“Chemical Agents of Control: Evaluation of Antiseptics by Filter paper disk Method”
This is certify that Project entitled” Chemical Agents of Control: Evaluation
of Antiseptics by Filter paper disk Method”
In the partial submission for the degree of Bachelor of Science in Biotechology.
This project has been prepared under our supervision and guidance. It is also
The project is the result of the candidate “Own work” & is of sufficient high
standard to warrant its presentation for the examination.
Submitted to: Mrs Jagriti Project Guide (Project guide) Lect. Biotech Deptt. Lect. Biotech Deptt.
Department of Biotechnology
Hindu Girls College – Jagadhri Session – 2006-07
I express my sincere thanks to my Biotech teachers Mrs. Sheetal & Mrs. Jagriti for
their guidance & inspiration throughout my project work & in the preparation of
I also thanks them for solving my queries & catching the minute mistakes that
hindered the arrival of results.
I am also grateful to my lab assistant Mr. Gulshan for providing the materials & for
immediately getting the instruments repaired whenever some fault occurs so that
our project could not suffer.
I am also grateful to our library staff for providing us with essential books and
journals. At last I express my deep gratitude to all those also were involved either
directly or indirectly in conceptualizing & conceiving the project work & report.
Isolation of Staphylococcus from Skin
Sub culturing to obtain pure broth culture.
Filter paper Disc method
AIM : To test the antimicrobial activity of different Antiseptics.
Many microbes grow abundantly , both on surface & inside the normal body &
those that are more or less regularly present without causing disease constitutes its
normal flora while those that are present only for few days or months form its
transient flora. However under certain condition, some member of normal flora
become parasites, these organisms are then known as opportunistic pathogens.
The normal flora of skin comprises Micrococcus spp, Staphylococcus sp.
Propionibacterium etc. Perspiration and sebum serves as source of nutrition for
these microbes. Staphylococcus aureus is the part of normal flora, but when injury
occurs,it is able to penetrate the deeper tissues & causes infection.
In order to study the antimicrobial activity of antiseptics , S. aureus is chosen as
test organism because of its abundance on human skin & capability of becoming
opportunistic pathogen also.
Originally the term antiseptic was applied to any that prevented sepsis or
putrefaction which is caused by growing microorganisms. The preparations of
chemicals that are meant to be applied to skin or other living tissues to decrease
the number of microbes are called antiseptics. Ethanol (ethyl alcohol) and
isopropanol (isopropyl alcohol) are alcohols used to reduce the number of
microbes on skin sites designation for hypodermic infections or for the withdrawal
of blood. Ethyl alcohol is occasionally used to disinfect electrodes, face masks, and
thermometers, which are first cleaned and then soaked in alcohol for 15-20
minutes. Dettol, savlon and alcohol are good antiseptics with bactericidal and
sporicidal property and are used for skin antisepsis.
Isolation of Staphylococcus strain from Skin
Principle : Mannitol salt agar medium is used for selective isolation of pathogenic
Staphylococci since most other bacteria are inhibited by high salt concentration.
The colonies are surrounded by yellow halo indicating mannitol fermentation. The
acid produced by fermentation changes the colour of indicator phenol red to
Mannitol salt agar plates
Autoclaved cotton swab
Autoclaved saline (10ml. 0.85%)
Preparation of Materials :
(i) Mannitol Salt agar (pH 7.4)
Beef extract 40mg
Phenol red pinch
Distilled water 40ml
Dissolve 85mg NaCl in 10ml d/w
1. Moisten a sterile cotton swab in saline aseptically.
2. Remove excess saline by pushing the swab against test tube wall.
3. Rub the swab over the skin surface of nose, elbow or any other area.
4. Spread the swab uniformly on the mannitol salt agar plates
5. Incubate the plates for 48 - 60 hrs at 37°C.
Large opaque colonies surrounded by yellow halos are observed. These are
colonies of Staphylococcus.
Subculturing (or picking off) technique
To obtain pure broth cultures
After incubation has been completed in streak-plate, pour-plate or spread
plate techniques & appearance of discrete well-separated colonies has
been examined, the next step is to subculture some of the cells from one of
the colonies to separate agar plates or nutrient broth.
Each of this new culture represents the growth of single species & is called
pure or stock culture.
Sub culturing term is used to describe the procedure of transferring of
microbes from their parent growth source to fresh medium. When the
transfer is from solid medium (agar) to liquid medium (broth) the term
picking off is used.
Sterile test – tubes
Nutrient broth (pH – 7. 0)
Beef extract 150mg
1. Loopful of culture of Staphylococcus is transferred from mannitol
salt agar plate to nutrient broth tube.
2. The tube was incubated overnight at 37°C.
Turbidity appears in the tube due to growth of culture.
The culture obtained is pure culture of Staphylococcus.
Testing Antimicrobial activity of different Antiseptics
By Filter-Paper Disc Method
Filter paper disc method (or zone of inhibition method) is employed to
evaluate effectiveness of antimicrobial agents against selected test
A sterile filter paper disc is impregnated with an antiseptic & placed on a
fresh heavily inoculated agar plate of test organism. Following incubation
the agar plate is looked for zone of inhibition. The presence of clear zone of
inhibition surrounding disc is indicative of inhibitory activity against microbe.
The diameter of zone of inhibition is directly proportional to the
Nutrient agar plates
Sterile filter paper discs
Solution of different antiseptics.
1. 1/5 Dettol
2. 1/10 Dettol
3. 1/5 Savlon
4. 1/10 Savlon
5. 100% Alcohol
6. 70% Alcohol
7. 50% Alchol
8. 30% Alcohol
Sterile test tubes
1. About 0.2ml of Staphylococcus broth culture is spread on surface
of respective nutrient agar plates by sterile spreader.
2. Filter paper discs are dipped in different soap solutions & placed
on Staphylococcus inoculated plats.
3. The plates are incubated overnight at 37°C.
4. Observe & measure the diameter of zone of inhibition around each
The diameters of zone of inhibition of different solutions are as follows :
1. Dettol 4.1 cm
2. Dettol 3.1 cm
3. Savlon 3.2 cm
4. Savlon 2.6 cm
Testing Antimicrobial activity of Alcohol Using Cotton Swab.
1. About 0.2 ml of staphylococcus broth culture is spread on surface of
respective nutrient agar plates by sterile spreader.
2. Cotton Swabs are dipped in different concentration of alcohol
solutions and move on agar plate divided in four quadrants.
3. The plates are incubated overnight at 370C.
4. Observe the Result.
Solutions 1/5 Dettol has highest antimicrobial.
Among alcohol solutions 10% alcohol has highest antimicrobial
Experiments in Microbiology by K.R. Aneja – New Age International
Textbook of Microbiology by Pelczar
Eco Science Journal of Science & Technology – Yamunanagar