To lay down the procedure to determine the efficacy of the disinfectants being used for house keeping activities.
This SOP is applicable to the Quality Control Department (Microbiology Lab).
5.1 Ensure that the availability of following material before start of analysis.
1) Pure cultures of Staphylococcus aureus ATCC 6538 &
Pseudomonas aeruginosa ATCC 9027
2) Sterilized Conical flasks ( 250 ml capacity)
3) Sterilized graduated pipettes ( 1 ml )
4) Sterilized Petriplates
5) Sterilized test tubes (18 x 150 mm)
6) Nutrient agar / Soya bean casein digest agar
7) Nutrient broth / Soya bean casein digest medium
8) Inactivating agent (Lecithin / Sodium thiosulfate/ Sodium thioglycollate)
5.2 Preparation of Inoculum:
5.2.1 Inoculate the organism in nutrient or soyabean casein digest medium and incubate at 37°C for 24 hours. Inoculate the same organism by transferring loop full of previous suspension in nutrient medium continuously two days.
5.2.2 Finally inoculate 100 ml of nutrient broth or Soyabean casein digest medium With one ml of the activated culture of both the organisms, individually and incubate At 37°C for 16-18 hrs. Keep the activated culture in refrigerator.
5.2.3 Estimate the viable count of bacteria per ml of the medium for both the organisms By pour plate method by diluting the culture appropriately to get 30 - 300 colonies Per plate.
5.3 Preparation of disinfectant :
5.3.1 Prepare the disinfectant solution in different concentration like 0.5%, 1.0%, 1.5% & 2.5% in 250 ml conical flask .
5.3.2 Add 1 ml of the cell suspension (concentration 10 5) of each culture, individually to the disinfectant solution, carefully and shake well.
5.3.3 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 of inactivator . Shake well.
5.3.4 Determine the number of viable bacteria per ml present in the solution by using pour plate method and incubate at 37°C for 48 hrs. Record the result
5.3.5 Use any one inactivator of the following depending on the type of disinfectant. The inactivators are Sodium thiosulphate 5 % w/ v solution.
6.0 ACCEPTANCE CRITERIA
Use only those concentration where there is 90% reduction of microbial count .