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Validation Protocol For Efficacy of Chemical Disinfectants
Validation Protocol For Efficacy of Chemical Disinfectants
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4.0 Pre-Requisites
Prior to conducting/executing the General validation protocol following things must be available
4.1 Microbial Standard Cultures
4.1.1 Bacillus subtilis
4.1.2 Escherichia coli
4.1.3 Staphylococcus aureus
4.1.4 Candida albicans
4.1.5 Aspergillus niger
4.1.6 Environment isolates
4.2 Disinfectants: All disinfectants used in the facility
4.3 Micropipettes
4.4 Sterilized tips and Petri plates
5.0 Responsibility
Responsibilities of Quality Assurance and Quality control Microbiology personnel involved in activities related to the General validation protocol are
defined below
5.1 Quality Assurance (Validation)
5.1.1 Approval of protocol
5.2 Quality Control
5.2.1 Preparation of protocol
5.2.2 Review of protocol
5.2.3 Execution of protocol
6.0 Validation Method
Qualification Tests
A) Suspension method (Validation of sanitizer)
B) Surface spray/immersion or wipe method (Validation of Sanitization method)
6.1 Suspension Method
6.1.1 Objective:
To establish the test concentration and the contact time a suspension test is generally applied. The suspension test 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 period
• Interfering substances (i.e. inorganic, organic matter)
6.1.2 Procedure:
6.1.2.1 Prepare the culture suspension from the original culture as per the SOP for preparation of Microbiological culture media.
6.1.2.2 Select the dilution which will yield 105 to 106 cells per ml.
6.1.2.3 Prepare 10 ml of test dilution to be tested with sterile distilled water.
6.1.2.4 Vortex the tube for 1 minute.
6.1.2.5 Add 0.1 ml of any one culture containing 105 to 106 cells per ml into the test disinfectant with the decided concentration.
6.1.2.6 The final concentration shall be 104 to 105 cells per the tube. Consider the preparation for 0 min.
6.1.2.7 Prepare like the same above for other 3 different sets for 3 different time intervals.
6.1.2.8 Give a contact time of 0 min, 5 min, 10 min and 15 min.
6.1.2.9 After the specified contact time, filter the samples through a 0.45 m membrane filter.
6.1.2.10 Give three washing of 100 ml each with 0.1 % sterile peptone water/ Sterile Water.
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6.1.2.11 After filtration with the help of a sterile forceps take the membrane filter and place it on a Soybean casein digest agar.
6.1.2.12 Incubate the bacterial culture at 30-35 °C for 24 to 48 hours and fungal cultures at 20-25 °C for 72 to 120 hours.
6.1.2.13 After incubation count the number of colonies present on the membrane.
6.1.2.14 Note down the number of colonies.
6.1.2.15 This will be the Observed count after the exposure.
6.1.2.16 Select the plates, which have least to Nil counts.
6.1.2.17 Proceed in the same manner taking all the cultures to be tested.
6.1.2.18 Contact time for the usage of the disinfectant will be set on the basis of the results, which will have least counts.
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11.0 Abbreviations
SOP: Standard Operating Procedure
° C: Degrees centigrade
% : Percentage
CFU: Colony forming units
SCA: Soybean casein digest agar
m: Micron
PU: Poly Urethane
Annexure –I
Results: Suspension Method
Annexure –II
Results: Surface Spray / Wipe Method
Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of pharmaguideline.com, a widely-read pharmaceutical
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