Disinfectant efficacy testing in microbiology

1.0 OBJECTIVE

To lay down a procedure for the testing of disinfectant efficacy.

2.0 SCOPE

This procedure is applicable to the procedure for disinfectant efficacy test in company Name

3.0 RESPONSIBILITY         

3.1 Microbiologist /Officer is responsible for the execution. 

3.2 HOD or designee is responsible for approval, effective implementation and compliance to the disinfectant efficacy test for execution.               

4.0 PROCEDURE 

4.1 Requirement

4.1.1 Pure cultures of Staphylococcus aureus ATCC 6538  & Pseudomonas aeruginosa  ATCC 9027.

4.1.2 Sterilized Conical flasks (250 ml capacity)

4.1.3 Sterilized graduated pipettes (1 ml).

4.1.4 Sterilized   Petriplates.

4.1.5 Sterilized test tubes (18 x 150 mm).

4.1.6 Nutrient agar / Soya bean casein digest agar.

4.1.7 Nutrient broth / Soya bean casein digest medium.

4.1.8 Inactivating agent (Lecithin 0.5% with tween 20 4.0% / Sodium thiosulfate /  Sodium thioglycollate)

4.2 Preparation of Inoculums’:

4.2.1 Inoculate the organism in Nutrient Broth or Soyabean Casein Digest Medium and   incubate at 30-35°C for 24 hours. 

4.2.2 After 24 hours again Inoculate the same organism by transferring loop full of previous suspension on soyabean casein digest agar slants continuously for two days to activate the culture.

4.2.3 Prepare the suspension in sterile normal saline to achieve 105 per ml.

4.2.4 Estimate the viable count of bacteria by serial dilution to get 10 to 100 Cfu / per plate.

4.2.5 Finally calculate the counts per ml in saline use the dilution having the count 105    per ml, Keep the activated culture in refrigerator. 

4.3 Preparation of  disinfectant:

4.3.1 Prepare the disinfectant solution in different concentration like 0.5%, 1.0%, 2.0%, 2.5% and 5.0% in 250 ml conical flask.

4.3.2 Add 1 ml of the cell suspension (concentration 10 5) of each culture, individually to the disinfectant solution, carefully and shake well.

4.3.3 After 10 minutes of contact time aseptically remove 1 ml of solution from the flask    and transfer to tubes containing 9 ml of a sterile solution with inactivator and shake well.

4.3.4 Determine the number of viable bacteria per ml present in the solution by using pour plate method.

4.3.5 Let the plate solidify and incubate at 30-35°C for 48-72 hrs. Record the result in Attachment #1

4.3.6 Use any one inactivator of the following depending on the type of disinfectant.

4.3.7 The inactivators are, Sodium thiosulphate 5 % w/ v solution, Lecithin 0.5% with  tween 20 4.0% and Sodium thioglycollate.

4.4 Acceptance criteria

Recovery should not be more than 10 %.