plasma (OAUGDP) reactors for surface treatment
applications. J Phys D Appl Phys 38:555567
21. Liu S, Neiger M (2001) Excitation of dielectric barrier discharges by unipolar submicrosecond
square pulses. J Phys D Appl Phys 34:16321638
22. Martens T, Brok WJM, Dijk J, Bogaerts A (2009) On the regime transitions during the forma-
tion of an atmospheric pressure dielectric barrier glow discharge. J Phys D Appl Phys
42:122002
23. Urabe K, Sakai O, Tachibana K (2011) Combined spectroscopic methods for electron-density
diagnostics inside atmospheric-pressure glow discharge using He/N2 gas mixture. J Phys D
Appl Phys 44:115203
24. Kalghatgi S, Kelly CM, Cerchar E, Torabi B, Alekseev O, Fridman A, Friedman G,

Azizkhan-Clifford J (2011) Effects of non-thermal plasma on mammalian cells. PLoS One
6:e16270
25. Schulz-von der Gathen V, Schaper L, Knake N, Reuter S, Niemi K, Gans T, Winter J (2008)
Spatially resolved diagnostics on a microscale atmospheric pressure plasma jet. J Phys D Appl
Phys 41:194004
26. Morfill GE, Shimizu T, Steffes B, Schmidt HU (2009) Nosocomial infections a new approach
towards preventive medicine using plasmas. New J Phys 11:115019
27. Eto H, Ono Y, Ogino A, Nagatsu M (2008) Low-temperature internal sterilization of medical
plastic tubes using a linear dielectric barrier discharge. Plasma Process Polym 5:269274
28. Moisan M, Barbeau J, Crevier MC, Pelletier J, Philip N, Saoudi B (2002) Plasma sterilization.
J Phys D Appl Phys 40:5907
30. Boudam MK, Moisan M, Saoudi B, Popovici C, Gherardi N, Massines F (2006) Bacterial
spore inactivation by atmospheric-pressure plasmas in the presence or absence of UV photons
as obtained with the same gas mixture. J Phys D Appl Phys 39:34943507
Hartmann B, Ottomann C, Fluhr JW, Hinz P, Hubner G, Lademann O (2009) Risk assessment
of the application of a plasma jet in dermatology. J Biomed Opt 14:054025
New J Phys 11:115013
33. Nastuta AV, Topala I, Grigoras C, Pohoata V, Popa G (2011) Stimulation of wound healing by
helium atmospheric pressure plasma treatment. J Phys D Appl Phys 44:105204
34. Friedman G (2010) Plasma pharmacology. In: Proceedings of the 3rd international conference
for plasma medicine, Greifswald, p 18
35. Karakas E, Koklu M, Laroussi M (2010) Correlation between helium mole fraction and plasma
bullet propagation in low temperature plasma jets. J Phys D Appl Phys 43:155202
36. Lu XP, Laroussi M (2010) Dynamics of an atmospheric pressure plasma plume generated by
submicrosecond voltage pulses. J Appl Phys 100:063302
37. Shi J, Zhong F, Zhang J, Liu DW, Kong MG (2008) A hypersonic plasma bullet train traveling
in an atmospheric dielectric-barrier discharge jet. Phys Plasmas 15:013504
38. Walsh JL, Iza F, Janson NB, Law VJ, Kong MG (2010) Three distinct modes in a cold atmo-
spheric pressure plasma jet. J Phys D Appl Phys 43:075201
39. Teschke M, Kedzierski J, Finantu-Dinu EG, Korzec D, Engemann J (2005) High speed photo-
graphs of dielectric barrier atmospheric pressure plasma jet. IEEE Trans Plasma Sci 33:310311
40. Mericam-Bourdet N, Laroussi M, Begum A, Karakas E (2009) Experimental investigation of
plasma bullets. J Phys D Appl Phys 42:055207
41. Sakiyama Y, Graves DB, Jarrige J, Laroussi M (2010) Finite element analysis of ring-shaped
emission profile in plasma bullet. Appl Phys Lett 96:041501
42. Ermolaeva SA, Varfolomeev AF, Chernukha MY, Yurov DS, Vasiliev MM, Kaminskaya AA,
Moisenovich MM, Romanova JM, Murashev AN, Selezneva II, Shimizu T, Sysolyatina EV,
Shaginyan IA, Petrov OF, Mayevsky EI, Fortov VE, Morfill GE, Naroditsky BS, Gintsburg AL
(2011) Bactericidal effects of non-thermal argon plasma in vitro, in biofilms and in the animal
model of infected wounds. J Med Microbiol 60:7583
43. Heinlin J, Morfill G, Landthaler M, Stolz W, Isbary G, Zimmermann JL, Shimizu T, Karrer S
(2010) Plasma medicine: possible applications in dermatology. J Dtsch Dermatol Ges
8:968976
44. Lloyd G, Friedman G, Jafri S, Schultz G, Fridman A, Harding K (2010) Gas plasma: medical
uses and developments in wound care. Plasma Process Polym 7:194211
45. Heinlin J, Isbary G, Stolz W, Morfill G, Landthaler M, Shimizu T, Steffes B, Nosenko T,
Zimmermann JL, Karrer S (2011) Plasma applications in medicine with a special focus on
dermatology. J Eur Acad Dermatol Venereol 25:111
46. Grigoras C, Topala I, Nastuta AV, Jitaru D, Florea I, Badescu L, Ungureanu D, Badescu M,
Dumitrascu D (2011) Influence of atmospheric pressure plasma treatment on epithelial regen-
eration process. Rom J Phys 56:5461
47. Grigoras C, Badescu L, Jitaru D, Florea I, Topala I, Dumitrascu N, Badescu M (2010) Helium
plasma effects on epidermal regeneration. Ann RSCB 15:135141
48. Machala Z, Jedlovsky I, Chladekova L, Pongrac B, Giertl D, Janda M, Sikurova L, Polvcic P
(2009) DC discharges in atmospheric air for bio-decontamination spectroscopic methods for
mechanism identification. Eur Phys J D 54:195204
Chapter 27
Non-equilibrium Air Plasma for Wound
Bleeding Control
Spencer P. Kuo, Cheng-Yen Chen, Chuan-Shun Lin,

and Shu-Hsing Chiang
Abstract A low temperature non-equilibrium air plasma spray is tested as a blood

coagulator. Emission spectroscopy of the plasma effluent indicates that it carries
abundant reactive atomic oxygen (RAO), which can activate erythrocyte platelet
interactions to enhance blood coagulation for plug formation. Tests of the device
for wound bleeding control were performed on pigs. Four types of wounds, straight
cut and cross cut in the ham area, a hole in an ear saphenous vein, and a cut to an
ear artery, were examined. The results showed that this plasma spray could effec-
tively stop the bleeding and reduced the bleeding time considerably. Post-Operative
observation of straight cut and cross cut wound healing was carried out. It was
found that the plasma treatment had a positive impact on wound healing, in par-
ticular, of the cross cut wound; its healing time was shortened by a half.
Bleeding, even from an external hemorrhage, may be life threatening if it is not

treated swiftly [1]. Most cases occur under emergency situations. For example,
Hemorrhage accounts for 30 to 40% of all fatalities, second only to central nervous
system injury as a cause of death in the battlefields [2]. New methods and devices,
which can effectively stop bleeding to save the life of an injured person, such as in
accident and battlefield situations, are of considerable interest.
The argon plasma coagulator (APC) is a high-frequency monopolar device used
for non-contact thermal coagulation. This device is used in particular in endoscope
S.P. Kuo (*) C.-Y. Chen

Department of Electrical and Computer Engineering, Polytechnic Institute of New York
University, 6 MetroTech Center, Brooklyn, NY 11201, USA
e-mail: skuo@duke.poly.edu
C.-S. Lin S.-H. Chiang
Department of Animal Science and Biotechnology, Tung Hai University,
Taichung, Taiwan ROC
348 S.P. Kuo et al.
surgery for bleeding control [35]. The heat carried by argon plasma, produced by
a high-frequency discharge between the tip of a probe and the target tissue, cauter-
izes and desiccates blood. However, it is difficult to treat larger external wounds. On
the other hand, reactive oxygen metabolites produced in non-equilibrium air plasma
could also induce blood clotting effect. Oxidants can affect several key steps of
platelet function to indirectly enhance platelet agglomeration through local increases
in platelet-activating factor (PAF) [6]. Furthermore, oxidants promote de novo syn-
thesis of tissue factor pro-coagulant activity [7]. Indeed, Kalghatgi et al. [8] showed
that a blood sample could be clotted by direct contact of the sample to non-thermal
atmospheric pressure plasma [9], produced by a dielectric barrier discharge (DBD).
Therefore, new devices that can deliver copious low temperature plasma, which will
not cause thermal damage on the tissue surrounding the wound, would be
desirable.
Chen et al. [10] and Kuo et al. [11] showed that a low temperature non-equilibrium
air plasma [12] could clot anti-coagulated whole blood samples in less than 20 s,
which is much less than 30 min for an untreated sample to reach complete coagula-
tion. It was also found via emission spectroscopy that this plasma spray produces
abundant reactive atomic oxygen (RAO) in its plasma effluent [13]. Activation of
erythrocyte platelet interactions by RAO to trigger blood coagulation was sug-
gested as a plausible coagulation mechanism.
However, those experimental results performed under well-controlled in-vitro
conditions may not describe what occurs in the much more complicated in-vivo
environment. In the present work, we use pigs as the animal model [14] to perform
in-vivo tests of blood coagulation by non-equilibrium air plasma [12]. The effective-
ness of this plasma spray to stop bleeding is studied. The results of these tests are
presented and discussed. Post-operative observation of straight cut and cross
cut wound healing has been conducted. The impact of plasma treatment on wound
healing is reported.
27.1 The Portable Air Plasma Spray
A non-equilibrium air plasma spray [12] is designed to generate low tempera-

ture air plasma for blood coagulation applications [10, 11, 14]. This plasma
generator consists of a pair of concentric electrodes, a ring-shaped permanent
magnet, a blower, and a power supply. The airflow (flow rate of 3 l/s and an
average flow speed of 24.5 m/s at the nozzle exit) supplied by a blower and the
magnetic field (~0.18 T) enforce the arc discharge path into a rotating (around
the central axis) elongated arc loop, forming a stable diffused arc plasma. The
generated air plasma is low temperature, which is about 75C for running at cw
mode, and can be reduced to be less than 55C for running at intermittent mode
(such as 2 s on and 2 s off). A nozzle is introduced to direct the flow of the
plasma effluent as well as to cover the electrodes for safety, so that the high
voltage (HV) central electrode is not exposed. The nozzle opening is positioned
27 Non-equilibrium Air Plasma for Wound Bleeding Control 349
Fig. 27.1 (a) A photo and (b) a schematic of the plasma spray; (c) voltage V, current I, and power
P of the discharge in one cycle; (d) the spatial distribution of the plasma emission intensity at
777.4 nm; the numerical value at each contour is the log10 of the intensity in Rayleigh; (e) a video
graph of the plasma plume
about 12.5 mm above the ring-shaped outer electrode. A photo of the device
with the generated plasma plume and its schematic are shown in Figs. 27.1a, and b,
respectively.
27.1.1 Electric Characteristics
The discharge is periodic at 60 Hz and in each AC cycle there are two discharges.
The ionization percentage of the plasma produced by the discharges is less than
one thousandth of one percentage of the airflow (i.e., <105); the plasma spray gas
is dominated by nitrogen and oxygen molecules. The time varying voltage V(t)
and current I(t) of the discharge were measured using a digital oscilloscope
(Tektronix TDS3012 DPO 100 MHz and 1.25 GS/s), where V is the voltage of the
central electrode of the device (outer electrode is grounded). Current I was
350 S.P. Kuo et al.
measured by a current loop (0.1 V/1A) around the electric wire connected to the
outer electrode. All of the discharge current passes through that wire. The product
of the V and I functions gives the instantaneous power function P(t). The V, I and
P in one cycle are presented in Fig. 27.1c. As shown in Fig. 27.1c, the positive
(PD) and negative (ND) discharges are quite different in their characteristics. ND
is a standard arc discharge with a rapid voltage drop at the start of the discharge;
on the other hand, PD is probably in the arc-glow transition, which has a higher
maintaining voltage to drive larger current and consumes more power. The peak
power in the positive discharge reaches 2 kW; the overall average power is about
350 W.
27.1.2 Emission Spectroscopy
The RAO flux of the plasma spray was determined via the emission spectroscopy of
the plasma plume [13]. We measured the spatial distribution of the 777.4 nm emis-
sions from the 5P state of atomic oxygen (OI). The image data were taken with an
Apogee Alta E47 thermoelectrically cooled CCD camera with a Nikon 50 mm f/1.2
lens and a 50 mm diameter 2 nm pass band interference filter with a center wave-
length of 777.4 nm. The choice of lens relative to detector size ensures that the tar-
get emission line passes through the filter over the entire field of view, allowing the
camera to measure the entire plasma plume simultaneously. Another advantageous
feature of the camera measurement is that, for a diffuse pixel-filling source such as
the plasma plume, the distance between the camera and the source does not change
the measured intensity per pixel.
The camera was calibrated to determine the bias counts, thermal noise, and sen-
sitivity by acquiring images of an integrating sphere calibration source traceable to
the National Institutes of Standards and Technology (NIST) at the exposure times
and aperture settings used in the experiment. A calibration table supplied with the
source and the filter transmission documented by the manufacturer provides a linear
relationship between pixel counts and the intensity in Rayleighs of 777.4 nm emis-
sions in the plasma plume for each aperture setting and exposure time, where 1
Rayleigh = 1010 photons/m2 s, weighing the apparent emission rate integrated along
a line of sight. The volume emission rate in photons m3 s1 can be determined by
dividing the apparent intensity in Rayleighs by the path length of the emitting
region.
Due to the short lifetime (~0.20.3 ms) of atomic oxygen [15], the axial distri-
bution of the emission intensity varied strongly and to have a sharp upward extent.
To capture the full axial extent of this distribution beyond the dynamic range of a
single 16-bit digital image (which is limited to four order of magnitude), multiple
images of the plasma were recorded separately with a moveable stage approxi-
mately 0.15 m square draped in black velvet cloth blocking the brighter portions of
the plasma effluent to allow operation of the camera at higher sensitivity and lon-
ger exposure times while avoiding saturation in the brighter parts of the plasma
plume. Due to the size of the barrier and the diffuse covering, the measurements
were made in the geometrical optics regime with diffraction negligible relative to
the resolution of the camera.
The imaging process started with the barrier completely lowered, the bright
emissions from the direct flames required the camera to be operated at its lowest
sensitivity, with the aperture stopped to f/16 and exposure time set to 0.1 s. Even at
this shortest exposure time of 0.1 s, the image was an average over 6 discharges. In
other words, the instantaneous fluctuation of the emission in the image due to the
60 Hz alternating current driving the plasma spray has been evened out. The cover
was then moved up by 3 mm sequentially in each image, with the aperture and
exposure time adjusted accordingly at each stage. It proceeded until the cover
blocked most of the flame region to detect the upward boundary of the emission
using the camera at high sensitivity with the aperture opened to f/1.2 and the expo-
sure time set at 1 s. The resulting images were then calibrated and combined into a
composite image giving contours of the intensity distribution, ranging from 105 to
109 Rayleighs, as shown in Fig. 27.1d. An image of the plasma plume of the spray
recorded by a video camera is included in Fig. 27.1e for reference. As shown,
the visible plasma plume extends out axially to about 40 mm from the nozzle of the
device, where the intensity of 777.4 nm emissions is about 105 Rayleigh; i.e., the
total photon emission from a slice of the plasma effluent at 40 mm away from
the nozzle is about 10 15 m 2 s1. Assuming no other emissions are present
within the filter passband, and incorporating the air flow speed of ~20 m/s (on
average at 40 mm away from the nozzle), the corresponding flux and density of 5P
state OI are estimated to be about 6 1013 m2 s1 (i.e., ~1015/[(4p) (4/p)]) and
3 1012 m3, respectively. A spectrometer was also used to scan the emission spec-
trum of the plasma from 300 to 900 nm, in which intensive lines contributed by
oxygen radicals appear also only around 777.4 nm. The UV radiation from 300 nm
to 400 nm was not detected.
There are three likely processes to produce atomic oxygen; one is to dissociate
an oxygen molecule into two oxygen atoms via the reaction e + O2 2O + e, which
has a reaction rate coefficient k1 = 4.2E9exp(5.6/Te) [16], where Te is in eV. This
reaction rate decreases rapidly with Te < 5.6 eV; thus it needs about 5 eV electrons to
effectively dissociate O2 into atomic oxygen. The second one is through recombina-
tion of charged particles e + O+ O, which may not need the presence of energetic
electrons. The third one is the dissociative attachment of electrons to molecular
oxygen e + O2 O + O; conservation of energy requires that the electron energy
exceeds a threshold level, which normally is 3.6 eV. Though this threshold level
decreases as the internal energy of molecular oxygen increases, laboratory experi-
mental results [17] show that this level can go down to 1 eV and less. Therefore, the
third process of dissociative attachment is likely the dominant process of OI genera-
tion by this low temperature plasma spray. The 5P state of the transition in OI has
rather high energy, about 10.74 eV, relative to the ground state, hence, the strong
line intensity outside the core of the plasma (i.e., outside the white area of the con-
tour plot) indicates that plasma is in a non-equilibrium state with a strong presence
of high-energy electrons (1 eV) and an abundant concentration of 5P state OI in
352 S.P. Kuo et al.
the plasma effluent, again assuming no other emissions are present within the finite
filter pass-band. Since OI in other states (in particular, in the ground state) may also
be produced, the total OI concentration in the plasma effluent may be much higher
than that of 5P state alone.
27.2 Preparations and Procedure of In-Vivo Experiments
Experiments were conducted to examine four types of wounds, straight cut

and cross cut in the ham area, a hole in an ear saphenous vein, and a cut to an
ear artery. Three 6 mouth-old male pigs weighing around 40 kg were used in
the experiments. The straight cut and cross cut wounds introduced on two
pigs were treated by plasma; the other pig was an untreated control stopping
bleeding by itself. The bleeding and healing times of the untreated control were
recorded to be the natural clotting (bleeding)/healing times. In the cases of ear
wounds, the left ears were the control group and the right ears the experimental
group.
Each pig was first injected with calmative-Stresnil and fastened on a table.
The pig was then anesthetized with Isoflurance-Fluothane which kept it in a nar-
cotized state. After the experiments, pigs were put into stainless experiment
cages for postoperative observation of recovery. The stainless cage prevents pig
to scratch an itchy part of the wounds against the wall during the recovery
period.
27.2.1 In Vivo Blood Coagulation Tests
The effectiveness of the plasma spray on stopping bleeding from a hole onto an ear
saphenous vein and a cut to an ear artery were examined. The natural clotting (bleed-
ing) times of the similar wounds on untreated controls were used as references for
comparisons.
Fig. 27.2 (a) A needle punching a hole in an ear saphenous vein, (b) blood flowing out of the hole,
(c) plasma treatment, and (d) bleeding stopped after 14 s (7 on/off runs) of plasma exposure
27.2.1.1 Hole in a Saphenous Vein
A saphenous vein from a pig ear was first identified; a needle was then used to
punch a hole in this vein as shown in Fig. 27.2a, in which the orange ring is an ID
tag. When blood flow started as shown in Fig. 27.2b, it was treated immediately by
the plasma spray with an exposure distance of 2.5 cm as shown in Fig. 27.2c. The
plasma spray was run with 2 s on and 2 s off alternately. After 7 runs, the bleeding
was stopped completely as demonstrated in Fig. 27.2d. The total exposure time to
the plasma spray was 14 s. In the untreated control, the bleeding time of the other
pig was measured to be about 88 s.
27.2.1.2 A Cut to an Artery
Before cutting an artery, the ear was tied with a tourniquet to slow down the blood
flow. A scalpel was then used to cut the ear small artery and then measure the natu-
ral coagulation time as well as the needed plasma treatment time. The processes of
the two cases, untreated and treated, are illustrated in Figs. 27.3a, and b, respec-
tively. The treatment was adopting an intermittent approach, plasma on-off alter-
nately with 2-s on 4-s off. It was found that it took 1 min to stop bleeding naturally.
On the other hand, bleeding was stopped after 6 runs of plasma on-off treatment.
Although the total treatment time was about 35 s, only about half of the natural clot-
ting time, the actual plasma treatment time was only 12 s.
Fig. 27.3 Photos showing the processes of cutting an artery, and (a) leaving the bleeding to stop
naturally and (b) stopping the bleeding by the plasma spray
354 S.P. Kuo et al.
27.3 Post-Operative Observation of Straight Cut and Cross

Cut Wound Healing After Plasma Treatment
The post-operative observation is important and feasible in vivo situation. It helps

to understand the plasma effluent effect upon the skin tissue surrounding the
treated wound and upon the progress of wound recovery. Straight cut and cross
cut wounds were introduced to three pigs which were 6-month-old and had a
weight of about 40 kg; one was untreated as a control and the other two were
treated by the plasma torch with an intermittent exposure approach. Each test was
repeated once. We then observed the recovering situation. After the tests pigs
were raised in stainless experiment cages to keep them from rubbing their skin.
The post-operative observation recorded the changes of the treated wounds in
14 days.
Two sets of running parameters (TE, TP, N, D) = (2, 4, 4, 3) and (2, 2, 5, 3) were
chosen for the intermittent treatments, where the torch running parameters TE, TP,
N, and D are the torch on time at each run, off (pause) time between two runs, num-
ber of runs in a treatment, and exposure distance, respectively. The respective heal-
ing progresses of treated straight cuts are presented in rows 1 and 2 of Fig. 27.5. As
seen, the scabs were peeling in the 6th day, and crusts were removed completely in
the 14th day in both cases. The progress of wound healing without plasma treatment
(natural healing) is presented in row 3 of Fig. 27.4 for a comparison. The peeling of
scab prolonged to the 8th day. The tissue was also repaired completely without any
crust in the 14th day.
On the other hand, the healing progresses of the cross cuts treated by plasma by
the two intermittent approaches are slightly different. The progresses of the recov-
ery in the two cases are presented in rows 1 and 2 of Fig. 27.5. It shows that the
scabs start to be peeling in the 6th day and 4th day, respectively; moreover,
the crusts disappear completely in the 10th day and 8th day. The progress of the
untreated cross cut is presented in row 3 of Fig. 27.5 for a comparison. A small
piece of the crust still remains in the wound area in the 14th observation day.
Fig. 27.4 Progress of the recovery of treated (rows 1 and 2) and untreated (row 3) straight cuts
Fig. 27.5 Progress of the recovery of treated (rows 1 and 2) and untreated (row 3) cross cuts
27.4 Discussion and Conclusion
In this study, we report in-vivo tests of blood coagulation assisted by a plasma spray.
The experimental results have demonstrated that this plasma spray could rapidly
stop bleeding. It was not easy to locate a saphenous vein in the body of the pig. Thus
an ear saphenous vein, which is a relatively small vein, was chosen in the test. With
this choice, it also reduced the blood loss of the pig used in the untreated control.
Yet, the plasma effluent reduced the bleeding time from 88 to 15 s. Since bleeding
from an artery is difficult to stop, again an ear artery was chosen for the test and the
ear was tied with a tourniquet before the cut. The results of the tests showed that the
plasma effluent can effectively clog the cut to the artery to stop bleeding; the bleed-
ing time was reduced to a half.
The experimental results have shown that this plasma spray could rapidly clot
blood to stop bleeding and made a positive impact on wound healing. The atomic
oxygen produced in the plasma effluent is likely the catalyst in the coagulation pro-
cesses. When interacted with H2O, atomic oxygen carried by the plasma effluent
can generate large amount of reactive oxygen species (oxygen ions, free radicals,
and peroxides). Studies have shown that platelets are a prime target for oxidants
produced or released in the vascular lumen and, at the same time, they are also
capable of endogenous generation of oxidants [18, 19]. It has also been shown that
oxidants can affect several key steps of platelet function to enhance platelet aggre-
gation [1921].
Hypoxia [22] acts a key factor to stimulus tissue repair by creating an oxygen
gradient from the hypoxic tissue of wound to the nearby unbroken tissue [23].The
central area of the wound is most hypoxic, and the oxygen gradient increase toward
the uninjured tissue progressively. However, with the supply of RAO from the
plasma spray, the amount of oxygen, consumed to generate H2O2, is reduced.
Consequently, more oxygen can be shared in other action such as producing super-
oxide (SOD), cell metabolism and raising tissue oxygen tension in the wound heal-
ing. RAO also provides the oxygen in the blood by reaction of catalase which plays
a protection role avoiding cells damaged by H2O2. In summary, RAO reduces the
demand of oxygen using in respiratory burst and increases oxygen content of tissue
356 S.P. Kuo et al.
indirectly. Both of reducing requirement and increasing supplement paths raise the
tissue oxygen tension in the wound site during the plasma treatment, as well as
provide the oxygen for wound healing and cell metabolisms
Acknowledgements We are grateful to Alessandro Betti for fabricating the plasma spray
device. This work was supported in part by a NYU-Poly seed Grant and in part by Adventix
Technologies Inc.
References
1. Jevon P, Cooper L (2008) First aid. Part 5. First-aid treatment for severe bleeding. Nurs Times
104:267
2. Perkins JG, Cap AP, Weiss BM, Reid TJ, Bolan CD (2008) Massive transfusion and nonsurgi-
cal hemostatic agents. Crit Care Med 36(Suppl 7):S32539
3. Vargo JJ (2004) Clinical applications of the argon plasma coagulator. Gastrointest Endosc
59:8188
4. Raiser J, Zenker M (2006) Argon plasma coagulation for open surgical and endoscopic appli-
cations: state of the art. J Phys D Appl Phys 39:35203523
5. Malick KJ (2006) Clinical applications of argon plasma coagulation in endoscopy. Gastroenterol
Nurs 29:386391
6. Ambrosio G, Oriente A, Napoli C, Palumbo G, Chiariello P, Marone G, Condorelli M,
Chiariello M, Triggiani M (1994) Oxygen radicals inhibit human plasma acetylhydrolase, the
enzyme that catabolizes platelet-activating factor. J Clin Invest 93:240816
7. Golino P, Ragni M, Cirillo P, Avvedimento VE, Feliciello A, Esposito N, Scognamiglio A,
Trimarco B, Iaccarino G, Condorelli M, Chiariello M, Ambrosio G (1996) Effects of tissue
factor induced by oxygen free radicals on coronary flow during reperfusion. Nature Med
2:3540
8. Kalghatgi SU, Fridman G, Cooper M, Nagaraj G, Peddinghaus M, Balasubramanian M,
Vasilets VN, Gutsol AF, Fridman A, Friedman G (2007) Mechanism of blood coagulation by
nonthermal atmospheric pressure dielectric barrier discharge plasma. IEEE Trans Plasma Sci
35:15591566
9. Fridman G, Peddinghaus M, Ayan H, Fridman A, Balasubramanian M, Gutsol A, Brooks AD,
Friedman G (2006) Blood coagulation and living tissue sterilization by floating electrode
10. Chen CY, Fan HW, Kuo SP, Chang JH, Pedersen T, Mills T, Huang CC (2009) Blood clotting
by low temperature air plasma. IEEE Trans Plasma Sci 37:993999
11. Kuo SP, Tarasenko O, Chang JH, Popovic S, Chen CY, Fan HW, Scott A, Lahiani M, Alusta P,
Drake JD, Nikolic M (2009) Contribution of a portable air plasma torch to rapid blood coagu-
lation as a method of preventing bleeding. New J Phys 11:115016, 17 pp
12. Kuo SP (2010) Portable plasma sterilizer. US Patent US7777151
13. Kuo SP, Pedersen T, Mills T (2008) Lateral distribution of atomic oxygen flux produced by an
array of three fan-shaped plasma torches. IEEE Trans Plasma Sci 36:10561057
14. Kuo SP, Chen CY, Lin CS, Chiang SH (2010) Wound bleeding control by low temperature air
plasma. IEEE Trans Plasma Sci 38:19081914
15. Oda T, Yamashita Y, Takezawa K, Ono R (2006) Oxygen atom behavior in the nonthermal
plasma. Thin Solid Films 506507:669673
16. Georg A, Engemann J, Brockhaus A (2002) Investigation of a pulsed oxygen microwave
plasma by time-resolved two-photon allowed laser-induced fluorescence. J Phys D Appl Phys
35:875881
17. Henderson WR, Fite WL, Brackmann RT (1969) Dissociative attachment of electrons to hot
oxygen. Phys Rev 183:157166
18. Finazzi-Agro A, Menichelli A, Persiani M, Biancini G, Del Principe D (1982) Hydrogen

peroxide release from human blood platelets. Biochim Biophys Acta 718:2125
19. Del Principe D, Menichelli A, De Matteis W, Di Giulio S, Giordani M, Savini I, Finazzi-Agro
A (1991) Hydrogen peroxide is an intermediate in the platelet activation cascade triggered by
collagen, but not by thrombin. Thromb Res 62:36575
20. Del Principe D, Menichelli A, De Matteis W, Di Corpo ML, Di Giulio S, Finazzi-Agro A
(1985) Hydrogen peroxide has a role in the aggregation of human platelets. FEBS Lett
185:1426
21. Pratic D, Iuliano L, Alessandri C, Camastra C, Violi F (1993) Polymorphonuclear leukocyte-
derived O2-reactive species activate primed platelets in human whole blood. Am J Physiol
264:H1582H1587
22. Davis JC, Hunt TK (1988) Problem wounds: the role of oxygen. Elsevier, New York
23. La Van FB, Hunt TK (1990) Oxygen and wound healing. Clin Plast Surg 17(3):46372
Part V
Plasma and Electric Fields in Medicine
Chapter 28
Subcellular Biological Effects of Nanosecond
Pulsed Electric Fields
Juergen F. Kolb and Michael Stacey
Abstract Membranes of biological cells can be charged by exposure to pulsed electric

fields. After the potential difference across the barrier reaches critical values on the
order of 1 V, pores will form. For moderate pulse parameters of duration and ampli-
tude, the effect is limited to the outer cell membrane. With the exposure to nanosecond
pulses of several tens of kilovolts per centimeter, a similar effect is also expected for
subcellular membranes and structures. Cells will respond to the disruption by different
biochemical processes. This offers possibilities for the development of novel medical
therapies, the manipulation of cells and microbiological decontamination.
Morphologies and functions of cells and their constituents can be changed by expo-
sures to electric fields. The response that is instigated depends on the strength of the
electric field and the duration it is acting upon the target. Short stimuli (on the order
of milliseconds and voltages of several tens of millivolts) that are imposed across
the cell membrane open voltage-gated channels and, in this way, regulate the trans-
port of ions such as potassium and sodium across the membrane [13]. Extended
exposures of several seconds or even minutes with several tens of volts per meter
provide enough energy to either denaturate proteins or even cause thermal damage
directly, i.e., burn tissues. Stimulations of the first kind are used in electrophysiolog-
ical studies of action potentials, for example. The second type of exposure, with the
goal of delivering energy to malignant tissues, is utilized in radio- or microwave
ablation therapies [4, 5]. Instead of these quasi-continuous exposures, temperatures
J.F. Kolb (*)

Leibniz Institute for Plasma Science and Technology e.V. (INP Greifswald),
e-mail: juergen.kolb@inp-greifswald.de
M. Stacey
Frank Reidy Research Center for Bioelectrics, Old Dominion University,
4211 Monarch Way, Norfolk, VA 23508, USA
362 J.F. Kolb and M. Stacey
that are no longer conducive to survival can also be achieved with a single pulsed
electric field of short duration, if the field strength is sufficiently high. More inter-
esting, however, are the more specific responses that can be accomplished with
short, pulsed electric field exposures that will not result in thermal damage, but are
already considerably stronger than stimuli that would merely trigger a physiological
reaction by membrane proteins. Biological effects that are instigated above this
threshold are generally caused by an initial increase in the permeability of mem-
branes. This effect is generally attributed to the formation of pores. Characteristics
and dynamics, i.e., pore diameter, their distribution along the cell surface, and life-
time, are critically dependent on the pulse duration and magnitude of the applied
electric fields [610]. In addition, many applications take advantage of a cumulative
effect on the membrane and rely on exposures with bursts of individual pulsed elec-
tric fields. Biological processes, and ultimately, the fate of a cell, depend on the
possibility that these pores will reseal, and on the transport of substances across the
membrane, either by migration or diffusion, while they are open [1113].
Consequently, the delicate balance of ion concentrations of sodium, potassium,
chlorine, calcium and others will be disturbed. Moreover, large molecules, such as
pharmaceutical agents and genetic material, can pass this otherwise-impenetrable
barrier [14, 15]. After membrane integrity has been restored, these additions to the
cytoplasm will then participate in the cells biochemistry.
This offers intriguing possibilities for new medical therapies and novel biotech-
nological approaches. For example, can chemotherapeutic agents that are poorly
membrane permeant, and consequently require large doses to be effective, now be
efficiently delivered into tumor cells directly [14, 16]. Another appealing applica-
tion is the introduction of genetic material into cells to change their function and
development. The method is currently being investigated for its potential as a vac-
cination against certain cancers, while the most prominent use of the technique is
probably still in cloning [17, 18]. Of course, if exposure parameters prevent resto-
ration of membrane integrity, cells will die [13]. Accordingly, pulsed electric fields
have also been used successfully to ablate tissues and to inactivate microorgan-
isms, particularly in liquids where most current applications focus on the decon-
tamination of water and food, such as milk and juices [1921]. In fact, the killing
of bacteria and yeast in water by this method was already reported by Sale and
Hamilton in 1967, which is also commonly assumed to be the first account of
membrane damage by pulsed electric field [22, 23]. The process was later described
as electroporation by Neumann [24]. The name summarizes theories that explain
the damage to the membrane and lead to an increase in permeability by the forma-
tion of pores under the influence of the electric field. When exposure conditions
allow for membrane recovery, they may still have a temporary effect on microor-
ganisms without necessarily killing them. For aquatic species, e.g., brine shrimp, a
temporary inactivation or stunning is observed that might last several minutes
but does not seem to permanently impair the organisms functions [25]. Details of
the mechanisms responsible for the observed transient inactivation of organisms of
higher order are even less well studied and understood than for single cells.
However, an obvious application of the method is the prevention of biofouling in
28 Subcellular Biological Effects of Nanosecond Pulsed Electric Fields 363
water treatment facilities or the treatment of coolant water taken from reservoirs
(lakes and rivers) without the need for chemical solutions, and therefore, no envi-
ronmental burden [26].
28.1 Basic Mechanisms of the Interaction of Pulsed

Electric Fields and Cells
Different applications and the ranges for pulse durations and pulse amplitudes nec-
essary to achieve a specific response not caused by thermal damage or by the phys-
iological activation of membrane proteins, are shown in Fig. 28.1. Most of these
applications assume that an increase in membrane permeability is the underlying
cause, and that this increase in permeability is primarily limited to the outer cell
membrane. Subsequent biological responses are a result of the different processes
that are set in motion by this damage. Whether pores form in the membrane depends
on the voltage difference that can be achieved across the membrane during the
exposure. Without damage, the cell membrane resembles a fairly good insulator.
Fig. 28.1 Parameter ranges for different applications of pulsed electric field exposures of cells.
A potential difference above a critical voltage, Vcr, will result in the formation of pores, which initi-
ate the subsequent response. For shorter pulse durations, increasingly higher voltages are required
to reach the critical voltage during exposure as indicated by the dash-dotted line. (The calculation
of exposure parameter for a given critical voltage, Vcr, is assuming a spherical cell of 10 mm,
suspended in a medium of 70 Wcm, equivalent to the conductivity of the cytoplasm.) The electrical
energy applied will also heat cells, and thermal damage becomes more prevalent with increasing
field strengths as indicated by the dashed line. (The calculation of the critical temperature increase,
DTcr, assumes an adiabatic heating of the cell volume filled with water)
Charges (sodium, potassium and other ions) accumulate along this barrier during
the application of an electric field, E, leading to a change in the transmembrane
potential, Vcell. When the membrane potential change reaches a critical value rang-
ing from several hundred millivolts to 1 V, pores will form. (The value varies
among cell types, but is close to 1 V for most cell membranes.) An extended expo-
sure primarily provides the energy to increase pore size and number [27].
For a spherical cell with diameter, D, the charging characteristic at the poles of
the cell (with respect to the direction of the field) is described by Eqs. 28.1 and 28.2
[28, 29]. (The angular dependency of the induced membrane potentials is described
by a cosine modulation of this maximum value not shown.) Hereby, the charging
time constant, tc, is again determined by the size of the cell, together with the spe-
cific membrane capacity, cm, and the conductivities of the cytosol, rc, and the cell
suspension, ra. Typical values for the resistivity of the cytoplasm for physiological
cell solutions are on the order of 70 Wcm, while values of 1 mF/cm2 have been deter-
mined for the specific membrane capacitance [30, 31].
D
| Vcell (t) | = 1.5 E (1 exp( t / c )) (28.1)
2
D
c = (c + 0.5 a ) c m (28.2)
2
More sophisticated analytical and numerical models have been developed,

striving for a more accurate description of exposure conditions, different mem-
brane structures and compositions (e.g., for bacteria), cell parameters and cell
shapes [3238]. However, key observations can be sufficiently evaluated with this
basic formula, particularly for spherical mammalian cells. According to Eqs. 28.1
and 28.2, for a cell with a diameter of 10 mm, an electric field of at least 1.34 kV/
cm needs to be applied to achieve a change of membrane potential by 1 V in the
steady state. Much higher electric fields are required for shorter exposure times
(Fig. 28.1). The analysis further shows that for a given electric field, larger cells
can reach critical transmembrane voltages faster than smaller cells. Accordingly,
smaller microorganisms, such as bacteria and yeast, require exposures to higher
electric fields to porate their membranes, and eventually inactivate them, than
mammalian cells do for the transient permeabilization required to introduce
drugs.
With the charging of the outer cell membrane, a counterfield develops inside the
cell, which effectively shields subcellular structures from further exposure to the
applied field. Potential differences that are adjusting across the internal membranes
of organelles, such as the nucleus, mitochondria, endoplasmic reticulum and others,
during the evolution towards steady state conditions, can be described by Eq. 28.3.
(The derivation of the equation assumes a spherical organelle of diameter d in the
center of a spherical cell. The organelle is exposed to an electric field, which is ini-
tially the same as the external field but is decreasing in the same manner as DVcell is
increasing. Again, the angular dependence is omitted.)
d t t
| Vorganelle (t) | = (1.5)2 E exp( t / c ) c exp (28.3)
2 c o
c o
The charging time constant for the organelle, to, is determined by an expression
similar to Eq. 28.2, by replacing cell diameter, D, with organelle diameter, d, the
conductivity of the cytoplasm, rc, with the conductivity of the organelle content, ro,
and the ambient conductivity of the suspension, ra, with the conductivity of the cyto-
plasm, rc. (It is also assumed that the dielectric properties of all cellular membranes
are identical to the characteristics of the outer cell membrane.) The comparison with
the development of transmembrane voltages across the outer membrane shows that,
for sufficiently high electric fields, subcellular membranes will also experience
potential differences that are on the order of, or exceed, typical critical voltages. For
a 5-mm diameter organelle (e.g., the nucleus) inside a 10-mm cell, and an exposure to
a field of 10 kV/cm, the voltage changes after 5 ns across the membranes are
|DVorganelle| = 0.93 V and |DVcell| = 0.68 V, respectively. The example demonstrates that
subcellular membranes can in fact charge faster than the outer cell membrane.
Accordingly, poration of organelle membranes and modifications of subcellular struc-
tures can be expected for strong applied electric fields. The example further shows that
membranes are charged to critical voltages in only a few nanoseconds. Since the
extent of the exposure, after critical values are met, primarily determines the further
increase in pore density, short exposures on the order of, or shorter than, the charging
time of the outer cell membrane could have a more pronounced effect on organelles
than on the cell membrane. Moreover, even a less extensive poration of subcellular
membranes might be sufficient to trigger an irreversible biological response.
Equations 28.1, 28.2, and 28.3 give a simplified description of the mechanisms
following the application of an electric field. However, the approach cannot account
for many parameters of the exposure, such as actual subcellular geometries, mem-
brane compositions, or non-linear events, such as the change in membrane conduc-
tion after pores have formed. Even the application of a field with an infinitely fast rise
time is an idealization. More elaborate approaches have been employed to describe
actual experimental conditions more accurately and gain further insight. Most inter-
estingly, a more detailed analysis of the exposure to intense short pulsed electric
fields with a fast rise time predicts that, in fact, the pores that can be generated under
these conditions are different from pores that can be created by longer pulsed expo-
sures of lower field strength as commonly used in electroporation techniques for the
outer membrane. In particular, all membranes (outer cell membrane and organelle
membranes) are uniformly porated within a few tens of nanoseconds into the expo-
sure [7, 35, 39, 40]. The number and density of pores is predicted to be several orders
of magnitude higher when compared with electroporation pulses. However, pores are
also expected to be much smaller, which would allow only small ions to pass through
but not larger molecules [6, 41]. Since many of the ions that are involved in the regu-
lation of cellular functions still could permeate cellular membranes, cell functions
are likely to be affected. As a result, intense ultrashort pulsed electric field exposures
offer a method for intracellular manipulation of cells (Fig. 28.1).
28.2 Intracellular Effects of Nanosecond Pulsed

Electric Field Exposures
The theoretical analysis shows that exposures that primarily affect subcellular
structures require pulsed electric fields with a duration that is short compared to the
charging time of the cell envelope. (For a cell of 10 mm in diameter and resistivities
for cell suspension and cytoplasm of 70 Wcm, the charging time constant is 52.5 ns
and the charging time is, accordingly, on the order of 200 ns.) In addition, the field
strengths need to rise as fast as possible to amplitudes in the range of several tens of
kilovolts per centimeter to expose also the subcellular space before it is shielded and
critical voltages can no longer be achieved in the remaining field. These parameters
cannot be provided using conventional electronic circuits at least not for the expo-
sure of relevant cell or tissue volumes between electrodes at least a few millimeters
apart. Alternatively, pulsed power technologies are employed [42]. Basic circuits
are based on pulse forming networks or pulse forming lines. When using a pressur-
ized spark gap as the switching element, rise times of 1 ns have been realized for
voltage pulses with amplitudes of 35 kV, corresponding to electric fields of 350 kV/
cm across the electrode gap of 1 mm of a standard electroporation-cuvette [43].
The equipment allows the exposure of 100 ml of cell suspensions, which is sufficient
for the post-exposure evaluation of biological and biochemical responses in particu-
lar. (For lower electric fields, larger volumes can be exposed.) Similar systems have
also been used in in vivo experiments. In this case, high voltage pulses were usually
delivered by needle electrodes instead of plane parallel electrodes [4446]. For the
observation of early processes during or immediately after the exposure, micro-
scope-mounted systems have been developed [4749].
The fastest mechanism that has been observed so far after the exposure to nano-
second pulsed electric fields is the charging of the outer cell membrane [47]. A fast
voltage sensitive fluorescent dye was used, reflecting changes in the electric field
across the membrane by characteristic changes in excitation and emission spectra.
These changes were recorded with a temporal resolution of 5 ns and quantified to
describe the development of the associated transmembrane potential. An example
of the measurements is shown in Fig. 28.2.
The highest change in voltage (about 1.6 V) was observed at the anodic pole of
the cell (the side facing the anode in a parallel plate exposure system). The change
corresponds approximately to the values expected from theoretical models [50].
However, a significant difference has been observed between the anode and cathode
poles, with voltages that are 1 V lower at the cathode pole. Transmembrane voltages
across the cathode side also develop more slowly than for the anode side, which
suggests significant contributions to the potential difference across the membrane
from dipole alignment. These dipoles are found in headgroups of phospholipids that
are embedded in the membrane. (In addition dipole moments might also be induced
by the electric field.) The restricted mobility of the molecules might account for the
observed differences between hemispheres [5153]. The time at which the peak
value in transmembrane potential is approached during the exposure depends on the
Fig. 28.2 Transmembrane voltage changes (absolute values) of Jurkat cells for the exposure to a
50-kV/cm electric field of 60-ns duration (indicated by the shaded area). Values at the anode pole
jump to more than 1 V immediately. The difference in the values across anode and cathode might
be accounted for by the alignment of phospholipid heads, which then affect the local electric field.
Pores gradually open across the membrane during exposure and, after reaching a peak value of
about 1.4 V, allow for significant discharge currents
amplitude of the applied electric field. After the peak voltage is reached, values start
to decrease again. Apparently pores are starting to open across the membrane very
early during the exposure. Their continuous increase in number would allow for a
limited migration of ions, which first impedes charging and eventually will lead to
a reversal and discharge the membranes through these leaks at an increasing
rate. That the observed charging mechanisms are in fact primarily determined by
physical parameters was recently confirmed by similar experiments conducted in
plant cells [54].
Due to the significance of the cell membrane as the interface for the cell to receive
and process outside stimuli, and to the readily available methods to study these
interactions, many studies have focused on the effects on the outer membrane [5561].
They have confirmed that the membrane initially becomes permeable for smaller
ions only. However, for relatively long exposures and relatively high electric fields,
it is possible that the membrane eventually becomes permeable for larger molecules
[60, 61]. Some experiments on the permeability for small ions show certain other
characteristics, such as ion selectivity or preferences of ion movements that cannot
be explained by simple diffusion through holes in the cell envelope alone [58, 62].
Likewise this model cannot sufficiently explain the observed translocation of phos-
phatidylserine to the outer cell surface [63, 64]. (The membrane protein is usually
found exclusively on the inside of healthy cells and expressed on the outside only
when cells undergo apoptosis.)
The reasonably good agreement between the measurements of transmembrane

potential voltages induced across the outer membrane and theoretical predictions
suggests that predictions for the poration of subcellular structures by nanosecond
pulsed electric fields are reasonable as well. Unfortunately, measurements are not
yet available on the development of transmembrane voltage changes across internal
membranes. Some experimental proof of the theoretical models was provided with
vesicle systems (artificial lipid bilayer spheres) [65]. Since the direct observation of
membrane potentials across internal membranes is challenging, the evaluation of
subcellular mechanisms has mostly been based on the observation of secondary
effects, which can often be explained by the poration of organelle membranes [66].
Further complications arise from the complexity of geometries and unknown char-
acteristics of subcellular structures, for example, whether mitochondria are envel-
oped by a double membrane, and the difference in composition between interior
membranes and the outer cell membrane. (The outer membranes of different cell
types also show considerable differences in the relative quantities of different mem-
brane proteins.) In addition, the dielectric properties of organelles and other subcel-
lular structures are often unknown. Finally, membranes are not the only constituents
that can be affected by nanosecond pulsed electric fields. Molecular structure of the
cytoskeleton and genetic molecules are highly charged and are likely to respond to
strong electric fields [67]. An example for the response of the cytoskeleton to the
exposure of 60-ns pulses is shown in Fig. 28.3. (The cytoskeleton is a network of
protein filaments running through the cytoplasm, which provide structure to the
cells and anchors for the organelles. It plays an active role in many cellular pro-
cesses.) In adherent-growing cell lines, a ruffled appearance of the membrane within
1 min after the exposure makes apparent the rapid disruption of the cytoskeleton.
Within a few minutes, the support structure of the cytoskeleton is gone and cells
become round and detach. In addition, exposed cells also showed a reduction in
telomere count; these are structures that tether the chromosomes to the nuclear
membrane [68]. Similar disruption of cytoskeleton and nuclear membrane was also
observed for plant cells [69]. Without support structures, cells struggle to survive
and become more susceptible to subsequent pulsed electric field exposures. This
could also explain why the survival rate is much lower in cells lacking an extensive
supporting cytoskeleton such as Jurkat cells. In these cells, the application of a
nanosecond pulsed electric field led to deterioration of the cytoskeleton within sec-
onds of exposure.
Nanosecond pulsed electric field exposures could also affect the nucleus and
nucleic acids directly [70, 71]. Changes in chromatid structures, such as gaps,
breaks, and the number of fragments, have been observed for nanosecond pulsed
electric field exposures, which are similar to damage that can be induced by ionizing
radiation [67, 72]. (Chromatids are the two main substructures making up a chromo-
some). Damage to DNA was assessed using comet assays or standard electrophore-
sis ladders, which showed much longer comet tails when compared to unexposed
cells, indicating DNA fragmentation, particularly for suspension cells. That this
damage is most likely caused by the electric field directly and is not a secondary cell
response triggered by the exposure is indicated by significant differences in the
Fig. 28.3 The cytoskeleton of HeLa cells was made visible by staining actin filaments (with
Oregon green 488 phalloidin). The cells were growing attached to polylysine-coated coverslips.
Image (a) shows the extensive and intact cytoskeleton typical of control cells (sham exposed).
Image (b) shows cells 1 min after exposure to a single 60-ns pulsed electric field of 60 kV/cm. The
membrane appears ruffled and filaments become less distinct. Four minutes after exposure, the
cytoskeleton structure disappears and bright actin spots appear instead. Simultaneously, the cell
shape becomes spherical and cells detach from the cover slip
DNA extracted immediately after exposure. Direct effects on the nucleus have also
been found with DNA markers, such as acridine orange [61]. The dye is used as a
nuclear stain, intercalating with the double strand structure of the DNA. Immediately
after the exposure to a nanosecond pulsed electric field, the recorded fluorescence
activity decreases. This observation can be interpreted either as a direct effect on the
binding sites between DNA molecules and dye, or as an outflow of dye through
pores that are forming in the nuclear envelope.
Notwithstanding the significant direct effect on macromolecules and struc-
tures, the exchange of molecules and ions across barriers through pores formed by
the exposure is still likely to be the most important mechanism to affect cell func-
tions. The concentrations of many of the ions released into the cytosol this way
are, under ordinary circumstances, carefully maintained. A sudden change in con-
centration will lead to a response with the goal of compensating for the imbal-
ance. Since changes in ion concentrations regulate cell functions, nanosecond
pulsed electric fields should likewise provide control of these mechanisms. First
proof of this concept was provided in an experiment conducted by Stephen
Buescher with neutrophil chemotaxis. When placed in a microreactor between two
electrodes, the cells move towards a chemoattractant. The directed movement
is temporarily interrupted by the application of a single 300 ns, 45 kV/cm
pulse [73].
One of the most important ions involved in intracellular signaling events and
chemical signal transmission between cells is calcium. It is stored inside cells in
the endoplasmic reticulum and mitochondria and is released in small quantities
for signaling events. Cell functions that are mediated by varying calcium concen-
trations include fertilization, muscle contraction and apoptosis. Calcium responses
Fig. 28.4 A single pulsed electric field of 60 ns instigates an immediate, transient release of cal-
cium from intracellular stores (as indicated by the fluorophore fluo-4). The magnitude of the fluo-
rescence response increases with field strength. For fields of 50 kV/cm, the induced signals are of
the same order of magnitude as subsequent naturally occurring fluctuations. However, for a field of
100 kV/cm, these random changes no longer occur, indicating sustained subcellular damage. This
seems to be confirmed by the instantaneous drop in fluorescence after several minutes, suggesting
loss of membrane integrity
induced with nanosecond pulsed electric fields have been observed in a variety of
cell types with different pulse parameters [7376]. A detailed analysis shows that
the release of calcium from internal stores occurs within only a few milliseconds
after exposure, again indicating that pores, rather than physiological pathways,
are responsible [77]. The calcium response is transient and qualitatively similar to
physiological signals, as observed for a normal, i.e., unperturbed, cell. This shows
that cells deal with the electrical stimulus in a manner similar to other trigger
mechanisms, and calcium is eventually returned to these intracellular stores
through calcium pumps. How much calcium is released depends on the pulse
parameters (Fig. 28.4). When only moderate increases in concentrations are insti-
gated, calcium activated channels do not seem to activate in the outer cell mem-
brane, and no calcium is taken up from outside. However, if the pulse amplitude
is large (with respect to pulse duration and cell type), calcium pathways can incur
significant damage. Calcium rushes in through the cell membrane, further increas-
ing calcium concentrations. In this case, cells wont recover from the stimulus; in
fact, after several minutes, loss of membrane integrity can be observed, which
indicates the death of the cell.
The manipulation of biochemical processes via the regulation of calcium levels

offers intriguing potential for applications. However, other pathways and messengers
might be affected in the same way. Some that have been investigated with respect to
triggering of apoptosis are caspases [55, 71, 78, 79].
28.3 Applications of Nanosecond Pulsed Electric

Field Exposures
As a tool, pulsed electric field exposures offer interesting possibilities for the treat-
ment of cells in medical and environmental applications. The potential to disrupt
cell membranes and subcellular components with the goal of killing cells is appar-
ent [19]. As such, the method is particularly interesting and is investigated to treat
contamination of liquid foods, e.g. milk and fruit juices, and also for the treatment
of waste water and drinking water [20, 25, 8085]. For nanosecond pulsed electric
fields, the stimulus can actually reach inside the cell and be used against pathogens
that are otherwise protected against agents acting on the cell membrane or chemi-
cals that need to permeate the membrane first [86, 87]. The lack of chemical residue
is a further advantage for environmental applications.
The unique strength of the method, however, lies in the possibility of instigating
more subtle responses [67, 72, 88, 89]. Sub-lethal exposure conditions still hold the
potential to affect internal structures and membranes. Many of the direct mecha-
nisms are speculative but it seems plausible that proteins, e.g., receptors, can be
directly affected, for example, by breaking individual molecular bonds or by induc-
ing charge shifts along macromolecules. Subsequently, cells will respond with a
characteristic signaling cascade and coping mechanism if the stimulus can mimic a
familiar stimulus, for example, from a chemical compound. Alternatively, protein
structures might actually be broken and cells will have to expend repair mecha-
nisms, possibly leading to unforeseen results in the attempt to repair or compensate
for the damage [90, 91]. Even the relatively simple process of releasing ions from
internal stores, in particular calcium [73, 74, 77], will first of all be interpreted as a
biochemical signal. Many of the cell functions that are controlled by intracellular
calcium concentrations will respond accordingly [73]. This offers a particular means
to control the behavior of specialized cells. These include excitable cells, and
accordingly, the effect of nanosecond pulsed electric fields on cells have been shown
for cardiac myocytes [92], skeletal muscle [93], motoneurons [94], and neurosecre-
tory cells [95].
When applied to platelets, pulsed electric field exposures have been shown to
instigate the same response as the enzyme thrombin. The process is also mediated
by an increase in intracellular calcium concentrations. As a result, platelets aggre-
gate [96]. (Platelets are specialized blood cells that are activated to aggregate in
wounds and contribute to coagulation.) Platelet rich plasma is used in surgical and
chronic wound care. The activation using the physical (electrical) stimulus avoids
complications that have been associated with the use of bovine thrombin and further
eliminate the dependency on the protein. Studies that have been conducted to deter-
mine the healing rate using platelet gels activated by pulsed electric fields show that
wounds heal at least as fast as when the gels are activated with thrombin. In addi-
tion, a bactericidal effect of the electric field activated gel was observed, which
would help to prevent infections.
Perhaps the most interesting application of nanosecond pulsed electric fields
involves their capacity to induce apoptosis in cancer cells. (Apoptosis is also known
as programmed cell death a process that is inhibited in cancer cells, leading to
uncontrolled cell proliferation.) The interaction mechanisms are still under investi-
gation, but in general, many different processes could be affected by exposure to an
electric field [41, 97]. Accordingly, different pathways have been investigated [57,
60, 61, 63, 67, 78, 98]. Apoptosis can be instigated by the effect on either the outer
membrane or on organelles, such as the mitochondria. Hallmarks of apoptosis, such
as activation of caspases, release of cytochrome c, phosphatidylserine externaliza-
tion and DNA fragmentation, have been observed and studied with respect to expo-
sure conditions [46, 78, 79, 99102].
More recently, the efficacy of pulsed electric fields alone as a tumor therapy has
been tested in in vivo experiments on different tumor types. Figure 28.5 shows first
results for B16 melanoma tumors grown in mouse skin. By applying 100 pulses of
300 ns duration and 10 kV amplitude in different locations across the tumor, a
significant reduction in tumor size could be achieved in only one day. No chemo-
therapeutic drugs were administered to enhance this effect. In subsequent studies,
the treatment conditions were optimized and eventually a complete remission of
tumors was achieved in a group of 17 animals with a treatment regimen applying
up to 100 pulses of 300-ns, on different days during a 2 week period [103]. (The
number of treatments depended on the individual tumor response.) While more of
half of the 18 control animals did not survive for more than 3 weeks (and few did
much beyond that), did all of the treated animals live for more than 120 days, the
commonly accepted point demonstrating long term survival. Tumor cells treated
in vivo showed many of the same hallmarks of apoptosis that were already observed
for cell suspensions and some other features, such as characteristic nuclear shrink-
age [44, 45, 104]. In addition, nanosecond pulsed electric fields showed the ability
to act not only on individual cells, but also systemically on the tumor by disrupting
the capability for angiogenesis, hence disrupting the tumors supply of nutrients
from blood [44, 45]. In the meantime, similar results and long term survival could
also be demonstrated on HEP1-6 liver tumors that were also grown subcutaneously
in a mouse model and treated with 100-ns pulses. Many other tumor cell lines have
been investigated for their susceptibility, at least in vitro, but some additional ones,
in vivo [46, 71, 79, 89]. In one case, a basal cell carcinoma was successfully treated
in a patient [46].
Although nanosecond pulsed electric field treatments do not require additional
agents, such as chemotherapeutics, to be effective, possible synergies might actually
enhance a sought-after effect, for example, by increasing the permeability of subcel-
lular membranes for chemical compounds [65, 105]. The possibility to reach into
the cell with an electric field could also offer a way to control cell functions remotely
Fig. 28.5 Results of a pilot study comparing untreated melanoma tumors (right column) and mel-
anoma tumors that were treated with a nanosecond pulsed electric field regimen (left column). One
million B16f murine melanoma cells were injected into the flanks of C57BL6 mice. When tumors
reached a diameter of about 5 mm, nanosecond pulsed electric fields were applied by inserting a
pair of needle electrodes (31 gauge hypodermic needles) on either side of the tumor. The electric
field was generated by applying a 10-kV pulse of 300-ns duration (30 ns rise time) from a Blumlein
pulse forming network. For the best possible exposure, the electrodes were relocated in 34 steps
(depending on tumor size) of 1 mm and the treatment repeated at the new location. In each loca-
tion, 100 pulses were applied. The upper left picture shows the tumor immediately after the first
treatment with injection sites clearly visible. The treatment was repeated on the next day. The
lower left picture shows the same tumor on the third day. The treated tumor is regressing rapidly,
while the control tumor almost doubles in size over the same period
by introducing otherwise inactive substances. A first attempt has been made using
carbon nanotubes, which were introduced into tumor cells and are expected to
respond strongly to an applied electric field due to their unique electrical character-
istics [106]. Even these newer developments, however, rely on electrode systems
that can be brought close to the tumors. Accordingly, targets that can be treated have
to be located close to the skin surface or they will require invasive surgery. A new
idea proposes to focus strong electric fields into a patient by using ultrawideband
antennas [107]. This appealing approach requires shortening the high voltage pulses
that have to be applied to the antenna into the picosecond range and increase the
amplitudes to several tens to hundreds of megavolts per centimeter. Accessing this
parameter range poses new challenges and opportunities for engineering and
research. Different physical phenomena and processes will be dominant for these
conditions, and as a consequence, will likely lead to different biological responses
as it has already been observed for cell viability [108].
Acknowledgment The insight and knowledge on effects and applications of pulsed electric
fields is the result of a dedicated group of researchers on this topic. I have been lucky to work
with many of them at the Frank Reidy Research Center for Bioelectrics or to have found them as
collaborators at Old Dominion University. Without their ideas and dedication, the field would
not have advanced as it has. Credit and my thanks for the opportunity to write this summary
therefore are extended to E. Stephen Buescher, Peter F. Blackmore, Michael Stacey, Stephen J.
Beebe, James R. Swanson, Christopher Osgood, Ravindra P. Joshi, Shu Xiao, M. Arif Malik,
Yeong-Jer Chen, Richard Heller, Loree Heller, Olga Pakhomova, Andrei Pakhomov, Barbara
Hargrave, Richard Nuccitelli, Angela M. Bowman, Betsy Gregory, W. Hunter Baldwin, Jennifer
Pomicter, Wolfgang Frey, Uwe Pliquett, Jue Zhang, Barbara Carroll (also for proofreading this
and many other manuscripts), Ruth Lyman, many students (too many to list them all) who spent
years on the actual experiments and in particular Karl H. Schoenbach for his vision and lead-
ership in this field.
References
1. Catterall WA (2008) From ionic currents to molecular mechanisms: the structure and function
of voltage-gated sodium channels. Neuron 26:1325
2. Catterall WA (2000) Structure and regulation of voltage-gated Ca2+ channels. Annu Rev Cell
Dev Biol 16:521555
3. Pongs O (1992) Molecular biology of voltage-dependent potassium channels. Physiol Rev
72:S69S88
4. Habash R, Elwood JM, Krewski D, Lotz WG, McNamee J, Prato F (2009) Recent advances
in research on radiofrequency fields and health: 20042007. J Toxicol Environ Health B
12:250288
5. Simon CJ, Dupuy DE, Mayo-Smith WW (2005) Microwave ablation: principles and applica-
tions. Radiographics 25:S69S83
6. Vasilkoski Z, Esser A, Gowrishankar T, Weaver J (2006) Membrane electroporation:
the absolute rate equation and nanosecond time scale pore creation. Phys Rev E
74:021904(12)
7. Gowrishankar TR, Weaver JC (2006) Electrical behavior and pore accumulation in a multi-
cellular model for conventional and supra-electroporation. Biochem Biophys Res Commun
349:643653
8. Neumann E, Kakorin S, Toensing K (1999) Fundamentals of electroporative delivery of drugs
and genes. Bioelectrochemistry 48:316
9. Teissie J, Golzio M, Rols M (2005) Mechanisms of cell membrane electropermeabilization:
a minireview of our present (lack of ?) knowledge. Biochim Biophys Acta (BBA) General
Subjects 1724:270280
10. Benz R, Zimmermann U (1980) Pulse-length dependence of the electrical breakdown in lipid
bilayer membranes. Biochim Biophys Acta 597:637642
11. Weaver JC (1993) Electroporation: a general phenomenon for manipulating cells and tissues.
J Cell Biochem 51:426435
12. Jaroszeski MJ, Gilbert R, Heller R (1997) Electrochemotherapy an emerging drug delivery
method for the treatment of cancer. Adv Drug Deliv Rev 26:185197
13. Davalos RV, Mir LM, Rubinsky B (2005) Tissue ablation with irreversible electroporation.
Ann Biomed Eng 33:223231
14. Gothelf A (2003) Electrochemotherapy: results of cancer treatment using enhanced delivery
of bleomycin by electroporation. Cancer Treat Rev 29:371387
15. Heller LC, Ugen K, Heller R (2005) Electroporation for targeted gene transfer. Expert Opin
Drug Deliv 2:255268
16. Sersa G, Stabuc B, Cemazar M, Jancar B, Miklavi D, Rudolf Z (1998) Electrochemotherapy

with cisplatin: potentiation of local cisplatin antitumour effectiveness by application of elec-
tric pulses in cancer patients. Eur J Cancer 34:12131218
17. Lucas ML, Heller R (2003) IL-12 gene therapy using an electrically mediated non-viral
approach reduces metastatic growth of melanoma. DNA Cell Biol 22:755763
18. Forsberg EJ, Strelchenko NS, Augenstein ML, Betthauser JM, Childs LA, Eilertsen
KJ et al (2002) Production of cloned cattle from in vitro systems. Biol Reprod
67:327333
19. Castro AJ, Barbosa-Canovas GV, Swanson BG (1993) Microbial inactivation of foods by
pulsed electric fields. J Food Process Preserv 17:4773
20. Nguyen P, Mittal G (2007) Inactivation of naturally occurring microorganisms in tomato
juice using pulsed electric field (PEF) with and without antimicrobials. Chem Eng Process
46:360365
21. Reina LD, Jin ZT, Zhang QH, Yousef AE (1998) Inactivation of Listeria monocytogenes in
milk by pulsed electric field. J Food Prot 61:12031206
22. Sale AJH, Hamilton WA (1967) Effects of high electric fields on microorganisms: I. Killing
of bacteria and yeasts. Biochim Biophys Acta 148:781788
23. Hamilton WA, Sale AJH (1967) Effects of high electric fields on microorganisms: II.
Mechanism of action of the lethal effect. Biochim Biophys Acta 148:789800
24. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982) Gene transfer into mouse
lyoma cells by electroporation in high electric fields. EMBO J 1:841845
25. Schoenbach KH, Peterkin FE, Alden RW, Beebe SJ (1997) The effect of pulsed electric
fields on biological cells: experiments and applications. IEEE Trans Plasma Sci
25:284292
26. Amr AG, Schoenbach KH (2000) Biofouling prevention with pulsed electric fields. IEEE
27. Abidor IG, Arakelyan VB, Chernomordik LV, Chizmadzhev YA, Pasushenko VF, Tarasevich
MR (1979) Electric breakdown of bilayer lipid membranes I. The main experimental facts
and their qualitative discussion. Bioelectrochem Bioenerg 6:3752
28. Schwan HP (1963) Electric characteristics of tissues. Biophysik 1:198208
29. Cole KS (1937) Electric impedance of marine egg membranes. Trans Faraday Soc
33:966972
30. Tien HT (1974) Bilayer lipid membrane: theory and practice. Marcel Dekker, New York
31. Ohki S (1969) The electrical capacitance of phospholipid membranes. Biophys J
9:11951205
32. Pucihar G, Kotnik T, Vali B, Miklavi D (2006) Numerical determination of transmem-
brane voltage induced on irregularly shaped cells. Ann Biomed Eng 34:642652
33. Pucihar G, Miklavi D, Kotnik T (2009) A time-dependent numerical model of transmem-
brane voltage inducement and electroporation of irregularly shaped cells. IEEE Trans Biomed
Eng 56:14911501
34. Gowrishankar TR (2003) An approach to electrical modeling of single and multiple cells.
Proc Natl Acad Sci 100:32033208
35. Gowrishankar T, Esser A, Vasilkoski Z, Smith K, Weaver J (2006) Microdosimetry for con-
ventional and supra-electroporation in cells with organelles. Biochem Biophys Res Commun
341:12661276
36. Krassowska W, Filev PD (2007) Modeling electroporation in a single cell. Biophys J
92:404417
37. Joshi R, Hu Q, Schoenbach K, Beebe S (2004) Energy-landscape-model analysis for irrevers-
ibility and its pulse-width dependence in cells subjected to a high-intensity ultrashort electric
pulse. Phys Rev E 69:051901(10)
38. Hu Q, Joshi RP (2009) Analysis of intense, subnanosecond electrical pulse-induced trans-
membrane voltage in spheroidal cells with arbitrary orientation. IEEE Trans Biomed Eng
56:16171626
39. Hu Q, Joshi R, Schoenbach K (2005) Simulations of nanopore formation and phosphatidylser-

ine externalization in lipid membranes subjected to a high-intensity, ultrashort electric pulse.
Phys Rev E 72:031902(10)
40. Hu Q, Viswanadham S, Joshi R, Schoenbach K, Beebe S, Blackmore P (2005) Simulations of
transient membrane behavior in cells subjected to a high-intensity ultrashort electric pulse.
Phys Rev E 71:031914(9)
41. Esser AT, Smith KC, Gowrishankar TR, Weaver JC (2009) Towards solid tumor treatment by
nanosecond pulsed electric fields. Technol Cancer Res Treat 8:289306
42. Kolb JF (2010) Generation of ultrashort pulses. In: Pakhomov AG, Miklav i D, Markov MS
(eds) Advanced electroporation techniques in biology and medicine (biological effects of
electromagnetics). CRC Press, Boca Raton, pp 341352
43. Kolb JF, Kono S, Schoenbach KH (2006) Nanosecond pulsed electric field generators for the
study of subcellular effects. Bioelectromagnetics 27:172187
44. Nuccitelli R, Pliquett U, Chen X, Ford W, Swanson RJ, Beebe S et al (2006) Nanosecond
pulsed electric fields cause melanomas to self-destruct. Biochem Biophys Res Commun
343:351360
45. Chen X, Kolb JF, Swanson RJ, Schoenbach KH, Beebe SJ (2010) Apoptosis initiation and
angiogenesis inhibition: melanoma targets for nanosecond pulsed electric fields. Pigment
Cell Melanoma Res 23:554563
46. Garon EB, Sawcer D, Vernier PT, Tang T, Sun Y, Marcu L et al (2007) In vitro and in vivo
evaluation and a case report of intense nanosecond pulsed electric field as a local therapy for
human malignancies. Int J Cancer 121:675682
47. Frey W, White JA, Price RO, Blackmore PF, Joshi RP, Nuccitelli R et al (2006) Plasma mem-
brane voltage changes during nanosecond pulsed electric field exposure. Biophys J
90:36083615
48. De Angelis A, Kolb JF, Zeni L, Schoenbach KH (2008) Kilovolt Blumlein pulse generator
with variable pulse duration and polarity. Rev Sci Instrum 9:044301(4)
49. Kuthi A, Gabrielsson P, Behrend MR, Vernier PT, Gundersen MA (2005) Nanosecond pulse
generator using fast recovery diodes for cell electromanipulation. IEEE Trans Plasma Sci
33:11921197
50. Smith K, Weaver J (2008) Active mechanisms are needed to describe cell responses to sub-
microsecond, megavolt-per-meter pulses: cell models for ultrashort pulses. Biophys J
95:15471563
51. Marrink S, Devries A, Tieleman D (2009) Lipids on the move: simulations of membrane
pores, domains, stalks and curves. Biochim Biophys Acta Biomembr 1788:149168
52. Bockmann R, Degroot B, Kakorin S, Neumann E, Grubmller H (2008) Kinetics, statistics,
and energetics of lipid membrane electroporation studied by molecular dynamics simula-
tions. Biophys J 95:18371850
53. Qin H, Sridhara V, Joshi RP, Kolb JF, Schoenbach KH (2006) Molecular dynamics analysis
of high electric pulse effects on bilayer membranes containing DPPC and DPPS. IEEE Trans
Plasma Sci 34:14051411
54. Flickinger B, Berghfer T, Hohenberger P, Eing C, Frey W (2010) Transmembrane potential
measurements on plant cells using the voltage-sensitive dye ANNINE-6. Protoplasma
247:312
55. Deng J, Schoenbach KH, Buescher ES, Hair PS, Fox PM, Beebe SJ (2003) The effects of
intense submicrosecond electrical pulses on cells. Biophys J 84:27092714
56. Vernier PT, Sun Y, Marcu L, Craft CM, Gundersen MA (2004) Nanosecond pulsed electric
fields perturb membrane phospholipids in T lymphoblasts. FEBS Lett 572:103108
57. Pakhomov A, Shevin R, White J, Kolb J, Pakhomova O, Joshi R et al (2007) Membrane per-
meabilization and cell damage by ultrashort electric field shocks. Arch Biochem Biophys
465:109118
58. Pakhomov AG, Bowman AM, Ibey BL, Andre FM, Pakhomova ON, Schoenbach KH (2009)
Lipid nanopores can form a stable, ion channel-like conduction pathway in cell membrane.
Biochem Biophys Res Commun 385:181186
59. Kennedy SM, Ji Z, Hedstrom JC, Booske JH, Hagness SC (2008) Quantification of electropo-
rative uptake kinetics and electric field heterogeneity effects in cells. Biophys J
94:50185027
60. Baldwin WH, Gregory BW, Osgood CJ, Schoenbach KH, Kolb JF (2010) Membrane perme-
ability and cell survival after nanosecond pulsed electric field exposure - significance of
exposure-media composition. IEEE Trans Plasma Sci 38:29482953
61. Chen N, Schoenbach KH, Kolb JF, Swanson RJ, Garner AL, Yang J et al (2004) Leukemic
cell intracellular responses to nanosecond electric fields. Biochem Biophys Res Commun
317:421427
62. Bowman AM, Nesin OM, Pakhomova ON, Pakhomov AG (2010) Analysis of plasma mem-
brane integrity by fluorescent detection of Tl + uptake. J Membr Biol 236:1526
63. Vernier PT, Sun Y, Marcu L, Craft CM, Gundersen MA (2004) Nanoelectropulse-induced
phosphatidylserine translocation. Biophys J 86:40404048
64. Vernier PT, Ziegler MJ, Sun Y, Gundersen MA, Tieleman DP (2006) Nanopore-facilitated,
voltage-driven phosphatidylserine translocation in lipid bilayersin cells and in silico. Phys
Biol 3:233247
65. Tekle E, Oubrahim H, Dzekunov SM, Kolb JF, Schoenbach KH, Chock PB (2005) Selective
field effects on intracellular vacuoles and vesicle membranes with nanosecond electric pulses.
Biophys J 89:274284
66. Schoenbach KH, Beebe SJ, Buescher ES (2001) Intracellular effect of ultrashort electrical
pulses. Bioelectromagnetics 22:440448
67. Stacey M, Stickley J, Fox P, Statler V, Schoenbach K, Beebe SJ, Buescher S (2003) Differential
effects in cells exposed to ultra-short, high intensity electric fields: cell survival, DNA dam-
age, and cell cycle analysis. Mutat Res/Genet Toxicol Environ Mutagen 542:6575
68. Stacey M, Fox PM, Buescher ES, Kolb JF (2011) Nanosecond pulsed electric field induced
cytoskeleton and nuclear membrane and telomere damage adversely impact cell survival.
Bioelectrochemistry 82(2):131134
69. Berghfer T, Eing C, Flickinger B, Hohenberger P, Wegner LH, Frey W et al (2009)
Nanosecond electric pulses trigger actin responses in plant cells. Biochem Biophys Res
Commun 387:590595
70. Chen N, Garner AL, Chen G, Jing Y, Deng Y, Swanson RJ et al (2007) Nanosecond electric
pulses penetrate the nucleus and enhance speckle formation. Biochem Biophys Res Commun
364:220225
71. Hall EH, Schoenbach KH, Beebe SJ (2005) Nanosecond pulsed electric fields (nsPEF) induce
direct electric field effects and biological effects on human colon carcinoma cells. DNA Cell
Biol 24:283291
72. Buescher ES, Schoenbach KH (2003) Effects of submicrosecond, high intensity pulsed elec-
tric fields on living cells - intracellular electromanipulation. IEEE Trans Dielect Elect Eng
10:788794
73. Buescher ES, Smith RR, Schoenbach KH (2004) Submicrosecond intense pulsed electric
field effects on intracellular free calcium: mechanisms and effects. IEEE Trans Plasma Sci
32:15631572
74. Vernier PT, Sun Y, Marcu L, Salemi S, Craft CM, Gundersen MA (2003) Calcium bursts
induced by nanosecond electric pulses. Biochem Biophys Res Commun 310:286295
75. Craviso GL, Chatterjee P, Maalouf G, Cerjanic A, Jihwan Y, Chatterjee I et al (2009)
Nanosecond electric pulse-induced increase in intracellular calcium in adrenal chromaffin
cells triggers calcium-dependent catecholamine release. IEEE Trans Dielect El In
6:12941301
76. White JA, Blackmore PF, Schoenbach KH, Beebe SJ (2004) Stimulation of capacitative cal-
cium entry in HL-60 cells by nanosecond pulsed electric fields. J Biol Chem
279:2296422972
77. Scarlett SS, White JA, Blackmore PF, Schoenbach KH, Kolb JF (2009) Regulation of intrac-
ellular calcium concentration by nanosecond pulsed electric fields. Biochim Biophys Acta
Biomembr 1788:11681175
78. Beebe SJ, Fox PM, Rec LJ, Willis LK, Schoenbach KH (2003) Nanosecond, high-intensity
pulsed electric fields induce apoptosis in human cells. FASEB J 17:14931495
79. Ren W, Beebe SJ (2011) An apoptosis targeted stimulus with nanosecond pulsed electric
fields (nsPEFs) in E4 squamous cell carcinoma. Apoptosis 16:382393
80. Gusbeth C, Frey W, Volkmann H, Schwartz T, Bluhm H (2009) Pulsed electric field treatment
for bacteria reduction and its impact on hospital wastewater. Chemosphere 75:228233
81. Beveridge JR, MacGregor SJ, Anderson JG, Fouracre RA (2005) The influence of pulse dura-
tion on the inactivation of bacteria using monopolar and bipolar profile pulsed electric fields.
82. Beveridge JR, Wall K, MacGregor SJ, Anderson JG, Rowan NJ (2004) Pulsed electric field
inactivation of spoilage microorganisms in alcoholic beverages. Proc IEEE 92:11381143
83. Perni S, Chalise PR, Shama G, Kong MG (2007) Bacterial cells exposed to nanosecond
pulsed electric fields show lethal and sublethal effects. Int J Food Microbiol 120:311314
84. Vernier PT, Thu MMS, Marcu L, Craft CM, Gundersen MA (2004) Nanosecond electroper-
turbation-mammalian cell sensitivity and bacterial spore resistance. IEEE Trans Plasma Sci
32:16201625
85. Wan J, Coventry J, Swiergon P, Sanguansri P, Versteeg C (2009) Advances in innovative
processing technologies for microbial inactivation and enhancement of food safety pulsed
electric field and low-temperature plasma. Trends Food Sci Technol 20:414424
86. Schoenbach KH, Joshi RP, Stark RH, Dobbs FC, Beebe SJ (2000) Bacterial decontamination
of liquids with pulsed electric fields. IEEE Trans Dielect El In 7:637645
87. Katsuki S, Moreira K, Dobbs F, Joshi RP, Schoenbach KH (2002) Bacterial decontamination
with nanosecond pulsed electric fields. In: Conference record 25th IEEE international power
modulator symposium and high voltage workshop, Hollywood, CA, 30 June3 July 2002
88. Beebe SJ, Blackmore PF, White J, Joshi RP, Schoenbach KH (2004) Nanosecond pulsed
electric fields modulate cell function through intracellular signal transduction mechanisms.
Physiol Meas 25:10771093
89. Beebe SJ, White J, Blackmore PF, Deng Y, Somers K, Schoenbach KH (2003) Diverse effects
of nanosecond pulsed electric fields on cells and tissues. DNA Cell Biol 22:785796
90. Coster HGL, Chilcott TC (2002) Electric field effects in proteins in membranes.
Bioelectrochemistry 56:141146
91. Ohshima T, Tamura T, Sato M (2007) Influence of pulsed electric field on various enzyme
activities. J Electrostat 65:156161
92. Wang S, Chen J, Chen MT, Vernier PT, Gundersen MA, Valderrbano M (2009) Cardiac
myocyte excitation by ultrashort high-field pulses. Biophys J 96:16401648
93. Rogers WR, Merritt JH, Comeaux JA Jr, Kuhnel CT, Moreland DF, Teltschik DG et al (2004)
Strength-duration curve for an electrically excitable tissue extended down to near 1 nanosec-
ond. IEEE Trans Plasma Sci 32:15871599
94. Pakhomov A, Kolb JF, Joshi RP, Schoenbach KH, Dayton T, Comeaux J et al (2006)
Neuromuscular disruption with ultrashort electrical pulses. Proc SPIE 6219:621903
95. Craviso GL, Choe S, Chatterjee P, Chatterjee I, Vernier PT (2010) Nanosecond electric
pulses: a novel stimulus for triggering Ca2+ influx into chromaffin cells via voltage-gated
Ca2+ channels. Cell Mol Neurobiol 30:12591265
96. Zhang J, Blackmore PF, Hargrave BY, Xiao S, Beebe SJ, Schoenbach KH (2008) The char-
acteristics of nanosecond pulsed electrical field stimulation on platelet aggregation in vitro.
Arch Biochem Biophys 471:240248
97. Schoenbach KH, Joshi RP, Kolb JF, Nianyong C, Stacey M, Blackmore PF et al (2004)
Ultrashort electrical pulses open a new gateway into biological cells. Proc IEEE
92:11221137
98. Ibey BL, Pakhomov AG, Gregory BW, Khorokhorina VA, Roth CC, Rassokhin MA et al
(2010) Selective cytotoxicity of intense nanosecond-duration electric pulses in mammalian
cells. Biochim Biophys Acta General Subjects 1800:12101219
99. Hall EH, Schoenbach KH, Beebe SJ (2007) Nanosecond pulsed electric fields have differen-
tial effects on cells in the S-phase. DNA Cell Biol 26:160171
100. Hall EH, Schoenbach KH, Beebe SJ (2007) Nanosecond pulsed electric fields induce
apoptosis in p53-wildtype and p53-null HCT116 colon carcinoma cells. Apoptosis
12:17211731
101. Vernier PT, Ziegler MJ, Sun Y, Chang WV, Gundersen MA, Tieleman DP (2006) Nanopore
formation and phosphatidylserine externalization in a phospholipid bilayer at high transmem-
brane potential. J Am Chem Soc 128:62886289
102. Vernier PT, Sun Y, Gundersen MA (2006) Nanoelectropulse-driven membrane perturbation
and small molecule permeabilization. BMC Cell Biol 7:37
103. Nuccitelli R, Chen X, Pakhomov AG, Baldwin WH, Sheikh S, Pomicter JL et al (2009)
A new pulsed electric field therapy for melanoma disrupts the tumors blood supply and
causes complete remission without recurrence. Int J Cancer 125:438445
104. Chen X, Swanson RJ, Kolb JF, Nuccitelli R, Schoenbach KH (2009) Histopathology of nor-
mal skin and melanomas after nanosecond pulsed electric field treatment. Melanoma Res
19:361371
105. Andr FM, Gehl J, Sersa G, Prat V, Hojman P, Eriksen J et al (2008) Efficiency of high- and
low-voltage pulse combinations for gene electrotransfer in muscle, liver, tumor, and skin.
Hum Gene Ther 19:12611272
106. Stacey M, Osgood C, Kalluri BS, Cao W, Elsayed-Ali H, Abdel-Fattah T (2011) Nanosecond
pulse electrical fields used in conjunction with multi-wall carbon nanotubes as a potential
tumor treatment. Biomed Mater 6:011002
107. Shu X, Altunc S, Kumar P, Baum CE, Schoenbach KH (2010) A reflector antenna for focus-
ing subnanosecond pulses in the near field. IEEE Anten Wirel Propag Lett 9:1215
108. Schoenbach KH, Shu X, Joshi RP, Camp JT, Heeren T, Kolb JF et al (2008) The effect of
intense subnanosecond electrical pulses on biological cells. IEEE Trans Plasma Sci
36:414422
Chapter 29
First Achievements and Opportunities
for Cancer Treatment Using
Non-thermal Plasma
Eric Robert, Marc Vandamme, Julien Sobilo, Vanessa Sarron,

Delphine Ries, Sbastien Dozias, Laura Brulle, Stphanie Lerondel,
Alain Le Pape, and Jean Michel Pouvesle
Abstract This paper summarizes the experimental results and plasma delivery
strategy developed in Orlans for the evaluation of antitumor action of dielectric
barrier discharge and plasma gun for cancer treatment. Detailed analysis of biologi-
cal effects following non thermal plasma application for both in vitro and in vivo
experiments reveals the role of ROS, DNA damage induction, cell cycle modifica-
tion and apoptosis induction. Recent characterization of plasma splitting and mixing
in different capillary geometries, using the plasma gun, together with preliminary
tolerance study dealing with lung and colon treatment indicate that endoscopic
plasma delivery may be a new and valuable therapy in cancerology.
29.1 Introduction
This work deals with the development of two non thermal plasma (NTP) sources
and their application in cancerology. A floating electrode dielectric barrier discharge
device (FE-DBD), close to that first proposed by Drexel Plasma Institute [1], and a
plasma jet generator, labelled plasma gun [2], are used for both in vitro and in vivo
E. Robert (*) V. Sarron D. Ries S. Dozias J.M. Pouvesle

GREMI, CNRS-PolytechOrlans, 14 rue dIssoudun, 45067 Orlans Cedex 2, France
e-mail: eric.robert@univ-orleans.fr
M. Vandamme
GREMI, CNRS-PolytechOrlans, 14 rue dIssoudun, 45067 Orlans Cedex 2, France
TAAM-CIPA, CNRS, 3B rue de la Ferollerie, 45071 Orlans Cedex 2, France
GERMITEC, 30 rue Mozart, 92110 Clichy, France
J. Sobilo L. Brulle S. Lerondel A. Le Pape
TAAM-CIPA, CNRS, 3B rue de la Ferollerie, 45071 Orlans Cedex 2, France
382 E. Robert et al.
assessment of non thermal atmospheric pressure plasma as a new therapy for cancer.
Antitumor activity of NTP was evaluated in vitro on U87 glioma cancer and HCT116
colorectal cancer cell lines. Cell cycle analysis, DNA damage characterization,
reactive oxygen species (ROS) quenching, and apoptosis quantification were per-
formed to allow the description of the processes involved in the plasma antitumor
action. Antitumor effect of plasma on in vivo U87 xenografs subcutaneously
implanted on mice legs was previously reported [3]. In this work, complementary
biological diagnostics have been used to allow, for the first time to our knowledge,
in vivo apoptosis and cell cycle alteration measurements after NTP treatment. The
agreement between in vitro and in vivo NTP induced effects are enlighten and sup-
port the development of the Plasma Gun for its optimization in plasma endoscopy.
Preliminary encouraging NTP endoscopy feasibility studies and tolerance assess-
ment are finally reported. In a first section, the two NTP sources developed in
GREMI are briefly presented. The Sect. 29. 2 summarizes the in vivo and in vitro
antitumor studies together with a first approach of possible process chain to explain
in vitro and in vivo antitumor action of NTP. The specific features of the Plasma
Gun associated with the propagation in thin, long and eventually branched capillar-
ies are documented in Sect. 29.3 before some concluding remarks.
29.2 NTP Sources Developed at GREMI
Figure 29.1 presents a synopsis and a picture of each of the two plasma sources
developed in GREMI. While being cheap, robust and rather easy to develop, the
FE-DBD source suffers from several limitations, inherent with its mode of opera-
tion, which do not preclude its use for NTP assessment but support the design of
new NTP source much more convenient and matched for in vivo applications. The
generation of NTP with FE-DBD devices, occurs when the gap between the dielec-
tric embedded powered electrode surface and the sample ranges from about 1 to
5 mm. The sample (cell culture dish or living tissue) acts as a floating potential
electrode through which the low amplitude (mA range) electrical current circulates.
When the gap thickness is larger than about 5 mm, the plasma generation stops for
the typical voltage, ranging from about 10 to 50 kV, used to sustain the discharge
through millimetric dielectric barrier layers. The safe use of FE-DBD for in vivo
applications was clearly demonstrated, together with the possibility to induce very
limited undesirable effects such as skin burns, local temperature increase, etc., if the
so called plasma dose is limited to a few J/cm2 [4]. Nevertheless, the application
of high voltage pulses, a few tens of kV in amplitude, across a dielectric barrier in
the very close vicinity of the biological target may require some specific safety
precaution to anticipate the possible dielectric layer disruption and the successive
arc burning from the powered electrode to the sample. The possibility to develop a
small volume DBD applicator, likely to be used for endoscopic treatment also seems
rather complex even if DBD plasma were recently designed through flexible capil-
lary [5]. In this latter case, the production of DBD plasma all along the capillary
29 First Achievements and Opportunities for Cancer Treatment 383
Fig. 29.1 Left: synopsis and photography of the FE-DBD source, the discharge operates in air.
Right: synopsis and photography of the Plasma Gun source, the discharge operates in neon
requires some rather sophisticated technological design and plasma generation in

dielectric assembly is associated with possible undesirable capillary wall erosion.
This may lead to plasma content modification through evaporated/ablated material
adjunction. The last limitation lies in the fact that a single, air or air-based mixture,
plasma is generated and applied to the sample. This induces to account for a com-
promise between the best plasma generation conditions, and the best plasma char-
acteristics (reactive species, radiation, spatial homogeneity) required for biomedical
experiments.
The plasma gun device is one among the very large variety of plasma jets, plasma
bullet generator developed during the past years especially in relation with the
plasma medicine [6] emergence or for material science [7]. In comparison with the
great variety of recently developed plasma jets, two unique features dealing with
the long distance plasma generation and the use of very moderate rare gas flow rate
down to a few sccm, appear particularly attractives to achieve plasma delivery in the
tumor vicinity and assess the toxicity and antitumor action of NTP. Both lung and
colorectal cancers have been selected considering their rather poor prognosis using
conventional therapies, while being especially challenging for in vivo studies using
mice as animal models for these pathologies. As depicted in Fig. 29.1, the plasma
gun associates two or three sub assemblies: a DBD reactor where primary plasma
generation occurs, a capillary for plasma transport from the reactor to the target, and
an optional plasma tailoring setup through which the primary plasma composition
could be modified whether during its propagation or in the zone of application on
the biological sample. The plasma delivery can thus occur at large distances, a few
tens of centimetres, away from the high voltage reactor, through flexible capillary of
various nature and diameters down to a few hundreds of microns. The plasma at the
capillary outlet may extend up to a few centimetres downstream where energy trans-
fers occur leading to a plasma composition changing. An important difference
between the two NTP sources lies in the decoupling of the plasma generation and
transport, with the plasma tailoring at the target surface.
29.3 In Vivo and In Vitro Studies
The first experiments were performed using the FE-DBD as an external plasma
applicator. For in vitro studies, two different cell lines U87 (human glyoblastome)
and HCT116 (colon cancer) were cultured and transferred into 24 well plates con-
taining 500 ml of culture medium. The gap between the DBD reactor and the upper
surface of the culture medium was set to 2 mm. The DBD was applied during a
preset period of few seconds at a repetition rate of 2 kHz, the voltage pulse ampli-
tude being of 25 kV, the pulse duration of 5 ms (fwhm).
Both cell lines are transfected to express firefly luciferase, an enzyme catalyzing
light production thanks to luciferine substrate, allowing in vitro and in vivo biolu-
minescence imaging. This imaging modality appears as a unique tool, complemen-
tary with the calliper measurement, as bioluminescence signal intensity is related
with the tumor metabolism rather than with the tumor volume which may include
necrotic areas. Bioluminescence signal is produced by alived cells, its intensity is
correlated with the tumor metabolism. The volume is inferred from the measure-
ment with the calliper of two perpendicular diameters of the tumor following the
international standard technique. In vivo experiments were performed using nude
mice, following the international animal care guidelines (EC directive 86/609/CEE,
French decree n 87848). Tumor xenografs were achieved by subcutaneous injec-
tion of tumor cell suspensions (106 cells in 0.1 ml 0.9% NaCl) into the hind legs.
The operation of the FE-DBD at low electrical power, and correspondingly mod-
erate plasma flux on the mouse skin surface, allows repetitive plasma delivering up
to 6 min during 5 consecutive days. Following this plasma application protocol,
neither systemic behaviour, cardiac and pulmonary rhythm alterations nor severe
skin burns were measured during tolerance studies [3].
A five day DBD plasma treatment, with a daily 6 min at 200 Hz plasma fraction,
was shown to induce a significant delay and growth rate reduction of U87 glioma
cancer, in comparison with non treated control group [3]. Both short term and long
term effects were observed and demonstrate, for the first time to our knowledge, the
NTP potentialities for in vivo cancer treatment approach. Following this encourag-
ing in vivo trial, a detailed analysis of plasma induced effects on in vitro cancer cells
was performed. As verified by different authors, the indirect treatment for which the
culture medium is exposed to plasma treatment before being transferred to wells
containing cells, was shown to induce a comparable cell death with that resulting
Fig. 29.2 U87 cell cycle distribution in control and plasma treated groups for in vitro (left) and
in vivo (right)
form the direct plasma protocol. This is an indication that active species, such as
ROS, are the main agent in the plasma responsible for cell death for in vivo applica-
tions. This was confirmed by using a ROS scavenger in the culture medium before
plasma treatment. In this case, the plasma action is quenched, the bioluminescence
signal being comparable with that of the control wells. As illustrated in Fig. 29.2a,
the plasma application leads to significant cell cycle distribution modification. With
respect to the control groups, the flow cytometry measurement reveals that cells
exposed to plasma present a slight decrease on the population in the primary G0/G1
phase, an increase of the cell number in the S phase and no statistical variation of
the distribution in the G2/M phase. Same analysis was performed on the tumor
xenografts treated by DBD. Following the 5 day treatment, tumor fragments were
collected and analyzed by flow cytometry. Figure 29.2b presents this in vivo cell
cycle distribution measurement for treated and control mice. This figure indicates
that cell cycle distribution pattern is modified following in vivo plasma treatment
and that there exists a good correlation between in vitro and in vivo results.
The in vitro observation of cell death amplification 24 h and 48 h after the very
short duration (a few seconds) plasma treatment, and the in vivo long term effect
measurements suggested that plasma action relies essentially in cell death triggering
rather than in a physical instantaneous damaging. Figure 29.3 presents the biolumi-
nescence signal measured in control and plasma treated groups 6, 24, 48 and 72 h
after treatment completion. In the control group, the cell activity is continuously
growing with a very important increase from day to day up to the third day when the
cells have colonized the whole dish. The plasma-treated cell bioluminescence signal
is slightly increased 24 h after treatment and vanishes later. This programmed cell
death induction, so called apoptosis, was already proven to be of major importance
in NTP applications. In this work, in vitro apoptosis was measured 24 h after plasma
treatment using annexin V detection kit while in vivo apoptosis was quantified by
immunohistochemistry detection of cleaved caspase 3. For in vivo apoptosis
diagnostics, both control and plasma exposed tumors were excised 24 h after the
end of the treatment and tissue slices were collected with reference to the plasma
application position.
1.01008
Bioluminescence (p/sec/cm) 8.01007 CTRL
30s
6.01007
4.01007
2.01007
0.0
6h 24h 48h 72h 6h 24h 48h 72h
Fig. 29.3 Kinetics of bioluminescence intensities for control and plasma treated HCT116 cell
lines. The DBD plasma was applied for 30 s
Figure 29.4 presents the apoptosis index in control and plasma treated groups for
in vitro and in vivo U87 cell lines. In both cases, significant apoptosis induction is
measured 24 h after plasma treatment. As underlined from cell cycle analysis, the
apoptosis enhancement is of the same magnitude for in vitro and in vivo samples
exposed to different number of DBD plasma impulsions. As an illustration, a three-
fold apoptosis induction is measured whether in vivo after application of 3.6105
plasma pulses or in vitro as the result of 3104 plasma pulse delivery. These num-
bers simply indicate that comparable apoptosis induction or cell cycle modification
can be measured as a result of DBD application during a few seconds for cultured
cells, or during a few minutes over the skin surface. The picture in Fig. 29.4c pres-
ents the apoptotic cell distribution in a tumor slice. It has been measured that apop-
totic cells were homogeneously distributed among the whole tumor volume. While
the processes at the origin of plasma-induced in vivo modifications have not yet
been determined, our study indicates that they can be induced through rather thick
tissue layers and that no diffusion-like processes seem to be clearly evidenced.
In vitro DNA damages were analyzed by assessment of g H2AX immunofluores-
cence by flow cytometry. A very early response to DNA damage consists in the
phosphorylation of H2AX histone, leading to the recruitment of the gH2AX protein
at DNA damaged sites. The phosphorylation of H2AX can be detected with a spe-
cific targeted antibody. The fluorescence of such antibody tagged with specific fluo-
rescent marquer is a very sensitive diagnostics tool. In this work, assessment of
gH2AX immunofluorescence (IF) by flow cytometry was performed 1 h after treat-
ment using gH2AX phosphorylation Assay Kit (Millipore) in accordance with man-
a 25 b 20
**
Apoptototic Cells (%)
Apoptotic index (%)

20 **
15
15
10
10
5
5
0 0
CTRL 30s CTRL NTP
c
CONTROL PLASMA TREATED
Fig. 29.4 (a) in vitro apoptosis quantification after 30 s DBD treatment. (b) In vivo apoptosis
measurement, and (c) micropictures of caspase 3 fluorescence (red dots for on-line version) in U87
tumor. Higher apoptotic cell density zones appear as dark areas on the black and white picture
Fig. 29.5 Immunofluore-

scence level in control and
plasma treated (30 s) U87
cell lines. Fluorescence level
measured 1 h after plasma
treatment, associated with
early DNA damage induction,
is normalized to the signal
from control group
ufacturer protocol. Briefly, after ethanol fixation and saponin cell permeabilization,
histone H2AX phosphorylated at serine 139 was detected through the fluorescence
of the anti-phospho-Histone gH2AX, FITC conjugate.
Figure 29.5 presents the immunofluorescence signal intensity measured in U87
control and plasma treated cell lines. NTP treatment is shown to induce a significant
40% increase of the immunofluorescence level following 30 s of DBD application.
Such effects were detected as soon as 1 h after plasma treatment, suggesting that the
major contribution to DNA strand break is a direct consequence of plasma treatment,
while DNA fragmentation associated with apoptosis induction would require longer
delays after treatment to be triggered.
As a summary, in vivo and in vitro analysis of plasma-induced antitumor effects
present striking common features. To the best of our knowledge, our study is the first
to demonstrate in vivo antitumor action of NTP together with the quantification of
cell cycle alteration and apoptosis induction. The major ROS implication for in vitro
experiments was clearly demonstrated and the early stage DNA damage measure-
ments allow to suggest a full plasma action scheme very close to that at the base of
the todays cancer treatment strategies, chemo and radio therapies. ROS chemo deliv-
ery or ROS generation through ionizing radiation targeting, induce lethal DNA dam-
ages leading successively to cell cycle modification and apoptosis triggering. It
appears that same chain processes occur following NTP application. The role of ROS
during in vivo treatments remains unclear, the main issue lying in the possibility for
these active species to pass through the mouse skin and diffuse to and inside the
tumor volume. Another scenario relies on the plasma-triggered, in situ, i.e. in the
tumor environment, ROS release which may be mediated by local charge density, pH
modification over the mouse skin under treatment. Such local pH modification was
for instance observed on the skin surface where acidification occurs while subcutane-
ously a slight increase of the pH value was measured. Such extracellular pH variation
may for instance influence the NO or hydroxyl radical production, and induce suc-
cessive DNA synthesis perturbation or apoptosis [8]. Besides the need for specific
study to determine whether in situ plasma triggered ROS release is a relevant assump-
tion to explain in vivo NTP mode of action, if ROS are the key agent for tumor cell
destruction, the tumor treatment will probably be greatly optimized by delivering
chemically active plasma in the close vicinity of the targeted cells. Tolerance studies
using FE-DBD on mouse colon were performed and demonstrate the possibility to
apply DBD plasma during periods of a few minutes. This is a proof that NTP could
be delivered even on rather fragile organs. The main targets of our study concern
colorectal and lung cancers for which organ access requires specific care and should
rather be operated through endoscopic treatment. The plasma gun, exposed in the
first section of this manuscript, may represent a unique device to evaluate the NTP
antitumor effect for these two challenging cancers.
29.4 Towards Plasma Gun Endoscopic Treatment
The plasma gun developed for cancer treatment consist in a compact, see Fig. 29.1, low
power, a few to a few tens of watt, pulse power generator sustaining a nanosecond DBD
developing across glass pipe flushed with rare gases and rare gas based mixtures. The
outlet of the glass pipe is connected to thin flexible silicon or polyimide tubes through
which fast travelling, so called plasma bullets [9] propagate at very high velocity
towards the biological target. Besides the determination of the physical processes at the
origin of plasma propagation at very high velocities, up to a few 108 cm s1, recent
experiments have been performed to study the plasma expansion through micro sized
Fig. 29.6 Five nanoseconds snapshots revealing plasma stream splitting in a T-shaped glass 4 mm
inner diameter. The plasma is generated in neon at atmospheric pressure. Time origin is set to 0 for
the first (left) picture, delay with respect to this origin are of 60, 80, 100, 120 and 140 ns from
second picture to the righter picture
in diameter silicon capillaries which can be used for mice tracheal intubation or colon
endoscopy. The plasma delivery can either be planed as a direct exposure of the tumor
surface or for less accessible targets through the plasma propagation in the organ
cavities leading to the cancerous location. The first technique requires implementing
non intrusive diagnostic to monitor the capillary outlet position while the second pro-
tocol imposes preliminary experiments on the possibility for plasma splitting, expan-
sion, shrinking, and connection as may be encountered in real organ topography. To
this end, the plasma splitting and mixing have been studied in multi branched glass
assemblies and in circular rings, using fast ICCD imaging. Both plasma splitting in
glass assembly presenting up to ten successive branchings, and mixing of two collid-
ing plasmas have been observed and characterized [10, 11]. The denomination of
Pulsed Atmospheric Plasma Streams (PAPS) was suggested to describe both the highly
emissive head of PAPS, so called bullet, and the very fast travelling filamentary
plasma region, appearing as a tail of the bullet, during the PAPS propagation [11].
Figure 29.6 presents, 5 ns duration, ICCD images showing the propagation and split-
ting of plasma streams in a T shaped glass expansion volume. These measurements
demonstrate the possibility to launch a plasma stream from a primary pipe and achieve
plasma propagation in successive bifurcations of a complex volume.
Plasma propagation in 200 mm inner diameter pipette was also characterized
together with the splitting of an initial plasma stream in three sister PAPS in a
cross shaped 200 mm inner diameter assembly as documented in Fig. 29.7. These
encouraging properties of atmospheric pressure plasma gun device, lead us to per-
form preliminary endoscopic experiment on mice both for lung and colon targets.
The propagation of plasma through small diameter flexible capillaries flushed at
moderate gas flow rate, 1050 cc/min, in the lung and colon of anesthetized mice
was recently successfully achieved together with preliminary evidence for a good
tolerance of both colorectal and lung tissues under plasma exposure up to 10 min.
The feasibility of such plasma application in combination with a proper matching of
the plasma gun characteristics allows in situ in vivo study and optimization of anti-
tumor action of NTP. Recent studies from other groups on the plasma jet action [12]
on colorectal cell lines confirms cell growth arrest [13] and report a selective apop-
tosis induction for different tumoral and normal cells [14].
Fig. 29.7 Ten nanoseconds snapshot of plasma stream splitting in a 200 mm inner diameter cross
shaped glass assembly. The dark central region consists in an opaque mechanical connection of
four glass pipettes. Plasma is launched in atmospheric pressure neon gas in the pipe on the left
hand side of the photography
29.5 Conclusion
In vivo and in vitro NTP action have been tested and characterized on cancerous cell
lines. This work summarizes the detailed analysis of in vitro cancer cell lines, U87
and HCT116, response to non thermal plasma treatment. For this study, a floating
electrode dielectric barrier discharge set up, delivering microsecond pulses, was used
as a plasma applicator in ambient air 2 mm above culture medium surface.
Complementary analysis of, previously reported [3] antitumor action of the same
plasma source on U87 tumor xenografts implanted on mice groups, have also been
performed. In vitro plasma action appears to be largely induced by ROS production
in the culture medium, which then triggers a rather conventional cascade leading
to the cell destruction. Thus, early DNA damages have been detected and quantified,
successive cell cycle alteration leading to an accumulation of cell in a premitotic
stage, and significant apoptosis induction were proven for both cell lines. Cell cycle
modification and apoptosis induction were also proven to occur following in vivo
U87 plasma treatment. These two plasma-induced biological effects probably associ-
ated with the measured tumor short term activity stabilization and longer term growth
slowing, exhibit striking similarities both in vitro and in vivo samples. This would
suggest a major implication of ROS for in vivo NTP action. Two mechanisms appear
likely to be at the origin of ROS stimulation in the tumor environment. Whether ROS
produced in the air plasma diffuse through the mouse skin and tissue towards the
tumor vicinity, or plasma acts as an external trigger for ROS release in the living
organism. The first process may be favorized by synergetic action of high amplitude
transient electric fields generated in the DBD streamers and ROS penetration.
The second assumption which is already known for instance in drug delivery for
melanoma treatment, or for arterial pressure control through NO release at selective
dosing, may be involved during NTP treatment. pH modifications or surface charg-
ing occurring during DBD plasma application, may be at the origin of such intra
organism ROS release triggering. Finally, the development of a specific plasma jet,
labeled Plasma Gun, is documented in two ways. First, atmospheric pressure plasma
propagation in micrometric flexible capillaries and plasma splitting in branching
geometries is presented and second, the first feasibility and tolerance studies dealing
with NTP delivery in colon and lung of anesthetized mice was performed. These are
the preliminary successful experiments dealing with NTP endoscopic applications.
The impact of plasma propagation on the surrounding tissues before reaching the
tumor environment has not yet been characterized, and will be one of the key issues
to implement a beneficial plasma endoscopic treatment. Plasma selectivity on different
tumor cells would be of great importance to address this problem.
Acknowledgements The authors are grateful to the Region Centre (APR Plasmed) and ANR
2010 BLAN 093003 PAMPA financial supports. MV is supported by Germitec, VS by Conseil
Gnral 45, DR by CNRS and Rgion Centre.
References
1. Fridman G, Peddinghaus M, Ayan H, Fridman A, Balasubramanian M, Gutsol A, Brooks A,

Friedman G (2006) Blood coagulation and living tissue sterilization by floating-electrode
2. Robert E, Barbosa E, Dozias S, Vandamme M, Cachoncinlle C, Viladrosa R, Pouvesle JM
(2009) Experimental study of a compact nanosecond Plasma Gun. Plasma Process Polym
6:795802
3. Vandamme M, Robert E, Dozias S, Sobilo J, Lerondel S, Le Pape A, Pouvesle JM (2011)
Response of human glioma U87 xenografted on mice to non thermal plasma treatment. Plasma
Med 1:2743
5. Ehlbeck J, Schnabel U, Polak M, Winter J, Von Woedtke T, Brandenburg R, Von dem Hagen
T, Weltmann KD (2010) Low temperature atmospheric pressure plasma sources for microbial
decontamination. J Phys D Appl Phys 44:031002
6. Favia P (2006) Biomedical applications of plasma processes, Special issue. Plasma Process
Polym 3:383
7. Szili EJ, Al-Bataineh SA, Bryant PM, Short RD, Bradley JW, Steele DA (2011) Controlling
the spatial distribution of polymer surface treatment using atmospheric-pressure microplasma
jets. Plasma Process Polym 8:3850
8. Shu Z, Jung M, Beger HG, Marzinzig M, Han F, Butzer U, Bruckner UB, Nussler AK (1997)
pH-dependent changes of nitric oxide, peroxynitrite, and reactive oxygen species in hepatocel-
lular damage. Am J Physiol Gastrointest Liver Physiol 273:G1118G1126
9. Laroussi M, Lu XP (2005) Room temperature atmospheric pressure plasma plume for bio-
medical applications. Appl Phys Lett 87:113902
10. Sarron V, Robert E, Dozias S, Vandamme M, Ries D, Pouvesle JM (2011) Splitting and mixing
of high velocity ionization wave sustained atmospheric pressure plasmas generated with the
plasma gun. DOI 10.1109/TPS.2011.2155099, to be published in IEEE Transactions on Plasma

Science, 6th Triennial Special Issue of Images in Plasma Science
11. Sarron V, Robert E, Dozias S, Ries D, Vandamme M, Pouvesle JM (2011) Propagation of two
symetrical pulsed atmospheric plasma streams generated by a pulsed plasma gun. In: ISPC
20th, Philadelphia
12. Yan X, Zou F, Zhao S, Lu X, He G, Xiong Z, Xiong Q, Zhao Q, Deng P, Hunag J, Yang G
(2010) On the mechanism of plasma inducing cell apoptosis. IEEE Trans Plasma Sci
38:24512457
13. Kim GC, Bahn JH, Lee SH, Kim GY, Jun SI, Lee K, Baek SJ (2010) Induction of cell growth
arrest by atmospheric non-thermal plasma in colorectal cancer cells. J Biotechnol
150(4):530538
14. Georgescu N, Lupu AR (2010) Tumoral and normal cells treatment with high-voltage pulsed
cold atmospheric plasma jets. IEEE Trans Plasma Sci 38(8):19491955
Chapter 30
Nitric Oxide Plasma Sources
for Bio-Decontamination
and Plasma Therapy
Victor N. Vasilets and Anatoly B. Shekhter
Abstract One of the main products generated in atmospheric plasma sources is

nitric oxide. The nitric oxide molecule is known as anti-bacterial agent on one hand
and the molecule providing signaling and regulation biological functions on the other
hand. Human body produces NO to kill invading pathogens. At the same time nitric
oxide works as a primary vasoregulator and anti-hypertensive agent. NO also
regulates: inflammation, collagen production, angiogenesis and apoptosis. Exogenous
NO generated by plasma devices could enhance bio-activity of NO-assisted processes
in human organism. Some applications of nitric oxide for bio-decontamination and
plasma therapy will be illustrated and discussed in the paper.
30.1 Introduction
One of the most exciting medical discoveries of the 1980s in the study of human
physiology was the realization that nitric oxide (NO) is a short-lived, endogenously
produced molecule that acts as a signaling molecule in the body. NO is a gas with one
unpaired electron and thus is a free radical (NO) that reacts with many biological
molecules. The discovery of signal transmission by a NO gas, produced by one cell,
which penetrates membranes and regulates the function of other cells was awarded by
Noble Prize in Physiology and Medicine for 1998 and was considered as an entirely
V.N. Vasilets (*)

Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences,
Prosp. Semenova 1/10, 142432 Chernogolovka, Moscow region, Russia
e-mail: vnvasilets@gmail.com
A.B. Shekhter
Russian Ministry of Health and Social Security, Sechenov First Moscow
State Medical University,
Moscow, Russia
394 V.N. Vasilets and A.B. Shekhter
new principle for signaling in the human organism. NO has important roles in the
function of many tissues and organs, from the cardiovascular system to the brain [1].
Physiology and biochemistry of NO have been discussed in more than 40,000 papers
since 1990. Wound healing processes are known to involve a sharp increase of nitric
oxide generation in wound tissue. This increase is caused by the activation of constitu-
tive isoforms of NO-synthase (eNOS and nNOS) and by a markedly enhanced synthe-
sis of inducible NOS (iNOS) [2]. These observations suggest a possible therapeutic
use of various NO donors for the acceleration of the wound healing. Our previous
studies [3] indicated that gaseous NO flow produced by air-plasma generator Plason
acts beneficially on the wound healing. To understand the mechanism of this effect the
NO levels in the tissues of rats were studied using exogenous NO spin traps, Fe2+
complexes with diethyldithiocarbamate (DETC)1 or N-methyl-D-glucamine dithio-
carbamate (MGD). NO binding with these traps results in the formation of paramag-
netic mononitrosyl iron complexes, MNICDETC or MNICMGD, which are soluble
in lipophilic or hydrophilic media, respectively [4]. As a result of these experiments
the sharp increase of MNICDETC and nitrosylhemeiron complexes in wound
tissue as a response to successive short-lasting exposures to gaseous NO flow. It was
shown that endogenous NO is responsible for the formation of these complexes, and
that this NO is synthesized by NOS enzymes. The EPR experiments gave direct proof
that gaseous NO treatment increased the stationary level of endogenous NO molecules
in the wound tissues. We propose that this beneficial effect could be caused by the
mechanism involving peroxynitrite as an intermediate. Small doses of peroxynitrite
may actually have protective action in cell cultures and tissues. These results were
attributed to a mobilization of various antioxidant defenses by the presence of per-
oxynitrite [5]. This mobilization could diminish the amount of superoxide anions in
wound tissue, thereby protecting endogenous NO molecules against superoxide attack
By implication, more endogenous NO molecules become available as signalling mol-
ecules to regulate the metabolic processes in wound tissue. Thus, the therapeutic effect
of the exogenous NO treatment is attributed to enhanced NO bioavailability.
Today NO is known also as a universal anti-microbial factor. The sterilization effect
of NO is proved for more than 45 bacteria including gram-negative, gram-positive,
spore-forming rods, alpha-hemolytic anaerobe, facultative anaerobe, cocci arranged in
clusters, anti-biotic-resistant bacteria as well as biofilms (S aureus) and other infections
[611]. Nitric oxide is also a very effective agent against fungi [1113] and parasites
(Leshmaniasis) [1416]. One of the main benefit of NO bio-decontamination is that no
identification of infecting microbe is necessary since NO treats all pathogens. It is
important to mention also that the topical antibacterial market for broad spectrum
agents that effectively kill drug resistant bacteria is >$1.4B only in US.
30.2 Nitric Oxide Generation
Exogenous NO gas for bio-decontamination or therapeutic applications could be

generated by several methods. The easiest way to obtain NO is chemical synthesis
in the reaction of ammonia oxidation: 4NH 3 + 5O2 = 4NO + 6H 2 O. However, the
30 Nitric Oxide Plasma Sources for Bio-Decontamination and Plasma Therapy 395
Fig. 30.1 Multi-purpose medical plasma chemical source Plason. (a) configuration in hot
mode applications (b) configuration in cold mode applications
storage of chemically synthesized NO gas for further applications is a complicated

problem because NO radicals are not stable and could be converted to NO2 gas in
the reaction of recombination in the presence of oxygen or nitrogen. Plasma chemi-
cal continuous synthesis in the flow of atmospheric air could be an alternative
method of NO generation for anti-bacterial and medical applications in situ.
Plasma device Plason (see Fig. 30.1) based on arc discharge was developed
for generation gas flow containing NO with different configurations of the exit
channels corresponding to the different medical applications: wound sterilization,
blood coagulation, tissue destruction, therapeutic manipulator/stimulator. Plasma
temperature and nitric oxide content at the anode exit differs in different configura-
tions of the manipulator, corresponding to different medical applications. The
Plason system is based on the jet of hot air plasma rapidly quenched and provid-
ing relatively high NO concentration (2002,500 ppm) with significant therapeutic
effect [17, 18].
The plasma device is used in two modes. In the first hot mode plasma jet is
used for rapid coagulation and sterilization of wound surfaces, destruction and
desiccation of dead tissue and pathologic growths, dissection of biological tissues
(Fig. 30.1a). In the second cold mode NO-containing plasma gas flow with
temperature of 2040C is used for wound sterilization, stimulation of regenera-
tive processes and wound healing (Fig. 30.1b). In the hot mode the temperature
in the manipulator 1 (see Fig. 30.2) was 3,000 K. In the cold mode the manipu-
lator 1 was inserted in the special cooler mounted in the device (see position b
on the Fig. 30.1). The temperature in the cooler output was 2040C depending
on the gas flow.
Main and common elements of the Plason system construction are the liquid-
cooled cathode, intra-electrode insert, and anode. Atmospheric air enters the manip-
ulator through the built-in micro-compressor, passes through the plasma arc, heats
up and thus accelerates, and exits through the hole in the anode of the plasma-
generating module. Plasma temperature at the anode exit differs in different
Fig. 30.2 Manipulators for destruction (1), therapy mode (generation of room temperature
NO-containing gas flow) (2), coagulation, wound sterilization (3)
configurations of the device, corresponding to different medical applications

(see Fig. 30.2). At high temperatures realized in the arc discharge (2,0004,000 K)
in humid air highly reactive radical species like atomic oxygen (O), hydroxyl radi-
cals (OH), atomic hydrogen (H) are formed in addition to NO, NO2 and other nitro-
gen and oxygen containing species. We have measured the concentrations of NO
and NO2 (see Fig. 30.3) and calculated the concentrations of the radicals O, OH and
H in the hot mode using the software package Chemical Workbench 3.6 (http://
www.kintechlab.com/products/chemical-workbench/). According to the results of
our simulation the (see Table 30.1) atomic concentrations of O > H > OH were com-
parable with the concentration of NO but were more then 3 orders of magnitude
higher then that for NO2 for the arc discharge in air with 2% H2O humidity at the
temperature 3,000 K.
With the increase of the distance from the anode, temperature drops rapidly, and
at 3050 mm from the anode, the flow is composed simply of the warm air, and the
plasma-generated stable molecules like NO, NO2, H2O2 and others. Nitric oxide as a
main therapeutic component in the cool gas flow is determined by the temperature in
plasma reactor and quenching rate. The necessary quenching rate for effective opera-
tion of the medical device is about ~107108 K/s. Commonly, the cooling rate of
plasma jets is on the order of ~106 K/s. Thus, to achieve the cooling rate of ~107
108 K/s, it is necessary to utilize additional configuration of the plasma jet, which has
been achieved by special construction of the cooler mounted inside Plason.
The therapeutic manipulator-stimulator configuration of the Plason discharge
system could be used both for bio-decontamination and for therapeutic treatment by
exogenous nitric oxide. The principle difference of this manipulator is that the air-
plasma jet does not freely exit into the atmosphere, but rather it exits the anode into
Fig. 30.3 Experimental concentrations of NO and NO2 depending on the distance from manipulator
1 (therapeutic concentrations 300500 ppm)
Table 30.1 Results of stimulation for neutral and radical particles in DC arc
discharge in air at the temperature 3,000 K
Species N2 O2 H2O Ar H
mol% 6.7E + 01 9.1E + 00 1.5E-01 8.9E-01 1.8E + 00
Species N O OH NO H2
mol% 1.8E 02 1.5E + 01 1.3E + 00 4.9E + 00 1.8E 02
the two-step cooling system, gas channels of which are created in a maze scheme to
force-cool the jet by the liquid circulating from the cooling system. This construc-
tion allows for obtaining NO-containing gas flow with sufficiently low temperature,
and optimal concentration of nitric oxide molecules, which makes it possible to
apply this manipulator for treatment of external body surfaces by using the cooling
hose of 150 mm length (temperature of NO at the exit ~36C). Of course, NO con-
tent in the gas flow depends on the distance from the exit channel (Fig. 30.3).
Additionally, for laparoscopic operation, a special manipulator of 350 mm length
and 10 mm diameter is utilized (Fig. 30.2, manipulator 2).
The possible operating regimes of the apparatus are defined by the characteristics
of the gas flow exiting from the manipulator, main parameters of which are its tem-
perature and the nitrogen oxide content. The typical concentration of NO used for
wound healing and sterilization varies in the range 300500 ppm (see Fig. 30.3).
The device Plason generating active species in air plasma was approved for
medical purposes by Russian Ministry of Health in 2000 and has been used for
medical applications in Russia more than 10 years for the treatment of various dis-
eases associated with inflammatory and destructive processes, regeneration distur-
bances and vascular dysfunctions. In 2009 Plason was also certified in Czech
Republic as medical device Class IIb (EC Certificate No 09 0637 QS/NB) and suc-
cessfully applied by the company ONKOCET in Slovakia [19].
30.3 Anti-bacterial Effect of Nitric Oxide
Since the discovery of antibiotics there has never been a time when there has been a
greater need for a new antimicrobial platform. The main reasons of this emergency
situation are:
the emergence of multi-drug resistant bacteria
the continued inappropriate use of antibiotics in humans and in animals
newer antibiotics are scarce and are often far more expensive and more toxic than
predecessors
there may soon be diseases once easy to cure that become untreatable
the announcement that payors will no longer cover hospital acquired infections
Bacterial Skin and Soft Tissue Infections (SSTI) are also a rapidly growing prob-
lem, with ever-increasing morbidity, due to the growing incidence of multi-drug
resistant bacteria. Over 22 million SSTIs were reported in 2009. In the US, there
were 15.6 million ambulatory visits and over 6.5 million non-ambulatory visits for
SSTIs, an increase of more than 100% from 2001 [11].
Nitric oxide novel approach uses a variation of the bodys normal defense against
pathogens, which delivers fast action and universal spectrum coverage, at a dose
that is lethal to all bacteria and fungus but is fully tolerated by human cells. NO is
used by the body to kill bacterial, viral, fungal and parasitic infections. The GSH
pool protects the bacteria from the attack by NO and H2O2. NO and H2O2 are two
endogenous mechanisms of pathogen control. (NO being released by macrophages,
GSH = Reduced Glutathione). In each case, the defense mechanism is based on
physiological NO levels. Our approach is to locally overwhelm the infection with
supra-physiological levels of NO which has a very short lifetime (69 s). Human
cells since they secrete NO to kill naturally have a much higher tolerance for NO
and are either minimally impacted or stimulated.
In vitro investigation of the NO bio-decontamination effect on Escherichia coli,
Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris, gram-negative
Pseudomonas aeruginosa, gram-positive methicillin-sensitive Staphylococcus aureus,
Clostridium difficile and Candida albicans, which are typically associated with many
hospital infections, showed that treatment by NO with the concentrations 200400 ppm
practically inactivate them all [611]. NO could be used also to kill antibiotic-resistant
(methicillin-resistant Staphylococcus aureus, MRSA), fungal (Tinea Pedis,
Coniothyrium minitans) [79] and parasitic infections (Leishmaniasis) [11, 1416].
In vivo investigation of NO anti-bacterial properties was done in animal experi-
ments using Plason plasma source. Two sets of experiments were carried out with
90 male rats. In the first set of experiments conditioned aseptic skin wounds (300 mm2)
were inflicted. A rings were inserted in the wound edges and covered with cello-
phane to prevent drying. After 2nd, 4th, 6th and 8th days wound surface and edges in
first experimental group (25 rats) were treated with NO-containing (300 ppm) flow
from Plason for 30 s (distance 20 cm, T = 40C). In the control (25 rats) the wounds
were exposed only to the warm air at the same temperature. In the second set of
Fig. 30.4 Conditioned Wound infection CFU/cm2, (% to initial value)

aseptic full-thickness plane
wounds vs. time of treatment
(Plason NO treatment
50
300 ppm, 4 sessions, 30 s)
40
30 Control
20
NO
10
0
Fig. 30.5 Infected 5

(Staphylococcus aureus),
full-thickness plane wounds
vs. time of treatment 4
(Plason NO treatment Control
300 ppm, 30 s; 500 ppm,
LgCFU/cm2
30 s; control) 3
500 ppm 300 ppm
0
0 3rd 7th 14th
experiments (40 rats), wounds were infected with Staphylococcus aureus culture
(109 bacteria per sample) for simulation of infected suppurative wound. The experi-
ment and the control were exactly the same as in the first set. But 20 rats were treated
with NO concentration 300 ppm and the other 20 rats were exposed by 500 ppm NO
concentration. On days 3rd, 7th, 14th and 21st the bacterial content in the wounds
was measured using traditional colony forming unit (CFU) method.
For the model of conditioned aseptic full-thickness plane wounds (Fig. 30.4.),
nitric oxide treatment (4 sessions, 30 s, 300 ppm) reduced bacterial level in the
experimental group up to 7% CFU/cm2 (more than ten times) in comparison with
the control group where the reduction was limited to 43%. The anti-bacterial effect
of NO-containing plasma gas was much more pronounced for the model of infected
(Staphylococcus aureus), full-thickness plane wounds (Fig. 30.5). The reduction of
CFU/cm2 value more than four orders of magnitude was observed for 300 ppm NO
concentration and about five orders of magnitude sterilization effect was obtained
for 500 ppm NO treatment procedure on the 21st day. For comparison the reduction
of bacteria load for the control group was about two orders of magnitude less than
that for 500 ppm NO-treatment (Fig. 30.5).
Concluding this part of the paper one can say that nitric oxide treatment has the
profile to become a first-line treatment for all skin and soft tissue infections, whether
caused by bacteria, fungus, or parasites.
30.4 Nitric Oxide Plasma Therapy
Effectiveness of the plasma NO-therapy is already at present shown with a number of

diseases in wound pathologies (trophic ulcers, diabetic foot ulcer), gynecology, trau-
matology, stomatology, ophthalmology, otorhinolaryngology, dermatology, gastroen-
terology, etc. [2022]. Some new specific aspects of the NO-plasma therapy with
Plason source were demonstrated recently for the treatment of extensive burns.
The process of burn healing by NO-plasma therapy is illustrated on the Fig. 30.6.
The first stage NO plasma therapy at the dosage 15 s/cm2 and concentration 500 ppm
was used for the sterilization of extensive burn before skin transplantation. On the
second stage after skin-split meshed graft transplantation.
NO-plasma therapy (every day session 15 s/cm2, concentration 500 ppm) was
used to improve and accelerate transplantation process. On the 3rd day after split-
skin meshed graft transplantation, clear and distinguishable growth of granulations
was observed as a result of plasma nitric oxide therapy (Fig. 30.6). On the 8th day
after grafting and plasma NO-therapy treatment, formation of epithelium and wound
healing with primary adhesion was detected.
30.5 Conclusions
Summarizing the results of 10 years applications of plasma NO therapy one could

assert that air plasma flow extensively stimulates wound healing particularly in
patients with complicated and chronic wounds as well as with extensive burns and
prevents suppuration and wound complications. NO is a specific component of
plasma gas flow and its functions as an active cell regulator and messenger is well
known [1]. The mechanism of wound healing by plasma generated NO we suggested
based on our previous experimental studies includes the following processes:
direct anti bacterial and static bacterial effects
activation of phagocytosis, wound cleansing from germs and necrotic detritus;
rapid transition to reparative phase of wound healing;
activation of macrophagal cytokine secretion and mast cells reaction;
stimulation of fibroblast proliferation, vascularization, granulation and tissue
growth;
loss and elimination of microcirculatory disturbances and inflammatory
manifestations.
In addition to NO air plasma gas contains the other biologically active molecules
like H2O2, O2, NO2 et cetera. Medical and biological effects of NO listed above
Fig. 30.6 The 3rd day after

split-skin meshed graft
transplantation (plasma
NO-therapy of the graft;
exposition 1015 s per cm2,
500 ppm NO)
could be enhanced by the synergy effects of NO/H2O2 and NO/O2 are known in the
regulation of cell apoptosis, antimicrobial macrophage defense processes and treat-
ment of primary pulmonary hypertension [2328].
Therefore, plasma NO-therapy is a promising technique for treatment and pre-
vention of inflammatory, ulcerative, necrotic and sclerotic processes as well as
vascular dysfunctions in the human body.
References
1. Moncada S, Palmer RM, Higgs EA (1991) Nitric oxide: physiology, pathophysiology and
pharmacology. Pharmacol Rev 43:109142
2. Schaffer MR, Tantry U, Barbul A (1997) Nitric oxide metabolism in wounds. J Surg Res
71:2531
3. Shekhter AB, Kabisov RK, Pekshev AV, Kozlov NP, Perov IL (1998) Experimental clinical
substantiation of plasma dynamic therapy of wounds with nitric oxide. Bull Exp Biol Med
(Rus) 126:210215
4. Serezhenkov VA, Vanin AF, Shekhter AB, Rudenko TG, Gavrilchak AV, Pekshev AV (2001)
Dynamics of the levels of endogenous and exogenous nitric oxide in wound and organ tissues
of rats: EPR assays. In: NO-therapy: theoretical aspects and clinical experience of exogenous
nitric oxide application in medicine (Moscow): 2735
5. Laude K, Thuillez C, Richard V (2002) Peroxynitrite triggers a delayed resistance of coronary
endothelial cells against ischemiareperfusion injury. Am J Physiol 283:H1418H1423
6. Kerr AR, Wei XQ, Andrew PW, Mitchell TJ (2004) Nitric oxide exerts distinct effects in local
and systemic infections with Streptococcus pneumoniae. Microb Pathog 36:303310
7. Morita H, Yoshikawa H, Suzuki T, Hisamatsu S, Kato Y, Sakata R, Nagata Y, Yoshimura T
(2004) Anti-microbial action against verotoxigenic Escherichia coli O157: H7 of nitric oxide
derived from sodium nitrite. Biosci Biotechnol Biochem 68:10271034
8. Satriano J (2004) Arginine pathways and the inflammatory response: interregulation of nitric
oxide and polyamines: review article. Amino Acids 26:321329
9. Fox S, Wilkinson TS, Wheatley PS, Xiao B, Morris RE, Sutherland A, Simpson AJ, Barlow
PG, Butler AR, Megson IL, Rossi AG (2010) NO-loaded Zn2+-exchanged zeolite materials: a
potential bifunctional anti-bacterial strategy. Acta Biomater 6:15151521
10. Daeschlein G, Woedtke T, Kindel E et al (2010) Antibacterial activity of an atmospheric

pressure plasma jet against relevant wound pathogens in vitro on a simulated wound environ-
ment. Plasma Process Polym 7:224230
11. http://www.nitricbio.com
12. Li B, Fu YP, Jiang DH et al (2010) Cyclic GMP as a second messenger in the nitric oxide-
mediated conidiation of the mycoparasite Coniothyrium minitans. Appl Environ Microbiol
76:28302836
13. Lushchak VI (2011) Adaptive response to oxidative stress: bacteria, fungi, plants and animals.
Comp Biochem Physiol C-Toxicol Pharmacol 153:175190
14. Chowdhury KD, Sen G, Biswas T (2010) Regulatory role of nitric oxide in the reduced sur-
vival of erythrocytes in visceral leishmaniasis. Biochem Biophys Acta General Aspects
1800:964976
15. Nahrevanian H, Jalalian M, Farahmand M et al (2010) Acetyl salicylic acid inhibits systemic
leishmaniasis via nitric oxide pathway in susceptible Balb/c model infected with Lelshmania
major. Nitric Oxide Biol Chem 22:S89S89
16. Lopez-Jaramillo P, Rincon MY, Garcia RG et al (2010) A controlled, randomized-blinded
clinical trial to assess the efficacy of a nitric oxide releasing patch in the treatment of cutaneous
leishmaniasis by Leishmania (V.) panamensis. Am J Trop Med Hyg 83:97101
17. Reshetov LV, Kabisov RK, Shekhter AB, Pekshev AV, Manelova MV (2000) The use of
Plason air plasma apparatus for coagulation and NO therapy in plastic reconstructive surgery
for oncology patients. Ann Plast Reconstr Esthetic Surg 4:2438
18. Lipatov KV, Sopromadze MA, Shekhter AB, Emelyanov AY, Grachev SV (2001) The use of
gas flow with nitric oxide (NO-therapy) in combined treatment of purulent wounds. Surgery
2:4143
19. http://www.onkocet.eu/en/produkty-detail/283/1/
21. Vasilets VN, Gutsol A, Shekhter AB, Fridman A (2009) Plasma medicine. High Energy Chem
43:229233
22. Shekhter AB, Pekshev AV, Guller AE, Vasilets VN (2010) 3rd international conference of
plasma medicine, Greifswald, Germany, Abstracts, p 43, 1924 Sept 2010
23. Chae HJ, Kim HR, Kwak YG, Ko JK, Joo CU, Chae SW (2001) Signal transduction of nitric
oxide donor-induced protection in hydrogen peroxide-mediated apoptosis in H9C2 cardio-
myoblasts. Immunopharmacol Immunotoxicol 23:187204
24. Farias-Eisner R, Chaudhuri G, Aeberhard E, Fukuto JM (1996) The chemistry and tumoricidal
activity of nitric oxide/hydrogen peroxide and the implications to cell resistance/susceptibility.
J Biol Chem 271:61446151
25. Kotamraju S, Tampo Y, Keszler A, Chitambar CR, Joseph J, Haas AL, Kalyanaraman B (2003)
Nitric oxide inhibits H2O2-induced transferrin receptor-dependent apoptosis in endothelial
cells: role of ubiquitin-proteasome pathway. Proc Natl Acad Sci USA 100:1065310658
26. Yoshioka Y, Kitao T, Kishino T, Yamamuro A, Maeda S (2006) Nitric oxide protects mac-
rophages from hydrogen peroxide-induced apoptosis by inducing the formation of catalase. J
Immunol 176:46754681
27. Pacelli R, Wink DA, Cook JA, Krishna MC, DeGraff W, Friedman N, Tsokos M, Samuni A,
Mitchell JB (1995) Nitric oxide potentiates hydrogen peroxide-induced killing of Escherichia
coli. J Exp Med 182:14691479
28. Chotigeat U, Khorana M, Kanjanapattanakul W (2007) Inhaled nitric oxide in newborns with
severe hypoxic respiratory failure. J Med Assoc Thai 90:266271
Chapter 31
Generation of Focused Shock Waves in Water
for Biomedical Applications
Petr Luke, Pavel unka, Petr Hoffer, Vitaliy Stelmashuk, Ji Bene,

Pavla Poukov, Marie Zadinov, and Jan Zeman
Abstract The physical characteristics of focused two-successive (tandem) shock

waves (FTSW) in water and their biological effects are presented. FTSW were
generated by underwater multichannel electrical discharges in a highly conductive
saline solution using two porous ceramic-coated cylindrical electrodes of different
diameter and surface area. The primary cylindrical pressure wave generated at each
composite electrode was focused by a metallic parabolic reflector to a common
focal point to form two strong shock waves with a variable time delay between the
waves. The pressure field and interaction between the first and the second shock
waves at the focus were investigated using schlieren photography and polyvi-
nylidene fluoride (PVDF) shock gauge sensors. The largest interaction was obtained
for a time delay of 815 ms between the waves, producing an amplitude of the nega-
tive pressure phase of the second shock wave down to 80 MPa and a large number
of cavitations at the focus. The biological effects of FTSW were demonstrated
in vitro on damage to B16 melanoma cells, in vivo on targeted lesions in the thigh
muscles of rabbits and on the growth delay of sarcoma tumors in Lewis rats treated
in vivo by FTSW, compared to untreated controls.
P. Luke (*) P. unka P. Hoffer V. Stelmashuk

Institute of Plasma Physics, Academy of Sciences of the Czech Republic,
Za Slovankou 3, 18200 Prague, Czech Republic
e-mail: lukes@ipp.cas.cz
J. Bene P. Poukov M. Zadinov J. Zeman
First Faculty of Medicine, Charles University,
Prague, Czech Republic
404 P. Luke et al.
31.1 Introduction
During the past several decades, non-equilibrium atmospheric pressure plasmas

generated by electrical discharges in gases and liquids were shown to be efficient
treatment method in a variety of environmental applications, such as air pollution
control (involving removal of NOx, SO2 and volatile organic compounds), removal
of organic contaminants from water, or treatment of solid surfaces [1, 2]. More
recently, research on the biological and medical applications of these plasmas
become a rapidly developing field with many exciting and promising results. New
successful or potential applications of plasmas in medicine have been demonstrated
in electrosurgery, the treatment of skin diseases and dental cavities, plasma-assisted
blood coagulation, the sterilization of surfaces and medical instruments, and many
other applications [35]. Depending on the type of plasma, five main plasma agents
(i.e. heat, electric field, ultraviolet radiation, charged particles, and neutral reactive
species) may be involved in the plasma interaction with living matter. Chemical
effects induced by the reactive oxygen species (e.g., atomic oxygen, metastable
oxygen molecules, ozone, and OH radicals) and reactive nitrogen species are gener-
ally accepted to play the dominant role in the atmospheric air plasma systems [46].
Physical processes, such as ultraviolet (UV) photolysis, are generally not believed
to participate significantly in the inactivation processes of these plasmas [6].
On the other hand, for underwater plasmas, physical processes, such as high
electric field, UV radiation and shock waves, may significantly contribute to the
biological effects (e.g., the inactivation of microorganisms in water) in addition to
the chemical effects that are largely attributed to OH radicals and hydrogen per-
oxide [710]. For underwater plasmas initiated by exploding fine wires, it was
shown that up to 28% of the energy transferred into the plasma (1.5 kJ/pulse) is
converted into UV radiation with a peak radiant power of 200 MW [11]. Ching
et al. [10] estimated the UV light intensity generated by the pulsed arc discharge
to be on the order 106 W/cm2 and determined the UV radiation to be the main
disinfection process in the discharge. The significant emission of UV light has
also been demonstrated for pulsed corona discharge in water, with a pulse energy
on the order of several J/pulse. UV radiation was estimated to contribute about
30% of the overall plasma inactivation of bacteria Escherichia coli induced by
electrical discharge in water [12].
In addition to UV radiation, a significant amount of the discharged energy in
underwater discharges (especially pulsed electrohydraulic sparks/arcs with the
pulse energies on the order of several kJ/pulse) is transformed into shock waves in
water. Gilliland and Speck [13] investigated the bactericidal action produced by
electrohydraulic shock waves generated by submerged high-voltage spark discharge
already in the 1960s. The authors concluded that there were no indications of death
caused by the mechanical disruption of cellular integrity. Later studies, however,
demonstrated significant role for shockwaves in the electrohydraulic discharge
treatment of water when used for sludge decontamination, killing of bacteria or
Zebra mussel removal from the intake pipes of water treatment facilities [14].
31 Generation of Focused Shock Waves in Water for Biomedical Applications 405
Li et al. [15, 16] observed the mechanical rupture of intracellular gaseous vacuoles
of cyanobacteria cells exposed to underwater streamer discharge, which was caused
presumably by the physical effects of shock waves induced by the discharge.
Underwater shock waves are also used in extracorporeal shockwave lithotripsy
(ESWL) to non-invasively disintegrate kidney stones and gallstones. In fact, ESWL
is one of the most successful applications of shock waves in medicine. Since the
mid-1980s, lithotripsy has been in clinical use worldwide for treating kidney stones
and continues to be the favored method for uncomplicated, upper urinary tract cal-
culi, even with the advent of percutaneous surgical methods. The treatment involves
focusing shock waves generated by the ESWL device (lithotripter) outside of the
patients body to disintegrate the stone at a depth in tissue. The targeted stone is
located at the shock wave focus, and hundreds to thousands of shock wave pulses
are delivered to comminute the stone. Acoustic coupling with the patient is achieved
through water baths or liquid-filled pillows. After the urinary stone treatment, the
stone debris passes through the urinary tract. In the case of gallstones, the stones are
chemically dissolved. A typical pressure waveform at the lithotripter focus in water
consists of a leading shock wave front (compressive wave) with a peak positive
pressure in the range of 30150 MPa and a phase duration of 0.53 ms, followed by
a tensile wave with a peak negative pressure down to 20 MPa and a duration of
220 ms [17].
Over 40 models of clinical lithotripters are commercially available worldwide
(electrohydraulic, electromagnetic and piezoceramic). In electrohydraulic litho-
tripters, an underwater high-current spark discharge between a pair of electrodes is
generated at the focus of the ellipsoidal reflector, and the emerging spherical shock
wave produced by the plasma at the spark gap is concentrated on the kidney stone
located at the second focus of the ellipsoid [18]. At the end of the 1980s, one of
modified versions of the electrohydraulic type of lithotripter was developed at the
Institute of Plasma Physics AS CR [1921]. These generators are used in the litho-
tripters Medilit (produced by the company MEDIPO, Brno, Czech Republic) at
approximately 20 hospitals in the Czech and Slovak Republics [22]. So far, more
than 120,000 patients have been successfully treated with these devices.
The success of the ESWL stimulated research on the applications of focused
shock waves (FSW) in other branches of medicine. Shock waves are used in orthopedy
for the fracture healing, the treatment of pseudarthrosis, tendinopathy and other
orthopedic diseases [23]. Great attention has been on to the role of cavitation in
ESWL-induced biological effects on soft tissues and the possible treatment of some
types of cancers. It was initially studied to identify possible side effects of the litho-
tripsy therapy, such as haematuria, renal colic, perirenal and intrarenal hematomas,
pancreatitis and arrhythmias [17]. The evidence suggests that cavitation is an impor-
tant factor in lithotripsy. Stones initially break by spall, and then erosion grinds the
fragments into a size suitable for the patient to pass [18]. Micro-inhomogeneities or
microbubbles in the fluid (e.g., water or urine) that are located in the vicinity of the
focal zone may produce cavitations, which may interact with the stone but also with
the cells/tissue. The threshold for cavitation in water is approximately 0.5 MPa; how-
ever, this depends on the duration of the tensile pulse [17].
406 P. Luke et al.
To accelerate stone comminution and/or reduce/enhance tissue damage, special

generators of shock waves and a focused high-intensity ultrasound (HIFU) are
being designed to modify the cavitation field and to control cavitation. The corre-
sponding methods manipulate the timing between pulses or modify the lithotripter
waveform by altering the reflector material in the electrohydraulic or electromag-
netic devices or by reversing the polarity in the piezoceramic devices [2429].
Several authors have reported controlling bubble growth and collapse by so-called
tandem shock waves, which intensify the collapse of cavitation bubbles by sending
a second shock wave at the moment before the bubbles produced by the first shock
wave begin to collapse [30, 31]. Most of these experimental studies demonstrated
a significant effect on stone comminution and/or tissue injury, attributed in large
part to the modified cavitation field. Shock waves have been shown to cause sig-
nificant cytotoxic effects in tumor cells both in vitro and in vivo. Furthermore, it
was discovered that shock waves induce necroses in tumors, which delay tumor
growth. However, despite the demonstrated effectiveness in experimental tumors,
the mechanism of interaction between ultrasonic shock waves and tumors is
unknown. Both positive and negative pressures, and inertial cavitation especially,
are thought to play major roles in the interaction of lithotripter-induced shock
waves with biological tissue [29]. The optimal shock wave profile and pulse com-
bination have yet to be established.
One reason is a fundamental difference in the acoustic impedance of kidney
stones and tissue. A kidney stone represents a relatively strong acoustical non-
homogeneity in comparison with the surrounding liquid and soft tissues. The non-
homogeneity localizes the action of the shock wave because the shock wave
propagates through soft tissue and liquid with a small attenuation almost without
interacting with them. In the case of cancer tissue, no acoustical non-homogeneities
exist between cancer and healthy tissues. The localized action of the shock waves
in an acoustically homogeneous medium (soft tissue) is attributed to the cavita-
tions produced by FSW or HIFU. Collapsing cavitations create strong secondary
shock waves of nanosecond duration (tens of micrometers in scale) that can inter-
act with cell scale-structures. Local thermal effects (on the order of mm dimen-
sions) accompanying cavitations collapse (sonoluminescence) and the production
of chemical radicals may also play a role in cell damage. In general, the intensity
of the secondary shock wave and the velocity of the microjet are assumed to depend
on the initial bubble radius. The larger the bubbles grow (>1 mm), the more violent
their collapse, which may be more suitable for stone or tissue damage. Small bub-
ble sizes (<200 mm) that are comparable to the size of cells may be used to enhance
shock wave-mediated drug delivery and gene transfer. In the case of HIFU, the
dynamics of cavitation can be varied by the frequency and amplitude of the wave.
In the case of tandem shock waves, the second wave can interact with the cavita-
tion produced by the first wave in a different phase of its evolution. Thus, by using
tandem shock waves, inertial bubble interactions may be tailored for different
biomedical applications [26].
To enhance the cavitations and the interaction of the shock waves with soft tis-
sue, we have developed a novel generator of focused shock waves in water, which is
based on the production of cylindrical pressure waves generated by underwater

multichannel electrical discharges using one or two porous ceramic-coated (com-
posite) cylindrical metallic electrodes of different diameter [3235]. The primary
pressure waves generated by the underwater plasma channels at the surface of the
composite electrode(s) are focused by a metallic parabolic reflector to a common
focal point and only close to the focus are transformed into a strong shock wave.
Depending on the power supply arrangement, we are able to generate either one
shock wave or two successive shock waves focused to a common focal point
(i.e., focused tandem shock waves, FTSW) with an adjustable delay between the
waves on the order of microseconds. We have found that at time interval of 815 ms
between the two shock waves, the second original pressure wave is strongly attenu-
ated at the focal region and reaches the focus as a rarefaction wave. The amplitude
of the pressure wave is up to 100 MPa, while the amplitude of the rarefaction wave
falls down to 80 MPa, producing thus a large number of cavitation bubbles at the
focus. The collapsing cavitation bubbles produce secondary, short-wavelength
shock waves and fast microjets that can interact with cell-scale structures and soft
tissues. In this work, characteristics of the focused tandem shock waves in water and
their biological effects are presented.
31.2 Apparatus and Methods
Figure 31.1 shows the scheme for a generator of focused tandem shock waves in
water. The generator is divided by an acoustically transparent membrane (Mylar
foil, thickness 100 mm) into two parts. The first part, which is filled with a highly
conductive saline solution (on the order of tens of mS/cm), consists of two compos-
ite metallic cylindrical high-voltage electrodes of different diameter d and length h
(A1: d = 90 mm, h = 17 mm; A2: d = 60 mm, h = 55 mm) placed along the axis of the
outer metallic parabolic reflector. The composite anode consists of a metallic cylin-
drical electrode covered with a thin porous ceramic layer deposited by thermal
plasma spraying. The role of the ceramic layer is to redistribute the electric field on
the electrode during the pre-discharge phase. Due to the differences in conductivity
and permittivity between the water and the ceramic layer, the electric field strength
on the surface of the composite electrode is many times enhanced in comparison
with a metallic electrode of the same dimensions. This setup allows for the simulta-
neous generation of a large number of filamentary discharge channels distributed
almost homogenously around the entire surface of the composite electrode at a
moderate applied voltage [36].
A pulsed high voltage of positive polarity was applied to the composite elec-
trodes using a pulse power supply that consists of two capacitors (C1, C2)
charged up to 30 kV and two triggered spark gaps (SG). The spark gap switches
were triggered either at the same time (switch S was closed) or with a variable
delay (switch S was open), allowing us to produce either a single shock wave or
two successive shock waves focused to a common focal point (i.e., focused tandem
408 P. Luke et al.
Fig. 31.1 The scheme of the generator of focused tandem shock waves in water
shock waves) with an adjustable delay between the waves. The spark gaps were
triggered by 12 kV/ms pulses produced by discharging the capacitors through the
thyratrons. The thyratrons were triggered by a dual-channel pulse generator with
a variable time delay (100 ns step) between the channels. The focal point of the
parabolic reflector is situated in the second part of the generator, which is filled
with tap water and is located 5 cm from the Mylar membrane. Each discharge
channel formed at the composite electrode creates a semi- spherical pres-
sure wave in the saline solution. By superposition of the waves, a cylindrical
pressure wave propagating from the anode is formed. The primary cylin-
drical pressure wave is focused by a reflector. Near the focus, this wave is
transformed into a strong shock wave. The amplitude and, to some extent, the
waveforms of the shock waves, at the focus can be controlled by the anode
surface areas, the discharge circuit parameters and the solution conductivity,
which play a decisive role in the characteristics of underwater plasmas. Higher
conductivity leads to higher power density in the discharge channel, and this
results in an increase in the plasma electron density, a higher plasma temperature
and more intensive pressure wave evolution from the discharge [32]. In this work,
saline solution conductivity of 18 mS/cm was used for the generation of the dis-
charge at the composite electrode, which was based on results from previous
work that demonstrated that the given experimental setup generates the highest
amplitude of the pressure wave at this conductivity [33].
Schlieren photography was used to acquire time-resolved images of the pressure
field induced by the shock wave(s) in the focal region. The Nd:YAG pulsed laser beam
[wavelength = 532 nm, pulse width = 10 ns full width at half maximum (FWHM)] was
expanded to a diameter of 60 mm and applied through a focal region as an illumina-
tion light source in a perpendicular direction to the shockwave propagation. A camera
was set up to acquire the Schlieren photographs of the focal region at various time
delays after the discharge ignition by the triggered spark gap switch SG1 and synchro-
nized with the firing of the laser beam via a delay generator. Consequently, the pres-
sure field and the pressure waveforms of the shock waves produced at the focus were
measured by polyvinylidene fluoride (PVDF) shock gauge sensors (Model S25_04,
Piezotech SA, France). The pressure field at the focal region was mapped point-by-
point, and the focal volume with the dimensions 2.5 mm 32 mm long (FWHM)
was determined [32, 33]. For observation of the dynamics of the cavitation at the focal
region, a microscope and a PCO 12-bit charge-coupled device (CCD) Sensicam cam-
era were used. Microscope magnifications of 10 to 25 were used. The focal area
was illuminated by a xenon lamp, which was fed by a high-voltage capacitor switched
by a thyratron. The ignition of the lamp was synchronized with the triggered spark gap
switch SG1 at various time delays. The exposure time of the PCO camera was 400 ns.
This exposure time was sufficient to obtain sharp and bright photos.
B16 melanoma syngeneic cells were grown in a tissue culture medium (6 106
cells). The viability of the B16 melanoma cells after exposure to FTSW was exam-
ined using the trypan blue staining method and by optical imaging microscopy.
Tumor growth delay experiments were performed with Lewis strain rats inoculated
subcutaneously with R5-28 malignant cells (2.5 105 cells/rat) [37]. The time of the
tumor growth latency and the volume of the growing sarcoma in the Lewis rats were
followed throughout the experiment and compared with the control (not exposed)
tumors (four animals in each group). The tumor size was measured with Vernier cali-
pers. The tumor volumes V at particular time intervals were calculated by the formula
V = a b2 p/6, where a and b are the tumor length and tumor width, respectively.
FTSW-induced lesions in rabbits thigh muscles were examined by magnetic reso-
nance imaging (MRI). Prior to exposure, the rabbits were anesthetized and depilated
on their abdomen. The scanning procedure was carried out on an MR tomograph
Siemens Magnetom Trio 3T at the Institute of Clinical and Experimental Medicine,
Prague, Czech Republic. The echo (TE) and repetition (TR) times were set as fol-
lows: TE = 20 ms and TR = 642 ms. The slice thickness of the MRI scans was set to
2 mm. The imaging procedure was carried out on the first, second and seventh day
after the exposure to shock waves. The rabbit was under anesthesia during imaging.
410 P. Luke et al.
31.3 Characteristics of Focused Tandem Shock Waves in Water
The pressure field at the focal region formed by the focused tandem shock waves
was visualized by Schlieren photography. Figure 31.2 shows the pressure field for
three different situations. Figure 31.2a, b show the pressure field when only the first
or the second waves were generated, respectively. The waves propagate from right
to left in these pictures. The white cross-like spot in the picture specifies the region
of the highest pressure of the focused shock wave with a sharp front of the shock
waves. The convergence angle corresponds to the reflector geometry and the posi-
tion of the composite electrode in the reflector. The circle in the picture indicates a
secondary short-wavelength spherical shock wave created by the collapsing cavita-
tion bubbles. Figure 31.2a, b show that both shock waves, if produced separately (or
with a long time delay between them), generated only a small number of cavitations
at the focus. Figure 31.2c shows the situation when two successive waves interacted
(time delay of 15 ms). The second wave is heavily damped in the focal region, and
this created a very complex pressure field with a large number of secondary short-
wavelength shock waves from the collapsing cavitation. A shock wave duration of
approximately 70 ns and a corresponding wavelength on the order of 0.1 mm were
estimated, which are capable of interacting with cell-scale structures.
Figure 31.2 demonstrates that the degree of interaction of the second shock wave
with the first one depended on the time delay between the waves. Figure 31.3 shows
the pressure wave-forms of two interacting waves arriving at the focus with differ-
ent time delays td. These measurements indicate that there are three different phases
in the interaction. The first and second phases are demonstrated in Fig. 31.3a.
A relatively short delay between the shocks (time interval of 815 ms) resulted in the
second pressure wave creating a large amplitude rarefraction wave at the focus
instead of an originally positive pressure wave. With increasing time intervals
between the waves, the amplitude of the second shock wave rapidly decreased. For
the interval 50 ms < td < 500 ms, the second shock wave was totally damped. The
second shock started to propagate as a pressure wave for td > 600 ms, and its
propagation was fully re-established only at time delays above 1 ms (Fig. 31.3b).
Fig. 31.2 Schlieren photographs of the focal region: (a) first shock wave only, (b) second shock
wave only, and (c) two interacting shock waves. The time interval between the waves was 15 ms.
The direction of the shock wave (SW) propagation was from the right to the left (as depicted by
the arrow)
Fig. 31.3 The pressure waveforms of the two interacting shock waves measured at the focus for
different time delays between the first and the second shock waves
Fig. 31.4 The dynamics of cavitations at the focal region after the interaction of the focused tan-
dem shock waves for a time delay of 10 ms between the waves
This scenario can be explained by assuming that the cavitation bubbles formed by
the first shock wave served as cavitation nuclei for the second shock wave.
Because the cavitation nuclei dissipate over time, the interval between the shock
waves affected the total number of cavitations and the amplitude of the negative
pressure. Figure 31.3a shows that the largest interaction between the first and second
wave was obtained for a time delay of 815 ms, resulting in the maximum negative
phase of the pressure wave of second shock wave and a large number of cavitation.
With increasing time delay between the waves, the second wave propagated through
the perturbed me-dium, and its energy was absorbed by the collapsing cavitations.
Therefore, the influen-ce of the first shock wave on the second wave was insignifi-
cant at longer time delays (td > 600 ms) when most of the cavitations produced by
the first wave were quenched.
An example of the evolution of the cavitation bubbles formed in water at the focus
after the interaction of the focused tandem shock waves for a time delay of 10 ms is
shown in Fig. 31.4. The growth of the bubbles lasted for about 200250 ms (reaching
a maximum bubble radius ~1.3 mm), and then the bubbles began to collapse.
412 P. Luke et al.
31.4 Biological Effects of Focused Tandem

Shock Waves in Water
The presented FTSW generator was used to investigate the biological effects of
focused tandem shock waves. A time delay between the waves of 10 ms was used
based on previous experiments, which showed that the largest interaction of the
waves occurred at this condition (i.e., td = 815 ms). The amplitude of the pressure
(positive) wave was 80 MPa with a duration of 0.7 ms. The amplitude of the rarefac-
tion (negative) wave was 80 MPa with a duration of 2 ms. These waves produced a
large number of cavitations at the focus (see Figs. 31.2, 31.3, and 31.4). The dimen-
sion of focal volume was 2.5 mm 32 mm long (FWHM) [32, 33]. The applied
voltage was 30 kV. The charging capacity was 0.8 mF. The pulse repetition fre-
quency of the applied tandem shock waves was 0.7 Hz.
Figure 31.5 shows optical micrographs of melanoma B16 cells exposed in vitro
to a different number of tandem shocks. Polypropylene Eppendorf vials with a sus-
pension of melanoma cells (1.5 ml) were placed at the focus of the generator and
exposed to the shock waves. Figure 31.5 demonstrates that exposure of carcinomas
cells to the tandem shocks resulted in cell membrane perforation and total damage
(fragmentation) of the cells at a higher dose. The subsequent analysis of the cell
viability of the B16 melanoma cells confirmed greater damage to the cells with an
increasing number of applied tandem shock waves (data not shown).
Consequently, effects of focused tandem shocks on soft tissue were examined
in vivo. Figure 31.6 shows MRI images of a targeted lesion in a rabbits thigh mus-
cle caused by applying 900 tandem shocks and its progress for 7 days after FTSW
exposure. The left side of each image is a control showing the non-exposed muscle.
The lesion in the tissue is clearly visible as a dark spot of hematoma surrounded by
a bright area of accompanying edema (marked by the white arrow). The differences
in the shape and size of the hematoma and edema over time can be seen. After
1 week, the hematoma disappeared, and the edema area was reduced, indicating
recovery of the injured tissue in the shock wave impact area. The larger size of the
hematoma (~ 5 mm) compared to the focal area of the FTSW (~ 2.5 mm) can be
explained by the changes in the acoustical properties of the treated tissue with an
increasing number of applied shocks, which leads to enhanced damage of the tissue
at the targeted area by the shock waves. Additional MRI analysis revealed that the
extent of the damage in the direction of the shock propagation is about 15 mm. The
subsequent histological examination of the injured femoral muscle showed an
extensive focus of granulation tissue in the field of subacute dystrophic changes in
the muscle fibers with a very sharp transition between the damaged and non-exposed
tissue (not shown). These results showed that tandem shocks are capable of causing
localized lesions at a predictable location deep within soft tissue at the focus without
damaging tissue located in front of the focal point. This finding is important in terms
of the potential biomedical applications of tandem shock waves, such as in treat-
ment of tumors that are inoperable or very difficult to be treated (e.g., many liver
metastases and pancreatic tumors).
Fig. 31.5 Optical micrographs of the melanoma B16 cells exposed in vitro to different numbers
of focused tandem shock waves: (a) control, (b) 200 shocks and (c) 400 shocks
Fig. 31.6 MRI images of targeted lesions in a rabbits thigh muscle (marked by the white arrow)
over time induced in vivo by applying 900 focused tandem shock waves. The left side in each
image is a control showing the non-exposed muscle. (a) 1st day, (b) 2nd day, and (c) 7th day after
FTSW exposure
Figure 31.7 shows the in vivo effect of FTSW on the growth of sarcoma tumors
in rats of the Lewis strain. R5-28 malignant cells of the syngeneic sarcoma were
subcutaneously applied caudally on the right and left sides of Lewis rats and
tumors were allowed to grow for 35 days, after which the tumors were treated by
FTSW. The tumors on the left sides of the rats were exposed to 240 shocks by
placing the anesthetized animals in the water bath of the FTSW generator. The
contralateral tumor served as a control. Shock waves were applied to the tumors
from two different angles with 120 shock waves at each angle. Figure 31.7 shows
that, in comparison with the intact controls, the growth of the exposed tumors was
significantly delayed.
Considering the tumor size and much smaller size of the shock wave focus area
(~ order of magnitude difference), the scanning of the tumor by shock waves can be
414 P. Luke et al.
Fig. 31.7 The growth of

sarcoma tumors in Lewis rats
after in vivo exposure to 240
tandem shock waves
used to increase the efficiency and to enlarge the localized effect of shock wave
exposure. Varying the number of applied shocks can also be used. In our case, a
higher dose (~400 shocks) resulted in mechanical damage to the tumor tissue. Thus,
repeated treatment of the tumor by a smaller number of shocks over several days
might be considered. Apart from mechanical effects, the shock waves may also
contribute to tumor treatment due to cavitation-induced sonochemical effects by the
intracellular formation of radicals. Combining the effect of shock waves with
anticancer drugs may also be possible (e.g., to enhance the chemotherapy efficiency
by increasing the permeability of tumor cells membranes to cytostatic drugs by the
shockwaves or through the sonodynamic activation of cytotoxic drugs by cavitation
induced by FTSW). Research on the possible synergetic effects treatment with
shock waves in conjunction with other therapeutic methods used in cancer treatment
is in progress.
31.5 Conclusions
The physical principles of a novel generator of focused tandem shock waves in

water have been presented. FTSW were generated with a variable time delay
between the waves using underwater multichannel electrical discharges in a highly
conductive saline solution using two porous ceramic-coated cylindrical electrodes
of different diameter and surface area. The largest interactions were obtained for a
time delay of 815 ms between the waves, producing an amplitude for the negative
pressure phase of the second shock wave down to 80 MPa and a large number of
cavitations at the focus. The biological effects of FTSW were demonstrated in vitro
on damage to B16 melanoma cells, in vivo on targeted lesions in the thigh muscles
of rabbits and on the growth delay of sarcoma tumors in Lewis rats treated in vivo
by FTSW compared to untreated controls.
Acknowledgements This work was supported by the Czech Science Foundation (project No.
202/09/1151) and the Ministry of Education, Youth and Sports of the Czech Republic (MSM
0021620808). The authors would like to thank Dr. V. Horak from the Institute of Animal
Physiology and Genetics AS CR for providing R5-28 malignant cells for the experiments with the
Lewis rats, Dr. V. Herynek and Dr. M. Dezortova from the Institute for Clinical and Experimental
Medicine, Prague, Czech Republic for the MRI analysis of the lesions in the rabbits thighs and
Dr. J. Kralova from the Institute of Molecular Genetics AS CR for optical micrographs of the
melanoma B16 cells.
References
1. Kim HH (2004) Nonthermal plasma processing for air-pollution control: a historical review,
current issues, and future prospects. Plasma Proc Polym 1:91110
2. Locke BR, Sato M, Sunka P, Hoffmann MR, Chang JS (2006) Electrohydraulic discharge and
nonthermal plasma for water treatment. Ind Eng Chem Res 45:882905
3. Stalder KR, McMillen DF, Woloszko J (2005) Electrosurgical plasmas. J Phys D: Appl Phys
38:17281738
plasma medicine. Plasma Proc Polym 5:503533
5. Dobrynin D, Fridman G, Friedman G, Friedman A (2009) Physical and biological mechanisms
6. Laroussi M (2005) Low temperature plasma based sterilization: overview and state of the art.
Plasma Proc Polym 2:391400
7. Sato M, Ohgiyama T, Clements JS (1996) Formation of chemical species and their effects on
microorganisms using a pulsed high-voltage discharge in water. IEEE Trans Ind Appl
32:106112
8. Efremov NM, Adamiak BYu, Blochin VI, Dadashev SJa, Dmitriev KI, Semjonov VN,
Levashov VF, Jusbashev VF (2000) Experimental investigation of the action of pulsed electrical
discharges in liquids on biological objects. IEEE Trans Plasma Sci 28:224229
9. Abou-Ghazala A, Katsuki S, Schoenbach KH, Dobbs FC, Moreira KR (2002) Bacterial decon-
tamination of water by means of pulsed-corona discharges. IEEE Trans Plasma Sci
30:14491453
10. Ching WK, Colusi AJ, Sun HJ, Nealson KH, Hoffmann MR (2001) Escherichia coli disinfec-
tion by electrohydraulic discharges. Environ Sci Technol 35:41394144
11. Robinson JW, Ham M, Balaster AN (1973) Ultraviolet radiation from electrical discharges in
water. J Appl Phys 44:7275
12. Lukes P, Clupek M, Babicky V, Sunka P (2008) Ultraviolet radiation from pulsed corona dis-
charge in water. Plasma Source Sci Technol 17:024012
13. Gilliland SE, Speck ML (1967) Mechanism of the bactericidal action produced by electrohy-
draulic shock. Appl Microbiol 15:10381044
14. Zastawny HZ, Romat H, vel Karpel Leitner N, Chang JS (2004) Pulsed arc discharges for
water treatment and disinfection. In: Electrostatics 2003. IOP Publishers, Bristol, p 325
15. Li Z, Sakai S, Yamada Ch, Wang D, Chung S, Lin X, Namihira T, Katsuki S, Akyiama H
(2006) The effects of pulsed streamerlike discharge on cyanobacteria cells. IEEE Trans Plasma
Sci 34:17191725
16. Li Z, Ohno T, Sato H, Sakugawa T, Akiyama H, Kunitomo S, Sasaki K, Ayukawa M, Fujiwara
H (2008) A method of water-bloom prevention using underwater pulsed streamer discharge.
J Environ Sci Health A 43:12091214
17. Coleman AJ, Saunders JE (1993) A review of the physical properties and biological effects of
the high amplitude acoustic fields used m extracorporeal lithotripsy. Ultrasonics 31:7589
416 P. Luke et al.
18. Bailey MR, Khokhlova VA, Sapoznikov OA, Kargl SG, Crum LA (2003) Physical mechanisms
of the therapeutic effect of ultrasound (a review). Acoust Phys 49:369388
19. Benes J, Sunka P, Kordac V, Barta Z, Stuka C, Figura Z, Jirsa M (1988) Apparatus for clinical
performance of extracorporeal lithotripsy. UK Patent GB2199249
20. Sunka P, Babicky V, Barta Z, Benes J, Kolacek K, Kordac V, Stuka C (1990) Method and
apparatus for adjusting the spark gap of a non-invasive lithotriptor. EU Patent EP0349915
21. Stuka C, Sunka P, Benes J (1995) New discharge circuit for efficient shock wave generation.
In: Brun R, Dumitrescu LZ (eds) Shock waves @ Marseille III. Springer, Berlin/Heidelberg,
pp 455458
22. MEDIPO-ZT, s.r.o. (Ltd.). http://www.medipo.cz/litotryptor.htm
23. Haupt G (1997) Use of extracorporeal shock waves in the treatment of pseudarthrosis, tendi-
nopathy and other orthopedic diseases. J Urol 158:411
24. Bailey MR, Blackstock DT, Cleveland RO, Crum LA (1998) Comparison of electrohydraulic
lithotripters with rigid and pressure-release ellipsoidal reflectors. I. Acoustic fields. J Acoust
Soc Am 104:25172524
25. Bailey MR, Blackstock DT, Cleveland RO, Crum LA (1999) Comparison of electrohydraulic
lithotripters with rigid and pressure-release ellipsoidal reflectors. II. Cavitation fields. J Acoust
Soc Am 106:11491160
26. Zhong P, Lin H, Xi X, Zhu S, Bhogte ES (1999) Shock wave-inertial microbubble interaction:
methodology, physical characterization, and bioeffect study. J Acoust Soc Am 105:19972009
27. Sokolov DL, Bailey MR, Crum LA (2001) Use of a dual-pulse lithotripter to generate a
localized and intensified cavitation field. J Acoust Soc Am 110:16851695
28. Sokolov DL, Bailey MR, Crum LA (2003) Dual-pulse lithotripter accelerates stone fragmenta-
tion and reduces cell lysis in vitro. Ultrasound Med Biol 29:10451052
29. Huber P, Debus J, Jochle K, Simiantonakis I, Jenne J, Rastert R, Spoo J, Lorenz WJ,
Wannenmache M (1999) Control of cavitation activity by different shockwave pulsing regimes.
Phys Med Biol 44:14271437
30. Loske AM, Prieto FE, Fernandez F, van Cauwelaert J (2002) Tandem shock wave cavitation
enhancement for extracorporeal lithotripsy. Phys Med Biol 47:39453957
31. Alvarez UM, Ramirez A, Fernandez F, Mendez A, Loske AM (2008) The influence of single-
pulse and tandem shock waves on bacteria. Shock Waves 17:441447
32. Sunka P (2001) Pulse electrical discharges in water and their applications. Phys Plasmas
8:25872594
33. Sunka P, Babicky V, Clupek M, Benes J, Pouckova P (2004) Localized damage of tissues
induced by focused shock waves. IEEE Trans Plasma Sci 32:16091613
34. Sunka P, Babicky V, Clupek M, Fuciman M, Lukes P, Simek M, Benes J, Majcherova Z, Locke
BR (2004) Potential applications of pulse electrical discharges in water. Acta Phys Slovaca
54:135145
35. Sunka P, Stelmashuk V, Babicky V, Clupek M, Benes J, Pouckova P, Kaspar J, Bodnar M
(2006) Generation of two successive shock waves focused to a common focal point. IEEE
36. Lukes P, Clupek M, Babicky V, Sunka P (2008) Pulsed electrical discharge in water generated
using porous ceramic coated electrodes. IEEE Trans Plasma Sci 36:11461147
37. Moravkova A, Malek O, Pokorna E, Strnadel J, Hradecky J, Horak V (2005) Immune charac-
terization of the Lewis rats inoculated with K2 sarcoma cell line and newly derived R5-28
malignant cells. Folia Biol 51:159165
Chapter 32
DBD Plasma Assisted Silver Functionalization
of Surgical Meshes
Jozef Rhe, Hana Polkov, Eva Jonov, Markta Hudcov,

Miroslav Zahoran, and Petr Nasadil
Abstract Atmospheric pressure diffuse barrier discharge in nitrogen was employed

to promote the formation sub-micron particles of silver salts of AgNO3, Ag2O, Ag2CO3
and AgCl on polypropylene and polyester warp-knitted surgical meshes. The methods
for producing and evaluation of coating are described in detail. All silver salts proved
their antimicrobial efficacy against gram-positive S. aureus and gram-negative E. coli.
In general the quantity of immobilized silver sufficient for antibacterial action was
found to be in the order of units of mg per gram of polymer mesh.
32.1 Introduction
32.1.1 Surgical Meshes
Surgical mesh can be seen as a reinforcement or patch, which is inserted to the weak
spot or defect in the patient tissue. The wound is healed after the surgery in a way
where the mesh acts as an external support through which the new tissue is grown,
J. Rhe (*)
Faculty of Science, Masaryk University, Kotlsk 2, 61137 Brno, Czech Republic
Department of Experimental Physics, Comenius University, Mlynsk dolina F2,
e-mail: rahel@mail.muni.cz
H. Polkov E. Jonov
Faculty of Science, Masaryk University,Kotlsk 2, 61137 Brno, Czech Republic
M. Hudcov P. Nasadil
Textile Testing Institute, Vclavsk 6, Brno, Czech Republic
M. Zahoran
Department of Experimental Physics, Comenius University, Mlynsk dolina F2,
418 J. Rhe et al.
providing a tension-free repair [1]. The most common types of surgical meshes are
hernia mesh, stress urinary incontinence slings and mesh for treating prolapse.
Hernias are very common medical condition which affects during the life time
510% of the population. The use of polymeric surgical meshes for hernia repair
was introduced by Francis Usher, who in 1958 began promoting the use of polyeth-
ylene and polypropylene meshes (commercial name Marlex) [1, 2]. Nowadays
lightweight knitted meshes of pore size larger than 1 mm are used preferably in
order to improve the biocompatibility by reduced amount of foreign tissue [3].
Polypropylene (PP) is the most widely used polymer for the manufacture of sur-
gical meshes. It is a cheap material offering a proper tension strength and high level
of chemical inertness, which is responsible for its extreme resistance to biological
degradation and high level of biocompatibility. Polyester (PES) meshes are less
frequent than polypropylene. An example is a multifilament Mersilene (Ethicon) or
CHS 100 (VUP). PES is a flexible, well adaptive mesh, but it has a higher risk of
infection and cause a greater degree of inflammatory response.
Infection is among one of the most serious complications associated with the inser-
tion of implant into the body [4]. According to Engelsman et al. [5] surgical mesh
implant is prone to infiltration by bacteria even 39 months after insertion into the
body. Gram-positive S. aureus, S. epidermidis and gram-negative E. coli are the most
common bacteria infecting surgical meshes [4]. Experiments showed that the multi-
filament implants are due to greater surface more susceptible to bacterial colonization
than monofilament [4, 6].
The risk of bacterial infection can be substantially reduced by an appropriate mesh
surface modification [5, 6]. Successful results on antimicrobial surface finishes of
meshes were achieved using various chemical compounds such as silver, gold, tita-
nium, chlorhexidine, triclosan, chitosan [4, 79]. Scheindbach et al. [10] studied influ-
ence of titanium on the surface of hard polypropylene mesh (titanium-coated Atrium).
Positive effect on the biocompatibility and the reduction of bacterial adhesion was
demonstrated. Saygun et al. [11] studied the effect of gold and gold with palladium
coatings. A significant reduction in bacterial colonization was shown, in particular
when combining gold and palladium.
Silver and its various forms (silver oxide, silver sulfadiazine, silver carbonate,
silver nitrate, silver iodide, etc.) is probably the most widely used antimicrobial
metal agent. Silver can be readily combined with other substances, which may
increase and prolong the desired antibacterial effect. Fox et al. [12] lists in his patent
various combinations of antimicrobial agents, focused primarily on the synergistic
effect of silver salts and chlorhexidine or chlorhexidine salts.
Table 32.1 summarizes the composition and functional finishing for some of the
most common commercially available PP and PES meshes. Surprisingly neither of
them employs silver as an antimicrobial agent. To our knowledge the only commer-
cially available surgical mesh employing silver is DUALMESH PLUS (WL Gore
and Associates) which is made from rather expensive expanded polytetrafluoroeth-
ylene (ePTFE). Its antimicrobial action is facilitated by the combination of silver
carbonate and chlorhexidine [4, 13]. The mesh is claimed to be resistant to bacteria
for 10 days after its implantation.
32 DBD Plasma Assisted Silver Functionalization of Surgical Meshes 419
Table 32.1 Commercially available surgical meshes [1, 4, 5, 7, 14, 15]

Trade name Material Manufacturer
Marlex PP C.R. Bard, USA
Prolene PP Ethicon Inc., USA
SurgiPro mesh PP US Surgical Corp., USA
Mersilene PES Ethicon Inc., USA
Parietex composite PES/collagen Sofradim, France
TiMesh PP/titan GfE Medizintechnik, Germany
SepraMesh PP/H/CMC Genzyme Biosurgery, USA
Proceed PP/ORC/PD Ethicon Inc., USA
PP polypropylene, PES polyester, CHH chlorhexidine, H hyaluronate, CMC
carboxymethylcellulose, PD polydioxanone
32.1.2 The Mechanism of Silver Action
Elementary (atomic) silver is not antimicrobially active. Only solvated forms of

silver ions Ag+ or small Ag0 clusters (e.g. colloidal silver) are biologically active
[16]. The mechanisms of actual silver-microbes interactions are still not fully under-
stood and are constantly under investigation. According to Klueh et al. [17] silver
affect the microbial cell in multiple pathways. In the water solution free unbound
silver ions interrupt the cell metabolism either reversibly or irreversibly depending
on their concentration. Silver ions bind with proteins through the sulfhydryl group
of cysteine and imidazole group of histidine. Silver ions form with SH groups in the
bacterial wall an insoluble compound. SH group is also part of numerous enzymes,
and thus the actions associated with these enzymes are influenced as well. Silver
ions can also penetrate into the cell and bind to bacterial DNA. Binding to nucle-
otide bases causes denaturation by displacing hydrogen bonds between adjacent
purines and pyrimidines. Silver ions also block respiratory chain of bacteria.
32.1.3 Silver Functionalization
There are several proved methods of preparing antimicrobial silver containing

fabric. Silver additives can be introduced directly to the fibers volume during the
melt extrusion, or silver can be applied as some finishing agents on final fiber or
textile surface. The later method offers better conservation of mechanical properties
of the polymer and faster dynamics of antimicrobial silver release. One of the most
straightforward methods offering the minimum use of potentially harmful chemicals
is direct exposure of fibers to the aqueous solution of AgNO3. This method can be
particularly beneficial in medicine where the risk of potential secondary complications
should be minimized.
In order to create metallic silver attached to the polymer surface, solvated
monovalent silver ion Ag+ needs to be reduced to the zero valent state Ag0. Efficient
420 J. Rhe et al.
reducing agents may be provided in the form of organic radicals created in the
suitably chosen solution by ionizing radiation, which is already proved method in
making metal nanocolloids [18]. A favorable energy balance then drives reduced
zero valent atoms to mutually dimerize or associate with the excess of ions which
eventually leads to the progressive coalescence into silver clusters Agmz+ with
nuclearity m and number of associated ions z.
32.1.4 Plasma Treatment
An alternate method to ionizing radiation is to create desired radicals directly on the

surface of polymer material so called surface activation. For the surface activation
without the risk of potentially harmful chemical residues, the treatment facilitated
by electrical discharge plasma is one of the most promising options.
In the study presented by Yuranova et al. [19], the combination of UV light and
RF plasma (13.56 MHz, 100 W, 0.8 Torr) was used to activate the textiles. Afterwards
the textile was placed into the AgNO3 solutions of different concentrations. AgNO3
concentration of 1.5 g.dm3 caused complete inhibition of E. coli bacterial growth.
A study that is more close to our work was published by Kostic et al. [20]. The
desired antimicrobial activity of silver functionalized cotton/PES fabrics against S.
aureus, E. coli and C. albicans was achieved by dielectric barrier discharge (DBD)
treatment operating in air at atmospheric pressure, followed by dip in 0.01 mol.dm3
AgNO3.
DBD discharge manifests itself as an assembly of numerous randomly appearing
non-thermal plasma channels of sub-milimetre diameter microfilaments. Non-
thermal plasma generated by DBD is known to offer high-energy electrons (tens of
thousands of Kelvin) at close to room temperature of working gas. Recently a possi-
bility of generating spatially uniform DBD plasma (diffuse DBD) without any micro-
filaments was discovered [21, 22]. The necessary conditions for operating plasma in
diffuse DBD regime is the presence of rare gas (He, Ar, Ne) or high purity N2.
The mechanism of surface activation mediated by plasma treatment is based on
the interaction of active working gas particles (radicals, electrons, ions, excited
atoms, UV photons, etc.) providing numerous physical and chemical changes oxi-
dation, polymerization, cross-linking, etching, etc. [23]. The kinetic model study of
changes induced by air plasma on PP surface made by Dorai et al. [24] suggests the
formation of alkyl radicals preferably from tertiary H extraction, which conse-
quently leads to the formation of these polar functional groups: COH (alcohol),
COOH (hydroperoxide), HCO (aldehyde), CO (carbonyl), COO (peroxy),
COCO (ester) HOCO (carboxyl) and NH2 (amine). The presence of oxygen con-
taining groups even when non-oxygen working gas is used can be attributed to the
exposure of treated samples to ambient air after the plasma treatment [25], and to
the residual gas and water adsorbed on the material surface.
One of the most important advantages of plasma treatment is that the polymer is
modified only at the surface (up to tens of nm), while its bulk properties remain
essentially unaffected.
In the following text we will present the results of our work on preparing silver
particles functionalized surgical meshes made of PP a PES and the evaluation of
their performance. To reduce the final cost of process being developed, surface
activation involving DBD at atmospheric pressure was adopted. Our study is focused
primarily on PP meshes functionalization, which is not only commercially more
important product, but due to its well-known chemical inertness it represents a
particular challenge for chemical surface modification.
32.2 Experiment
32.2.1 DBD Plasma Treatment
Volume dielectric barrier discharge (DBD) transparent electrode system was assem-
bled from two water filled transparent polycarbonate blocks, separated by two
dielectric barriers plates made of 1.1 (0.05) mm thick borosilicate glass D263 T
(er = 6.7, suppl. by Praezisions Glas and Optik, Germany). The particular combina-
tion of low surface roughness and high permittivity value of D263 T was found to
be beneficial to stabilizing DBD in its diffuse mode of operation.
The inter-electrode gap was adjusted by 3 screws located underneath of the bottom
electrode to 0.7 mm. Electrode system was housed in a transparent gas-tight reactor, to
allow gas containment control. The reactor was operated at atmospheric pressure in
flowing gas at q = 4 dm3/min. N2 of technical purity and compressed air were used. The
reactor was purged with the working gas for 2 min prior any treatment run.
The active dimensions of the electrodes were 130 128 mm. Regular tap water
(specific conductivity 100 mS/cm) was used simultaneously both as an electric con-
ductor and as a cooling medium. The whole experimental set-up for plasma treat-
ment is shown in Fig. 32.1.
The electrodes were energized by a sinusoidal voltage from a VF 1500 ozone
generator power supply, made by Lifetech (Czech Rep.). The maximum operating
power was 300 W, operating frequency was 203 kHz. Two TEKTRONIX P6015A
(1,000) high-voltage probes were used to control applied voltage to electrodes.
Pearson current monitor, model 4100 (1:1, 35 MHz, 10 ns usable rise time), was
used for the electrical current measurements. Visual appearance of the plasma was
recorded using digital camera CASIO EX-F1 (6.0 Mpx). Signals from the HV
probes and current monitor were recorded using a LeCroy WaveRunner 6100A
oscilloscope (2 GHz Bandwidth, 10 GS/s max sample rate).
32.2.2 Material and Methods
Knitted synthetic surgical meshes made of polypropylene monofilament (PP MESH

standard 50 0.2 g/m2) and polyester filament yarn (CHS100 68 0.4 g/m2) were
supplied by VUP a.s. Czech Rep. AgNO3 water solutions of various concentration
422 J. Rhe et al.
Fig. 32.1 Experimental setup: MOD T2 water filled electrodes, HV high voltage probe, CM cur-
rent monitor, Ck variable capacitor, OSC oscilloscope, PC computer, GVF power supply, CIRC
water circulation system, CCD digital camera
was used to prepare primary silver deposit. Water solutions of NaOH, Na2CO3 and
NaCl with concentrations of 1 or 0.5 M were used to transform primary silver
deposit into silver salts of Ag2O, Ag2CO3 and AgCl respectively. Concentrated
HNO3 was employed for preparing the titration extract from AgNO3, Ag2O and
Ag2CO3. Titration extract from AgCl was prepared by 5% sodium thiosulfate. All
chemical used were of p.a. purity.
Qualitative testing of the antibacterial effect was done according to the ISO
20645 standard [26]. The test is based on the assessment of bacterial growth around
and under the textile specimen that was laid into the particular bacterial strain
inoculated agar. The test will result in three possible outcomes for antimicrobial
activity insufficient effect (bacterial growth in agar under the specimen), limit of
efficacy (growth almost totally suppressed, restricted to small colonies); good effect
(no bacterial growth underneath the specimen, presence of inhibition zone around
the specimen). The purpose of test is to identify the minimum concentration of
antibacterial agent in order to achieve final assessment of good effect.
Gram-positive S. aureus ATCC 6538 and gram-negative E. coli ATCC 11229
were evaluated. Cultivation took place on plate count agar (PCA) and nutrient agar
(NA). Ten milliliters of pure, sterile agar was poured into plastic Petri dishes and let
to solidify. Afterwards 5 ml of 45C bacteria inoculated agar was added (1 5.108 CFU/
ml). This temperature was found to be sufficient for keeping agar in a flowing state
without killing bacteria. Mesh specimen was laid into the inoculated agar. After incu-
bating samples for 24 h at 37C the evaluation was carried out by the assessment of
bacterial growth under the tested specimen and by the size of inhibition zone around
the specimen. Each cultivation test was made for two separate samples.
Quantitative analysis of amount of immobilized silver was done by the potentio-
metric titration. Orion 4-Star Benchtop pH/ISE meter (Thermo Fisher Scientific)
equipped with the PerfectIONTM combination Ag/S electrode (Mettler-Toledo) was
used. Silver coated polymer meshes were immersed for 1 h into concentrated HNO3
(65%) at 60C to dissolve immobilized silver. Consequently the solute was diluted
to approx 1:10 by deionized water and titrated with the water solution of NaCl in the
absence on light. To verify the obtained data, the concentration of silver in solute
was determined also by atomic absorption spectrometry using UNICAM AAS 939
spectrometer. Flame atomizer or graphite tube electrothermal (ETA-AAS) atomizers
was used for high and low concentration of Ag respectively. The difference between
both methods was found be less than 20%, which is within the stated accuracy of
used AAS spectrometer.
Morphology of silver particles immobilized of meshes was studied by SEM
using VEGA II SBH (fy TESCAN, Brno, Czech Rep.) offering the resolution of
2 nm. Prior the SEM analysis all samples were coated by gold using magnetron-
sputtering device.
32.3.1 AgNO3 Functionalization
The use of transparent DBD reactor revealed immediately poor contact of generated
plasma with individual mesh fibers when treating in air gas (Fig. 32.2a). DBD
microfilaments were formed preferentially in relatively large inter-fibrous spaces.
To address this issue plasma treatment using N2 diffuse DBD was adopted.
Figure 32.2b illustrates the visual uniformity of N2 diffuse DBD in contact with PES
fiber. The improved treatment efficiency was confirmed by shortening of time nec-
essary for complete water wettability of polymer mesh to approx. half of the time
needed in air. In the following text all plasma activation were made in N2.
Immediately after plasma treatment samples of PES and PP meshes were
immersed into light-tight PP Erlenmeyer flask filled with AgNO3 water solution of
various concentration. The solution with samples was magnetically stirred at
250 rpm for 24 h at room temperature. After removing from the solution samples,
the excess of water in inter-fibrous space was shaked off and samples were let to dry
at the room temperature.
Table 32.2 summarizes the results of bacterial efficacy for various initial concen-
tration of AgNO3 solution for 2 min lasting plasma treatment. The formation of the
inhibition zone around the Ag functionalized PES sample is apparent from Fig. 32.3.
Initial AgNO3 concentration of 0.05 M was found to be sufficient to provide
desired antibacterial effect for both PES and PP meshes. The actual relative mass of
immobilized silver from this solution was measured to be (6.0 0.1) mg/g for PES
mesh and (3.8 0.1) mg/g for PP mesh. Smaller amount of Ag immobilized on PP
can be attributed to its higher chemical inertness and smaller fiber surface area (see
Fig. 32.4). It is interesting to note, that it was possible to approximately double
these values when the immersion time in AgNO3 solution is cut from 24 to 1 h. The
results obtained then were (14 3) mg/g for PES and (5.0 0.4) mg/g for PP mesh.
The exact nature of competing process responsible for the removal of immobilized
silver back to the solution has not been identified yet.
424 J. Rhe et al.
Fig. 32.2 Plane view on plasma treated PES samples of 4 4 cm: (a) DBD in air; (b) Diffuse DBD
in N2. exposure time 1 s for both pictures
Table 32.2 Cultivation tests of various concentrations AgNO3 deposited on polymer

meshes activated for 2 min by diffuse N2 DBD plasma
E. coli S. aureus
Inhibition Inhibition
Sample zone [mm] Assessment zone [mm] Assessment
PP untreated 0 Insufficient 0 Insufficient
PP 0.01 M 0 Insufficient 0 Insufficient
PP 0.05 M 1 Good 2 Good
PP 0.10 M 2 Good 2 Good
PES untreated 0 Insufficient 0 Insufficient
PES 0.01 M 1 Good 1 Good
The positive effect of plasma treatment was obvious for both PES and PP samples.
PES sample immersed to AgNO3 without prior plasma treatment resulted in the
silver relative mass of (3.3 0.6) mg/g. This is only 55% of Ag mass immobilized
on plasma-activated samples. The mass of 3.3 mg/g corresponds well with the mass
of Ag present in fluid adhering to the mesh immediately after its removal from the
deposition flask. Our conclusion is that solid state Ag compound on untreated PES
is formed by precipitation during the course of water evaporation (drying).
Plasma activation of chemically more inert PP samples resulted in similar rela-
tive enhancement although smaller in absolute numbers. We were able to increase
the absolute amount of Ag by extending the exposure time in N2 DBD plasma. The
results obtained were: (1.8 0.1) mg/g for untreated PP mesh; (3.8 0.1) mg/g for
2 min treatment; (5.8 0.2) mg/g for 4 min treatment and (7.7 0.3) mg/g for 10 min
lasting plasma treatment.
Fig. 32.3 The result of ISO 20645 cultivation test for E. coli inoculated agar: (a) Heavy growth
under the untreated PES mesh insufficient effect; (b) No growth under the PES 0.01 M mesh and
the growth inhibition zone of 1 mm around the mesh good effect
Fig. 32.4 Morphology of Ag2O coating on investigated polymer meshes. View field of 100 mm for
both photographs
The chemical nature of immobilized silver was identified by immersing coated

samples for 1 h into 37C deionized water, which results into complete removal
(i.e. beyond detectable limit) of silver deposit. We conclude that silver deposit on
the surface is in the form of AgNO3, since it is the only silver salt known to be solv-
able in water. Further evidence supporting this explanation was the observed darken-
ing of freshly prepared AgNO3 samples when exposed to the UV light. The effect is
426 J. Rhe et al.
attributed to the known photoreduction of AgNO3 to metallic silver. Therefore a

simplified formula describing the process of AgNO3 fictionalization can be written as:
R* + AgNO3(aq) R - AgNO3(s) (32.1)
where R* denotes an active site on the polymer surface. We assume that this
process is occurring in two steps. In the first step is the Ag+ reduction to
zero-valence state on the polymer surface radical. In the second step excess of
NO3 present in the solution contributes to its conversion to solid precipitate of
AgNO3. Nevertheless more detailed experiments on the process kinetics still
need to be done.
32.3.2 Ag2O, Ag2CO3 a AgCl Derivatives on PP Meshes
After producing mechanically stable but water-soluble AgNO3 functionalization, we

have attempted to convert bonded AgNO3 to somewhat less soluble silver salts, namely
Ag2O, Ag2CO3 and AgCl. Both the possibility of conversion and the existence of
antibacterial effect on examined water-insoluble Ag salts were investigated.
Dried AgNO3 samples prepared on PP (2 min plasma exposure +24 h at 0.05 M
AgNO3 solution) were placed for 4 h into magnetically stirred (250 rpm) water
solution of NaOH (1 M), Na2CO3 (0.5 M) and NaCl (1 M). Following chemical
reaction were expected to take place:
2 AgNO3 + 2 NaOH (aq) 2 AgOH + 2 NaNO3 Ag2 O(s) + H 2 O + 2 NaNO3 (32.2)
2 AgNO3 + Na 2 CO3(aq) Ag 2 CO3(s) + 2 NaNO3 (32.3)
AgNO3 + NaCl(aq) AgCl(s) + NaNO3 (32.4)
Successfully accomplished transformation of silver salts was immediately appar-

ent from the observed color change of meshes. Ag2O exhibited dark brown color-
ation. Initially yellow-green Ag2CO3 was slowly converted to brown color upon
exposure to the sunlight. White AgCl coating rapidly converted to deep violet and
black color after exposing to the sunlight.
Table 32.3 summarizes results of antibacterial efficacy of individual samples
against E. Coli and S. aureus. All of our tested silver salts exhibited good anti-
microbial effect. In addition to that Table 32.3 lists the relative mass of immo-
bilized silver salts. Somewhat smaller value of Ag2CO3 and AgCl comparing to
the initial amount of immobilized AgNO3 was attributed to partial dissolution of
Ag2CO3 in Na2CO3 and AgCl in NaCl. This issue can be in future easily addressed
by reducing the reagent concentration and/or by reducing the immersion period
in it.
Table 32.3 Bacterial efficacy against E.coli and S. aureus of various Ag salts on PP mesh
E. coli S. aureus
(Ag)/(PP) Inhibition Inhibition
Sample [mg/g] zone [mm] Assessment zone [mm] Assessment
PP untreated 0 0 Insufficient 0 Insufficient
PP + AgNO3 3.8 0.1 1 Good 2 Good
PP + Ag2O 5.3 0.7 1 Good 1 Good
PP + Ag2CO3 1.5 0.5 1.5 Good 2 Good
PP + AgCl 2.0 0.5 1 Good 1 Good
32.3.3 The Effect of Plasma Treatment

on Adhesion Improvement
Successful conversion of primary created AgNO3 to water-insoluble Ag2O was uti-

lized to estimate the stability of surface coating due to the plasma treatment.
Samples of Ag2O were chosen for its absence of photosensitivity which may inter-
fere with the test. After being shaken for 1 h in deionized water of 37C, the amount
of Ag2O on plasma activated PP mesh has been reduced from 3.0 to 1.6 mg/g, i.e.
54% of the initial value. The same wash-off experiment for samples without plasma
treatment resulted in Ag2O reduction from 1.9 to 0.6 mg/g (i.e. 32%). One may
conclude that plasma activation is capable of enhancing not only the absolute
amount of immobilized silver compound, but also the final bonding strength of
deposited silver salts.
32.3.4 Surface Morphology of Ag Functionalized Samples
SEM magnified view of Ag2O deposited on PP and PES meshes are shown in
Fig. 32.4. The same qualitative pictures were obtained also for the rest of investi-
gated functionalizations. In the case of PES submicron crystals of Ag salts are
deposited preferentially in areas between adjacent filaments of yarn. This prefer-
ence may turn be of practical importance, as bacteria in our cultivation experiments
are colonizing preferentially exactly the same locations. For PP silver salts are
deposited directly on the monofilament surface with relatively large degree of het-
erogeneity. We can observe spots completely covered with grown microcrystal, as
well as areas sparsely covered with submicron monocrystals. Further optimization
of deposition process could certainly address the observed heterogeneity; neverthe-
less as was shown in Table 32.3 even such heterogeneous surface is able to provide
sufficient antibacterial action.
428 J. Rhe et al.
32.4 Conclusion
We have presented an easy and straightforward method of depositing silver containing

compounds on PP and PES surgical meshes. The simplicity of the method is of
practical importance for medical applications, where the risk of potential harmful
side effects invoked by various chemical residua should be minimized.
All investigated samples with AgNO3, Ag2O, Ag2CO3 and AgCl functionaliza-
tion have proved their good antibacterial efficiency towards S. aureus and E. coli.
The quantities of immobilized silver found to be sufficient for antibacterial action
are in the order of units of mg per gram of polymer mesh.
Employing plasma treatment for Ag functionalization resulted in more than 50%
improved relative uptake of silver ions from the solution for both PES and PP
meshes. Chemically more inert PP meshes requires substantially longer plasma
treatment in order to achieve the same total uptake as PES samples. In addition to
that plasma treated samples are less prone to the wash off effect of deposited Ag
salts. This is important in order to ensure lasting bacterial resistance after the
implantation.
To our knowledge there is currently no commercially available PP or PES mesh
that would take advantage of any silver antimicrobial agent. The study presented
here suggests a viability of such product. Nevertheless a substantial research work
still needs to done, involving optimization of the deposition process, shortening the
fabrication time, cost reduction, improvement of deposit homogeneity, but also to
getting much better understanding and control on amount of silver ions released to
surrounding by the deposit.
Acknowledgments This research has been supported by the project KAN101630651

Nanotechnologies for Society, Academy of Sciences of the Czech Republic and by the project
R&D center for low-cost plasma and nanotechnology surface modifications CZ.1.05/2.1.00/03.0086
funded by European Regional Development Fund.
References
1. Pandit AS, Henry JA (2004) Design of surgical meshes an engineering perspective. Technol
Health Care 12:5165
2. Read RC (1999) Francis C. Usher, herniologist of the twentieth century. Hernia 3:167171
3. Hollinsky C, Sandberg S et al (2008) Biomechanical properties of lightweight versus heavy-
weight meshes for laparoscopic inguinal hernia repair and their impact on recurrence rates.
Surg Endosc 22:26792685
4. Engelsman AF, van der Mei HC et al (2007) The phenomenon of infection with abdominal
wall reconstruction. Biomaterials 28:23142327
5. Engelsman AF, van der Mei HC et al (2008) Morphological aspects of surgical meshes as a
risk factor for bacterial colonisation. Br J Surg 95:10511059
6. Engelsman AF, van Dam GM et al (2010) In vivo evaluation of bacterial infection involving
morphologically different surgical meshes. Ann Surg 251:133137
7. Carbonell AM, Matthews BD et al (2005) The susceptibility of prosthetic biomaterials to
infection. Surg Endosc 19:430435
8. Cobb WS, Paton BL et al (2006) Intra-abdominal placement of antimicrobial-impregnated

mesh is associated with noninfectious fever. Am Surg 72:12051209
9. akmak A, irpanli Y et al (2009) Antibacterial activity of triclosan chitosan coated graft on
hernia graft infection model. Int J Pharm 381:214219
10. Scheidbach H, Tannapfelb A et al (2004) Influence of titanium coating on the biocompatibility
of a heavyweight polypropylene mesh. Eur Surg Res 36:313317
11. Saygun O, Agalar C et al (2006) Gold and gold-palladium coated polypropylene grafts in a S.
epidermidis wound infection model. J Surg Res 131:7379
12. Fox CL (1991) Infection-resistant compositions, medical devices and surfaces and methods for
preparing and using same. US Patent 5019096
13. Gaertner WB, Bonsack ME et al (2010) Visceral adhesions to hernia prostheses. Hernia
14:375381
14. Robinson TN, Clarke JH et al (2005) Major mesh-related complications following hernia
repair. Surg Endosc 19:15561560
15. Emans PJ, Schreinemacher MHF et al (2009) Polypropylene meshes to prevent abdominal her-
niation. Can stable coatings prevent adhesion in the long term? Ann Biomed Eng 37:410418
16. Atiyeh BS, Costagliola M et al (2007) Effect of silver on burn wound infection control and
healing: review of the literature. Burns 33:139148
17. Klueh U, Wagner V et al (2000) Efficacy of silver-coated fabric to prevent bacterial coloniza-
tion and subsequent device-based biofilm formation. J Appl Biomater 53:621631
18. Belloni J, Mostafavi M et al (1998) Radiation-induced synthesis of mono- and multi-metallic
clusters and nanocolloids. New J Chem 22:12391255
19. Yuranova T, Rincon AG et al (2003) Antibacterial textiles prepared by RF-plasma and
vacuum-UV mediated deposition of silver. J Photochem Photobiol A 161:2734
20. Kostic M, Radic N et al (2009) Silver-loaded cotton/polyester fabric modified by dielectric
barrier discharge treatment. Plasma Process Polym 6:5867
21. Massines F, Segur P et al (2003) Physics and chemistry in a glow dielectric barrier discharge
at atmospheric pressure: diagnostics and modeling. Surf CoatTechnol 174175:814
22. Brandenburg R, Maiorov VA et al (2005) Diffuse barrier discharges in nitrogen with small
admixtures of oxygen: discharge mechanism and transition to the filamentary regime. J Phys
D: Appl Phys 38:21872197
23. Fang Z, Xie X et al (2009) Comparison of surface modification of polypropylene film by
filamentary DBD at atmospheric pressure and homogeneous DBD at medium pressure in air.
J Phys D: Appl Phys 42:19
24. Dorai R, Kushner MJ (2003) A model for plasma modification of polypropylene using
atmospheric pressure discharges. J Phys D: Appl Phys 36:666685
25. Simor M, Rahel J et al (2003) Atmospheric-pressure plasma treatment of polyester nonwoven
fabrics for electroless plating. Surf CoatTechnol 172:16
26. ISO 20645 (2004) Textile fabrics determination of antibacterial activity agar diffusion
plate test
Part VI
Plasma for Food Security
Chapter 33
Prospects for Treating Foods with Cold
Atmospheric Gas Plasmas
Gilbert Shama and Michael G. Kong
Abstract In this review the potential applications of cold atmospheric gas plasmas
are presented with particular reference to the problems of contamination of foods
by biological agents. In addition to the accidental contamination of food, the very
real threat arising from the deliberate contamination of the human food chain is
also considered. The evidence that has been gained for the efficacy of cold plasmas
in inactivating a wide range of biological agents is briefly surveyed. This is fol-
lowed by an examination of previous work in which various types of foodstuffs
have been successfully treated using cold gas plasmas. The need to demonstrate
that the quality attributes of treated foods is not adversely affected is stressed.
Finally, the role which gas plasmas may have in decontaminating food processing
equipment is considered.
33.1 Introduction
The World Health Organisation (WHO) claims that 1.5 million children under five
years of age die each year due to diarrhoea primarily as a result of contaminated
drinking water and food. WHO also estimates that globally there are about two
billion cases of diarrhoeal disease annually [50]. This is not a phenomenon con-
fined to the developing world; Mead et al. [24] stated that in the United States
there were 76 million incidences of foodborne disease resulting in 323,000 hospi-
talizations and 5,200 deaths annually. Moreover, this pattern is repeated in all
G. Shama (*)
Department of Chemical Engineering, Loughborough University,
Ashby Road, LE11 3TU Loughborough, Leics, UK
e-mail: g.shama@Lboro.ac.uk
M.G. Kong
Department of Electronic and Electrical Engineering, Loughborough University,
Ashby Road, LE11 3TU Loughborough, Leics, UK
434 G. Shama and M.G. Kong
industrialized countries. In England and Wales in the year 2000 for example, there
were 1,338,772 cases of foodborne infections resulting in 20,759 hospitalizations
and 480 deaths [2].
Increasingly more of the food we consume is subject to processing of one sort or
another. Some form of processing must inevitably take place immediately following
harvest or slaughter, but in industrialized countries food has become subject to ever
more processing to meet the changing demands of consumers for convenience
foods. There is mounting evidence to show that so-called ready to eat (RTE) foods
such as fresh cut salads for example are increasingly becoming associated with
incidences of foodborne disease [38]. This trend towards increased consumption of
convenience foods is inevitably influencing the way in which the food industry pro-
duces such products. Production is increasingly becoming centralised into fewer,
but larger, processing facilities. This in itself increases the impacts which isolated
incidences of contamination of food products may have on the population as a
whole. A telling example of this is an incident associated with the US dairy indus-
try; a Salmonella outbreak in a single product liquid ice cream resulted in some
224,000 people [41] being affected. A more recent example concerns the Kellogg
company. Salmonella-infected peanut butter obtained from one of the companys
suppliers became incorporated into some of its snack products. In the ensuing epi-
demic of foodborne disease that followed 714 persons became infected in 43 states
and there was evidence to suggest that infection from these food products may have
been a contributory factor in 6 deaths. Moreover, the incident led to the recall of
seven million cases of Kelloggs products [7].
Whilst there have been improvements in the microbiological safety of foods,
few, if any foodborne pathogens have been contained over a sustained period of
time. Bovine spongiform encephalitis (BSE) is still being detected in cattle, and
the possibility cannot be entirely ruled out that BSE might have infected sheep fed
the same contaminated feed supplements that caused the epidemic in cows [15].
Even apparent successes against particular agents hold little long term comfort; an
increase in salmonellosis took place throughout the 1980s that was largely due to
Salmonella Enteritidis Phage Type 4 which was found predominantly in poultry
and eggs. Concerted action was taken in a number of countries which involved the
wholesale slaughter of infected flocks coupled with extensive programmes of
immunisations, and this proved successful as the incidences of this disease showed
a significant decline during the 1990s. However, there are indications that since the
year 2000 incidences are once more on the increase. The culprits are now different
phage types of S. Enteritidis, and it appears that as one pathogen is effectively
removed from a particular niche others evolve to take its place [27].
Another recent concern regarding food is the potential target it offers to terrorist
organisations. Although Manning et al. [23] cite a source claiming that since 1912
there have only been 12 documented cases of deliberate attempts to introduce patho-
genic agents into foods or livestock, the current perceived threat level remains high.
A further consequence of the centralisation of food processing is that it serves to
increase the impact which terrorist attacks directed towards the food supply chain
33 Prospects for Treating Foods with Cold Atmospheric Gas Plasmas 435
might have. Although there have been no concerted recent attempts made against
national food supplies, examples exist that serve to expose both the vulnerability of
the food supply chain and provide an indication of what the consequences of the
deliberate contamination of elements of the supply chain could be. This is illus-
trated by an incident of contaminated Worcester sauce that occurred in the UK in
2006. One of the ingredients that was added to a batch of the sauce was chilli pow-
der to which had been added a dye, Sudan Red I. The dye is not approved for food
use and is in fact a Category 3 carcinogen. The sauce subsequently became widely
distributed in a very large number of prepared foods and when the (deliberate) con-
tamination of the chilli powder was discovered the UK Food Standards Agency
ordered the withdrawal of over 600 different products from retail outlets [16]. The
motives in this case were financial rather than political or ideological but the exten-
sive permeation of a single ingredient throughout a nations food chain should serve
as a salutary warning.
Another area of increasing concern in the food industry is the removal of traces
of allergens from food processing facilities and equipment. If this could be achieved
it would allow different products to be processed using the same equipment thus
affording processors both versatility in the way they process foods and also cost
savings. According to a review by Kagan [19] between 1% and 2% of adults and
between 5% and 7.5% of children in the United States are subject to food allergies
of one sort or another. The food industry as a whole expends a great deal of effort in
attempting to remove allergens from the surfaces of food processing equipment and
has also to label its products using a rather convoluted system based on terms such
as may contain, free from, suitable for which are apt to confuse consumers and
may ultimately lead a proportion of them to take risks [48]. In essence this attests to
the difficulty of assuring total removal of allergens.
In what follows below the emphasis will be on the potential that atmospheric,
non-thermal gas plasmas have for the treatment of food in order to ensure its micro-
bial safety. Reference to the challenges that have to be faced for this to be achieved
will also be made as required. In addition to foods themselves, it is appropriate to
consider the environments and the equipment used during the processing of food.
The emphasis here will be on the opportunities for the application of plasma tech-
nology rather than on the technology itself. For the latter the excellent recent review
by Ehlbeck et al. [13] is strongly recommended.
33.2 The Action of Atmospheric Gas Plasmas

Against Biological Agents
There are about 200 known agents that are capable of causing disease when ingested
[1]. These include chemical, physical and microbial agents. The latter comprise bac-
teria, fungi, parasites, viruses and prions and allergens. To date all of the biological
agents that have been tested have proved themselves susceptible to the effects of
plasma treatment. Naturally with reference to foods the aim must be to maximise the
damage inflicted on the disease-causing agents whilst minimising any deleterious
impacts on the food itself. The latter are quite complex assessments to make, but include
damage to key nutrients, or changes in e.g. the texture or appearance of the food.
An understanding of the mechanisms by which gas plasmas bring about death or
lethal injury to pathogenic agents could potentially lead to the enhancement of
future treatment strategies through the manipulation of plasma conditions to maxi-
mise inactivation of pathogens. Perhaps the most-studied organisms to date have
been the bacteria. Both Gram positive and Gram negative bacteria, biofilm-formers
and bacterial spores have all been shown to be inactivated by gas plasmas [11, 29,
31, 44]. It is also amongst this group of organisms that the greatest body of knowl-
edge is being accumulated regarding mechanisms of inactivation. Moisan et al. [25]
attempted to infer information about possible mechanisms of inactivation of Bacillus
subtilis spores by examining the form of survival curves and SEMs of spores post
treatment. These workers concluded that UV photons played a significant role in the
inactivation of the spores and were aided in this by oxygen atoms. Later, Perni et al.
[30] used E. coli mutants to show that the inactivation of these bacteria was primar-
ily due to oxygen atoms and that the contributions of UV photons and OH radicals
were relatively minor. Although these accounts might appear contradictory, the
experimental data upon which they are based was conducted with different organ-
isms, using different plasma systems and under different conditions. It will evi-
dently be some time before the mechanism of inactivation of living cells can be
unified. However, studies such as those cited above will hopefully continue to pro-
vide useful information and allow deeper insights into the principal mechanisms.
Moving away from bacteria, gas plasmas have also been used to inactivate fungal
spores [45] and more recently, Avramidis et al. [3] were able to show that plasmas
caused deformation and disruption to the hyphae of growing fungi belonging to the
genera Ascochyta and Fusarium. Previously Perni et al. [31] had demonstrated the
plasma-induced inactivation of yeast of the species Saccharomyces cerevisiae.
Parasites have received rather less attention from the gas plasma community.
Two important pathogens are Giardia lamblia and Cryptosporidium parvum [40].
These organisms form oocysts frequently simply referred to as cysts that are
somewhat akin to bacterial and fungal spores in being able to withstand environ-
mental hardships. Both are common waterborne organisms that resist chlorination
the currently employed methods for disinfecting water.
The number of foodborne incidents due to viruses is less well-documented owing
to the difficulties in detecting viruses in foods. Notwithstanding, Roberts and
Antonoplos [35] were able to show that a hydrogen peroxide gas plasma steriliza-
tion unit was effective against a range of viruses including HIV type 1, hepatitis A
virus, respiratory syncytial virus, vaccinia virus, herpes simplex virus type 1, and
poliovirus type 2. Using a similar system, Vickery et al. [43] obtained total inactiva-
tion of duck hepatitis virus as a model for human hepatitis B virus. Of the viruses
selected in these studies, only hepatitis A is a foodborne virus, but the broad range
of viral types inactivated by gas plasma suggests that other foodborne viruses such
as Norovirus would also be susceptible to plasma treatment.
The term biological agent not only includes living organisms but also their
components and their products. One important category in this regard is the various
types of proteins produced by living organisms. Deng et al. [12] demonstrated the
ability of gas plasmas to both destroy and physically remove a protein, bovine serum
albumin (BSA), from the surface of stainless steel. This suggests a potential role for
gas plasmas in inactivating prions which are the proteins responsible for bovine
spongiform encephalopathy (BSE) and variant Creutzfeldt Jacob disease, (vCJB).
More recently, Bayliss et al. [5] used a pulsed radio frequency plasma jet to bring
about the destruction and removal of amyloid fibrils from mica sheets. The former
are viewed as a realistic non-infectious prion model.
The results obtained using these model proteins also suggest that it may be pos-
sible to remove food allergens from the surfaces of food processing plant. The food
processing industry has been obliged to take seriously the possibility of inducing
allergenic reactions in the population at large and has adopted an approach based
around that of Good Manufacturing Practices (GMP) that aims to assure segregation
of allergenic ingredients and declaration of the possible existence of allergens as was
mentioned above. Current decontamination practices centre on conventional clean-
ing techniques using detergents and sanitizers coupled with in some cases swab-
based assays based on the detection of adenosine triphosphate (ATP) which can be
used as a proxy indicator of the presence of protein on surfaces [47].
33.3 Atmospheric Gas Plasmas in the Food Industry
The most obvious application of gas plasmas in the food industry would be to
employ them directly to decontaminate foods. Evidence has steadily been accumu-
lating to show that gas plasmas can be used to effect reductions in microbial counts
for a wide range of different foods covering virtually all food types including meat,
dairy products and plant foods such as fruits, salad crops and nuts. A compilation of
such results for a range of foods is shown in Table 33.1.
Whilst the number of foods that are being subjected to plasma treatment contin-
ues to increase and in many cases results in acceptable reductions in bacterial counts,
a significant proportion of the studies listed in the table did not take into consider-
ation whether treatment resulted in any deleterious effects in the food. This would
include changes that might affect the quality, or the nutritional value, or even the
appearance of the foodstuff itself. The exceptions to this statement are the investiga-
tions of Vleugels et al. [44] with bell peppers, Basaran et al. [4] on various types of
nut, Niemira and Sites [28] on apples, Ragni et al. [34] on shell eggs, and Kim et al.
[20] for bacon. It cannot be stressed how important such considerations are as the
chemical species produced in the plasmas possess the potential of bleaching the
natural colour of foods and/or damaging key nutrients such as vitamins. Treatment
could conceivably result in the peroxidation of lipids in fatty foods. Kim et al. [20]
attempted to conduct assays to establish whether plasma-treated bacon showed evi-
dence of peroxidation but their results were inconclusive. In addition to what might
Table 33.1 Foods treated with atmospheric gas plasmas

Targeted micro-
Foodstuff organisms Treatment details Reference
Almonds E. coli 5 log reductions after 30 s Deng et al. [12]
Apples E. coli O157:H7 Salmonella; 2.93.7 log Niemira and Sites [28]
Salmonella Stanley reductions after 3 min
E. coli O157:H7
2.63 log reductions after
3 min
Apples E. coli O157:H7 >2 log reductions after Critzer et al. [8]
2 min
Bacon Listeria 4.6 log reductions after Kim et al. [20]
monocytogenes 1.5 min (results reported
Salmonella as total aerobic counts)
Typhimurium
E. coli
Cheese Listeria >8 log reductions in 2 min Song et al. [42]
monocytogenes
Eggs Salmonella Enteritidis S. Enteritidis 4.5 log Ragni et al. [34]
Salmonella reductions after 90 min.
Typhimurium S. Typhimurium ~3.7 log
reductions after 90 min
Ham Listeria 1.7 log reductions after Song et al. [42]
monocytogenes 2 min
Lettuce Listeria 1 log reduction after 1 min Critzer et al. [8]
monocytogenes
Mango E. coli P. agglomerans and Perni et al. [31]
Saccharomyces G. liquefaciens > 3 log
cerevisiae reductions after 2.5 s
Pantoea agglomerans E. coli > 3 log reductions
Gluconobacter after 5 s
liquefaciens S. cerevisiae > 3 log
reductions after 30 s
Melon Salmonella (unspeci- >2 log reductions after Critzer et al. [8]
(Cantaloupe) fied serovars) 1 min
Melon E. coli P. agglomerans and G. Perni et al. [31]
(honeydew) Saccharomyces liquefaciens > 3 log
cerevisiae reductions after 2.5 s
Pantoea agglomerans E. coli > 3 log reductions
Gluconobacter after 5 s
liquefaciens S. cerevisiae > 3 log
reductions after 10 s
Pork E. coli 6 log reductions after Moon et al. [26]
0.5 min
Nuts; hazelnuts, Aspergillus parasiticus 1 log reduction after 5 min Basaran et al. [4]
peanuts and 5 log reductions in the
pistachios presence of SF6
be termed chemical effects, the gas flows used in the plasmas may result in
moisture losses from the food undergoing treatment. These effects can all be quanti-
fied, but the ultimate test is consumer acceptance which needs to be assessed using
specially trained panels.
Perhaps the next most important application of plasmas would be in the treatment
of food processing equipment. Micro-organisms that attach to the surfaces of such
equipment have been shown to cause the cross contamination of foods that subse-
quently come into direct contact with the contaminated surface [9]. Arguably the
most notorious case being that of the epidemic of typhoid caused in Aberdeen,
Scotland in the summer of 1964. A catering-sized can of corned beef from
S. America contaminated with Salmonella was sliced using a food slicing machine
in a grocers premises in Aberdeen. The contaminated blade resulted in the cross
contamination of other food products subsequently sliced using the machine and
this resulted in an epidemic in which 500 people were hospitalised and a virtual
national state of panic [39]. This mechanism of microbial transfer has been investi-
gated in studies conducted by Sheen and Hwang [37] for ham slicing and by Perni
et al. [32] for fresh fruit cutting. Leipold et al. [22] proposed an innovative solution
to this problem. They designed a dielectric barrier discharge (DBD) plasma system
in which the circular cutting blade of a food slicing machine constituted one of the
electrodes. With this they were able to demonstrate the efficient inactivation of
Listeria innocua sprayed onto the blade. This represents a most interesting approach
to the concept of decontaminating food processing machinery and points the way to
extending the concept to other operations such as conveying etc.
An unconventional approach to food preservation was taken by Fernandez-
Gutierrez et al. [14] who attempted to coat the surface of apples with vanillin using
a plasma-based thin film deposition technique to protect the food against fungal
spoilage. The conditions chosen by these researchers did not result in a continuous
coating but rather in what they referred to as nodules. The concept is however an
interesting one and could be potentially applicable to other foods.
Another potential area where plasmas might make an impact is in food packag-
ing. Although relatively little work appears to have been conducted using atmo-
spheric gas plasmas, Heise et al. [17] demonstrated the efficacy of DBD against
bacterial and fungal spores deposited onto polymer foils.
The physical environment in which food processing takes place can occasion-
ally become colonised by micro-organisms that appear to survive cycles of
decontamination treatments and which are referred to as resident organisms [33].
This may occur because some organisms are only exposed to sublethal concentra-
tions of chemical disinfectants and thus are able to develop resistance against them
[46]. Although as stated above, the precise mode of action of gas plasmas has yet to
be fully elucidated, it seems likely that more than one plasma species will be
involved. This would lessen the possibility of organisms developing resistance.
The threats to the safety of foods resulting from potential terrorist action were
briefly mentioned above. Certain sectors and elements of the food industry have been
identified as being particularly vulnerable. Wein and Liu [49] for example constructed
a mathematical model to predict the consequences of the deliberate release of
botulinum toxin into the milk supply chain in California, USA. The worst possible
case envisaged the poisoning of up to some 5 105 people but this required a substan-
tial quantity of toxin (of the order of 100 g). Nevertheless, the implications are unset-
tling. A possibly more readily available toxin is ricin. Ricin is a potent toxin that is
found in the castor oil plant. Its threat lies in part from the fact that the plant grows
widely throughout the world and that it is relatively easy to extract from the castor
bean mash that remains after pressing for oil. Jackson et al. [18] studied the thermal
inactivation of ricin in reconstituted infant formula. They found that the some of the
thermal treatment regimes currently in place were insufficient to inactivate the toxin.
Existing measures to safeguard foods against microbial contamination may therefore
not be sufficient to render harmless such toxins. Could plasmas be used to provide
protection against such toxins? The literature would appear to indicate that the use of
gas plasmas in this context has not been investigated, and therefore this warrants some
attention by researchers in the field. A possible indication that they could is provided
by the work of Birmingham and Hammerstrom [6] who showed that plasmas possess
the ability to destroy a mycotoxin in aerosolised form. However, considerably more
work would be needed to extend their findings to the cases mentioned above.
33.4 The Future
The range of foods subject to plasma treatment will doubtless continue to grow.
If gas plasmas are ever to escape the confines of the laboratory and assume accep-
tance in the food industry a number of factors aside from their proven ability to
inactivate pathogens, will need to be taken into account. This would include exhaustive
testing to ensure that foods that have been treated with plasmas do not undergo any
undesirable changes that would render them harmful, unpalatable or otherwise
unsaleable. Absolute proof that no harmful products have been produced is actually
very difficult, if not impossible, to demonstrate as the list of potential toxic by-products
is a very long one. Ensuring the quality of treated foods is a rather more straightfor-
ward undertaking. This would include tests for key nutrients such as vitamins, tex-
tural studies, colour etc. The issue of public perception of foods treated with gas
plasmas is also one that needs addressing. Ill-informed publicity could impact
negatively on the chances of this technology being adopted by the food industry.
Mention was made above of the increasing popularity of RTE foods and the
problems associated with minimally processed foods such as salads etc. One assess-
ment of the current state of affairs is that the available technology for disinfection
has not kept pace with the changing eating habits of consumers and that an effective
and adaptable new decontamination technology is sorely needed. Adaptability will
be essential, as not only must the technology prove itself efficacious against current
pathogens, but it will need to provide assurance that it can operate equally effec-
tively against emerging pathogens. Climate change will constitute one driver of
change. That it will influence global food production can surely not be in doubt
[36], more uncertain however will be its impact on food pathogens. Increases in
temperature will have some direct effects including increasing the growth rate of
most of the common foodborne pathogens. But it will also influence eating behav-
iour. Warmer temperature are associated with increased consumption of salads and
barbecued foods both of which have been heavily implicated in incidences of
foodborne disease [21].
One assessment of the true scale of the problem faced by any decontamination
technology may be gained from a telling statement from the work of Newell et al.
[27]; over millennia all food-borne pathogens have developed efficient and effec-
tive strategies, which exploit, wholly or in part, food as a vehicle to transfer from
one human gut host to another, or from an animal to a human. The mechanisms
involved are complex and varied but all are able to survive intervening periods in the
environment, and then avoid the human innate gut defences to colonize and multi-
ply rapidly before enabling effective dispersal, frequently through fluid faeces, back
into the environment to progress again through each cycle.
References
1. Acheson DWK (1999) Foodborne infections. Curr Opin Gastroenterol 15:538545

2. Adak GK, Long SM, OBrien SJ (2002) Trends in indigenous foodborne diseases and deaths,
England and Wales:1992 to 2000. Gut 51:832841
3. Avramidis G, Stwe B, Wascher R, Bellmann M, Wieneke S, von Tiedemann A, Vil W (2010)
Fungicidal effects of an atmospheric pressure gas discharge and degradation mechanisms. Surf
Coat Technol 205:S405S408
4. Basaran P, Basaran-Akgul N, Oksuz L (2008) Elimination of Aspergillus parasiticus from nut
surface with low pressure cold plasma (LPCP) treatment. Food Microbiol 25:626632
5. Bayliss DL, Walsh JL, Shama G, Iza F, Kong MG (2009) Reduction and degradation of amy-
loid aggregates by a pulsed radio-frequency cold atmospheric plasma jet. New J Phys 11,
Article number: 115024
6. Birmingham JG, Hammerstrom DJ (2000) Bacterial decontamination using ambient pressure
nonthermal discharges. IEEE Trans Plasma Sci 28:5155
7. CDC (Centers for Disease Control) (2009) Investigation update: outbreak of Salmonella
Typhimurium infections, 20082009. http://www.cdc.gov/salmonella/typhimurium/update.html
8. Critzer FJ, Kelly-Wintenberg K, South SL, Golden DA (2007) Atmospheric plasma inactiva-
tion of foodborne pathogens on fresh produce surfaces. J Food Protection 70:22902296
9. den Aantrekker ED, Boom RM, Zwietering MH, van Schthorst M (2003) Quantifying recon-
tamination through factory environments a review. Int J Food Microbiol 80:117130
10. Deng S, Ruan R, Mok CK, Huang G, Lin X, Chen P (2007) Inactivation of Escherichia coli on
almonds using nonthermal plasma. J Food Sci 72:M62M66
11. Deng XT, Shi JJ, Shama G, Kong MG (2005) Effects of microbial loading and sporulation
temperature on atmospheric plasma inactivation of Bacillus subtilis spores. Appl Phys Lett 87,
Article number: 153901
12. Deng XT, Shi JJ, Kong MG (2007) Protein destruction by a helium atmospheric pressure glow
discharge: capability and mechanisms. J Appl Phys 101, Article number: 074701
13. Ehlbeck J, Schnabel U, Polak M, Winter J, von Woedtke T, Brandenburg R, von dem Hagen T,
Weltmann K-D (2011) Low temperature atmospheric pressure plasma sources for microbial
decontamination. J Phys D Appl Phys 44, Article number: 013002
14. Fernandez-Gutierrez SA, Pedrow PP, Pitts MJ, Powers J (2010) Cold atmospheric pressure
plasmas applied to active packaging of apples. IEEE Trans Plasma Sci 38:957965
15. Fryer HR, Baylis M, Sivam K, McLean AR (2007) Quantifying the risk from ovine BSE and
the impact of control strategies. Proc R Soc B 274:14971503
16. FSA (Food Standards Agency, UK) (2005) Sudan 1 consolidated product list from February
2005 recall. http://www.food.gov.uk/safereating/chemsafe/sudani/sudanlistno. Accessed 27
Jan 2005
17. Heise M, Neff W, Franken O, Muranyi P, Wunderlich J (2004) Sterilization of polymer foils
with dielectric barrier discharges at atmospheric pressure. Plasmas Polymers 9:2333
18. Jackson LS, Tolleson WH, Chirtel SJ (2006) Thermal inactivation of ricin using infant formula
as a food matrix. J Agric Food Chem 54:73007304
19. Kagan RS (2003) Food allergy: an overview. Environ Health Perspect 111:223225
20. Kim B, Yun H, Jung S, Jung Y, Jung H, Choe W, Jo C (2011) Effect of atmospheric pressure
plasma on inactivation of pathogens inoculated onto bacon using two different gas composi-
tions. Food Microbiol 28:913
21. Lake IR, Gillespie IA, Bentham G, Nichols GL, Lane C, Adak GK, Threlfall EJ (2009) A
re-evaluation of the impact of temperature and climate change on foodborne illness. Epidemiol
Infect 137:15381547
22. Leipold F, Kusano Y, Hansen F, Jacobsen T (2010) Decontamination of a rotating cutting tool
during operation by means of atmospheric pressure plasmas. Food Control 21:11941198
23. Manning L, Baines RN, Chadd SA (2005) Deliberate contamination of the food supply chain.
Br Food J 107:225245
24. Mead PS, Slutsker L, Dietz V, McCraig LF, Bresee JS, Shapiro C, Griffin PM, Tauxe RV
(1999) Food-related illness and death in the United States. Emerg Infect Dis 5:607625
25. Moisan M, Barbeau J, Crevier M-C, Pelletier J, Philip N, Saoudi B (2002) Plasma sterilization.
26. Moon SY, Kim DB, Gweon B, Choe W, Song HP, Jo C (2009) Feasibility study of the steriliza-
tion of pork and human skin surfaces by atmospheric pressure plasmas. Thin Solid Films
517:42724275
27. Newell DG, Koopmans M, Verhoef L, Duizer E, Aidara-Kane A, Spromng H, Opsteegh M,
Langelaar M, Threfall J, Scheutz F, van der Giessen J, Kruse H (2010) Food-borne diseases
the challenges of 20 years ago still persist while new ones continue to emerge. Int J Food
Microbiol 139:S13S15
28. Niemira BA, Sites J (2008) Cold plasma inactivates Salmonella Stanley and Escherichia coli
O157:H7 inoculated on Golden Delicious apples. J Food Prot 71:13571365
29. Opretzka J, Benedikt J, Awakowicz P, Wunderlich J, von Keudell A (2007) The role of chemical
sputtering during plasma sterilization of Bacillus atrophaeus. J Phys D Appl Phys 40:28262830
30. Perni S, Shama G, Hobman JL, Lund PA, Kershaw CJ, Hidalgo-Arroyo GA, Penn CW, Deng
XT, Walsh JL, Kong MG (2007) Probing bactericidal mechanisms induced by cold atmo-
spheric plasmas with Escherichia coli mutants. Appl Phys Lett 90, Article number: 073902
31. Perni S, Liu DW, Shama G, Kong MG (2008) Cold atmospheric plasma decontamination of
the pericarps of fruit. J Food Prot 71:302308
32. Perni S, Shama G, Kong MG (2008) Cold atmospheric plasma disinfection of cut fruit surfaces
contaminated with migrating microorganisms. J Food Prot 71:16191625
33. Proudy I, Bougle D, Leclercq R, Vergnaud M (2008) Tracing of Enterobacter sakazakii iso-
lates in infant milk formula processing by BOX-PCR genotyping. J Appl Microbiol
105:550558
34. Ragni L, Berardinelli A, Vannini L, Montanari C, Sirri F, Guerzoni ME, Guarnieri A (2010)
Non-thermal atmospheric gas plasma device for surface decontamination of shell eggs. J Food
Eng 100:125132
35. Roberts C, Antonoplos P (1998) Inactivation of human immunodeficiency virus type 1, hepa-
titis A virus, respiratory syncytial virus, vaccinia virus, herpes simplex virus type 1, and polio-
virus type 2 by hydrogen peroxide gas plasma sterilization. Am J Infect Control 26:94101
36. Schmidhuber J, Tubiello FN (2007) Global food security under climate change. Proc Natl
Acad Sci 104:1970319708
37. Sheen S, Hwang C (2008) Modeling transfer of Listeria monocytogenes from slicer to deli
meat during mechanical slicing. Foodborne Pathog Dis 5:135146
38. Sivapalasingam S, Friedman CR, Cohen L, Tauxe RV (2004) Fresh produce: a growing cause
of outbreaks of foodborne illness in the United States, 1973 through 1997. J Food Prot
67:23422353
39. Smith DF (2007) Food panics in history: corned beef, typhoid and risk society. J Epidemiol
Community Health 61:566570
40. Smith JL (1993) Cryptosporidium and Giardia as agents of foodborne disease. J Food Prot
56:451461
41. Sobel J, Khan AS, Swerdlow DL (2002) Threat of a biological terrorist attack on the US food
supply: the CDC perspective. Lancet 359:874880
42. Song HP, Kim B, Choe JH, Jung S, Moon SY, Choe W, Jo C (2009) Evaluation of atmospheric
pressure plasma to improve the safety of sliced cheese and ham inoculated by 3-strain cocktail
Listeria monocytogenes. Food Microbiol 26:432436
43. Vickery K, Deva AK, Zou J, Kumaradeva P, Bissett L, Cossart YE (1999) Inactivation of duck
hepatitis B virus by a hydrogen peroxide gas plasma sterilization system: laboratory and in
use testing. J Hosp Infect 41:317322
44. Vleugels M, Shama G, Deng XT, Greenacre E, Brocklehurst T, Kong MG (2005) Atmospheric
33:824828
45. Vohrer U, Trick I, Bernhardt J, Oehr C, Brunner H (2001) Plasma treatment an increasing
technology for paper restoration? Surf Coat Technol 142:10691073
46. Walsh C, Fanning S (2008) Antimicrobial resistance in foodborne pathogens a cause for
concern? Curr Drug Targets 9:808815
47. Wang X, Young OA, Karl DP (2010) Evaluation of cleaning procedures for allergen control in
a food industry environment. J Food Sci 75:T149T155
48. Ward R, Crevel R, Bell I, Khandke N, Ramsay C, Paine S (2010) A vision for allergen manage-
ment best practice in the food industry. Trends Food Sci Technol 21:619625
49. Wein LM, And Liu Y (2005) Analyzing a bioterror attack on the food supply: the case of botu-
linum toxin in milk. PNAS 102:99849989
50. WHO (World Health Organisation) (2009) Diarrhoel disease. http://www.who.int/mediacentre/
factsheets/fs330/en/index.html. Accessed 27 Jan 2009
Chapter 34
Decontamination of Bacillus subtilis Spores
in a Sealed Package Using a Non-thermal
Plasma System
Kevin M. Keener, J.L. Jensen, V.P. Valdramidis, E. Byrne, J. Connolly,

J.P. Mosnier, and P.J. Cullen
Abstract The safety of packaged food and medical devices is a major concern to
consumers and government officials. Recent inventions (PK-1 and PK-2) based on
the principles of non-thermal, atmospheric plasma has shown significant reduction
in bacterial contamination inside a sealed package.
The objective of this study was to evaluate the PK-1 and PK-2 systems in the
reduction of Bacillus subtilis spores using packages containing air or modified
atmosphere (MA) gas (65% O2/30% CO2/5% N2). The experimental design con-
sisted of the following parameters: (1) two voltage conditions: 13.5 kV with 1.0 cm
electrode gap (PK-1) and 80 kV with 4.5 cm electrode gap (PK-2), (2) two treatment
conditions: inside and outside the field of ionization, (3) PK-1 and PK-2 optimized
treatment times: 300 and 120 s, respectively, and (4) two package gas types: air and
modified atmosphere (MA) gas (65% O2/30% CO2/5% N2). Measurements included:
(1) bacterial reductions of Bacillus subtilis var. niger (B. atrophaeus), (2) ozone,
nitrous oxides (NOx), and carbon monoxide concentrations, and (3) relative humidity.
K.M. Keener (*) J.L. Jensen

Department of Food Science, Purdue University,
745 Agriculture Mall Drive, West Lafayette, IN 47907, USA
e-mail: kkeener@purdue.edu
V.P. Valdramidis
School of Food Science and Environmental Health, Dublin Institute of Technology,
Cathal Brugha St, Dublin 1, Ireland
Biosystems Engineering, School of Agriculture, Food Science and Veterinary Medicine,
University College Dublin, Belfield, Dublin 4, Ireland
E. Byrne P.J. Cullen
School of Food Science and Environmental Health, Dublin Institute of Technology,
Cathal Brugha St, Dublin 1, Ireland
J. Connolly J.P. Mosnier
School of Physical Sciences and NCPST, Dublin City University,
Glasnevin, Dublin 9, Ireland
446 K.M. Keener et al.
Bacillus subtilis (1.7 106/strip) were loaded into sterile uncovered petri dishes and
treated with ionization generated in packages using air or MA gas blend. Samples
were treated for 300 s (PK-1) or 120 s (PK-2) and stored at room temperature for
24 h. Results documented relative humidity (RH) ranged from 20% to 30%. After
300 s of PK-1 treatment (13.5 kV/44 W/1.0 cm gap), ozone concentrations were
6,000 ppm (air) and 7,500 ppm (MA). After 120 s of PK-2 treatment
(80 kV/150 W/4.5 cm), ozone concentrations were 7,500 ppm (air) and 12,000 ppm
(MA). Ozone and NOx concentrations were non-detect (ND) after 24 h. PK-1 car-
bon monoxide levels were <20 ppm (air) and <100 ppm (MA) after 24 h. The PK-2
carbon monoxide levels were <20 ppm (air) and <400 ppm (MA) after 24 h.
Treatments showed reductions in spores of greater than 6 log10 after 24 h. Reductions
were maintained without additional re-growth at 72 h. These results indicate that the
PK-1 and PK-2 systems have the capacity to reduce Bacillus subtilis spores in an
in-package ionization process.
34.1 Introduction
Biological decontamination and surface sterilization is crucial throughout society: in

military applications such as the decontamination of equipment and facilities exposed
to deadly biological agents, or in a broad array of civilian applications including
medical applications, food production and consumer goods [1, 2]. Presently chemi-
cal, heat, high-energy electron beams, x-ray or gamma-ray irradiation systems are
commercially used for treatments; however, utilization of these systems in a combat
situation is not realistic due to the cost, efficiency, immobility, electric power require-
ments, toxic waste, personal hazard, and time required to decontaminate items.
Over the last decade, considerable research has been conducted in using atmo-
spheric plasmas as a decontamination method [3, 4]. Atmospheric plasmas have the
ability to generate unique radiolytic profiles. Research has documented the ability
of these reactive gas species generated to inactivate bacteria, spores, and biological
contaminants from food and non-food surfaces [1, 57]. Studies have shown that
biological contaminants exposed to atmospheric plasmas can be sterilized in sec-
onds to minutes [8, 9]. Research found that 60 s of ALT-P treatment achieved greater
than 5 log10 reductions of Escherichia coli O157:H7, Listeria monocytogenes, and
Salmonella spp. on the surface of agar media [9]. Atmospheric plasmas are fairly
easy to produce and the equipment needed is relatively inexpensive. There are no
hazardous wastes and the gaseous by-products can be locally controlled. Up to this
time, utilization of atmospheric plasmas has been through sealed chambers and jets.
An in-package plasma technology called in-package ionization has been developed
[8, 10]. This technology utilizes the dielectric properties of the sealed package to
create a localized plasma field inside the package. Based on the selected operating
conditions, the item in the sealed container can be directly exposed to plasma field
or reactive, stable species inside or outside of the plasma field. The in-package
plasma process allows for very rapid decontamination and extended plasma species
exposure for the product enclosed. A published study on packaged spinach
34 Decontamination of Bacillus subtilis Spores in a Sealed Package 447
documented reactive oxygen species (i.e., ozone) will remain for up to 12 h in a

sealed package [8]. The research reported in this paper highlights the capabilities of
the in-package technology to inactivate Bacillus subtilis spores under two different
plasma voltages (13.5, 80 kV) and gas compositions (air, MA) inside and outside
the plasma field. In addition, plasma diagnostics have been collected and compari-
sons made to data generated from plasma jets and plasma chambers.
34.2.1 Experimental Design
An experimental design was selected that utilized two voltage conditions:

13.5 kV/44 W/1.0 cm gap (PK-1 ionization system) and 80 kV/150 W/4.5 cm gap
(PK-2 ionization system); 3 treatment conditions: infield ionization, out of field
ionization, and no ionization; one treatment time of 300 s (PK-1) and 120 s (PK-2);
room temperature; two package gas types: air (78% N2, 22% O2) and modified
atmosphere, MA (65% O2, 30% CO2, 5% N2); in triplicate.
34.2.2 Fill Gas
Air (78% N2, 22% O2) and modified atmosphere (MA) gas (65% O2, 30% CO2, 5% N2)
were purchased from a local gas supplier at specified concentrations with a certificate
of analysis. These gas composition(s) were then metered into sealed package at a rate
of 2.1 L/min. using a flow meter (Model 2260, Gilmont Instruments, Inc., Barrington,
IL, USA) yielding final fill volume of 1.5 L with average fill time of 45 s.
34.2.3 Storage Bags
Clear, 3.78 L Ziploc (SC Johnson and Son, Inc., Racine, WI, USA) heavy duty
freezer bags were obtained from a local grocery store. Bags consisted of low-density
polyethylene (LDPE) and were 1.6 mm thick. All treatments were double bagged
for storage time of 24 h at room temperature.
34.2.4 Biological Indicator/Spore Strips
Bacillus subtilis var. niger (B. atrophaeus) spore strips (NAMSA, Northwood, OH,
USA) with size of 3.2 cm 0.6 cm, each containing Bacillus populations of 1.7 106/
strip or 6.23 log10 were loaded into open sterile petri dish inside treatment package
and then used in ionization treatments. For infield ionization with PK-1 system, one
end of each spore strip was secured with transparent tape to the inside of the storage
bag within electrode gap space prior to treatment.
34.2.5 In-Package Ionization Systems
Treatments were carried out utilizing either the PK-1 system developed in the Food
Technology Development Laboratory at Purdue University or the PK-2 system
(Phenix Technologies, Accident, MD). The PK-1 system is capable of generating
ozone inside a sealed package at various geometries and is a patent-pending tech-
nology [8]. The PK-1 system was operated at 44 W and 60 Hz generating 13.5 kV
of potential between the electrodes (1.0 cm gap). Electrodes consisted of coils of
wire wound around a dielectric base with a treatment area of 92.4 cm2
(7.7 cm 12 cm). The PK-2 system was operated at 150 W and 60 Hz generating
80 kV of potential between the high voltage circular stainless electrodes (15 cm
diam., 4.5 cm gap) with polypropylene (pp) insulation layers: top (11.1 mm) and
bottom (3.2 mm) between electrodes. Treatment of all samples occurred at room
temperature. The storage bags containing spore samples were filled with the work-
ing gas (air or MA) and purged three times to ensure purity of the gas in the bag.
Electrodes were placed above and below the bag, oriented directly above each other
to allow for maximum ionization of plasma field. Electrodes rested on top of each
other with the bag in between having an approximate gap distance of 1.0 cm (PK-1)
and 4.5 cm (PK-2). Hollow plastic spacers were inserted inside the bag to maintain
proper gap (PK-1).
34.2.6 Temperature Measurement
Temperature of the electrodes and treated storage bags was measured prior to and
immediately after treatment using an infrared thermometer (Omega Engineering,
Inc., Stamford, CT, USA). Temperatures of treated samples registered at room tem-
perature for both ionization systems immediately after treatment. The electrodes of
the PK-1 system were allowed to cool (2530C) for 300 s between treatments for
uniform treatment temperature conditions.
34.2.7 Reactive Oxygen Species: Ozone, Nitric Oxides,

and Carbon Monoxide
Ozone and nitric oxide concentrations were measured immediately following the
300 or 120 s treatments as well as after 24 h storage. This was completed by using
Draeger Short-Term Detector tubes (Draeger Safety AG & Co., Luebeck,

Germany). These particular tubes were chosen for ease of use with the given
experimental setup and their rapid measurement capabilities. The tubes contain a
reagent which changes color upon coming into contact with the specified gas and
are calibrated for specific sampling volumes. Tubes were connected to a bellows
hand pump, Accuro Gas Detector Pump (Draeger Safety AG & Co., Luebeck,
Germany), and calibrated such that one pump equals 100 mL of gas. Ozone tubes
(part no. CH21001) had an indicated range of 20300 ppm. Nitrous oxides (part
no. 6724001) tubes had an indicated range between 20 and 500 ppm. A cross-
sensitivity of 5 ppm NOx per 100 ppm ozone was identified. Carbon monoxide
tubes (part no. 6733051) had an indicated range between 25 and 300 ppm. It was
noted that carbon monoxide tubes had an interference with ozone. Thus, no car-
bon monoxide measurements could be taken with ozone present. In order to deter-
mine ozone values when measuring very high concentrations, smaller gas sample
volumes were collected in 5 or 20 mL syringes. The syringe was connected to the
detection tube by means of flexible tubing. A syringe volume was expelled into
the detection tube and then removed allowing total flow volume of 100 ml to
occur. The observed gas concentration was then multiplied by the volume ratio of
the detection tube volume over the syringe volume. The Draeger portable gas
detection system had a precision of 15% (Draeger Safety AG & Co., Luebeck,
Germany).
34.2.8 Spore Recovery and Enumeration
Spore recoveries and aseptic methods were followed per manufacturer (NAMSA,
Northwood, OH, USA) for population verification of Bacillus subtilis spore strips.
After ionization treatment and 24 h storage, each strip was aseptically removed
from bag and transferred into sterile 20 150 mm test tube containing 10 mL of
0.1% sterile peptone. Ten sterile 6 mm glass beads were then added to each test
tube. Each test tube was vortexed (model vortexer 59, Denville Scientific, Inc.,
Metuchen, NJ, USA) on high speed for 120 s or until the spore strip was fully mac-
erated into loose fibers. Test tubes were then heat shocked in order to fully germi-
nate spore population by placing into a 500 mL beaker with 300 mL of water heated
to 90C and maintained at 8085C for 10 min. Test tubes were transferred to a cold
tap water bath momentarily (2 min), and then to ice water bath to rapidly cool test
tubes to 04C. Test tubes were then removed from ice bath and further serial dilu-
tions were performed including 102, 103, 104, and/or 105 based on treatments or
recoveries of positive (+) controls (Bacillus populations of 1.5 106/strip, 6.18
log10). The required aliquot volumes from corresponding serial dilutions were then
plated into respective petri dishes (100 15 mm) containing sterile Tryptic Soy Agar
(TSA) prepared per Difco Manual specifications for spore colony enumeration [11].
TSA plates were incubated at 3031C and colony growth and recoveries were
monitored at 24, 48, and 72 h.
34.2.9 Atomic Emission Spectroscopy
Optical emission spectral analysis was performed using the PK-1 System
(12.0 kV). The plasma emission was captured via a 0.22 NA optical fiber and
analyzed using a low-resolution UV-VIS spectrometer operated in the wavelength
range 2001,000 nm. The optical fibre was aligned at the centre of the plasma at
a distance of 6 cm.
34.2.10 Relative Humidity (RH)
Relative humidity and temperatures inside the storage bags were measured using a
Springfield Precise Temp relative humidity sensor (Taylor Precision Products,
Oak Brook, IL, USA) recorded at 0 and 24 h storage.
34.2.11 Statistical Analysis
Bacillus subtilis populations and gas concentrations were analyzed using t-test
comparisons (P < 0.05) and standard deviations in Microsoft Excel (2010 Version).
Figure 34.1 shows the optical emission spectra for an air plasma obtained with the
PK-1 system operating at 12 kV. The spectra clearly shows emission data at wave-
lengths previously characterized for air plasma species as noted by other researchers
[4]. The species include N2, N2+, O2+, and O (see table of assignments for the main
peaks as an insert in Fig. 34.1). The spectra for MA gas was similar (data not shown)
with the same major peaks. Interestingly, there were no further differences noted;
although reactive oxygen species generation rates were significantly different
between air and MA gas.
Figures 34.2 and 34.3 document the reactive oxygen species generation during
in-package ionization at the specified times for both 13.5 and 80 kV. It can be seen
from these data that high levels of reactive oxygen species can be generated for both
air and MA gas. However, reactive oxygen species generation rates were signifi-
cantly different between air and MA gas. At 13.5 kV an ozone generation rate of
1,200 and 1,500 ppm per minute were observed for air and MA gas, respectively.
At 80 kV an ozone generation rate of 3,750 and 6,250 ppm per minute were observed
for air and MA gas, respectively. These results suggest that ionization voltage
increases reactive oxygen species generation rate, even at constant voltage gradient.
In air, the nitrous gas concentrations did not significantly change with ionization
Optical Emission Spectroscopy: PK-1 Ionization System (12.0 kV)
336 nm N2
357 nm N2
380 nm N2
390 nm N2+
405 nm N2+
426 nm N2+
673 nm O2+
686 nm O2+
776 nm O
Fig. 34.1 Optical emission spectroscopy of air plasma generated using PK-1 ionization system
(12.0 kV)
Fig. 34.2 Gas concentrations generated using PK-1 ionization system (13.5 kV)
voltage. Both voltages (13.5 and 80 kV) achieved maximum nitrous gas concentration
of approximately 1,000 ppm. Conversely, the MA gas nitrous gas concentrations
reached a significantly higher level with increased ionization voltage. Nitrous gas
concentrations at 80 kV reached over 4,000 ppm at 120 s treatment.
Fig. 34.3 Gas concentrations generated using PK-2 ionization system (80 kV)
Both ozone and nitrous oxides levels decayed to zero within 24 h of treatment.
However, there was a measureable carbon monoxide concentration in MA gas at
24 h post-treatment with levels 200 and 400 ppm for the 13.5 and 80 kV at treatment
times of 300 and 120 s, respectively. As stated in Material and Methods, the current
carbon monoxide measurement technique does not allow measurement in presence
of ozone (i.e., time zero).
Figures 34.4 and 34.5 illustrate the spore reductions with ionization treatment.
It is observed that in-package ionization both inside and outside of the ionization
field at 13.5 and 80 kV can eliminate Bacillus subtilis spores. At 13.5 kV, treatment
times for MA gas spore elimination were 180 and 300 s for outside and inside field,
respectively. At 13.5 kV, treatment times for air spore elimination were 300 s out-
side of the ionization field with insignificant spore reductions (<1.2 log) inside the
ionization field. Similar results were reported by researchers utilizing a sealed enve-
lope containing Bacillus cereus spores and air in a DBD system operating at 30 kV
at 1.3 kHz [12]. At 80 kV, complete elimination of spores was shown in 15 s or less
with no measureable difference in spore reduction rates between air and MA gas.
It was observed that the inside the field, high voltage treatment times did show
slightly increased spore populations recoveries at 48 h compared to 24 h; however,
no additional organisms were recovered at 72 h. These results demonstrate that
using an 80 kV in-package ionization process, air or MA gas can provide complete
Fig. 34.4 Spore reductions generated using PK-1 ionization system (13.5 kV)
Fig. 34.5 Spore reductions generated using PK-2 ionization system (80 kV)
elimination of Bacillus subtilis spores in less than 15 s or less. For these studies, dry
air was used; and all samples were maintained at between 20% and 30% relative
humidity at room temperature. It is expected that elevated humidity may provide an
even greater spore reduction rate.
34.4 Conclusions
Atmospheric plasma has numerous advantages over existing technology to quickly

remove microorganisms from surfaces. These results clearly demonstrate the
sterilization capability of in-package ionization for Bacillus subtilis spores. Higher
ionization voltages and MA gas lend themselves to shorter sterilization times.
A complete elimination of spores was observed in less than 15 s or less for air and
MA gas at 80 kV. In addition, at 13.5 kV spore elimination can be achieved with
MA and air in 300 s or less.
Further research is needed to elucidate mechanisms of bactericidal reduction
both inside and outside the ionization field for the in-package process. Further, it is
well established in literature that relative humidity plays a crucial role in species
generated during ionization and bactericidal effects. Future studies will also exam-
ine the role RH plays in reactive oxygen species generation and bactericidal effects
with the in-package ionization process.
References
1. Ramos E (2008) Use of non-thermal, atmospheric-pressure plasma for reduction of bacterial

food pathogens. Master of Science thesis, Southern Illinois University, Carbondale, IL USA
2. Sohbatzadeh F, Hosseinzadeh A, Mirzanejhad C, Mirzanejhad S, Mahmodi S (2010) E. coli,
P. aeruginosa, and B. cereus bacteria sterilization using afterglow of non-thermal plasma at
atmospheric pressure. Appl Biochem Biotechnol 160:19781984
3. Gaunt LF, Beggs CB, Georghiou GE (2006) Bactericidal action of the reactive species produced
by gas-discharge nonthermal plasma at atmospheric pressure: a review. IEEE Trans Plasma Sci
34(4):12571269
spores using cold atmospheric plasmas. IEEE Trans Plasma Sci 34(4):13101316
5. Deng S, Ruan R, Mok CK, Huang G, Lin X, Chen P (2007) Inactivation of Escherichia coli on
almonds using nonthermal plasma. J Food Sci 72:M62M66
6. Vojkovska H, Slamova J, Kozakova Z, Krcma F (2010) Sterilization effect of dielectric barrier
discharge on eukaryotic microorganisms. In: 25th summer school and international sympo-
sium on the physics of ionized gases Donji Milanovac, Serbia, Publication of Astron Obs
Belgrade No. 89, pp 331334, 30 Aug3 Sept 2010
7. Trompeter F, Neff WJ, Franken O, Heise M, Neiger M, Liu S, Pietsch GJ, Saveljew AB (2002)
Reduction of Bacillus subtilis and Aspergillus niger spores using nonthermal atmospheric gas
discharges. IEEE Trans Plasma Sci 30:14161423
8. Klockow P, Keener KM (2009) Safety and quality assessment of packaged spinach treated
with a novel ozone generation system. Lebensm Wiss Technol 42(6):10471053
9. Johnson FM (2004) Atmospheric plasma inactivation of foodborne pathogens on fresh

produce surfaces. MS thesis, University of Tennessee-Knoxville, TN
10. Connolly J, Mosnier JP, Valdramidis V, Byrne E, Cullen PJ, Keener KM (2010) Diagnostic of
reactive species in a dielectric barrier discharge air/helium plasma for the treatment of a
pre-packed food simulant. In: 37th EPS conference on plasma physics, Dublin, Ireland
11. DIFCO Manual (1984) Difco manual of dehydrated culture media and reagents for microbio-
logical and clinical laboratory procedures, 10th edn. Difco Laboratories, Detroit
12. Dobrynin D, Fridman G, Mukhin YV, Wynosky-Dolfi MA, Rieger J, Rest RF, Gutsol AF,
Fridman A (2010) Cold plasma inactivation of Bacillus cereus and Bacillus anthracis (Anthrax)
spores. IEEE Trans Plasma Sci 38(8):18741884
Chapter 35
Impact of Atmospheric Plasma Generated
by a DBD Device on Quality-Related Attributes
of Abate Fetel Pear Fruit
Annachiara Berardinelli, Lucia Vannini, Luigi Ragni,

and M. Elisabetta Guerzoni
Abstract The effects of gas plasma generated by a Dielectric Barrier Discharge

(DBD) device on Abate Fetel fresh pears were assessed following exposure times
from 10 to 90 min. In particular the decontamination efficacy towards the indige-
nous microflora naturally occurring on the surface of the fruit was evaluated. The
main results showed that total mesophilic bacteria, yeasts and moulds had different
inactivation dynamics. However, maximum cell decreases of 2.5 Log CFU/fruit
were achieved for all the microbial groups after 90 min of treatment at a relative
humidity level of 60% (22C). Immediately after the treatments, no significant
effects were observed on the measured quality traits. After storage for 5 days at
20C significant changes were detected only in the peel (colour and antioxidant
capacity) of fruit samples treated for 90 min. The Magness-Taylor flesh firmness
(MTf), the soluble solid content (SSC) and the antioxidant capacity of fruits were
unaffected by the tested treatments.
35.1 Introduction
Traditional methods for surface decontamination of fruits are based on the use of
chemical biocides such as chlorine and washing procedures [1]. In order to reduce
the use of the chemical products, the water, the chemical residuals in the environment,
A. Berardinelli (*) L. Ragni

Agricultural Economics and Engineering Department, University of Bologna,
P.za G. Goidanich 60, 47521 Cesena, Italy
e-mail: annachi.berardinelli@unibo.it
L. Vannini M.E. Guerzoni
Food Science Department, University of Bologna, Bologna, Italy
Centro Interdipartimentale di Ricerca Industriale Agroalimentare,
University of Bologna, Bologna, Italy
458 A. Berardinelli et al.
and to improve the treatment efficacy and postharvest quality, new physical methods
are currently being researched. The application of UV radiations [2] and gaseous
ozone [3] are undoubtedly the most studied technologies for fresh fruit and vegetables.
Non-thermal gas plasma could also represent an alternative to the conventional
methods used for fruit surface decontamination. Non-thermal or cold plasma, char-
acterized by an electronic temperature much higher than the macroscopic one, can
be produced at atmospheric pressure and by using air as working gas [4]. These
conditions make the technique suitable for food decontamination when the quality
of the products must be preserved.
Non-thermal atmospheric plasma can be generated by means of AC or DC power
supply, by microwaves or by RF sources and the choice of the frequency exciting
mode influences the plasma characteristics (electrons and heavy species behaviour)
[5]. The dielectric barrier discharge (DBD), the resistive barrier discharge (RBD) and
the atmospheric pressure plasma jet (APPJ) are the most researched methods [6].
During the last years several researches were conducted on the use of cold plasma
for the decontamination of food products. The DBD method was successfully used
on shell eggs deliberated inoculated with Salmonella Enteritidis and S. Typhimurium;
negative effects of the treatment were not observed both on the eggshell and in the
internal components [7]. The lethal efficacy of the cold atmospheric plasma was
also investigated on surface cut sections of different types of fruit and vegetables
previously inoculated with food-borne pathogens (Escherichia coli, Salmonella and
Listeria monocytogenes) and spoilage organisms (S. cerevisiae, P. agglomerans and
G. liquefaciens) [8, 9]. Experiences on the entire vegetable surface were conducted
on apples inoculated with Escherichia coli and Salmonella [10], on almonds inocu-
lated with Escherichia coli [11], and on grains and legumes infected with Aspergillus
spp. and Penicillium spp. [12]. Even if the results showed important reductions
according to the treatment time and used method, the above cited researches were
conducted only on previously inoculated samples and did not refer about possible
negative effects on the product quality traits such as changes in the peel colour and
chemical composition.
The present research aims to assess effects of non-thermal atmospheric air plasma
towards the indigenous microflora and the quality traits of fresh Abate Fetel pears,
a major pear cultivar in Italy. Possible negative effects on fruit quality traits were
analysed both immediately after the treatments and after storage for 5 days at 20C.
35.2.1 The DBD Device
The decontamination treatments were conducted in a hermetic chamber characterized

by a maximum available volume of about 70 dm3. The atmospheric discharge was
generated between three pairs of parallel plate electrodes made of brass (Fig. 35.1).
35 Impact of Atmospheric Plasma Generated by a DBD Device 459
Fig. 35.1 Abate Fetel

pears during a superficial
treatment and a detail of the Power switchin gtransistors
electrodes used to generate
the discharge (gap
spacing between HV transformers
electrodes = 1.5 mm). The
fruits were placed at about Electrodes
7 cm under the electrodes
One of the two electrodes was covered by a 5 mm thick glass. The voltage at the
electrodes was produced by three high voltage transformers and power switching
transistors. The generated discharge was driven towards the fruits, placed at about
7 cm under the electrodes, by means of three fans mounted over the electrodes.
The discharge produced by the described configuration was electrically and
chemically characterized in a previous work [7]. Main results indicated that with an
input DC voltage of 19 V (chosen for the following microbiological and quality
tests) a potential difference of about 15 kV (peak-to-peak) was measured at the
electrodes. The plasma emission spectra showed the presence of very reactive spe-
cies such as the positive ion N2+ and NO and OH radicals. The emission of OH radi-
cals appeared to increase by increasing the humidity level of the air (RH).
The temperature profiles of the electrodes components (glass and brass) were
acquired for about 10 min by means of an infrared camera (FLIR, A325, FLIR
Systems) placed at about 40 cm from the discharge and operating in the range of
7.513.0 mm. The temperature of the electrodes components can indirectly give an
idea about the temperature reached by the gas plasma discharge generated between
the two parallel plates.
35.2.2 Fruit Characteristics
The Abate Fetel fruits were collected from the same farm immediately after har-
vest (Emilia Romagna region, Italy). The fruits were then stored at about 0C for
approximately 5 months followed by 2 weeks at 4C. The fruits were characterized

by a mean mass of 212 g (30 g) and mean dimensions of 109 mm (15 mm)
(height) and 66 mm (4 mm) (max width).
35.2.3 Assessment of Decontamination Efficacy
The efficacy of the device in reducing the superficial natural occurring microflora
was evaluated by exposing fruit samples to gas plasma for 10, 20, 30, 45, 60 and
90 min at RH of 60% (22C). For each treatment time five fruits were considered.
The plasma emission spectra at the cited atmospheric conditions were acquired
from 200 to 450 nm and at the distance of about 20 mm from the emission by using
a fibre optic probe (Avantes, FC-UV400-2) and a spectrometer (Avantes,
AVASpec-2048, resolution of 2.4 nm).
Following each treatment, pears were transferred into a sterile sampling bag
(International PBI S.p.A., Milan, Italy) and 100 ml of sterile saline solution (NaCl
0.9%) was added. Fruits were then hand-rubbed through the bag for 3 min to detach
the bacteria according to [13] and a 1-ml aliquot was used to prepare decimal serial
dilutions.
Enumeration of the surviving cells of total mesophilic bacteria, moulds and
yeasts was done by surface plating, in triplicate, 100 ml of the appropriate dilutions
onto Plate count Agar, Potato Dextrose Agar and agarized Sabouraud media with
added chloramphenicol (200 ppm), respectively. Plates were incubated at 30C for
48 h for bacteria and at 28C for 35 days for fungi.
35.2.4 Assessment of Fruit Quality Traits
The possible negative effects of the gas plasma on Abate Fetel pears quality traits
were assessed after 45 and 90 min of treatment at RH either of 23% and 60% and
22C (20 fruits/group). Immediately after the treatments possible weight losses and
changes occurring in the peel colour were quantified. The peel colour was measured
by means of a reflectance colorimeter in three different points of each fruit (Minolta
Chroma Meter CR-400, Minolta Italia S.p.A).
The peel colour was also measured after storage for 5 days at 20C with respect
to an untreated sample and together with the assessment of the Magness-Taylor
flesh firmness (MTf), the flesh soluble solids content (SSC) (Brix), and the antioxi-
dant capacity of peel and flesh (immediately under the peel). The MTf and SSC
measurements were carried out on opposite sides of each fruit by means of a manual
penetrometer (with 8 mm diameter probe) and a digital refractometer (PR-1, ATAGO
Co. Ltd.), respectively (only for the highest RH level). The antioxidant capacity was
assessed on the basis of the absorbance of the ABTS+ radical cation at 734 nm
(UV-Visible Spectrophotometer IE CARY, Varian) and the results were expressed in
terms of Trolox equivalent antioxidant capacity (g Trolox/l) [14]. The fruit

antioxidant capacity is mainly due to the presence of carotenoids, ascorbic acid and
phenolic compounds which act as singlet oxygen quenchers or scavengers of
reactive oxygen species such as free radicals [15].
Significant differences (at p-level < 0.05) between means of the measured quality
indices were explored by means of the analysis of variance (ANOVA); the Mann
Whitney test was used if significant differences emerged between variance means at
the Levene test (SPSS, version 14.0).
35.3.1 Thermal Profiles and Plasma Emission Spectra
The analysis of the thermal profiles of the electrodes components acquired by means
of an IR camera during the discharge generation showed temperature values of the
brass and glass, which are in direct contact with the plasma, lower than 40C
(Fig. 35.2). It is reasonable to suppose that the discharge temperature is not too far
from this value. This low temperature is not surprising because of the presence of
the fans placed over of each pair of electrodes. In addition to directing the plasma
reactive species towards the product, the fans are also able to cool the electric
components.
The emission spectra of the discharge acquired at RH of 60% (22C) showed
dominant peaks of the N2 second positive system (l = 290440 nm) and the pres-
ence of the positive N2+ ion (l = 391 nm) (Fig. 35.3). Emissions of OH (l = 306 nm;
l = 280 nm; l = 285 nm;) and NO (l = 226248 nm) radicals were also observed.
35.3.2 Assessment of Decontamination Efficacy
The efficacy of the gas plasma treatments for superficial decontamination of Abate
Fetel pears has been evaluated towards the main microbial groups naturally occur-
ring on such fruit, i.e. total mesophilic aerobic bacteria, yeasts and moulds.
Data on viable cells detected immediately after exposure to gas plasma are
shown in Fig. 35.4. Comparison of all the microbiological data on treated fruits
evidenced that gas plasma treatments can effectively inactivate the fruit-associated
microflora although differences in resistance and dynamic were observed among
bacteria and fungi.
The relationship between the time of exposure to the plasma and the extent of the
reduction in viable cells was not linear regardless the microbial group. In general
yeasts and moulds seems to be more sensitive to the treatments and cell load
decreases of about 1 Log CFU/fruit were observed after a 1020 min treatment.
Fig. 35.2 Thermal profiles of the electrodes components after 10 min of treatment (A glass,
B brass)
70
N2 second positive system
60
50
Irradiance (W/cm2)
40
30
20 NO systems OH A-X N2+B-X

A-X
10
0
200 250 300 350 400 450
10 Wavelength (nm)
Fig. 35.3 Emission spectra of the plasma glow with a discharge voltage at electrodes of about
15 kV (RH = 60% at 22C)
The efficacy of the gas plasma generator increased by increasing the treatment time
and maximum reductions of 2.5 Log CFU/fruit were achieved both for yeasts and
moulds following the longest treatment.
On the contrary, the inactivation curve of total aerobic bacteria showed an initial
shoulder and no significant changes in viable cells were detected up to 30 min.
7
Mesophiles
6 Yeasts
Moulds
Cell load (Log CFU/fruit)
0
0 10 20 30 45 60 90
Exposure time (min)
Fig. 35.4 Effect of exposure time on cell viability of total mesophilic aerobic bacteria, yeasts and
moulds on the surface of Abate Fetel pears
However, their population was reduced for longer treatments resulting in the same
maximum decontamination level as yeasts and moulds.
These results were obtained in the presence of humid air (RH = 60%, at 22C).
By using this RH level, Ragni et al. 2010 [7] evidenced that the decontamination
efficacy towards shell eggs is significantly influenced by the levels of OH radicals.
In particular, by means of the same afterglow device and voltage at electrodes, a
significant improvement of the decontamination power was observed for
S. Enteritidis by increasing the level of OH radicals obtained by modulating the
RH level from 35% to 65% (at 25C). In fact, maximum cell load reductions of
about 2.5 and 4.5 Log CFU/eggshell were obtained for RH levels of 35% and 65%,
respectively.
Since the measurements on fresh pears were conducted at high RH levels (60%
at 22C), the observed decontamination effect can be in part explained by the
presence of OH radicals which react with the fruit surface.
Guidelines for packing fresh or minimally-processed fruits and vegetables
generally specify a washing or sanitizing step to remove dirt, pesticide residues and
microorganisms responsible for quality loss and decay [16]. However, washing
procedures with water or chemical sanitizers typically result in only a 12 log
decrease in microbial counts [17]. Although chlorine is the most widely used disin-
fectant in industry to enhance the efficacy of the washing phase, recent outbreaks
associated with pathogen contamination in fresh-cut vegetables raised the concerns
about the efficacy of chlorine treatment in assuring the safety of the products [1].
Moreover, due to the environmental and health risks posed by the use of chlorine,
there is a trend in eliminating such chemical product in addition to the need for the
food industry to minimizing water consumption and wastewater discharge [18].
On the other hand during gas plasma treatments active species are present only
when the discharge is driven and disappear some milliseconds after the discharge
has been turned off. Such features assure that it is an almost harmless operation for
operators, consumers and materials, and some Authors reported that no toxic
residues remain on the sterilized foods [19].
The use of gas plasma for the decontamination of fruit pericarps from spoilage
and pathogenic microorganisms has already been proposed [9]. Although efficient
decontamination levels below the detection limit were achieved for S. cerevisae,
P. agglomerans and G. liquefaciens and E. coli deliberately inoculated onto the
pericarps of mango and melon, no study on the indigenous microflora contaminating
fruit has been performed. This aspect is quite important as natural contaminating
microflora can develop adaptation mechanisms which generally make it much more
resistant to decontamination procedures than the deliberately inoculated pathogens
or spoilage microorganisms.
35.3.3 Assessment of Fruit Quality Traits
No significant differences emerged between means for the peel colour and the mass
of the fruits analysed before and after the treatments (Table 35.1). It is likely that
differences in mass were not detectable due to the sample variance.
After a storage of 5 days at 20C, significant differences appeared for the peel
colour and the peel antioxidant capacity (Table 35.2). These differences were found
only after 90 min of treatment with respect to the untreated and 45 min treated samples.
L* parameter of fruits treated for 90 min at high RH level significantly decreased of
about 10% respect to the control, while the relative a* parameter significantly increased
of about 19% and 64% for low and high RH levels, respectively. The antioxidant
capacity of the peel appeared significantly lower (about 9%) in fruit samples treated
for 90 min both at low and high RH levels with respect to the untreated one. For the
flesh antioxidant capacity, MTf (N) and SSC (Brix) parameters (Table 35.3), no sig-
nificant differences between the tested samples were observed.
A possible reason for the observed pear darkening could be associated to
enzymatic activities and particularly to the polifenoloxidase (PPO), peroxidase and
phenylalanine ammonia-lyase (PAL) activities. In particular PAL is an important
plant enzyme that converts L-phenylalanine into trans-cinnamic acid, which in turn
is the precursor of various phenylpropanoids, such as lignins, flavonoids, and
coumarins. The phenolic compounds can then be oxidized by PPO producing brown
polymers that contribute to tissue browning in fruits. It is also reported that PAL
activity increases in response to several kinds of biotic and abiotic stresses including
wounding, exposure to ethylene, fungal infection [20].
Since 90 min treatments at both 23% and 60% RH levels (at 22C) appeared to
affect the peel colour and the peel antioxidant capacity, the increase in OH levels at
high RH values can not explain itself the oxidative processes occurring onto the fruit
surface during this kind of treatment. It is reasonable to suppose that also others
species detected in the emission spectra such as NO radicals can reach the fruit
surface and can play a role in the oxidative processes.
Table 35.1 Influence of the gas plasma treatments on the quality of pear fruits immediately after
the treatments
Peel colour Mass
Sample L* a* b* g
45 min bt 61.1a(4.3) 2.8a(1.5) 41.2a(3.0) 199.8a(23.8)
RH = 23% at 59.7a(4.6) 3.7a(1.2) 40.7a(3.7) 199.7a(23.8)
a a a
45 min bt 63.0 (2.9) 2.5 (1.6) 42.1 (1.5) 218.8a(16.3)
RH = 60% at 61.5a(3.5) 3.3a(1.2) 42.0a(2.0) 218.6a(16.3)
90 min bt 61.5a(2.9) 3.7a(1.3) 42.3a(2.2) 222.7a(45.0)
RH = 23% at 61.0a(3.0) 4.1a(1.5) 42.5a(2.5) 222.4a(45.0)
a a a
90 min bt 60.7 (4.3) 6.7 (1.5) 41.4 (3.0) 206.6a(31.5)
RH = 60% at 60.1a(4.2) 6.6a(1.6) 42.1a(2.8) 206.3a(31.4)
a
Differences between means with the same exponent, before and after the same treatment, are not
significant at p-level value < 0.05 (bt before treatment, at after treatment). Standard deviations are
in brackets. The Peel colour was in terms of CIE L*a*b* colour space: L* lightness coordinate,
a* red coordinate, b* yellow coordinate. The fruit mass in measured in grams (g)
Table 35.2 Influence of the gas plasma treatments on the quality of pear fruits after storage for
5 days at 20C
Peel colour Antiox. capacity
Peel Flesh
Sample L* a* b* g Trolox/l g Trolox/l
a a a a
Control 63.0 (4.3) 5.8 (2.9) 41.2 (2.0) 0.76 (0.04) 0.67a(0.03)
a a a a
45 min RH = 23% 60.9 (4.2) 5.4 (4.0) 40.9 (3.4) 0.74 (0.03) **
45 min RH = 60% 61.3a(2.9) 5.9a(4.3) 41.6a(1.9) 0.76a(0.02) 0.68a(0.01)
90 min RH = 23% 60.1a(3.1) 6.9b(3.0) 42.1a(3.0) 0.69b(0.03) **
90 min RH = 60% 56.5b(3.1) 9.5c(3.9) 39.3a(5.3) 0.69b(0.03) 0.67a(0.01)
a,b,c
Differences between means with the same exponent within the column are not significant at
p-level value < 0.05 (for 3 g of peel and 5 g of flesh; ** not measured). Standard deviations are in
brackets. The Peel colour was expressed in terms of CIE L*a*b* colour space: L* lightness coordi-
nate, a* red coordinate, b* yellow coordinate. The antioxidant capacity was expressed in terms of
TROLOX (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic Acid) equivalent antioxidant
capacity (grams of Trolox per liter)
Table 35.3 Influence of the gas plasma treatments on the MTf

and SSC of flesh pear fruits after storage for 5 days at 20C
MTf SSC
Sample N Brix
Control 23.0a(5.3) 14.2a(1.0)
45 min RH = 60% 23.0a(4.3) 14.0a(1.4)
90 min RH = 60% 23.0a(4.2) 14.3a(1.0)
a
Differences between means with the same exponent within the
column are not significant at p-level value < 0.05. Standard
deviations are in brackets. Magness Taylor flesh firmness (MTf)
was expressed in Newton (N) while the soluble solids content
(SSC) in Brix (grams of soluble solid content in 100 g of the
product)
35.4 Conclusions
The obtained experimental data showed that the DBD device used within this work
has a good potential in decontaminating Abate Fetel fruits. In fact, natural con-
taminating yeasts and moulds were reduced by 1 Log CFU/fruit after a 1020 min
exposure, while mesophilic bacteria resulted to be slightly more resistant to the
treatment. The decontamination efficacy of the gas plasma generator was enhanced
by using longer exposure treatments, and maximum reductions of 2.5 Log CFU/
fruit were achieved for yeasts, moulds and bacteria after 90 min. Despite the long
treatment times, no side effects on the quality traits such as peel colour, mass,
Magness-Taylor flesh firmness and flesh soluble solids content of the fruits were
detected immediately after the treatment and during storage. Such results are prob-
ably linked to the low temperatures reached during the treatment which makes such
a technology suitable for the decontamination of heat sensitive food products.
Although the temperature of the plasma was not be directly measured for the
unknown value of the emissivity of the produced discharge, the measurement of the
thermal profiles of the electrodes components proved that the temperature of both
the glass and brass does overcome 40C.
On the other hand, significant differences due to the gas plasma treatment were
detected for the pears treated for the longest time (90 min) in the presence of humid
air and then stored for 5 days. Even of the decontamination was achieved in after-
glow, the observed changes for the parameters representing the lightness (L*) and
redness (a*) and for the peel antioxidant capacity may suggest that the reactive spe-
cies produced in the gas plasma can interact with some enzymes, e.g. polifenoloxi-
dase, peroxidase and phenylalanine ammonia-lyase, or phenolic compounds and
vitamin C. On the other hand, it should be emphasised that pears peel can be easily
damaged due to chemico-physical, mechanical and thermal stresses.
Although the present study showed the potentialities of cold gas plasma as a
decontamination method alternative to fruit washing with chemicals (e.g. chlorine),
further studies are required in order to optimise the processing conditions to improve
the decontamination efficiency while reducing the side effects. In particular, the
working gas and distance of the products from the electrodes can be appropriately
chosen and modulated in order to avoid the losses observed for some superficial
quality traits after the longest exposures.
References
1. Seymour IJ (2003) Surface preservation for fruits and vegetables. In: Russell NJ, Gould GW
(eds) Food preservatives. Kluwer Academic/Plenum Publisher, New York, p 240
2. Allende A, Arts F (2003) UV-C radiation as a novel technique for keeping quality of fresh
processed Lollo Rosso lettuce. Food Res Int 36:739746
3. Palou L, Smilanick JL, Crisosto CH, Mansour MF (2001) Effect of gaseous ozone exposure on
the development of green and blue molds on cold stored citrus fruit. Plant Dis 85:632638
4. Moreau M, Orange N, Feuilloley MGJ (2008) Non-thermal plasma technologies: new tools for
bio-decontamination. Biotechnol Adv 26:610617
5. Tendero C, Tixier C, Tristant P, Desmaison J, Leprince P (2006) Atmospheric pressure plasmas:

a review. Spectrochim Acta B 61:230
7. Ragni L, Berardinelli A, Vannini L, Montanari C, Sirri F, Guerzoni ME, Guarnieri A (2010)
Non-thermal atmospheric gas plasma device for surface decontamination of shell eggs. J Food
Eng 100:125132
8. Critzer FJ, Kelly-Winterberg K, South SL, Golden DA (2007) Atmospheric plasma inactiva-
tion of foodborne pathogens on fresh product surfaces. J Food Prot 70:22902296
9. Perni S, Liu DW, Shama G, Kong M (2008) Cold atmospheric plasma decontamination of the
pericarps of fruit. J Food Prot 71:302308
10. Niemira BA, Sites J (2008) Cold plasma inactivates Salmonella Stanley and Escherichia coli
O157:H7 inoculated on golden delicious apples. J Food Prot 71:13571365
11. Deng S, Ruan R, Mok CK, Huang G, Chen P (2005) Non-thermal plasma disinfection of
Escherichia coli on almond. ASAE meeting presentation. Paper number: 056149
12. Selcuk M, Oksuz L, Basaran P (2008) Decontamination of grains and legumes infected with
Aspergillus spp. and Penicillium spp. by cold plasma treatment. Bioresour Technol
99:51045109
13. De Reu K, Grijspeerdt K, Messens W, Heyndrickx M, Uyttendaele M, Debevere J, Herman L
(2006) Eggshell factors influencing eggshell penetration and whole egg contamination by dif-
ferent bacteria, including Salmonella enteritidis. Int J Food Microbiol 112:253260
14. Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A (1993) A novel method for
measuring the antioxidant capacity and its application to monitoring the antioxidant status in
premature neonates. Clin Sci 84:407412
15. Hamauzu Y, Sakai I (2002) Changes in polyphenolic compounds and antioxidant functions in
Bartlett pear fruit during storage and postharvest ripening. Food Preservation Sci 28:2532
16. Sapers GM (2006) Washing and sanitizing treatments for fruits and vegetables. In: Sapers GM,
Gorny JR, Yousef AE (eds) Microbiology of fruits and vegetables. Taylor and Francis/CRC
Press, Boca Raton, p 375
17. Sapers GM (2001) Efficacy of washing and sanitizing methods for disinfection of fresh fruit
and vegetable products. Food Technol Biotech 39:305311
18. lmez H, Kretzschmar U (2009) Potential alternative disinfection methods for organic fresh-
cut industry for minimizing water consumption and environmental impact. LWT- Food Sci
Technol 42:686693
19. Moisan M, Barbeau B, Moreau S, Pelletier J, Tabrizian M, Yahia LH (2001) Low-temperature
mechanism. Int J Pharm 226:121
20. Lopez-Galvez G, Saltveit M, Cantwell M (1996) Wound-induced phenylalanine ammonia
lyase activity: factors affecting its induction and correlation with the quality of minimally
processed lettuces. Postharvest Biol Technol 9:223233
Chapter 36
Fungicidal Effects of Plasma and Radio-Wave
Pre-treatments on Seeds of Grain Crops
and Legumes
Irina Filatova, Viktor Azharonok, Alexander Shik,

Alexandra Antoniuk, and Natalia Terletskaya
Abstract An influence of RF plasma and RF electromagnetic field pre-treatments

on level of fungal infection of some important agricultural plants has been studied.
It is shown that pre-sowing plasma and radio-wave seeds treatments contribute to
their germination enhancement and plant productivity improvement owing to stimu-
lative and fungicidal effect of plasma and RF electromagnetic field irradiation.
36.1 Introduction
Plasma and electromagnetic fields treatments have been successfully applied over
the recent years for activation and decontamination of surfaces. Nowadays non-
thermal plasmas are considered functionally, energetically and ecologically as the
most efficient tool for pathogenic bacteria inactivation due to their high chemical
activity, short-time treatment and minimal materials destruction [1, 2].
Recently years these methods have also been successfully applied in agriculture
for improving the sowing quality of seeds. It has been shown in a number of previ-
ous studies that plasma and electromagnetic field pre-treatments of seeds stimulate
their germination and sprouting process, lead to suppression of fungal and bacterial
pathogens that cause various plant diseases [311]. At the same time the nature of
I. Filatova (*) V. Azharonok

Laboratory of Physics of Plasma Accelerators, The State Scientific Institution
B.I. Stepanov Institute of Physics of The National Academy of Sciences of Belarus,
Nesavisimosti Ave. 68, 220072 Minsk, Belarus
e-mail: filatova@imaph.bas-net.by
A. Shik A. Antoniuk N. Terletskaya
The Polessian Agrarian-Ecological Institute NAS Belarus, Brest, Belarus
470 I. Filatova et al.
plasma and electromagnetic fields interactions with biological substances still

remains unclear. It is obvious, that plasma treatment influences morphological and
sowing characteristics of seeds in different ways [6] due to the complexity of
plasma producing fluxes of various neutral or ionized active species as well as
energetic photons interacting with treated samples. Seeds are an extremely com-
plex biological system and vitally important biochemical processes may be affected
by plasma or electromagnetic treatment in a number of different ways. Moreover,
seeds appear to be the most likely source of the finished product contamination,
although conditions at the sprouting facility may also impact on the product safety.
The initial microflora on seeds increases during vegetation period in the field con-
dition because of potential sources of contamination that include irrigation water,
pathogens which survive in soil, inadequate worker hygiene etc. [12]. Therefore,
sanitary treatment is more effective for reducing contamination on seeds than on
sprouted seeds.
A considerable part of investigations have been performed using 13.56 MHz
and microwave plasma sources and the influence of electromagnetic fields on bio-
logical objects has been examined mainly in the microwave (300 MHz300 GHz)
or in the low frequency (50100 Hz) regions. In this paper, low-pressure 5.28 MHz
capacitively coupled plasma as well as 5.28 MHz electromagnetic field treatments
used for enhancement of seeds of some important grain crops and legumes. The
efficacy of treatments against some plant diseases caused by pathogenic fungi
and their influence on seed germination was also investigated in laboratory and
field trials.
36.2 Experiment
36.2.1 Experimental Set-up and Conditions
The principal scheme of the experimental set-up and the applied technique is shown
in Fig. 36.1. Tested species were processed with 5.28 MHz air plasma at a pressure
40 Pa. A capacitively coupled discharge was operated between two plane-parallel
water-cooled copper electrodes with a diameter of 120 mm placed in a stainless
steel vacuum chamber with the inner volume of 53.2 m3. The distance between
electrodes was varied between 20 and 40 mm according to the number and size of
the treated seeds. A supplied full specific RF power could be changed in a range
0.10.7 W/cm3 in dependence on treatment conditions.
A Petri dish with seeds to be treated by the plasma was put on the grounded
electrode before the vacuum chamber pumping. The exposure duration was 2, 5, 7,
10, 15 and 20 min. Under the experimental conditions the gas temperature did not
increase beyond 310C. Optical emission spectra were obtained with a Compact
Wide-Range Spectrometer S100 SOLAR LS in the optical range from 190 to
1,100 nm with average spectral resolution of 1 nm to identify the species present in
plasma during the treatment.
36 Fungicidal Effects of Plasma and Radio-Wave Pre-treatments on Seeds 471
Fig. 36.1 Experimental set-up: 1 alternator, 2 induction coil, 3 voltmeter, 4 vacuum chamber,
5 and 5 RF and grounded electrodes, 6 Petri dish, 7 quartz window, 8 lens, 9 monochromator,
10 camcorder, 11 personal computer
The experimental conditions for electromagnetic field treatment were as follows:

the alternator frequency was 5.28 MHz, the root-mean-square value of magnetic H
and electric E components of the electromagnetic field strength was 590 A/m
(B 1 mTl) and 12,700 V/m accordingly, the amplitude values H * = 2H and
E* = 2 E reached 835 A/m (B 1.5 mTl) and 17,960 V/m respectively. The field
was localized in a water-cooled spiral-shaped three-coil inductor with the inner
diameter of 80 mm and a length of 90 mm connected to the exit of the alternator as
an inductive load [8]. A glass dish with seeds to be treated was put in the axial zone
of the inductor onto a dielectric prop. The treatment was performed in air at atmo-
spheric pressure. The duration of seeds exposure to electromagnetic field was 5, 10,
15 and 20 min. The temperature of the seeds was controlled using a thermocouple.
In all our experiments it corresponded to the room temperature.
To obtain statistical data each portion of control and treated samples in the
laboratory and field trials contained 100 seeds. All treatments for all experimental
conditions and seed species were replicated three times. A plot with the area of
50 m2 was used for the field test.
36.2.2 Materials and Methods
Seeds of some important agricultural grain-crops and legumes such as wheat

(Triticum aestivum), spring barley (Hordeum vulgare), blue lupine (Lupinus angus-
tifolius), soy (Glycine soja) and field pea (Pisum arvense) were chosen for investi-
gations. The effectiveness of pre-sowing plasma and radio-wave seed treatments
was examined by means of evaluation of the laboratory/field germination ability,
seed vitality, morphometric characteristics of sprouts and roots as well as a level of
fungal infection on sprouting seeds for treated and untreated (control) samples.
In the laboratory tests seeds were grown on a moist filter paper in sterile Petri
dishes that were kept for 57 days (before the first sprout occurrence) in a
thermostat at 2021C under a lightdark regime. The seed germination power
(seed vitality) was estimated after 3 days and germination laboratory test was
performed after 7 days. Each seed was considered to be germinated when it had
a radicle of 1 mm at least. The phytotoxicity test for control and treated seeds
was carried out after 7 days according to [13]. The STATISTICA 6.0 program
package was used for data statistical processing, inclusive of correlation and
regression analysis [14].
For analysis of seed health, the untreated and treated samples were incubated
on Potato Dextrose Agar (PDA) in Petri dishes at 23C to stimulate the develop-
ment of fungal colonies. The different fungi were then identified taking into
account their morphological and cultural characteristics according to [15]. The
analysis of seed-born infection of seedlings was carried out using the blotter rolls
method. The surface sterilized grains (kept for 3 min in 70% ethyl alcohol and
then washed in sterile water) were incubated in wet blotter rolls for 718 days
before the disease depth was estimated. Similarly, pathogenic diseases were
evaluated in the field conditions at the plant tillering stage. We tested 10 plant lots
(10 plants per plant lot) for each investigated culture during the field sanitary
inspections. The pieces of stems and leafs of infected plants with typical for each
fungal infection signs were taken for analysis throughout the plot area. Selected
samples were washed in sterile water, cut and plated in Petri dishes on PDA with
the cut side facing down on the nutrient medium. The infection was tested after
7 days of incubation.
The level of fungal infection P in the field tests was evaluated as the ratio between
the number of infected plants n and the total number N of investigated samples in
plant lot:
n
P= 100%. (36.1)
N
The disease severity R was estimated as follows:
( n b )
i
R= i
100%, (36.2)
N K
where ni is a number of infected plants with the same level of fungal lesion that
was corresponded to a mark b estimated in points (0 healthy, 1 weakly, 2
moderately and 3 deeply damaged), K is the highest mark of disease scale
(04).
A part of seeds were treated with appropriate fungicide vincit forte (manufac-
tured on the base of three reactants: flutriafol 37.5 g/l, thiabendazole 25 g/l,
imazalil 15 g/l) to study the effectiveness of plasma and radio-wave treatments in
comparison with common chemical methods of plant protection against fungal
infection.
36.3 Plasma and Radio-Wave Seed Pre-treatments
36.3.1 Germination Test
The laboratory and field germination characteristics of treated and control seeds of
blue lupin and field pea in dependence on exposure time are presented in Figs. 36.2
and 36.3 correspondingly. Significant correlations were found between the results
of laboratory and field tests. Both RF plasma and radio-wave pre-treatments did not
influence negatively on the sowing characteristics of seeds, but even improved their
germination within the optimal treatment conditions. The effect is less pronounced
in field tests that connected with influence of environmental conditions on germina-
tion and seed vitality during vegetation period.
The optimal exposure time was varied between 7 and 15 min in dependence on
sort of species differing in seed size, seed coat etc. Further increase of the treatment
duration (t > 20 min) did not lead to the positive effect and in some cases, for radio-
wave treatment led to germination decrease. Germination of seeds in field trials
after the electromagnetic field exposure was higher for optimal time than that in
control as follows: field pea by 13%, soy 8%, blue lupin 6% and barley 4%.
This effect for plasma treated seeds was lower (6% for field pea and 3% for blue
lupin) or statistically insignificant (for barley and wheat for example).
In spite of negligible enhancement of seed germination, a considerable increase
(up to 30%) of weight of 100 shoots was observed after the plasma treatment. It was
established that plasma treatment did not influence the length of the main root and
shoot of seedlings, but stimulated their branching especially on the 7th10th days of
ontogenesis (Fig. 36.4). Thus, the shoot mass accretion Dm for blue lupin at the
optimal exposure duration t = 7.5 min (Fig. 36.3a) amounted to 98% during the
period between the third and the 10th day for plasma treated seeds, while for control
samples it amounted to only 57%.
Therefore, the positive effect of plasma pre-treatment on seed germination
parameters becomes apparent not on the first days of ontogenesis but after a period
Fig. 36.2 Laboratory

germination of seeds of blue
lupin and field pea as a result
of plasma and radio-wave
seeds treatments
Fig. 36.3 Field germination and seed vitality of blue lupin (a) and field pea (b) as a result of
plasma and radio-wave seeds treatments
Fig. 36.4 The mean value of root/shoot length (a) and root/shoot mass (b) of sprouting seeds of
Lupinus angustifolius for control and plasma treated samples (20 seeds were tested in three repeti-
tions) in dependence on exposure duration measured on the 3rd, 7th and 10th days of ontogenesis
of delay. We consider this phenomenon as an adaptation of seed to the environmen-

tal conditions after a stressor impact of plasma irradiation. We suppose that during
the adaptation seeds accrue the peculiar biologic safety properties that become
apparent at the later phases of ontogenesis. The period of seed adaptation after the
stress factors application is characterized by internal physiological changes con-
nected with protein and isoenzymes synthesis that are responsible for metabolism of
the seedlings in post-stress conditions. The treatment time is one of the key param-
eters influences the seed stress tolerance ability and, apparently, there is an optimal
duration of seed exposure to plasma under the experimental conditions for improv-
ing seed germination characteristics. An increase of treatment duration beyond the
optimal one can lead to insignificant effect or germination suppression as it was
observed for 20-min exposure. This tendency for blue lupin was revealed after the
15-min treatment (Figs. 36.2, 36.3, and 36.4).
Plasma and radio-wave treatments did not influence germination of spring barley
and wheat, but improved distinctly the seedlings growth force indexes (the shoot
Fig. 36.5 The field

germination, seed vitality and
shoot number of barley as a
result of plasma treatment
number and shoot weight) (Fig. 36.5). The best result was observed for 2.5-min
treatment. Moreover, sprouting seeds after the treatment developed better than the
controls and their vitality increased. The plant vitality that characterized the harvest
potency was evaluated as a ratio of number of ripe grown crops to the germinated
ones. It was established that radio-wave treatment of seeds increased the plant vital-
ity by 8% for wheat and barley owing to the larger number of productive stems per
unit area.
It was revealed that the efficiency of plasma and radio-wave seed treatments
depended on the degree of plant species domestication for agriculture with less
impact being achieved on the more domesticated species. In particular, the most
efficient effect of plasma and radio-wave treatments on seeds sowing characteristics
was observed for blue lupin and field pea as less domesticated cultures in compari-
son with wheat or barley.
36.3.2 Fungicidal Effect of Plasma and Radio-Wave Treatment
The results of the laboratory tests have shown that seeds of blue lupin were infected
mainly with Fusarium and Alternaria, seeds of field pea with Fusarium, Alternaria
and Stemphilium, barley seeds with Fusarium. Both the plasma and radio-wave
treatments leaded to reduction of seeds infection with pathogenic fungi. The most
effective results for all tested species were observed after treatment for 10 and 15 min.
So, the field pea seeds exposure to plasma for 10 min resulted in decrease of Fusarium,
Alternaria and Stemphylium infections by 4%, 24% and 3% correspondingly
(Figs. 36.6 and 36.7). The Fusarium infection level in blue lupin seeds diminished by
9% after 15 min of plasma treatment. The longest exposure time was not effective in
seed enhancement because of influence on germination and seed vitality.
In the field conditions Anthracnose, Fusarium, Alternaria blackspot and
Stemphilium were tested as blue lupin diseases. Plasma and radio-wave seed
Fig. 36.6 The level of fungal infection of field pea (a) and blue lupin (b) seeds in dependence on
plasma treatment duration (laboratory test)
Fig. 36.7 The effect of plasma treatment on the level of fungal infection of field pea seeds (labora-
tory test)
pre-treatments during 1015 min leaded to decrease by 614% of the level of the
most harmful fungal infection (Fig. 36.8a). At the same time no fungicidal effect was
observed against Anthracnose. Plasma treatment for 515 min of soy seeds sup-
pressed by 4.87.2% Fusarium and Alternaria blackspot at early stages and advanced
growth stages (Fig. 36.8b). Similar results were observed for radio-wave treatment.
Seed pre-treatments for 1015 min were the most effective for both seed germination
enhancement and fungal infection reduction on sprouting seeds. Therefore, the fun-
gicidal effect for optimal conditions of seeds exposure to plasma and electromag-
netic field was comparable with the chemical fungicide pre-treatment of seeds. No
further decrease of the level of fungal contamination was observed in the field tests
for the treatment duration beyond 20 min practically for all tested species.
We suppose that the fungicidal effect of air plasma provides with the UV radia-
tion, seed surface bombardment with atomic oxygen and radicals formed in plasma
due to the oxygen contained molecules dissociation, and the formation of functional
groups on treated surface (Fig. 36.9) [13, 16].
Fig. 36.8 Level of fungal infection of blue lupin (a) and soy (b) estimated in field trials as a result
of radio-wave (a) and plasma (b) treatments for different exposure durations
Fig. 36.9 Emission spectrum of RF air plasma recorded under conditions of plasma surface
decontamination
The effects of electromagnetic fields on biological objects are still insufficiently

studied. Electromagnetic field treatment improves viability of a seed and causes
physiological and biochemical changes in seeds related to initiation of endocellular
processes. It is shown that exposure to magnetic fields activates seed enzyme com-
plexes, which ensures faster growth of the germ, increases germination energy and
speeds up root formation [17]. The enzymatic activity may also change, as well as
the conformational dynamics of proteins and the viscosity of lipids [7], which are
components of membranes and cell protoplasm. It has also been noted that the elec-
tromagnetic irradiation causes intracellular magnetophoretic displacements in
starch-containing structures, as well as in cytoplasmic ionic currents. All these
effects may cause changes in the transport properties of cellular plasmatic mem-
branes, which play an extremely important role in cell regulatory functions. The
stimulative or inhibitory effect of electromagnetic fields on seeds closely depends
on the conditions of treatment: energy and frequency of field, irradiation time and
biological peculiarities of treated samples [7]. Therefore, to control the effect of

electromagnetic field treatment it is necessary to find the optimal parameters for
achievement of a positive biostimulation in seeds characterized by different
biological and chemical properties.
36.4 Conclusions
This study demonstrated that low-pressure RF air plasma and RF electromagnetic

field pre-treatments of plant seeds improved their sowing quality due to both seed
germination enhancement and reduction of seed contamination with pathogenic
fungi. The significant increase of vitality and seed germination (by 47%) was
observed in field tests for legumes exposed to electromagnetic field for 1015 min.
Plasma treatment stimulates strength and branching of shoots and roots of seeds.
This effect became apparent on the 7th10th days of ontogenesis due to the seed
adaptation after the stressor impact of plasma irradiation. The plasma-assisted
improvement of root and shoot development ensures better plant viability and good
growth of plant at later stages for treated samples in comparison with the control.
The results obtained show that seed germination depends on treatment duration
which optimal values vary from 7 to 15 min for different species. The increase of
exposure time beyond the optimal one can lead to insignificant effect or even to
germination suppression. We can not explain this phenomenon now because of very
poor experimental data for understanding the influence of plasma or radio-wave
treatment on seed germination. We consider the treatments as stressor impact caused
seed hormonal activity that influences the seedling adaptation ability to the environ-
mental conditions. Plasma and radio-wave seed pre-treatments led to decrease by
614% of the level of fungal infection such as Fusarium, Alternaria black spot and
Stemphylium, but the treatments had no impact on Anthracnose.
Therefore, pre-sowing plasma and radio-wave seeds treatments owing to their
stimulative and fungicidal effect on plant seeds can be considered as an alternative
method for effective preparation of seeds for industrial crop cultivation.
Acknowledgments This work has been supported partly by the State Committee on Science and
Technology of the Republic of Belarus (grant No. 11CP-015) and the Belarusian Republican
Foundation for Fundamental Research (grant No. T10-063).
References
1. Moisan M, Barbeau J, Moreau S, Pelletier J (2001) Low-temperature sterilization using gas

plasmas: a review of the experiments and an analysis of the inactivation mechanisms. Int J
Pharm 226:121
2. Fridman G, Friedman G, Gutsol A, Shekhter AB, Vasilets VN, Fridman A, Fridman G (2008)
Applied plasma medicine. Plasma Process Polym 5:503533
3. Selcuk M, Oksuz L, Basaran P (2008) Decontamination of grains and legumes infected with
Aspergillus spp. and Penicillum spp. by cold plasma treatment. Biores Technol 99:51045109
4. Dubinov A, Lazarenko E, Selemir V (2000) Effect of glow discharge air plasma on grain crops
seed. IEEE Trans Plasma Sci 20:180183
5. er B, Strak V, er M, Tich M, patenka P (2008) Germination of Chenopodium album
in response to microwave plasma treatment. Plasma Sci Technol 10:506511
6. Ser B, Spatenka P, Ser M, Vrchotov N, Hruskov I (2010) Influence of plasma treatment on
wheat and oat germination and early growth. IEEE Trans Plasma Sci 38:29632968
7. Penuelas J, Llusia J, Martinez B (2004) Diamagnetic susceptibility and root growth responses
to magnetic fields in Lens culinaris, Glycine soja, and Triticum aestivum. Electromagn Biol
Med 23:97112
8. Azharonok VV, Goncharik SV, Filatova II, Shik AS, Antonyuk AS (2009) The effect of the
high frequency electromagnetic treatment of the sowing material for legumes on their sowing
quality and productivity. Surf Eng Appl Electrochem 45:317327
9. Lynikiene S, Pozeliene A, Rutkauskas G (2006) Influence of corona discharge field on seed
viability and dynamics of germination. Int Agrophys 20:195200
10. Pietruszewski S, Muszyski S, Dziwulska A (2007) Electromagnetic fields and electromag-
netic radiation as non-invasive external stimulants for seeds (selected methods and responses).
Int Agrophys 21:95100
11. Aguilar CH, Dominguez-Pacheco A, Carballo AC, Cruz-Orea A, Ivanov R, Lopez-Bonilla JL,
Valcarcel-Montanez JP (2009) Alternating magnetic field irradiation effects on three genotype
maize seed field performance. Acta Agrophysica 14:717
12. Caetano-Anolles G, Favelukes G, Bauer WD (1990) Optimization of surface sterilization for
legume seed. Crop Sci 30:708712
13. Naumova N (1970) Analyz semjan na gribnuju i bakterialnuju infekciju (The analysis of
seeds on fungal and bacterial infection). Kolos, Moscow (In Russian)
14. Dospechov B (1979) Metodika polevogo opyta: s osnovami statisticheskoi obrabotki (Field
method: with the basics of statistical processing). Kolos, Moscow (In Russian)
15. Bilaii V, Gvozdjak I, Skripal R (1988) Vozbuditeli boleznei rastenii (Plant deseases patho-
gens). Naukova Dumka, Kiev (In Russian)
16. Sun P, Sun Y, Wu H et al (2011) Atmospheric pressure cold plasma as an antifungal therapy.
Appl Phys Lett. doi:10.1063/1.3530434
17. Carbonell M, Martinez E, Raya A (2000) Effects of 125 mT stationary magnetic field in the
initial stages of growth of wheat. Agric Eng Res Pap 32:510
Subject Index
A Bacillus globigii, 301

Acidification, 31, 67, 293 Bacillus subtilis, 17, 79, 149,
Active species, 31, 67, 293 301, 445
Afterglow, 93, 231 Bacillus subtilis spores, 231, 265, 445
Agar plate, 3, 17, 31, 45, 149, 311 Bacteria, 3, 17, 31, 45, 67, 79, 93, 121,
Air plasma, 31, 57, 67, 149, 293, 301, 381, 135, 149, 163, 179, 191, 215, 231,
393, 445, 457, 469 251, 265, 281, 301, 311, 381, 393,
air plasma spray, 347 417, 445, 457
Alternaria, 57, 469 bacterial colonization, 45
Antibacterial effect, 361, 393, 417 bacterial inactivation, 93, 301
Antifungal susceptibility, 201 bacterial decontamination of liquids, 361
Antimicrobial coating, 417 bactericidal activity, 163, 361
Antioxidative potential, 281 Bacteriophage inactivation, 79, 251
Antiseptic, 191, 281 Bio-decontamination, 3, 17, 31, 45, 57, 79, 93,
Apoptosis, 361, 381 121, 135, 149, 163, 179, 191, 201, 215,
Arc discharge, 393 231, 251, 265, 281, 301, 311, 361, 393,
Argon, 3, 45, 67, 121, 163, 191, 215, 281, 417, 445, 457, 469
311, 321 Biofilm, 31, 135, 149, 163, 179, 191, 201
argon humid, 93 biofilm inactivation, 31, 135, 149, 163,
Aspergillus 191, 201
Aspergillus niger spores, 231 biofilm disinfection, 179
Aspergillus oryzae, 57 Bio-macromolecules, 17
Atmospheric pressure plasma, 3, 17, 31, 45, Bleeding, 347
57, 67, 79, 93, 107, 179, 191, 281, 301, Blood coagulation, 347
311, 321, 381, 417, 445, 457 Bonding strength, 215
atmospheric pressure plasma jet, 3, 17, Burkholderia cenocepacia, 163
135, 281, 301, 335
Atomic
atomic force microscopy (AFM), 135 C
atomic oxygen, 347 Cancer therapy, 361, 381, 403
Candida
Candida albicans, 191, 201, 251, 311
B Candida glabrata, 201
Bacillus, 251 Candida krusei, 201
Bacillus atrophaeus spores, 3, 45 Carbon dioxide, 445
Bacillus cereus spores, 31 Cavitation, 403
DOI 10.1007/978-94-007-2852-3, Springer Science+Business Media B.V. 2012
482 Subject Index
Cell Enterococcus faecalis, 179

cell cycle, 381 Escherichia coli, 17, 31, 45, 67, 79, 93, 121,
cell integrity, 321 149, 251, 301, 311, 381, 457
cell membrane, 31, 149, 361 Excilamp, 251, 265
Chemical burn, 335 KrCl, 251
Chlamydia trachomatis, 163 XeBr, 251
Chloroxylenol spray, 301 XeI, 265
Chromobacterium violaceum, 135 Ex vivo, 179, 293
Chronic wound, 281, 311, 321
Cladosporium sphaerospermum, 57
Colony forming units, 3, 17, 31, 45, 57, 67, F
79, 93, 135, 149, 163, 179, 251, 265, Fast imaging, 335
301, 393, 457 Floating
Colorectal cancer, 381 floating electrode, 381
Confocal laser scanning microscopy (CLSM), floating potential, 163
179, 191 Fluorescent labeling, 79, 135, 163
Corona discharge, 57, 79 Food
streamer corona discharge, 31 food decontamination, 417, 445,
Cytotoxicity, 321 457, 469
food industry, 417
food package, 445
D food treatment, 417
DC discharges, 31 Fourier transform infrared spectroscopy
DC microjet, 201 (FTIR), 17, 215
Decontamination, 3, 57, 445 Fungi, 57, 201, 231, 469
bio-decontamination, 3, 17, 31, 45, 57, 79, fungicidal effect, 57, 469
93, 121, 135, 149, 163, 179, 191, 215, fungal spores, 57
265, 301, 311, 361, 393, 417 Fusarium, 469
of food processing equipment, 417
Dental composite restoration, 215
Dentin, 179, 215 G
Dentistry, 179, 191, 201 Germicidal range, 265
Dermatosis, 201 Glioma cancer, 381
Dielectric barrier discharge, 3, 67, 79, 93, Glycine soja, 469
179, 191, 251, 265, 301, 335, 381, Guanine, 107
417, 445, 457
Direct plasma treatment, 31, 231
DNA H
DNA absorbance, 215 Hailey-Hailey disease, 311
DNA damage, 17, 79, 93, 335, 381 Heat, 231
DNA fragmentation, 93, 361 HeLa cells, 361
DNA oxidation, 107 Helium, 17, 107, 135, 179, 201, 335
helium plasma jet, 335
HET-CAM (hens egg test on the
E chorioallantois membrane), 321
Effluent separation, 17 Hordeum vulgare, 469
Electron Hydrogen peroxide, 93, 293
electron spin resonance spectroscopy, 201
electron velocity distribution function, 45
Electrostatic precipitation, 79 I
Emission spectroscopy, 45, 93, 121, 149, 163, ICCD imaging, 335, 381
201, 215, 231, 251, 265, 281, 311, 347, Inactivation kinetics, 135
445, 457 Indirect plasma treatment, 31
Endodontic treatment, 179 Inhibition of bacterial colonization, 45, 201
Subject Index 483
Intracellular O
intracellular manipulation, 361 Octenidine, 281
intracellular parasites, 163 OH radical, 93, 201, 457
In vitro, 17, 121, 179, 321, 347, 361, 381, Optical emission spectroscopy, 45, 93, 121,
393, 403 149, 163, 201, 215, 231, 251, 265, 281,
In vivo, 17, 79, 251, 281, 321, 347, 361, 381, 311, 347, 445, 457
393, 403 Oxidative stress, 31
Irritative and imflammative potential, 321 Oxygen, 17, 107, 121, 179, 201, 347, 445
Ozone, 107, 445
K
Klebsiella peumoniae, 251 P
Pear fruit, 457
Penicillium crustosum, 57
L Peri-implantits, 191
Lactobacillus acidophilus, 215 Peroxynitrite, 67
Laser scanning microscopy, 281 Phototherapy, 251
Lesion-403 Pisum arvense, 469
Low pressure, 121, 469 Plaque forming unit, 79
Lupinus angustifolius, 469 Plasma
non-thermal plasma, 3, 17, 31, 45, 57, 67,
79, 93, 107, 121, 135, 149, 163, 179,
M 191, 201, 215, 281, 293, 301, 311, 321,
Medical 335, 347, 381, 417, 445, 457, 469
medical device, 3 plasma bullets, 149, 335, 381
medical therapy, 311, 361, 381, 393, 403 plasma chemistry, 67
Melanoma cells, 403 plasma cloud, 149
Mercury lamp, 231 plasma decontamination (sterilization),
Mesofilic bacteria, 457 3, 17, 31, 45, 57, 79, 93, 121, 135,
Microcathode sustained discharge, 107 149, 163, 179, 191, 201, 215, 231,
Microorganisms, 3, 17, 31, 45, 57, 67, 79, 93, 251, 265, 281, 301, 311, 361, 393,
121, 135, 149, 163, 179, 191, 201, 215, 417, 445, 457, 469
231, 251, 265, 281, 301, 311, 381, 393, plasma dentistry, 179, 191, 201, 215
417, 445, 457, 469 plasma diagnostics, 45, 93, 121, 149, 163,
Microplasma, 107 201, 215, 231, 251, 265, 281, 311, 335,
microplasma jet, 17, 45, 107, 201 347, 445, 457
Microwave plasma, 3, 121, 163 plasma disinfection, 179, 281, 301, 393
Microwave plasma jet, 3, 163, 311 plasma gun, 381
Mould, 57, 231, 457, 469 plasma inactivation, 17, 31, 67, 79, 93,
121, 135, 149, 163, 179, 215, 231, 281,
311, 417, 445, 457, 469
N plasma jet, 3, 135, 179, 191, 301, 321, 335,
Nanosecond pulsed electric fields, 361 381, 393
Neon, 381 plasma-liquid interaction, 67
Nitrate, 67 plasma medicine, 57, 281, 293, 311, 321,
Nitric oxide (NO), 107, 121, 293, 393, 445 335, 347, 381
NO therapy, 393 plasma micro jet, 17, 107, 201
Nitrite, 67 plasma splitting, 381
Nitrogen, 121, 135, 417 plasma torch, 347
Non-thermal plasma, 3, 17, 31, 45, 57, 67, 79, plasma-water interaction, 31, 67, 403
93, 107, 121, 135, 149, 163, 179, 191, Polihexanide, 321
201, 215, 281, 293, 301, 311, 321, 335, Post discharge, 93
347, 381, 417, 445, 457, 469 Psoriasis, 251
Nucleotide damage, 17 Protein damage, 17, 79
484 Subject Index
Pseudomonas aeruginosa, 135, 163, 251 Spore, 57, 231, 265, 445
Pulsed spore inactivation, 31, 45, 231
pulsed discharge, 31, 45 Sporulation, 57
pulsed electric field, 361, 403 Staphylococcus aureus, 3, 163, 251, 301,
393, 417
Stemphilium, 469
Q Sterilization, 3, 57, 79, 121, 231, 265
Quality traits, 457 Streptococcus
Streptococcus mutans, 31, 191, 215
Streptococcus sanguinis, 191
R Superoxide anion (O2-), 201, 293
Radical, 31, 93, 201, 281 Surface cleaning, 265
Radiofrequency Surgical mesh, 417
radiofrequency (RF) Synergy, 31, 67, 191, 231, 321
electromagnetic field, 469
radiofrequency (RF) plasma, 469
radiofrequency (RF) plasma jet, T
3, 135, 321 Teeth, 31, 179, 191, 201, 215
Raman microscopy, 281 Temperature measurement, 31, 45, 135, 149,
Reactive 163, 215, 281, 301, 321, 335, 445, 457
reactive nitrogen species (RNS), Tissue, 293, 335
17, 67, 293 tissue model, 293
reactive oxygen species (ROS), 17, 31, 67, tissue tolerable plasma (TTP), 281, 321
107, 293, 311, 347, 381 Titanium implant, 191
Remote exposure, 93, 231 Transepithelial electrical resistance
Risk assessment, 281 (TEER), 321
Root canal disinfection, 179 Transmission electron microscopy (TEM), 135
RNA damage, 17 Triticum aestivum, 469
Tumor treatment, 361, 381, 403
S
Saliva, 179, 191 U
Salmonella Ultraviolet
Salmonella enteritidis, 457 ultraviolet (UV) irradiation, 231, 281
Salmonella typhimurium, 31 ultraviolet (UV) radiation, 17, 163
Sarcina, 251 ultraviolet (UV) sterilization, 231, 251, 265
Scanning electron microscopy (SEM), 121, Underwater plasma, 403
179, 191, 201, 215
Schlieren photography, 403
Sealed package, 445 V
Seed Vacuum ultraviolet (VUV) radiation, 17, 163,
seed germination, 469 231, 251, 265
seed treatment, 469 Viable-but-non-culturable (VBNC), 135
Separation of plasma agents, 17, 31 Virulence test, 135
Shock waves, 403
Silver functionalization, 417
Singlet delta oxygen, 107, 201 W
Skin Water, 31, 57, 67, 201, 403
skin diseases, 251 water decontamination, 31, 265
skin disinfection, 281, 301, 393 Wound healing, 311, 321, 335, 347, 393
Spark, 31, 149, 293, 347
AC spark, 347
transient spark, 31 Y
Spectral sensitivity, 231 Yeast, 201, 457

Plasma For Bio-Decontamination, Medicine and Food Security

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Plasma For Bio-Decontamination, Medicine and Food Security

Uploaded by

Copyright:

Available Formats

Plasma for Bio-Decontamination, Medicine

and Food Security

A. Chemistry and Biology Springer

Series A: Chemistry and Biology

Published in Cooperation with NATO Emerging Security Challenges Division

Library of Congress Control Number: 2011945683

ISBN 978-94-007-2909-4 (PB)

Printed on acid-free paper

All Rights Reserved

Plasmas, especially non-thermal plasmas maintained close to room temperature at

doubt that multidisciplinary approach of plasma physicists, microbiologists,

must be given to the implementation of plasma applications in food technology and

Zdenko Machala, Karol Hensel and Yuri Akishev, the editors.

List of Corresponding Authors ...................................................................... xv

Part I Plasma Bio-decontamination, Water Chemistry

1 Atmospheric Pressure Plasmas for Decontamination

6 Plasma-Liquid Interactions: Chemistry

Part II Plasma Biofilm Inactivation and Dentistry Applications

11 Battling Bacterial Biofilms with Gas Discharge Plasma ..................... 135

14 A Sub-microsecond Pulsed Plasma Jet for Endodontic

Part III Plasma-Based UV Sterilization

18 Features of the Sterilization by VUV/UV Irradiation

Part IV Plasma Tissue Treatment and Wound Healing

21 Antisepsis of the Skin by Treatment with Tissue-Tolerable

22 Cold Microsecond Spark Discharge Plasma Production

Part V Plasma and Electric Fields in Medicine

28 Subcellular Biological Effects of Nanosecond Pulsed

32 DBD Plasma Assisted Silver Functionalization

Part VI Plasma for Food Security

33 Prospects for Treating Foods with Cold Atmospheric

Subject Index ................................................................................................... 481

Yuri Akishev Low Temperature Plasma Department, SRC RF TRINITI, Troitsk,

Chunqi Jiang Department of Electrical Engineering Electrophysics, Viterbi

Victor F. Tarasenko Laboratory of Optical Radiation, High Current Electronics

Klaus-Dieter Weltmann, Jrn Winter, Martin Polak, Jrg Ehlbeck,

Abstract Atmospheric pressure plasma sources produce a multiplicity of different

Recent improvements in medical science mostly go along with the enhancement or

K.-D. Weltmann (*) J. Winter M. Polak J. Ehlbeck T. von Woedtke

1.2 Materials and Methods

Fig. 1.3 Schematic

1.3 Results and Discussion

1.3.1 Atmospheric Pressure Plasma Jet

treatment with air admixture results in a higher amount of microorganism-free sections,

1.3.2 Dielectric Barrier Discharge

5 carrier gas: 1.5 slm argon

maximum surviving CFU

spores using a contamination method described in [13]. Since the contamination of

1.3.3 Microwave Driven Discharge

Fig. 1.12 Photo of a microwave driven discharge used in this work

This autarkic property of microwave driven discharges allows an easy implementation

Jan-Wilm Lackmann, Simon Schneider, Franz Narberhaus, Jan Benedikt,

Abstract Atmospheric pressure plasma jets effectively inactivate bacteria on surfaces

J.-W. Lackmann F. Narberhaus J.E. Bandow (*)

Atmospheric pressure plasmas are known to be capable of inactivating bacteria. The

2.2 Separation of Plasma Radiation

2.3 Impact of Plasma and Its Components

2.4 Plasma Impact on Biological Macromolecules

complex machinery that produces enzymes based on genetic information unequivocally

2.4.1 DNA Damage by Plasma

controlled test conditions, it is paramount to compare such experiments with in vivo

2.4.2 Plasma Damage to RNA

damage by induction of single strand breaks or plasma-based etching due to

2.4.3 Protein Etching with the X-Jet