1.0 Equipment Required
LAFMeasuring Cylinder
Sterile Petri Plates
2.0 Material Required
DisinfectantSterile Saline solution
Sterile & Molten cooled Soybean casein digest agar
Test organism – E. coli, Staph. aureus, Ps. aeruginosa, C. albicans, Salmonella.
3.0 Procedure
3.1 From a recently grown stock culture of each of the test organisms; subculture on the surface of respective media slants as given below.
S.No.
|
Organism
|
Type
|
Medium
|
Incubation temperature and duration
|
1
|
Candida albicans
|
MTCC 227
|
Sabouraud
Dextrose Agar
|
20-25°C for 48 hours
|
2
|
E.coli
|
MTCC4313
|
Soybean Casein
Digest Agar
|
30-35°C for 24 hours
|
3
|
Ps. aeruginosa
|
MTCC1688
|
Soybean Casein
Digest Agar
|
30-35°C for 24 hours
|
4
|
S. aureus
|
MTCC737
|
Soya bean Casein
Digest Agar
|
30-35°C for 24 hours
|
5
|
Salmonella
|
MTCC 3858
|
Soya bean Casein
Digest Agar
|
30-35°C for 24 hours
|
3.2 Using sterile solution about 10 ml of sterile normal saline with peptone (0.1 % w/v), harvest the cultures and dilute suitably with the sterile saline solution to bring the count to about 1 X 108 per ml
3.3 Determine the number of cfu per ml in each suspension by a ten-fold serial dilution.
3.4 Transfer 1.0 ml of culture suspension to a tube containing 9.0 ml of sterile saline solution to obtain 10-1 dilution.
3.5 Transfer 1.0 ml of the 10-1 dilution to a tube containing 9.0 ml of sterile saline solution to obtain test dilution 10-2.
3.6 Similarly serially dilute the culture suspension to obtain dilution of 10-3, 10-4, 10-5, 10-6, 10-7 and 10-8.
3.7 Plate 1.0 ml from test dilution 10-6, 10-7 and 10-8 in duplicate into 90 mm sterile Petri plates.
3.8 Pour approximately 20 to 25 ml of sterile molten cooled soybean casein digest agar in each plate.
3.9 Carry out the same dilution method for all the cultures and label accordingly with dilution number and test organism.
3.10 Allow to solidify the plates at room temperature, invert and incubate individually as specified in above table.
3.11 Count the number of colonies and find out the concentration of culture suspension and tabulates the results in the specified format.
3.12 Apply the above-known culture cells (approx 108 cfu) individually to a surface of 25 cm2.
3.13 Allow to air dry the applied cultures at room temperature.
3.14 Prepare the disinfectant solution to be tested as per SOP for Preparation of Disinfectant Solution and apply on the surface of the artificially contaminated area.
3.15 Take a swab immediately at 0 min, after 5 min, 10 min, 20 min and after 30 minutes, and then proceed the swab testing as per SOP for Swab Testing.
3.16 Incubate the plates as specified in above table and tabulate the result in the specified format.
3.17 The tested sample is effective at specified concentration when there is no growth in swab tested after 20 and 30 minutes for each culture.4.0 Precaution
4.1 Artificially contaminated area should be cleaned immediately after completion of work.4.2 Use hand gloves and nose mask during testing.
4.3 All the glassware used for dilution and testing, immediately clean and sterilize as per SOP.
Related: Microbial Resistance against Disinfectants
5.0 Frequencies
For each consignment6.0 Abbreviation
SOP: Standard Operating ProcedureQA: Quality Assurance
QC: Quality Control
NA: Not Applicable
WFI: Water for Injection
LAF: Laminar Air Flow
Also see: Validation of Efficacy of Disinfectants
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what volume of culture suspension and disinfectant do you apply on the surface?
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