Clin Oral Invest (2015) 19:97107
DOI 10.1007/s00784-014-1224-3
ORIGINAL ARTICLE
In situ antimicrobial activity on oral biofilm: essential
oils vs. 0.2 % chlorhexidine
Victor Quintas & Isabel Prada-Lpez &
Juan Carlos Prados-Frutos & Inmaculada Toms
Received: 8 July 2013 / Accepted: 3 March 2014 / Published online: 1 April 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract
Objectives This study aims to evaluate the in situ antibacterial
activity of a mouthwash containing essential oils (M-EO) on
undisturbed de novo plaque-like biofilm (PL-biofilm) up to
7 h after its application.
Patients and methods An appliance was designed to hold six
glass disks on the buccal sides of the lower teeth, allowing PL-
biofilm growth. Fifteen healthy volunteers wore the appliance
for 48 h and then performed a M-EO. Disks were removed
after 30 s and at 1, 3, 5, and 7 h later. After a washout period,
the same procedure was repeated with a M-WATER and a
M-0.2 % chlorhexidine. After PL-biofilm vital staining, sam-
ples were analyzed using a confocal laser scanning
microscope.
Results At 30 s after M-EO, the levels of bacterial vitality
were 1.18 %, significantly lower than that of the basal sample
(p<0.001). After 7 h, the antibacterial effect of essential oils
was still patent with a 47.86 % difference in bacterial vitality
compared to the basal sample (p<0.001).
Conclusion A single M-EO presents high antibacterial imme-
diate activity and penetration capacity in situ and a
substantivity which lasts for at least 7 h after its application
over de novo biofilm. These results were better than those
observed with 0.2 % chlorhexidine under the same conditions.
Clinical relevance A single M-EO is an effective measure
against the de novo biofilm, presenting a good alternative to
V. Quintas : I. Prada-Lpez : I. Toms (*)
Oral Sciences Research Group, School of Medicine and Dentistry,
Santiago de Compostela University, C./ Entrerrios s/n,
15872 Santiago de Compostela, Spain
e-mail: inmaculada.tomas@usc.es
J. C. Prados-Frutos
Department of Stomatology, Faculty of Health Sciences, Rey Juan
Carlos University, Madrid, Spain
clorhexidine such as a preoperative rinse, in periodontal
procedures or post-treatment applications.
Keywords Essential oil . Chlorhexidine . Mouthwash .
Substantivity . Plaque-like biofilm . Laser scanning confocal
microscopy
Introduction
Nowadays, Listerine is the most popular combination of
essential oils [1] and represents the oldest antigingivitis and
antiplaque agent used clinically in dentistry. It is considered
safe and effective by the Committee of Experts in Oral Health
of the FDA [2]. Listerine contains a fixed combination of
four essential oils (EO) as the active ingredients (thymol
0.064 %, eucalyptol 0.092 %, methyl salicylate 0.060 %,
menthol 0.042 %). EO kill microorganisms by disrupting their
cell walls and inhibiting their enzymatic activity. They prevent
bacterial aggregation, slow down bacterial multiplication, and
extract endotoxins [3].
Recognizing that biofilm bacteria may be ten to 1,000
times more resistant to antimicrobial agents than planktonic
cells [4], a more predictive assessment of mouthwash efficacy
may be better achieved with biofilm tests. Studies have been
performed on the activity of essential oils on oral biofilm, both
in vitro and in situ. The former, using artificial models, have
helped us gain a better understanding of oral biofilms in spite
of not being predictive of clinical activity [46], as they
involve a limited number of species and they are conducted
under conditions which do not reflect the physiological oral
status [79]. For this reason, several authors have stated that
results obtained from this type of study must be carefully
interpreted [7, 10, 11].
With respect to in situ studies, they have greater value
when establishing the antiseptic efficacy of several
98
mouthwashes since their activity is tested under in vivo clin-
ical conditions [1216]. In this type of study, differences have
been found between those performed on disturbed dental
plaque [1214] and those performed on undisturbed dental
plaque [15, 16]. In the first type, the plaque is analyzed after
being removed from the dental surface [1214]. Due to this, it
is not possible to assess either the original architecture of the
plaque or the penetration power of the antibacterial agent.
Therefore, methodologies which permit biofilm formation
under real clinical conditions are needed so that plaque dis-
turbance is not necessary for analysis. As a consequence,
several types of removable devices capable of holding multi-
ple sorts of substratum have been devised. These results in a
biofilm which is presumably similar to dental plaque, gener-
ated under similar conditions and set over an artificial sub-
stratum; this has been called a plaque-like biofilm (PL-
biofilm).
Historically, various microscopy techniques have been
used to visualize the PL-biofilm microstructure, including
optic microscopy, transmission microscopy, and scanning
electron microscopy [17]. Samples are distorted using these
techniques, making correct analysis very difficult, especially
in fluid-filled structures [18]. These problems have been elim-
inated or at least reduced with confocal laser scanning
microcopy (CLSM). Its main advantage is permitting PL-
biofilm analysis without altering its delicate structure [19];
in addition, this technique facilitates observation in real
time. It also permits acquiring very thin optical sections
(0.52 m) and examining the XZ and XY relationships
existing between the bacteria and its environment, significant-
ly improving, at the same time, the lateral resolution [20].
In contrast to traditional microbial quantification methods,
systems based on fluorescence have gained increasing impor-
tance since they are accepted as a simple, precise, reproduc-
ible, and highly sensitive procedure for quantifying adhered
microorganisms [21]. Furthermore, although there is not a
standard classification of the different bacterial states of vital-
ity, the staining capacity of the dyes present in the live/dead
assays seems to match with the physiological state of the
bacteria, though there are still intermediate colors with un-
known interpretation [22].
As a result, fluorescence staining has been incorporated to
analyze biofilm structure and vitality using a wide variety of
dye combinations having been used in PL-biofilm studies in
situ [8, 2326] due to their ability to stain live and dead
bacteria in a selective manner. The combination of SYTO 9
and propidium iodide has been posed as a reliable alternative
[27] to traditional blend of fluorescein diacetate with ethidium
bromide, mainly because of the destructive properties of this
combination and the toxicity and instability of ethidium bro-
mide [27]. This staining method allows for a visual demon-
stration of bactericidal activity as well as its quantification
using computerized image analysis [12].
Clin Oral Invest (2015) 19:97107
There are few studies in the literature in which the effects of
essential oils on in situ undisturbed PL-biofilm have been
measured by applying CLSM together with bacterial vitality
techniques [16]. The aim of the present study was to evaluate
the in situ antibacterial activity of an essential oil mouthwash
on undisturbed de novo PL-biofilm up to 7 h after its appli-
cation using CLSM and a dual-stain fluorescence solution.
Patients and methods
This was a randomized, double-blind, crossover study of the
antibacterial efficacy of essential oils on an in situ model of
PL-biofilm growth.
Selection of the study group
The study group was composed of 15 systemically healthy
adult volunteers between 20 and 45 years old and who pre-
sented a good oral health status: a minimum of 24 permanent
teeth with no evidence of gingivitis or periodontitis (commu-
nity periodontal index score = 0) [28] and an absence of
untreated caries at the beginning of the study. The following
exclusion criteria were applied: smoker or former smoker,
presence of dental prostheses or orthodontic devices, antibi-
otic treatment or routine use of oral antiseptics in the previous
3 months, and presence of any systemic disease that could
alter the production or composition of the saliva. Professional
tooth cleaning was performed on all volunteers before starting
the study.
This project was approved (number 2012/394) by the
Clinical Research Ethics Committee of Galicia. Written in-
formed consent was obtained from all participants in the study.
Production of the Intraoral Device of Overlaid Disk-holding
Splints (IDODS)
After considering a number of previously described in situ
models [8, 19, 24, 25], an individualized splint of the lower
arch was created for each volunteer, which was able to hold
six glass disks (6 mm in diameter, 1 mm thickness) and
polished at 4,000 grit. The characteristics of this splint have
been previously described by other authors [29], although a
new variety of splint has now been introduced; the old one
was a complete individual inferior arch splint, which has now
been broken in two, going from the last molar to the
homolateral canine (Figs. 1 and 2). These splints are formed
with two sheets, an internal 1-mm soft vinyl sheet where the
disks are attached and an external 1-mm rigid one made of
polyethylene terephtalate that is fenestrated.
The splints with the glass disks were worn by the volun-
teers for 48 h to favor growth of the PL-biofilm, withdrawing
it from the oral cavity only during meals (it was stored in an
Clin Oral Invest (2015) 19:97107
Fig. 1 Design of the intraoral device of overlaid disk-holding splints
(partial model). 1 Inner splint in contact with the lower dental arch with
holes on the disk area. 2 Glass disk. 3 Outer splint with holes on the disk
area
opaque container in humid conditions) and to perform oral
hygiene procedures, using only mechanical removal of bacte-
rial plaque with water without the use of any toothpaste or
mouthwash.
Application of the essential oil to PL-biofilm
After 48 h, the glass disks were withdrawn one by one from
the splint from each volunteer (from right to left in a distal
mesial direction) at baseline, 30 s, and 1, 3, 5, and 7 h after
performing the following mouthwashes under supervision:
1. A single, 30-s mouthwash with 20 mL of sterile water
(negative control) (M-WATER) or
2. A single, 30-s mouthwash with 10 mL of 0.2 % chlorhex-
idine (Oraldine Perio, Johnson and Johnson, Madrid,
Spain) (positive control) (M-0.2 % CHX) or
3. A single, 30-s mouthwash with 20 mL of essential oils in
a hydroalcoholic solution (Listerine Menthol, Listerine,
Johnson & Johnson, Madrid, Spain) (M-EO)
On the day of the experiment, the volunteers were not
allowed to eat or drink during the course of the tests.
Collection of the different PL-biofilm samples started at
11:50 AM (baseline sample) and finished at 7:00 PM (the final
sample was obtained 7 h after performing the mouthwash).
Using an Internet-based balanced randomization system
(Dallal GE www.randomization.com) indicating the
mouthwash each subject would use first, second, and third,
all volunteers performed the three mouthwashes, with a rest
period of 2 weeks between each test.
Fig. 2 Clinical images of the
partial splint model
99
Processing of the samples of PL-biofilm
The characteristics of LIVE/DEAD BacLight fluorescence
solution (Molecular Probes, Leiden, The Netherlands), as well
as its preparation, have been described by authors in a previ-
ous study [30]. The glass disks were withdrawn from the
splint and were immediately submerged in 100 L of fluores-
cence solution and kept in a dark chamber at room tempera-
ture for 15 min. Microscopic observation was performed by a
single investigator, who was unaware of the study design,
using a Leica TCS SP2 laser scanning spectral confocal mi-
croscope (Leica Microsystems Heidelberg GmbH,
Mannheim, Germany) with an HCX APOL 63x/0.9 water
immersion lens.
Four selected fields or XYZ series in the central part of
each disk were evaluated. These fields were considered as
representative of the whole sample after the observers general
examination. Fluorescence emission was determined in series
of XY images in which each image corresponded to each of
the Z positions (depth). The optical sections were scanned in
1-m sections from the surface of the biofilm to its base,
measuring the maximum thickness of the field and subse-
quently the mean thickness of the biofilm of the corresponding
sample. The maximum thickness of biofilm field was defined
as the distance between the substrate and the peaks of the
highest cell clusters [31]. The maximum biofilm thickness of
each field was divided into three zones or equivalent layers:
outer layer (layer 1), middle layer (layer 2), and inner layer
(layer 3).
Quantification of bacterial vitality in the series of XY
images was determined using cytofluorographic analysis
(Leica confocal software). In this analysis, the images of each
fluorochrome were defined as channels (SYTO 9 occupies
the green channel and PI the red channel), obtaining values for
the area (m2) occupied by each channel, the total area
occupied by the biofilm, and the corresponding percentage
of vitality. Determination of the mean percentage of bacterial
vitality in each field required sections with a minimum area of
biofilm of 250 m2, and the mean percentage of bacterial
vitality of the biofilm was calculated for the corresponding
sample and for each biofilm layer.
Statistical analysis
The results were analyzed using the PASW Statistics Base
18 package for Windows (IBM, Madrid, Spain). Data on
100
thickness and bacterial vitality in PL-biofilms are expressed as
mean and standard deviation of the mean. All values from the
quantitative variables analyzed (biofilm thickness and bac- lence of live bacteria was significantly lower in the middle and
terial vitality percentage) presented a normal distribution,
which was determined using the KolmogorovSmirnov test.
One-way ANOVA with repeated measures was used for intra-
mouthwash comparisons using all the PL-biofilm samples.
Two-way ANOVA with repeated measures was used for
intra-mouthwash (differentiating between the three biofilm
layers) and inter-mouthwash comparisons using all the PL-
biofilm samples. Three-way ANOVA with repeated measures
was used for inter-mouthwash (differentiating between the
three biofilm layers) comparisons using all the PL-biofilm
samples. Pairwise comparisons (with Bonferroni adjustment)
were used for the analysis of intra- and inter-mouthwash
results (including differentiating between the three biofilm
layers). Statistical significance was taken as a p value less
than 0.05.
Results
Influence of 0.2 %-CHX and M-EO on PL-biofilm thickness
The mean PL-biofilm thickness at baseline was 22.1 m
(range 1228 m). Significant differences were not found
over time after M-EO with regard to basal thickness. On the
other hand, after M-0.2 % CHX, lower PL-biofilm thickness
values were obtained in comparison to both the basal thick-
ness and the M-EO thickness (Table 1).
Influence of 0.2 %-CHX and M-EO on PL-biofilm bacterial
vitality
The basal vitality in PL-biofilm was 73.6 % (4494 %). The
M-WATER mouthwash did not have any significant effect on
PL-biofilm vitality compared to the basal level. The results
after M-0.2 % CHX and M-EO showed significant differences
compared to their respective basal levels from 30 s after
mouthwash use to 7 h later (Fig. 3). In comparison with the
values obtained, 30 s after M-0.2 % CHX and M-EO, a
significant recovery of the bacterial population was observed
in the later PL-biofilm samples (after 3 and 5 h, respectively).
Comparing M-0.2 % CHX and M-EO, M-EO presented lower
percentages of bacterial vitality up to 7 h after application,
obtaining significant differences from 1 to 5 h post-
mouthwash (Fig. 4).
Differentiating between the three biofilm layers, the prev-
alence of live bacteria under basal conditions was higher in the
outer layers with respect to deeper layers, reaching statistical
significance in the majority of comparisons (Table 2).
In comparison with M-WATER, the prevalence of live
bacteria was significantly lower in the three biofilm layers in
Clin Oral Invest (2015) 19:97107
all the biofilm samples taken after M-EO (p<0.05 in all
comparisons). In comparison with M-0.2 % CHX, the preva-
inner layers from 1 h after mouthwash use to 7 h later
(Table 2).
Discussion
Methodological approach
There has been marked inter-individual variability detected
regarding the characteristics of PL-biofilm [7, 18, 19, 26, 32].
In the present study, a sample group of 15 individuals was
selected. This sample group is bigger than in similar studies in
which the number of volunteers ranged from 3 to 10 [15, 16,
19, 33, 34]. With regard to the type of removable appliance
used to collect the supragingival dental plaque, devices such
as the Leeds in situ device [9, 18, 35, 36], bilateral mandibular
stents [32, 37, 38], and different types of individualized acryl-
ic splints [7, 8, 10, 19, 23, 25] have been previously described.
In the present series, two individualized splints formed from
two sheets were designed for each volunteer. The splint was
composed of an internal vinyl sheet to which three disks were
attached, with an external polyethylene terephtalate sheet that
was fenestrated to permit contact between the vestibular sur-
face of the disks and the saliva whilst protecting the surface
from the action of the cheeks and tongue. The disks were
positioned on each hemiarch and inserted towards the inter-
dental area between two adjacent teeth in order to imitate an
approximate PL-biofilm, which is only minimally influenced
by the shear forces of the oral soft tissues. This particular
design ensured that the biofilm was not touched or disturbed
during removal or repositioning of the appliance [29]. In
contrast to previous designs [7, 8, 19], where the splints
referred to a complete model, the partial model of IDODS
represents a new approach in the way of making a better in situ
biofilm model. This redesign was more comfortable for the
participants when talking and wearing the splints due to the
fact that their incisors were not covered. At the same time, the
extraction of the disks was easier because it was not necessary
to remove the whole inferior arch splint but only the hemiarch
corresponding to the analysis, keeping the other undisturbed.
A number of solid substrates of different characteristics
have been used in published studies on PL-biofilm, including
human enamel [18, 24, 25, 38], bovine enamel [10, 23, 34],
bovine dentine [26, 34], hydroxyapatite [15], polished glass
[7, 8, 19, 24], and titanium [16]. Although the roughness of
the surface of the substrate and its free energy are considered
to be important factors for the in vivo growth of PL-biofilm
[7], Netuschil et al. [24] found no major differences in the
thickness of 2-day PL-biofilm using enamel or glass disks; on
the other hand, due to the known autofluorescence of enamel,
Table 1 Measurement of PL-Biofilm thickness, as well as intra-mouthwash and inter-mouthwash comparisons, before the different mouthwashes (baseline) and after (30 seconds, 1 hour, 3 hours, 5 hours,
and 7 hours)

PL-Biofilm THICKNESS MeanStandard Deviation (m)

BASAL 30 SEC 1H 3H 5H 7H
M-WATER 19.35.4 18.02.6 22.35.4 21.05.1 23.94.8 23.93.9
M-0.2 % CHX 23.48.3 15.81.9 13.52.5 15.42.9 17.53.9 15.62.3
M-EO 23.64.7 20.93.6 20.24.3 21.04.5 18.84.4 21.62.8
INTRA-MOUTHWASH ANALYSIS
Clin Oral Invest (2015) 19:97107

BASAL vs. 30 SEG BASAL vs. 1 H 30 SEC vs. 1 H BASAL vs. 3 H 30 SEC vs. 3 H BASAL vs. 5 H 30 SEC vs. 5 H BASAL vs. 7 H 30 SEC vs. 7 H
M-WATER p<0.05 p<0.05 p<0.05
M-0.2 % CHX p<0.05 p<0.05 p<0.05
M-EO
INTER-MOUTHWASH ANALYSIS
BASAL 30 SEC 1H 3H 5H 7H
M-WATER vs. M-EO p<0.05
M-0.2 % CHX vs. M-EO p<0.001 p<0.001 p<0.05 p<0.001

Not a statistically significant difference.

M-WATER=a single, 30-second mouthwash with 20 mL of sterile water; M-0.2 % CHX=a single, 30-second mouthwash with 10 mL of 0.2 % chlorhexidine; M-EO=a single, 30-second mouthwash with
20 mL of essential oil solution.
BASAL=Biofilm sample collected under basal conditions; 30 SEC=Biofilm sample collected 30 seconds after the application of the different mouthwashes; 1 H=Biofilm sample collected 1 hour after the
application of the different mouthwashes; 3 H=Biofilm sample collected 3 hours after the application of the different mouthwashes; 5 H=Biofilm sample collected 5 hours after the application of the
different mouthwashes; 7 H=Biofilm sample collected 7 hours after the application of the different mouthwashes.
101
102
Fig. 3 Representative images of the PL-biofilm bacterial vitality under basal
conditions and at 30 s and 7 h after the application of different mouthwashes.
ac Basal samples collected before different mouthwashes with M-WATER,
using glass is recommended to avoid any optical disturbance,
mainly in the deepest layers of the biofilm [24]. On the basis
of these findings, in the present series, glass disks were used
for in vivo growth of the 2-day PL-biofilm.
In a recent paper, Tawakoli et al. [27] stated that the
BacLight system was a reliable alternative when assessing
bacterial vitality in a 120-min-old natural dental biofilm, in
which there are already several types of bacteria present. The
LIVE/DEAD BacLight fluorescence assay stains the bac-
teria in red or green depending on the permeability of their
membrane; given that the tested antiseptics act mostly at this
cellular element, this vital staining method is suitable for this
type of study.
Furthermore, Hannig et al. [39] considered that live/dead
staining methods were reliable when analyzing antimicrobial
Clin Oral Invest (2015) 19:97107
M-0.2 % CHX, and M-EO, respectively. df Images taken 30 s after M-
WATER, M-0.2 % CHX, and M-EO, respectively. gh Images taken 7 h
after M-WATER, M-0.2 % CHX, and M-EO, respectively
agents activity, although they continued to ask the question
how dead is dead? due to several stages of vitality which
have been discussed and described in the literature (viable and
culturable, viable but non-culturable, dormant, non-viable and
pre-lytic, and avital dead bacteria). The exact differentiation of
these stages is still one of the greatest challenges in modern
microbiology [40].
In addition, Tawakoli et al. [27] said that it was not possible
to compare properly the vitality assessed with fluorescence
staining solutions with traditional plaque cultures because of
the widely known limitations of this later method (among
others, only 50 % of oral bacteria are culturable), which
emphasizes the necessity of using vitality assays. On the other
hand, the authors recognize the convenience of contrasting
and complementing the data obtained with BacLight
Clin Oral Invest (2015) 19:97107
Fig. 4 PL-biofilm bacterial vitality percentage under basal conditions and at 30 s and 1, 3, 5, and 7 h after a single water mouthwash (M-WATER), 0.2 %
chlorhexidine mouthwash (M-0.2% CHX), or essential oil (M-EO)
fluorescence solution with other molecular or bacteriological
techniques such as the plate efficiency.
M-EO: immediate effect, substantivity, and influence
on PL-biofilm thickness
Studies which have analyzed in situ PL-biofilm have empha-
sized the great variation detected in PL-biofilm thickness
between individuals [7, 25, 33]; this was also observed in
the present study (mean value of PL-biofilm thickness after
2 days was 22.10 m, ranging from 12 to 28 m), indicating
that, in agreement with previous studies, the height of the oral
biofilms formed depended on the plaque-forming rate of the
individual donors [25]. Our mean value of PL-biofilm was
consistent with that obtained by Dong et al. [15] under similar
conditions, which was 27.55 m.
In the present series, the bacterial vitality of PL-biofilm was
approximately 73 %. These results are consistent with previ-
ous studies which reported mean bacterial vitality of PL-
biofilm between 60 and 77 % over 2- and 3-day periods [8,
19, 41]. Consequently, vital micro-organisms were located on
103
and embedded in dead layers, which may be responsible for
further plaque growth [24].
In some series, large inter-individual differences were
found among the subjects in their PL-biofilm vitality distribu-
tion [26], so no general pattern for the bacterial vitality distri-
bution could be described [26, 41]; in the present study, the
PL-biofilm vitality ranged from 44 to 90 %. However, it has
been suggested that a relatively constant ecological environ-
ment exists in each volunteer, which obviously leads to a
microbial identity pattern [19]. In this sense, Arweiler et al.
[19] detected great variation in the bacterial vitality values in a
2-day PL-biofilm for the different biofilm layers, identifying
three vitality patterns. In this study, despite the high degree of
variability detected in the bacterial vitality distribution, a
vitality pattern could be identified, which was based on a
low vitality percentage observed in the layers nearest to the
substrate, increasing in higher layers. This finding confirms
the importance of the dead cellular material in the initial states
of PL-biofilm development. This will particularly help its
growth, and this material will protect it from antibacterial
agents in the oral cavity [24, 25].
104 Clin Oral Invest (2015) 19:97107

Table 2 Mean percentages of bacterial vitality in PL-biofilm under basal between the three biofilm layers, as well as intra-mouthwash and inter-
conditions and in the samples collected at 30 s and 1, 3, 5, and 7 h after a mouthwash comparisons
single mouthwash of sterile water and 0.2 % chlorhexidine differentiating

BASAL 30 SEC 1H 3H 5H 7H

PL-biofilm bacterial vitality divided in layers, meanstandard deviation (%)

M-WATER
Layer 1 85.46.6 85.313.1 88.39.6 90.88.9 89.08.5 90.95.9
Layer 2 79.87.3 73.115.1 78.416.6 84.410.3 81.312.8 85.27.1
Layer 3 66.827.3 45.833.4 49.429.8 56.731.5 55.424.9 62.024.0
M-0.2 %CHX
Layer 1 80.06.2 5.216.2 15.115.4 35.415.5 21.719.8 27.022.6
Layer 2 82.27.8 5.066.4 16.515.6 36.716.4 24.820.6 28.720.8
Layer 3 71.817.4 5.05.0 15.210.2 35.214.8 27.513.5 40.020.4
M-EO
Layer 1 78.710.7 1.71.6 5.04.3 13.714.0 15.813.2 37.519.1
Layer 2 69.016.5 1.11.2 2.93.3 6.010.2 5.45.3 12.59.9
Layer 3 49.136.0 0.70.6 4.16.8 5.08.2 3.67.0 3.22.9
Intra-mouthwash analysis
M-WATER
Layer 1 vs. Layer 2 p<0.05 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001
Layer 1 vs. Layer 3 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001
Layer 2 vs. Layer 3 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001
M-CHX 0.2 %
Layer 1 vs. Layer 2
Layer 1 vs. Layer 3 p<0.05
Layer 2 vs. Layer 3 p<0.05 p<0.05
M-EO
Layer 1 vs. Layer 2 p<0.05 p<0.05 p<0.05 p<0.001 p<0.05 p<0.001
Layer 1 vs. Layer 3 p<0.05 p<0.05 p<0.001
Layer 2 vs. Layer 3 p<0.05 p<0.05
Inter-mouthwash analysis
M-WATER vs. M-EO
Layer 1 vs. Layer 1 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001
Layer 2 vs. Layer 2 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001
Layer 3 vs. Layer 3 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001
M-CHX 0.2 % vs. M-EO
Layer 1 vs. Layer 1 p<0.05
Layer 2 vs. Layer 2 p<0.05 p<0.05 p<0.001 p<0.05
Layer 3 vs. Layer 3 p<0.05 p<0.05 p<0.001 p<0.001 p<0.001

not a statistically significant difference, M-WATER a single, 30-s mouthwash with 20 mL of sterile water, M-0.2% CHX a single, 30-s mouthwash with
10 mL of 0.2 % chlorhexidine, M-EO a single, 30-s mouthwash with 20 mL of essential oil solution, BASAL biofilm sample collected under basal
conditions, 30 SEC biofilm sample collected 30 s after the application of the different mouthwashes, 1 H biofilm sample collected 1 h after the application
of the different mouthwashes, 3 H biofilm sample collected 3 h after the application of the different mouthwashes, 5 H biofilm sample collected 5 h after
the application of the different mouthwashes, 7 H biofilm sample collected 7 h after the application of the different mouthwashes, Layer 1 outer layer,
Layer 2 middle layer, Layer 3 inner layer

Although some papers have been published on EO antimi-
crobial activity on biofilm in vitro [36, 42, 43], studies on the
in situ effects of EO on PL-biofilm applying CLSM and
bacterial vitality techniques are very scarce. In this study, the
results with M-0.2 % CHX were taken as a positive control
since our research group has deeply analyzed and discussed
0.2 % CHX antimicrobial activity on in situ PL-biofilm in
previous publications.
Up to now, there have been few papers which have studied
the in situ antimicrobial effect on biofilm after a single
Clin Oral Invest (2015) 19:97107
application of EO. In two cases, the treatment was practiced
ex vivo, which means that no mouthwash was done [15, 16].
In the other three [1214], the studied plaque was completely
disturbed by the recollection method (paper points or cu-
rettes). Furthermore, only one paper has been published in
which the authors compared EO and CHX antimicrobial
activity after a single application (application ex vivo), and
they only measured the immediate antimicrobial effect [16].
Consequently, the present results have been compared with
those obtained in other studies that applied different
methodologies.
Immediate effect and substantivity
With a similar basal vitality between the three mouthwashes
(mean of 73.6 %), a clear and immediate post-mouthwash
effect was detected after EO application. This immediate
activity was very high if compared to that obtained in a similar
study reported by Gosau et al. [16] since, in the previous
study, bacterial vitality after M-EO was around 20 %, while
in the present series, the vitality was 1 % 30 s after M-EO. The
methodological differences should be noted; in the previous
case, ex vivo disk immersion was performed, while in the
present series, the volunteers themselves used the mouthwash,
providing in vivo antiseptic application. This is evidently a
more reliable approximation of the clinical situation.
Therefore, it was found that moving the essential oil solution
around the mouth and the force imposed by the cheeks when
projecting it onto the PL-biofilm is probably of prime impor-
tance for reducing bacterial biofilm vitality. In the same way,
this could be also related to improved deep penetration com-
pared to when the mouthwash was not actively applied. As
expected, the negative control (M-WATER) had no antibacte-
rial activity compared to M-EO. In comparison with the
positive control (M-0.2 % CHX), a single application of M-
EO was more effective, consistent with previous results ob-
tained by other groups [3, 16].
In the present series, EO antimicrobial activity was detect-
able until 7 h after mouthwash application, when the reduction
in vitality was still 61 %. In this study, it was also appreciated
that a single M-EO application was effective for maintaining
low bacterial vitality levels in PL-biofilm. As shown in Fig. 4,
there were no statistical differences in bacterial vitality until
5 h compared to the mouthwash sample after 30 s, which
indicates powerful antibacterial activity at high levels until
that moment. Up to now, there have been two studies on the
substantivity of essential oils in oral biofilm. The first found
21.3 % vitality 30 min post-mouthwash [12], while the other,
conducted by Fine et al. [14], showed a vitality reduction of
88 % after 12 h. Both studies used a disturbed dental plaque
in vivo model, so these results are not fully comparable.
Compared with the positive control, there were significant
differences from 1 to 5 h post-mouthwash, and the EO
105
mouthwash was more effective at maintaining low values of
bacterial vitality. These findings are not described in the
available literature due to the lack of studies measuring the
substantivity of EO in oral biofilm in comparison with CHX.
Another interesting aspect of this study is the higher pen-
etration capacity into the PL-biofilm of EO compared to 0.2 %
CHX. Statistical differences were found in bacterial vitality
reduction in layer 3 (the deepest one) between both antisep-
tics, starting from the immediate and 1-h samples, but more so
when time passed and until 7 h post-mouthwash. This indi-
cates that the penetration capacity of a single M-EO applica-
tion is greater than that shown by a single application of
M-0.2 % CHX. As Pan et al. [12] previously described, these
results confirm the ability of the essential oil mouthwash to
rapidly penetrate plaque and exert its bactericidal activity in
situ. Apart from that, the M-EO maintains its antimicrobial
activity in the deepest layers (closest to the theoretical tooth
surface) for a longer time.
There have been other in vivo studies demonstrating the
efficacy of a single mouthwash application in other oral eco-
systems, such as the saliva. It has been shown that a single M-
EO or M-0.2 % CHX application can significantly reduce the
levels of recoverable salivary bacteria compared to negative
control mouthwashes for periods of 57 h [31, 44]. Both
studies showed higher bacterial vitality than in PL-biofilm,
in comparison with the present series, so a longer antiseptic
(both EO and 0.2 % CHX) substantivity period was detected
in the PL-biofilm.
Thickness reduction
As the results of the present series show, a single M-EO
application is not effective in reducing PL-biofilm thickness.
These results are consistent with those previously described
by Dong et al. [15], who did not find significant differences in
biofilm thickness with regard to the basal sample after apply-
ing M-EO. These results are also consistent with an in vitro
study conducted by Sliepen et al. [3] who concluded that EO
caused nearly no changes in biofilm structure, thickness, and
surface coverage. With respect to M-0.2 % CHX, statistically
significant differences were found compared to M-EO at 30 s
and 1, 3, and 7 h after mouthwash use, which could suggest a
possible antiplaque effect of 0.2 % CHX after a single anti-
septic application.
Moreover, the group of Charles [13] concluded that the
clinical effectiveness of a single M-EO application against
plaque and gingivitis may be attributable to its bactericidal
effect and penetration into the dental plaque. Subsequently,
other authors demonstrated that performing daily mouth-
washes with an EO solution has a considerable antiplaque
effect [45, 46]. However, two recent literature reviews [47,
48] concluded that daily essential oil mouthwash use has a
lower antiplaque effect than using 0.2 % CHX, although the
106
gingival inflammation levels were quite similar in the long
and short term. Based on these findings, it would be very
interesting to analyze the antiplaque effect associated with
continuous EO use in a 4-day in situ undisturbed PL-
biofilm model as the next step in this research.
Conclusion
A single application of essential oil mouthwash presents high
antibacterial immediate activity and penetration capacity in
situ and substantivity which lasts, at least, for 7 h after its
application over de novo biofilm. These results are better than
those observed with 0.2 % chlorhexidine under the same
conditions.
Acknowledgments This work was supported by project PI11/01383
from Carlos III Institute of Health (General Division of Evaluation and
Research Promotion, Madrid, Spain), which is integrated in National Plan
of Research, Development and Innovation (PN I + D + I 2008-2011). This
project was co-financed by European Regional Development Fund
(ERDF 20072013). The funders had no role in the study design, data
collection and analysis, decision to publish, or preparation of the
manuscript.
Conflict of interest The authors declare that they have no conflict of
interest.
References
1. Vlachojannis C, Winsauer H, Chrubasik S (2012) Effectiveness and
safety of a mouthwash containing essential oil ingredients. Phytother
Res 27(5):685691. doi:10.1002/ptr.4762
2. FDA (2003) Oral health care drug products for over-the-counter
human use; antigingivitis/ antiplaque drug products; establishment
of a monograph; proposed rules part III. Vol 21 CFR
3. Sliepen I, Van Essche M, Quirynen M, Teughels W (2010) Effect of
mouthrinses on Aggregatibacter actinomycetemcomitans biofilms in
a hydrodynamic model. Clin Oral Investig 14(3):241250. doi:10.
1007/s00784-009-0286-0
4. Fine DH, Furgang D, Barnett ML (2001) Comparative antimicrobial
activities of antiseptic mouthrinses against isogenic planktonic and
biofilm forms of Actinobacillus actinomycetemcomitans. J Clin
Periodontol 28(7):697700. doi:10.1034/j.1600-051x.2001.
028007697.x
5. Filoche SK, Coleman MJ, Angker L, Sissons CH (2007) A fluores-
cence assay to determine the viable biomass of microcosm dental
plaque biofilms. J Microbiol Methods 69(3):489496
6. Pan PC, Harper S, Ricci-Nittel D, Lux R, Shi W (2010) In-vitro
evidence for efficacy of antimicrobial mouthrinses. J Dent 38(Suppl
1):S16S20. doi:10.1016/S0300-5712(10)70006-3
7. Auschill TM, Hellwig E, Sculean A, Hein N, Arweiler NB (2004)
Impact of the intraoral location on the rate of biofilm growth. Clin
Oral Investig 8(2):97101. doi:10.1007/s00784-004-0255-6
8. Auschill TM, Hein N, Hellwig E, Follo M, Sculean A, Arweiler NB
(2005) Effect of two antimicrobial agents on early in situ biofilm
formation. J Clin Periodontol 32(2):147152. doi:10.1111/j.1600-
051X.2005.00650.x
Clin Oral Invest (2015) 19:97107
9. Watson PS, Pontefract HA, Devine DA, Shore RC, Nattress BR,
Kirkham J, Robinson C (2005) Penetration of fluoride into natural
plaque biofilms. J Dent Res 84(5):451455
10. Al-Ahmad A, Wunder A, Auschill TM, Follo M, Braun G, Hellwig
E, Arweiler NB (2007) The in vivo dynamics of Streptococcus spp.,
Actinomyces naeslundii, Fusobacterium nucleatum and Veillonella
spp. in dental plaque biofilm as analysed by five-colour multiplex
fluorescence in situ hybridization. J Med Microbiol 56(Pt 5):681
687. doi:10.1099/jmm.0.47094-0
11. Hannig C, Hannig M (2009) The oral cavitya key system to
understand substratum-dependent bioadhesion on solid surfaces in
man. Clin Oral Investig 13(2):123139. doi:10.1007/s00784-008-
0243-3
12. Pan P, Barnett ML, Coelho J, Brogdon C, Finnegan MB (2000)
Determination of the in situ bactericidal activity of an essential oil
mouthrinse using a vital stain method. J Clin Periodontol 27(4):256
261. doi:10.1034/j.1600-051x.2000.027004256.x
13. Charles CH, Pan PC, Sturdivant L, Vincent JW (2000) In vivo
antimicrobial activity of an essential oil-containing mouthrinse on
interproximal plaque bacteria. J Clin Dent 11(4):9497
14. Fine DH, Furgang D, Sinatra K, Charles C, McGuire A, Kumar LD
(2005) In vivo antimicrobial effectiveness of an essential oil-
containing mouth rinse 12 h after a single use and 14 days use. J
Clin Periodontol 32(4):335340. doi:10.1111/j.1600-051x.2005.
00674.x
15. Dong WL, Zhou YH, Li CZ, Liu H, Shang SH, Pan BQ (2010)
Establishment and application of an intact natural model of human
dental plaque biofilm. Shanghai Kou Qiang Yi Xue 19(2):196201
16. Gosau M, Hahnel S, Schwarz F, Gerlach T, Reichert TE, Burgers R
(2010) Effect of six different peri-implantitis disinfection methods on
in vivo human oral biofilm. Clin Oral Implants Res 21(8):866872.
doi:10.1111/j.1600-0501.2009.01908.x
17. Hannig C, Hannig M, Rehmer O, Braun G, Hellwig E, Al-Ahmad A
(2007) Fluorescence microscopic visualization and quantification of
initial bacterial colonization on enamel in situ. Arch Oral Biol 52(11):
10481056
18. Wood SR, Kirkham J, Marsh PD, Shore RC, Nattress B, Robinson C
(2000) Architecture of intact natural human plaque biofilms studied
by confocal laser scanning microscopy. J Dent Res 79(1):2127. doi:
10.1177/00220345000790010201
19. Arweiler NB, Hellwig E, Sculean A, Hein N, Auschill TM (2004)
Individual vitality pattern of in situ dental biofilms at different loca-
tions in the oral cavity. Caries Res 38(5):442447. doi:10.1159/
000079625
20. Wright SJ, Wright DJ (2002) Introduction to confocal microscopy.
Methods Cell Biol 70:185
21. Hahnel S, Rosentritt M, Burgers R, Handel G (2008) Surface prop-
erties and in vitro Streptococcus mutans adhesion to dental resin
polymers. J Mater Sci Mater Med 19(7):26192627. doi:10.1007/
s10856-007-3352-7
22. Berney M, Hammes F, Bosshard F, Weilenmann HU, Egli T (2007)
Assessment and interpretation of bacterial viability by using the
LIVE/DEAD BacLight Kit in combination with flow cytometry.
Appl Environ Microbiol 73(10):32833290. doi:10.1128/AEM.
02750-06
23. Arweiler NB, Lenz R, Sculean A, Al-Ahmad A, Hellwig E, Auschill
TM (2008) Effect of food preservatives on in situ biofilm formation.
Clin Oral Investig 12(3):203208. doi:10.1007/s00784-008-0188-6
24. Netuschil L, Reich E, Unteregger G, Sculean A, Brecx M (1998) A
pilot study of confocal laser scanning microscopy for the assessment
of undisturbed dental plaque vitality and topography. Arch Oral Biol
43(4):277285. doi:10.1016/S0003-9969(97)00121-0
25. Auschill TM, Arweiler NB, Netuschil L, Brecx M, Reich E, Sculean
A (2001) Spatial distribution of vital and dead microorganisms in
dental biofilms. Arch Oral Biol 46(5):471476. doi:10.1016/S0003-
9969(00)00136-9
Clin Oral Invest (2015) 19:97107
26. Zaura-Arite E, van Marle J, ten Cate JM (2001) Confocal microscopy
study of undisturbed and chlorhexidine-treated dental biofilm. J Dent
Res 80:14361440. doi:10.1177/00220345010800051001
27. Tawakoli PN, Al-Ahmad A, Hoth-Hannig W, Hannig M, Hannig C
(2013) Comparison of different live/dead stainings for detec-
tion and quantification of adherent microorganisms in the
initial oral biofilm. Clin Oral Investig 17(3):841850. doi:10.
1007/s00784-012-0792-3
28. WHO (1997) Oral health surveys, basic methods, 4th edn. WHO,
Geneva
29. Toms I HB, Diz P, Donos N (2010) In vivo oral biofilm analysis by
confocal laser scanning microscopy: methodological approaches. In:
A M-V (ed) Microscopy. Science, technology, applications and edu-
cation. Formatex, Badajoz (Spain), pp 597-606
30. Toms I, Garca-Caballero L, Cousido MC, Limeres J, lvarez M,
Diz Dios P (2009) Evaluation of chlorhexidine substantivity on
salivary flora by epifluorescence microscopy. Oral Dis 15(6):428
433. doi:10.1111/j.1601-0825.2009.01570.x
31. Roberts SKBC, Brading M, Lappin-Scott H, Stoodley P (1999)
Biofilm formation and structure; whats new? In: Newman HNWM
(ed) Dental plaque revisitedoral biofilms in health and disease.
BioLine, Cardiff, pp 136
32. Diaz PI, Chalmers NI, Rickard AH, Kong C, Milburn CL, Palmer RJ
Jr, Kolenbrander PE (2006) Molecular characterization of subject-
specific oral microflora during initial colonization of enamel. Appl
Environ Microbiol 72(4):28372848. doi:10.1128/AEM.72.4.2837-
2848.2006
33. Dige I, Nyengaard JR, Kilian M, Nyvad B (2009) Application of
stereological principles for quantification of bacteria in intact dental
biofilms. Oral Microbiol Immunol 24(1):6975. doi:10.1111/j.1399-
302X.2008.00482.x
34. Jung DJ, Al-Ahmad A, Follo M, Spitzmuller B, Hoth-Hannig W,
Hannig M, Hannig C (2010) Visualization of initial bacterial coloni-
zation on dentine and enamel in situ. J Microbiol Methods 81(2):
166174. doi:10.1016/j.mimet.2010.03.002
35. Wood S, Nattress B, Kirkham J, Shore R, Brookes S, Griffiths J,
Robinson C (1999) An in vitro study of the use of photodynamic
therapy for the treatment of natural oral plaque biofilms formed
in vivo. J Photochem Photobiol B 50(1):17
36. Robinson C, Strafford S, Rees G, Brookes SJ, Kirkham J, Shore RC,
Watson PS, Wood S (2006) Plaque biofilms: the effect of chemical
environment on natural human plaque biofilm architecture. Arch Oral
Biol 51(11):10061014. doi:10.1016/j.archoralbio.2006.04.010
37. Palmer RJ Jr, Gordon SM, Cisar JO, Kolenbrander PE (2003)
Coaggregation-mediated interactions of streptococci and actinomy-
ces detected in initial human dental plaque. J Bacteriol 185(11):
34003409. doi:10.1128/JB.185.11.3400-3409.2003
107
38. Chalmers NI, Palmer RJ Jr, Du-Thumm L, Sullivan R, Shi W,
Kolenbrander PE (2007) Use of quantum dot luminescent probes to
achieve single-cell resolution of human oral bacteria in biofilms. Appl
Environ Microbiol 73(2):630636. doi:10.1128/AEM.02164-06
39. Hannig C, Follo M, Hellwig E, Al-Ahmad A (2010) Visualization of
adherent micro-organisms using different techniques. J Med
Microbiol 59(Pt 1):17. doi:10.1099/jmm.0.015420-0
40. Decker EM (2001) The ability of direct fluorescence-based, two-
colour assays to detect different physiological states of oral strepto-
cocci. Lett Appl Microbiol 33(3):188192. doi:10.1046/j.1472-765x.
2001.00971.x
41. von Ohle C, Gieseke A, Nistico L, Decker EM, DeBeer D, Stoodley
P (2010) Real-time microsensor measurement of local metabolic
activities in ex vivo dental biofilms exposed to sucrose and treated
with chlorhexidine. Appl Env Microbiol 76(7):23262334. doi:10.
1128/AEM.02090-09
42. Pan PH, Finnegan MB, Sturdivant L, Barnett ML (1999)
Comparative antimicrobial activity of an essential oil and an amine
fluoride/stannous fluoride mouthrinse in vitro. J Clin Periodontol
26(7):474476
43. Fine DH, Furgang D, Barnett ML, Drew C, Steinberg L, Charles CH,
Vincent JW (2000) Effect of an essential oil-containing antiseptic
mouthrinse on plaque and salivary Streptococcus mutans levels. J
Clin Periodontol 27(3):157161. doi:10.1034/j.1600-051X.1999.
260710.x
44. Jenkins S, Addy M, Wade W, Newcombe RG (1994) The magnitude
and duration of the effects of some mouthrinse products on salivary
bacterial counts. J Clin Periodontol 21(6):397401. doi:10.1111/j.
1600-051X.1994.tb00736.x
45. Stoeken JE, Paraskevas S, van der Weijden GA (2007) The long-term
effect of a mouthrinse containing essential oils on dental plaque and
gingivitis: a systematic review. J Periodontol 78(7):12181228. doi:
10.1902/jop.2007.060269
46. Cortelli SC, Cortelli JR, Wu MM, Simmons K, Charles CA (2012)
Comparative antiplaque and antigingivitis efficacy of a multipurpose
essential oil-containing mouthrinse and a cetylpyridinium chloride-
containing mouthrinse: a 6-month randomized clinical trial.
Quintessence Int 43(7):e82e94
47. Van Leeuwen MP, Slot DE, Van der Weijden GA (2011) Essential
oils compared to chlorhexidine with respect to plaque and parameters
of gingival inflammation: a systematic review. J Periodontol 82(2):
174194. doi:10.1902/jop.2010.100266
48. Neely AL (2012) Essential oil mouthwash (EOMW) may be equiv-
alent to chlorhexidine (CHX) for long-term control of gingival in-
flammation but CHX appears to perform better than EOMW in
plaque control. J Evid Based Dent Pract 12(3 Suppl):6972. doi:10.
1016/S1532-3382(12)70017-9
Copyright of Clinical Oral Investigations is the property of Springer Science & Business
Media B.V. and its content may not be copied or emailed to multiple sites or posted to a
listserv without the copyright holder's express written permission. However, users may print,
download, or email articles for individual use.