ANTIMICROBIAL ACTIVITY TESTING OF ANTISEPTIC

 Sterilize all glassware’s including Petri plates and pipettes in dry heat sterilizer at 180 oC
for 60 minutes.
 Prepare media’s by autoclaving at 121 oC for 15 minutes ( includes TSA, TSB, lactose and
peptone water test tubes).
 Take stock ATCC cultures and sub culture them in TSB and lactose and incubate it.
Incubation period for bacteria is 3 days at 32.5 + 2.5 oC while for fungus incubation
period will be 5 days at 22.5 + 2.5 oC.
 When growth occur streak on respective Petri plates containing TSA / SDA media in
order to get isolated colonies, incubate it (TSA for bacteria & SDA for fungus).
 After incubation period, take heavy loop full of cultures and inoculate it into test tube
containing peptone water that is dedicated for that particular organism.
 Next step is to make serial dilution in peptone water test tubes from 1: 10 to 1:10 8
 Then take 1 ml from each tube and transfer it into Petri plate that was specifically mark
for that particular organism.
 Then Pour 15 -20 ml TSA/ SDA in each plate and incubate it.(Incubation period for
bacteria is 3 days at 32.5 + 2.5 oC while for fungus incubation period will be 5 days at
22.5 + 2.5 oC).
 Keep all the test tubes in refrigerator.
 Follow the same procedure for all 5 ATCC cultures.
 After incubation period read plates and count no of Colony forming unit at each dilution
factor.
 Record all the results.
 Take sample antiseptic solution and use Dettol as standard. We use them in different
concentrations (Direct, 1:10, and 1:100)
 Next step is to take culture from TSB and lactose and pour it into melted TSA/SDA agar
i.e. (40 0C – 45 0C). Mix it and transfer it in sterile Petri plates. Cover the plate and leave
it to solidify. (Same procedure is applied for each organism used).Incubate all plate for 1
day.
 The test will be performed in duplicate.
 Next day take out all plates and make 4 wells in each petri plate with the help of sterile
borer.
 Then pour sample antiseptic and standard (Dettol) in their respective marked plates.
 After that incubate all these plates for 3 – 5 days as per requirement of bacteria and
fungus.
 Read zone of inhibition of both the antiseptic and dettol at every dilution factor with the
help of Vernier Caliper.
 Record the results and compare zone of inhibition of sample antiseptic with standard
dettol solution.

ANTIMICROBIAL ACTIVITY TESTING OF MOUTH WASH

 Select 6 persons within organization that have dirty teeth.
 Take out sterile swabs and take sample from teeth of selected individuals by swabbing
method.
 Sterilize all glassware’s including Petri plates and pipettes in dry heat sterilizer at 180 oC
for 60 minutes.
 Prepare media’s by autoclaving at 121 oC for 15 minutes (includes TSA, TSB and peptone
water test tubes).
 Prepare petri plates containing TSA media, leave it to solidify.
 After that mark plates with the name of individuals.
 Make loan of sample using swab stick of that particular person onto the respective agar
plate.
 Also inoculate sample into the TSB broth.
 Incubate both TSB and plates for 2- 3 days at 32.5 + 2.5 oC.
 After the incubation period take one colony from plate with the help of wire loop make
slide of it and identify the spirochetes using microscope.
 Take loop full of spirochete colony and transfer it into test containing peptone water.
 Next step is to make serial dilution from it from 1:10 to 1:10 8
 Then take 1 ml from each tube and transfer it into Petri plate that was specifically mark
for spirochetes
 Then Pour 15 -20 ml TSA in each plate and incubate for 3 days at 32.5 + 2.5 oC.
 Keep all the test tubes in refrigerator.
 After incubation period read plates and count no of Colony forming unit at each dilution
factor.
 Record all the results.
 Take sample Mouth wash and use it different concentrations (Direct, 1:10, and 1:100)
 Next step is to take culture from TSB and pour it into melted TSA agar i.e. (40 0C – 45 0C).
Mix it and transfer it in sterile Petri plates. Cover the plate and leave it to solidify.
 Incubate all plate for 1 day at 32.5 + 2.5 oC.. The test will be performed in duplicate.
 Next day take out all plates and make 4 wells in each petri plate with the help of sterile
borer.
 Then pour sample Mouth wash in wells. After that incubate all these plates for 2 – 3
days at 32.5 + 2.5 oC.
 Read zone of inhibition of Mouth wash at every dilution factor with the help of Vernier
Caliper.
 Record the results and compare zone of inhibition of sample Mouth wash.