1.0 PURPOSETo lay down the stepwise procedure for validation process for checking effectiveness of disinfection / cleaning procedure
2.0 SCOPEThis is applicable to Microbiology lab
4.0 ACCOUNTABILITYHead of Department
5.0 PROCEDURE5.1 Suspension method
5.1.1 To determine the test concentration and the contact time a suspension method is generally applied. The suspension method estimate the in vitro bactericidal activity of the disinfectant under precise experimental conditions including
Preparation of inoculum
Volume of inoculum vs. disinfectant
TemperatureDisinfectant concentration and contact time
Interfering substances (if any)
5.1.2 Preparation of culture suspension
Prepare the dilution of the following organisms Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Candida albicans, Aspergillus niger and environment isolates which will yield 105 to 106 cells per ml starting from the original culture suspension
5.1.3 Suspension testing
126.96.36.199 Prepare 10 ml of test dilution to be tested with sterile distilled water.
188.8.131.52 Vortex the tube for 1.0 minute.
184.108.40.206 Add 0.1 ml of any one culture into the test sample containing 105 to 106 cells which final concentration shall be 104 to 105 cells per tube.
220.127.116.11 Test from tube one consider it as ‘0’ minutes and give the contact time of 5 min, 10 min and 15 min.
18.104.22.168 After the specified contact time, filter the samples through a 0.45 m membrane filter.
22.214.171.124 Give three washing of 100 ml each with 0.1 % sterile peptone water/ Sterile Water.
126.96.36.199 After filtration with the help of a sterile forceps take the membrane and place it on a soybean casein digest agar.
188.8.131.52 Incubate the bacterial culture at 30-35 °C for 48 hours and fungal cultures at 20-25 °C for 3 to 5 days.
184.108.40.206 After incubation count the number of colonies present on the membrane.
220.127.116.11 Note down the number of colonies.
18.104.22.168 This will be the final count of the exposed culture.
22.214.171.124 Select the plates, which have least to Nil counts.
126.96.36.199 Proceed in the same manner taking all the cultures to be tested.
188.8.131.52 Contact time for the usage of the disinfectant will be set on the basis of the results, which will have least counts.
5.1.4 Surface spray / Wipe method
184.108.40.206 To determine the efficacy of the antimicrobial activity a spray / wipe method are used. Following surface shall be used for testing
PU paint wall
5.1.5 Preparation of culture suspension
Refer point No. 5.1.2
5.1.6 Determining the efficacy of the Disinfectant by Surface spray / Wipe method
220.127.116.11 Take plate of different surfaces such as SS, Epoxy, Panel, Floor present in the clean room having a surface area of 25 cm2
18.104.22.168 From the previously determined suspension having 105 to 106 cells per ml inoculate one culture on different surfaces mentioned above.
22.214.171.124 With the help of a sterile spatula spread the culture on the surface.
126.96.36.199 Keep it on the LAF bench for drying.
188.8.131.52 After the exposed duration for drying (a) spray the disinfectant (b) disinfect the surface by wipe method.
184.108.40.206 Allow the surface to be with the sanitize for the time generally kept in the clean room during sanitation (The exact procedure for sanitation followed in the clean room should be followed).
220.127.116.11 With the help of a sterile moistened swab, swab the surface gently covering all the area of the surface.
18.104.22.168 Place the swab sticks in a test tube having sterile saline solution and analyze immediately.
22.214.171.124 Vortex the test tube gently for 5.0 minutes.
126.96.36.199 Aseptically filter the samples through a 0.45 m membrane.
188.8.131.52 Give three washing of 100 ml each with 0.1 % sterile peptone water/sterile water.
184.108.40.206 After the filtration with the help of a sterile forceps take the membrane and place it on a soybean casein digest agar.
220.127.116.11 Incubate the bacterial culture at 32.5 ± 2.5 °C for 2 days and fungal cultures at 22.5 ± 2.5 °C for 5 days.
18.104.22.168 After incubation count the number of colonies present on the membrane.
22.214.171.124 Proceed in the same manner taking all the cultures to be tested and the various sanitizes.
6.0 ABBREVIATIONS6.1 SOP - Standard Operating Procedure
6.2 % - Percent
6.3 °C - Degree centigrade
Also see: Validation Protocol for Efficacy of Disinfectants
Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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