SOP for Checking Effectiveness of Disinfection/ Cleaning Procedure

1.0 PURPOSE

To lay down the stepwise procedure for validation process for checking the effectiveness of disinfection/ cleaning procedure

2.0 SCOPE

This is applicable to Microbiology lab

3.0 RESPONSIBILITY

Microbiologist

4.0 ACCOUNTABILITY

Head of Department

5.0 PROCEDURE

5.1 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

• Microbial strain
• Preparation of inoculum
• Volume of inoculum vs. disinfectant
• Temperature
• Disinfectant 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

5.1.3.1 Prepare 10 ml of test dilution to be tested with sterile distilled water.
5.1.3.2 Vortex the tube for 1.0 minute.
5.1.3.3 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.
5.1.3.4 Test from tube one consider it as ‘0’ minutes and give the contact time of 5 min, 10 min and 15 min.
5.1.3.5 After the specified contact time, filter the samples through a 0.45 m membrane filter.
5.1.3.6 Give three washing of 100 ml each with 0.1 % sterile peptone water/ Sterile Water.
5.1.3.7 After filtration with the help of a sterile forceps take the membrane and place it on a soybean casein digest agar.
5.1.3.8 Incubate the bacterial culture at 30-35 °C for 48 hours and fungal cultures at 20-25 °C for 3 to 5 days.
5.1.3.9 After incubation count the number of colonies present on the membrane.
5.1.3.10 Note down the number of colonies.
5.1.3.11 This will be the final count of the exposed culture.
5.1.3.12 Select the plates, which have least to Nil counts.
5.1.3.13 Proceed in the same manner taking all the cultures to be tested.
5.1.3.14 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

5.1.5.1 To determine the efficacy of the antimicrobial activity a spray/ wipe method is used. The following surface shall be used for testing

• Stainless Steel
• Epoxy
• Panel
• Floor
• PU paint wall
• Glass surface

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
5.1.6.1 Take a plate of different surfaces such as SS, Epoxy, Panel, Floor present in the clean room having a surface area of 25 cm²
5.1.6.2 From the previously determined suspension having 10⁵ to 10⁶ cells per ml inoculate one culture on different surfaces mentioned above.
5.1.6.3 With the help of a sterile spatula spread the culture on the surface.
5.1.6.4 Keep it on the LAF bench for drying.
5.1.6.5 After the exposed duration for drying (a) spray the disinfectant (b) disinfect the surface by wipe method.
5.1.6.6 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).
5.1.6.7 With the help of a sterile moistened swab, swab the surface gently covering all the area of the surface.
5.1.6.8 Place the swab sticks in a test tube having a sterile saline solution and analyze immediately.
5.1.6.9 Vortex the test tube gently for 5.0 minutes.
5.1.6.10 Aseptically filter the samples through a 0.45 m membrane.
5.1.6.11 Give three washing of 100 ml each with 0.1 % sterile peptone water/sterile water.
5.1.6.12 After the filtration with the help of a sterile forceps take the membrane and place it on a soybean casein digest agar.
5.1.6.13 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.
5.1.6.14 After incubation count the number of colonies present on the membrane.
5.1.6.15 Proceed in the same manner taking all the cultures to be tested and the various sanitizes.

6.0 ABBREVIATIONS

6.1 SOP - Standard Operating Procedure
6.2 % - Percent
6.3 °C - Degree centigrade
Also see: Validation Protocol for Efficacy of Disinfectants

Get ready to use editable documents in MS-Word FormatView List

Share

Tweet

Share

Share