PROCEDURE FOR VALIDATION OF DISINFECTANT

1. PURPOSE:

This Sop Describes the Procedure for Validation of Disinfectant

• SCOPE:

This Sop Is Applicable to the Procedure for Validation of Disinfectant.

• RESPONSIBILITY:

 Microbiologist

• ACCOUNTABILITY:

Head Quality Control.

• PROCEDURE
  • Disinfectant Used
    • Dettol 2.5%
    • Savlon 2.5%
    • Isopropyl Alcohol 70%
  • Equipments Required
    • LAF
    • Measuring Cylinder
    • Sterile Petri Plates
  • Material Required
    • Disinfectant
    • Sterile Saline solution
    • Sterile nutrient agar
    • Test organism

S. No	Name of the culture
01.	Staphylococcus aureus
02.	Escherichia coli
03.	Candida albicans
04.	Aspergillus niger
05.	Clostridium sporogens
06.	Bacillus subtilis
07.	Pseudomonas aeruginosa

• Procedure:
  • Preparation of Culture Suspension:
    • Take the culture with sterile inoculums loop and suspended in 100ml of sterile normal saline 0.9% and mix.
    • Stand for suspension for 1 hour for proper mix.
  • Determination of Population of Culture in Culture Suspension:
    • Take 10 sterile test tubes; take 9ml of normal saline 0.9% in each and every test tube with following process.
    • Culture Suspension 10^-1:Take 1ml from culture suspension and add in 1 in 9ml of test tube of Normal saline test tubes 1 and mix for 3 – 5 min.
    • Culture Suspension 10^-2 : Take 1ml from normal saline test tube 1 and add in 1 in 9ml of test tube of Normal saline test tubes 2 and mix for 3 – 5 min.
    • Culture Suspension 10^-3 : Take 1ml from normal saline test tube 2 and add in 1 in 9ml of test tube of Normal saline test tubes 3 and mix for 3 – 5 min.
    • Culture Suspension 10^-4 : Take 1ml from normal saline test tube 3 and add in 1 in 9ml of test tube of Normal saline test tubes 4 and mix for 3 – 5 min.
    • Culture Suspension 10^-5 : Take 1ml from normal saline test tube 4 and add in 1 in 9ml of test tube of Normal saline test tubes 5 and mix for 3 – 5 min.
    • Culture Suspension 10^-6 : Take 1ml from normal saline test tube 5 and add in 1 in 9ml of test tube of Normal saline test tubes 6 and mix for 3 – 5 min.
    • Culture Suspension 10^-7 : Take 1ml from normal saline test tube 6 and add in 1 in 9ml of test tube of Normal saline test tubes 7 and mix for 3 – 5 min.
    • Culture Suspension 10^-8 : Take 1ml from normal saline test tube 7 and add in 1 in 9ml of test tube of Normal saline test tubes 8 and mix for 3 – 5 min.
    • Take 1- 1 ml culture suspension from serial dilution in sterile petri plate and pore nutrient agar in each and every plates.
    • Incubate for 30 – 35⁰C for 3 day for bacterial observation and 20 -25⁰C for 5 days for fungal observation.
    • After completion of incubation count the colony by colony counter and note in Formate.
  • Validation of Disinfectant:
    • Take 9 ml of Dettol solution 2.0%, 2.5% & 3.0% in sterile test tube and 1 ml of culture suspension observed about 100 cfu/ml.
    • Stand for 30 minute and take 1 – 1ml for dettol solution 2.0%, 2.5% & 3.0% in sterile petri plate in duplicate, Incubate for 30 – 35⁰C for 2 days and further incubate for 20 – 25⁰C for 3 days.
    • After incubation count the colony by colony counter and note.
    • Acceptance Criteria: Should be colony observed not more than 10% from initial colony.
    • Performed the test for Savlon 2.0%, 2.5% & 3.0% & Isoprpyl alcohol 50%, 70% & 90.0% from step 5.4.3.1. to 5.4.3.4.
  • Determination of Recommended Concentration by Surface Swab Method:
    • Apply the above known culture cells (approx 10^-1 to 10^-6 cfu) individually to a surface of 25 cm2.
    • Allow to air dry the applied cultures at room temperature.
    • Prepare the disinfectant solution to be tested as per SOP for Preparation of Disinfectant Solution and apply on the surface of artificially contaminated area.
    • Take a swab immediately at 0 min, after 5 min, 10 min, 20 min and after 30 minutes, and then proceed the swab testing as per SOP for Swab Testing.
    • Acceptance Criteria: Should be colony observed not more than 10% from initial colony 20 – 30 min.
    • Incubate the plates as specified in above table and tabulate the result.
  • Preparation of Nutrient Agar & Disinfectant Solution:
    • Take 28 g of nutrient suspended in 1000ml of purified water and sterile at 121⁰C & 15 lbs for 15 minutes.
    • Dettol 2.0%: Take 20 ml of Dettol and diluted to 1000ml of purified water.
    • Dettol 2.5%: Take 25 ml of Dettol and diluted to 1000ml of purified water.
    • Dettol 3.0%: Take 30 ml of Dettol and diluted to 1000ml of purified water.
    • Savlon 2.0%: Take 20 ml of Savlon and diluted to 1000ml of purified water.
    • Dettol 2.5%: Take 25 ml of Savlon and diluted to 1000ml of purified water.
    • Dettol 3.0%: Take 30 ml of Dettol and diluted to 1000ml of purified water.
    • Isopropyl Alcohol 50%: Take 50 ml of Isopropyl alcohol and diluted to 100ml with purified water. And filter with 0.2µm pore size nylon filter paper.
    • Isopropyl Alcohol 70%: Take 70 ml of Isopropyl alcohol and diluted to 100ml with purified water. And filter with 0.2µm pore size nylon filter paper.
    • Isopropyl Alcohol 90%: Take 90 ml of Isopropyl alcohol and diluted to 100ml with purified water. And filter with 0.2µm pore size nylon filter paper.
    • Maintain the preparation of nutrient Agar & disinfectant solution in Format.
  • Precaution
    • Artificially contaminated area should be cleaned immediately after completion of work.
    •  Use hand gloves and nose mask during testing.
    • All the glassware’s used for dilution and testing, immediately clean and sterilize as per SOP.
    • Acceptance Criteria: NLT 90% population is killed.
• TRAINING:

Trainer   : Manager – Quality Control

Trainees : Staff of the microbiology departments

• DISTRIBUTION:

Master Copy                  :           QA Department

Controlled Copy            :           Microbiology Department

Display Copy                 :           Microbiology Department (If Required)

• REFERENCES:

In-House

1. ABBREVIATIONS:

Abbreviations	Extended Form
SOP	Standard Operating Procedure
NA	Not Applicable
MB	Microbiology
Sr. No.	Serial Number
QA	Quality Assurance
IPA	Isopropyl Alcohol
%	Percentage

1. REVISION HISTORY OF CHANGE:

Sr. No.	Date	Revision Details	Revision No.
01	NA	NEW SOP	NA
02	NA	SOP Periodical  Review	NA