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ROLL NO. 15
CLASS: MSc PART II MICROBIOLOGY
RESEARCH PROPOSAL
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LIST OF ABBREVIATIONS
Standards
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SERIAL NO. TOPIC PAGE NO.
1 PROJECT TITLE 3
2 INTRODUCTION 3
3 RATIONALE 7
4 LITERATURE REVIEW 9
5 AIM AND OBJECTIVES 13
6 PLAN OF WORK 13
7 MATERIALS AND METHODS 14
8 EXPECTED RESULTS 21
9 REFERENCES 22
10 APPENDIX
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1. PROJECT TITLE: Formulation of Herbal sanitizers and determining
their antimicrobial activities against skin pathogens.
2. INTRODUCTION:
The word hygiene derives from the ancient Greek goddess Hygeia, the
goddess of healing. Hygiene refers to behaviors that can improve
cleanliness and lead to good health, such as frequent hand washing, face
washing and bathing with soap and water. In many areas of world, practicing
personal hygiene etiquette is difficult due to lack of clean water and soap.
Many diseases can be spread if the hands, face, or body are not washed
appropriately at key times. Thousands of people die every day around the
world from infections acquired while receiving health care. Hands are the
main pathways of germ transmission during health care. Hand hygiene is
therefore the most important measure to avoid the transmission of harmful
germs and prevent health-care associated infections.
Washing hands with soap and water is the best way to reduce the
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number of microorganisms (germs) on hands. When soap and water are not
readily available, alcohol based hand sanitizers or rubs are acceptable but
only if the hands are not visibly soiled. The effectiveness of alcohol-based
hand sanitizers is reduced when used on hands that are visibly soiled or
dirty. Visible contamination and dirt should be removed before using hand
sanitizers and can be achieved by using a hand wipe.
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ingredients alongside ethanol or isopropanol. For example, humectants, such
as glycerin, work as moisturizing agents. Humectants draw moisture from
the surrounding environment and hold it close to the skin. Thickening
agents, such as polyacrylic acid, may also be used to give hand sanitizers a
gel like texture, which aids in the application and spreading of the product
on the hands. A newer development in hand sanitizers is the use of fragrance
oils to help lessen the smell of alcohol when applying the product.
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A proper protocol for hand sanitizing (Babeluk, R. et al., 2014)
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3. RATIONALE:
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reported (Kawaii, K. et al., 2000).The essential oil extracted from
tulsi (Ocimum tenuiflorum) showed antimicrobial activity against S.
aureus (including MRSA) and E. coli, but was less active against P.
aeruginosa. Tulsi essential oil could be a valuable topical antimicrobial
agent for management of skin infections caused by these organisms.
(Yamani, H.A. et al.,2016). So such different medicinal plants can be used
for formulation of Herbal Sanitizer.
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4. LITERATURE REVIEW:
Hand washing with soap removes the body's own fatty acids from the
skin, which may result in cracked skin that provides an entry portal for
pathogens (Larson, E.L. et al., 1998). In contrast, high-quality hand
disinfectants contain additional skin care products, like emollients.
(Lauharanta, J. et al., 1991). They also do not require the use of water, which
makes the application easy and uncomplicated. Unfortunately, clean water is
not always available. In these cases the CDC recommends using an alcohol-
based hand sanitizer, which is capable of killing most germs. (Sherwood, C.,
2017)
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of reducing bacterial load. Desderman pure gel, Sterillium and Lavit reduced
the bacterial count to 6.4%, 8.2% and 28.0% respectively. After education of
the correct use of each hand rub, the bacterial load was reduced even further,
demonstrating the value of education in improving hand hygiene. Efficacy,
followed by skin compatibility was found to be the two most important
attributes of a hand disinfectant (Babeluk, R. et al., 2014).
But these types of sanitizers are costly as complex chemicals are use
and it can affect our skin as such products depend upon skin compatibility.
So in such case herbal products gets a more demand as they are less costly as
well more effective with no side effects.
Like, in one of the research main purpose of study was to help to create
a product which is cheaper and to help people who do not have enough
money to buy expensive hand sanitizer. The procedure in making this
product is simple. They scrape the gel inside the Aloe Vera leaves and put
into a plastic container and added 1 cup of ethyl alcohol and 2 teaspoons of
glycerin. Next, they grated the orange to get the zest and added it to the
mixture, then, mix till well blended. Lastly, the group recycled a small bottle
that was used as a container for the mixture. The researchers concluded that
the aloe vera gel can be used to make the hand sanitizer more jelly and the
zest of the orange more hydrating to the skin. It is easy to make since the
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materials used are found in stores and have the cheapest price. The group is
confident that the product is effective because of the ethyl alcohol.
(Bernabe, et al., 2013)
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Some other herbal hand sanitizer, incorporating the leaves extracts of
Ocimumcanctum Linn.(Tulsi) and Eucalyptus globulus (Nilgiri), the well-
known herbal combination with multidimensional activities was formulated
and their respective antimicrobial efficacy were studied. The formulation
was evaluated against the specified microorganism by culture sensitivity
test. The significance was found to be more. (Wani, N. S. et al., 2013)
This study was aimed to evaluate the antimicrobial efficacy and safety
of PureHands Herbal Hand Sanitizer in healthy volunteers and also on
inanimate objects. The present study was an open, non-comparative
prospective study. Sterile cotton swab sticks were used to take swabs from
both hands and swabs were inoculated on the petri dishes marked before
application (before) in both aerobic and anaerobic media. Approximately 0.5
ml of PureHands Herbal Hand Sanitizer was squeezed out on the palms of
the subjects and they were asked to rub the gel thoroughly on the palms,
back of the hands, fingernails until the hands became dry and inoculation
was done on the respective dishes, in the part marked (after). The same
procedure was repeated for seven consecutive days on all subjects. For
evaluating the efficacy and safety of PureHands Herbal Hand Sanitizer on
inanimate objects preparation similar procedure was followed. PureHands
Herbal Hand Sanitizer was found to be effective, safe and less likely to
cause adverse skin reactions and saves time and human resources. It was
concluded that PureHands Herbal Hand Sanitizer has a significant
bacteriostatic effect on the bacteria present on the hands surface and on the
surface of inanimate objects. (Mondal, S. and Kolhapure, S.A, 2004)
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5. AIM: To formulate Herbal sanitizers and determining their antimicrobial
activities against skin pathogens.
3. To formulate herbal sanitizer from extract of neem, tulsi and lemon juice.
7. PLAN OF WORK:
4. Preparation of sanitizer.
B. MIC determination
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8. MATERIALS:
For extraction:
90% Methanol
For phytochemicals:
For Sanitizer:
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For Antibiotic Sensitivity Test:
Mueller-Hinton agar
Antibiotic disc
Chloramphenicol, C- 10mcg
Mueller-Hinton agar
9. METHODOLOGY:
Dry the Tulsi and Neem Leaves in oven at 50C and coarsely grind to
get a powder. This leaf powder is to be stored in fridge for further use.
For methanol extraction, 10g of leaves powder and 90% methanol is
added in 250ml flask (maceration) and kept on shaker for 48 hours.
After maceration, filtered the sample by whatman No.1 filter paper.
Heat this filtrate on water bath at 60C till thick sticky, dark coloured
extract is obtained. This extract is stored at 4C for sanitizer
preparation.
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ii. Preparation of Lemon extract:
Wash Lemon, cut into half and squeeze to get a juice. Store this juice at
4C for further use.
iii. Test for phenols and tannins (lead acetate test): To 2-3 ml of
alcoholic extract, add 0.5 ml of 1% lead acetate and the
formation of white precipitate indicate the presence of tannins
and phenolic compounds.
v. Test for Saponins (Foam test): Dilute the extraxt with distilled
water and shake well in a graduated cylinder for 15 min. The
persistent foam to a length of 1cm indicates the presence of
Saponins.
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vi. Test for Terpenoids: Add 4 mg of extract treated with 0.5 ml
of acetic anhydride and 0.5 ml of chloroform. Then add
concentrated solution of sulphuric acid slowly and red violet
color will observe for terpenoid.
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v. Preparation of sanitizer:
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method (AWDM) by standard protocol. A standard method of
National Committee for Clinical Laboratory Standards NCCLS.
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11. REFERENCES:
1. Hassan, A., Usman, J., Kaleem, F., Omair, M., Khalid, A., Iqbal,M.
(2011) Evaluation of different detection methods of biofilm formation in
the clinical isolates Brazilian Journal of Infectious Diseases volume 15,
issue 4, pg. 305-311.
2. Bernabe, Desierto, N.C., Maliclic, G.A., Manglangit, C. A.,
Marasigan, T. M., Austin. (2013) Citrus fruits as a bacterial hand
sanitizer.
3. Bolon, M.K. (2016) Hand Hygiene: An Update Infectious disease clinics
of North America, volume 30, issue 3, pg. 591607.
4. Kolhapure, S.A., Mondal, S. (2004) Evaluation of the antimicrobial
efficacy and safety of PureHands herbal hand sanitizer in hand hygiene
and on inanimate objects volume 101, issue 2, pg. 55-57.
5. Larson, E.L., Hughes, C.A., Pyrek, J.D., Sparks, S.M., Cagatay, E.U.,
Bartkus, J.M. (1998) Changes in bacterial flora associated with skin
damage on hands of health care personnel American Journal of infection
control volume 26, issue 5, pg. 513-521.
6. Yamani, H.A., Pang, E.C.,Deighton,M.A. (2016) Antimicrobial Activity
of Tulsi (Ocimum tenuiflorum) Essential Oil and Their Major
Constituents against Three Species of Bacteria volume 7.
7. Londhe, J., Jagtap,S.D., Doshi, C., Jagade, D. (2015) Formulations of
Herbal Hand Wash with Potential Antibacterial Activity International
Journal of Research in Advent Technology.
8. Lauharanta,J., Ojajarvi, J., Sarna, S., Makela, P. (1991) Prevention of
dryness and eczema of the hands of hospital staff by emulsion cleansing
instead of washing with soap Journal of hospital infection volume 17,
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issue 3, pg. 207-215.
9. Maruti, J., Dhanavade, Chidamber, B., Jalkute, Jai, S., Ghosh and
Sonawane, K. D., (2011) Study Antimicrobial Activity of Lemon (Citrus
lemon L.) Peel Extract British Journal of Pharmacology and Toxicology,
volume 2, issue 3 pg. 119-12.
10. Maruthamuthu., and Kandasamy., (2016) Ferric reducing anti-
oxidant power assay in plant extract Bangladesh Journal of
Pharmacology volume 11, pg. 570-572.
11. Thombare,M.A., Udugade,B.V., Hol,T.P., Mulik,M.B, Pawade, D.A.
(2015) Formulation and evaluation of novel herbal hand sanitizer Indo
American Journal of Pharmaceutical Research, volume 5, issue 01, pg.
483-488.
12. Wani, N.S., Bhalerao, A.K., Ranaware, V.P., Zanje, R. (2013)
Formulation and Evaluation of Herbal Sanitizer International Journal of
PharmTech Research Volume 5, issue 1, pg. 40-43.
13. Mathur, P. (2011) Hand hygiene: Back to the basics of infection control
The Indian journal of Medical research, volume 134, Issue 5, pg. 611
620.
14. Raut, R. R., Sawant, A. R., Jamge, B. B. (2014) Antimicrobial activity
of Azadirachta indica (Neem) against Pathogenic Microorganisms
Journal of Academia and Industrial Research (JAIR),Volume 3, Issue 7,
pg. 327-329.
15. Babeluk, R., Jutz, S., Mertlitz, S., Matiasek, J., Klaus, C. (2014)
Hand Hygiene Evaluation of Three Disinfectant Hand Sanitizers in a
Community Setting Journal.pone.0111969 Volume 9, Issue 11.
16. Choudhari,S., Sutar,M., Chavan, M. (2016) Formulation, evaluation
and antibacterial efficiency of herbal hand wash Indo American Journal
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of Pharmaceutical Research volume 6, issue 4, pg. 5202-5209.
17. Abbas, S.Z., Hussain, K., Hussain, Z., Ali, R., Abbas, T. (2016) Anti-
Bacterial Activity of Different Soaps Available in Local Market of
Rawalpindi (Pakistan) against Daily Encountered Bacteria Volume 7,
Issue 11.
18. Mishra,U.S., Murthy, P.N., Pasa,G., Nayak, R. K. (2011)
Formulation & Evaluation of Herbal Gel Containing Methanolic Extract
of Ziziphus Xylopyrus Asian Journal of Biochemical and Pharmaceutical
Research, Volume 1, Issue 4 , pg. 207-218.
19. Jain, V.M., Karibasappa, G.N., Mali, G.V. (2016) Comparative
assessment of antimicrobial efficacy of different hand sanitizers: An in
vitro study Dental research journal volume 13, issue 5, pg. 424-431.
20. Afsar, Z., Khanam, S. (2014) Antimicrobial activity of the extracts of
cassia fistula and milletia pinnata against ampicillin resistant strains of
clinical origin Asian Journal of Phytomedicine and Clinical Research
volume 2, issue 1, pg. 22-29.
21. Afsar, Z., Khanam, S. (2016) Formulation and evaluation of
Polyherbal soap and hand sanitizer International research journal of
pharmacy volume 7, issue 8.
22. Abelson,P.H. (1990) Medicine from plants volume 247, Issue 4942, Pg.
513.
23.Kawaii,S., Yasuhiko,T., Eriko,K., Kazunori,O., Masamichi,Y.,
Meisaku, K., Chihiroito, Hiroshi,F. (2000) Quantitative study of
flavonoids in leaves of Citrus plants Journal of Agricultural and Food
Chemistry volume 48, Pg. 3865-3871.
24. Sherwood, C. (2017) How Does Hand Sanitizer Kill Bacteria?
http://www.livestrong.com/article/177415-facts-about-hand-sanitizers
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APPENDIX-I
Composition of media
Agar 2.5gm
D/W 100ml
pH 7.50.2
2. Mueller-Hinton Agar
Starch 0.15gm
Agar 2.5gm
D/W 100ml
pH 7.30.1
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3. Trypticase Soy Broth with 1% Glucose
Glucose 1gm
D/W 100ml
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APPENDIX-II
Composition of Reagents
1. 90% Methanol:
Mix 10ml of distilled water with 90ml of Methanol.
2. Fehlings A Solution:
Dissolve 34.64gm of crystalline copper sulphate pentahydrate in water
and dilute up to 500ml volume.
3. Fehlings B solution:
Dissolve 173gm of potassium sodium tartrate tetra hydrate and 50gm
sodium hydroxide in water and dilute to 500ml volume.
4. 95% Ethanol:
Mix 5ml of distilled water with 95ml of Ethanol.
5. 0.2M Sodium phosphate buffer (pH 6.6):
Take 3.12gm of NaH2PO4.2H2O in 100ml D/W and 7.17gm of
Na2HPO4 in 100ml D/W.
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