Disinfectant Validation

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The objective of this validation protocol is to demonstrate the efficacy of a disinfectant solution which are being used for the surface and area sanitization of controlled and critical clean rooms by using following test –

  1. Use Dilution Test – Screening of disinfectants for their efficacy at various concentration and contact time against a wide range of standard test organisms and environmental isolates.
  2. Surface Challenge Test – Using standard test microorganisms and microorganisms that are typical environmental isolates, applying disinfectants to surfaces at the selected use concentration with a specified contact time, and determining the log reduction of the challenge microorganisms.

METHODOLOGY

  • Preparation of challenge organisms.
  • Preparation of disinfectant solution of various concentrations.
  • Preparation of neutralizing agent (Dey /Engley (D/E) broth) –
  • Use dilution test for testing of efficacy of disinfectant solution.
  • Surface challenge test for determination of log reduction of the challenge organisms.

Validation Procedure –

Use Dilution Test

Preparation of Challenge Organisms –

  • Take out the working cultures from the refrigerator 30 minutes prior to the testing so as to acclimatize with the working environment and place it under laminar airflow unit.
  • Red hot the loop and cool it, take a loop full of culture and inoculate it into 100 mL of Soyabean Casein Digest Medium (initial suspension).
  • Perform this exercise with all the challenge organisms.
  • Incubate the culture suspension of all the challenge organisms for a period of 12 hrs at 32.5 ± 2.5°C for bacterial cultures and 22.5 ± 2.5°C for yeast and mold to yield a minimum population of 0.5 x 108 CFU/mL.
  • After incubation gently vortex it so as to make a uniform suspension.
  • With the help of micropipette take 1.0 mL from the Initial suspension and inoculate it into 9 mL of 0.9 % Normal saline (1:10 dilution).
  • Transfer 1.0 mL from the above dilution to another tube containing 9 mL of 0.9 % Normal saline.
  • Perform the serial dilutions in the same manner ranging from 1:10 to1: 100000000.
  • Perform the whole exercise with all the challenge organisms, which are being used for validation.
  • Enumerate the challenge organism by pour plate method and add 1.0 mL of the dilutions into sterile Petri plates.
  • Pour sterile molten Soyabean Casein Digest Agar Medium to all the plates containing respective dilution for bacterial cultures and Sabourauds Dextrose Agar Medium to all the yeast and mold cultures into the plates.
  • Incubate the plates at 32.5 ± 2.5°C for 48 – 72 hrs for bacterial cultures and 22.5 ± 2.5°C for 72 – 120 hrs for yeast and mold.
  • Do not discard the initial culture suspension after plating the cultures.
  • Transfer 10 ml of the initial culture suspension into another sterile test tube and store it in the refrigerator at temperature 2 – 8°C.
  • Incubate the remaining initial culture suspension of all the challenge organisms for a period of 18 hrs at 32.5 ± 2.5°C for bacterial cultures and 22.5 ± 2.5°C for yeast and mold culture.
  • Perform the exercise from step 6.1.1.5 to 6.1.1.14 for a period of 18, 24, 36 and 48 hrs.
  • After the specified incubation period enumerate the number of colonies.
  • After enumerating the colonies calculate the concentration of initial culture suspension.
  • Preserve the culture suspension in refrigerator, which is having minimum population of 0.5 x 108 CFU/mL.

Preparation of disinfectant solution –

  • Prepare the disinfectant solution in water for injection according to the manufacturer recommended concentration.
  • Preparation of disinfectant solution is to be carried out as per SOP No. – N/QC/064.
  • Also prepare disinfectant solution + 0.5 % & – 0.5 % from the manufacturer’s recommended concentration to establish the efficacy of the disinfectant.
  • Filter the prepared disinfectant solution using 0.2-µ membrane filters.
  • Distribute 1.0 mL of filtered disinfectant solution into sterile test tubes.
  • Incase dilution is not recommended by the manufacturer then disinfectant is to be validated only on manufacturer recommended concentration.

 Preparation of neutralizing agent (Dey /Engley (D/E) broth) –

  • Prepare the Dey / Engley (D/E) broth which contains wide range of neutralizing agents.
  • Distribute 9.0 mL of Dey / Engley (D/E) broth into sterile test tubes.

Preparation of Disc (Inoculated carrier) –

  • Prepare a culture suspension of each challenge organism as describe in section 6.1.1.
  • Prepare filter paper discs of size 1/4” and sterilize the disc in the autoclave as per SOP No. – N/QC/093.
  • For each challenge organism to be tested inoculate sufficient sterile discs with 20 mL of the prepared culture suspension and allow the inoculated disc to air dry under laminar air flow unit.
  • For each challenge organisms prepare separate discs.

Neutralizing Agent Validation –

Validation of the neutralizing agent demonstrates the efficacy of the neutralizing agent in the test procedure.

Solution Preparations –

  • Test Control – Prepare sterile test tubes with 1.0 mL of the filtered disinfectant solution (+ 0.5 % from the manufacturer recommended concentration) or at manufacturer recommended concentration (if dilution is not recommended) add 9.0 mL of the Dey/Engley (D/E) broth, which contains wide range of neutralizing agent.
  • Negative Control – Distribute 10.0 mL of 0.9% Normal Saline in sterile test tubes.
  • Positive Control – Distribute 10.0 mL of 0.9% Normal Saline in sterile test tubes.

Test Control –

  • For each challenge organisms, place an inoculated carrier (Disc) into each test tube containing 1.0 mL filtered disinfectant solution and 9.0 mL Dey/Engley (D/E) broth, which contains neutralizing agent mixture for one minute (1 minute).
  • After one minute (1 minute), macerate the inoculated carrier using glass beads and vortex mixture.
  • These tubes are considered the 10 – 1
  • Transfer 1.0 mL from the above dilution to another tube containing 9.0 mL of the 9% Normal Saline.
  • Perform the serial dilutions in the same manner ranging from 1:10 to1: 100000000.
  • After serial dilution plate out in duplicate 1.0 mL aliquot from each tube.
  • Pour sterile molten Soyabean Casein Digest Agar Medium for bacterial cultures and Sabourauds Dextrose Agar Medium for all the yeast and mold cultures into the plates.
  • Incubate all the plates at 32.5 ± 2.5°C for 48 – 72 hrs for bacterial cultures and 22.5 ± 2.5°C for 72 – 120 hrs for yeast & mold.
  • After completion of incubation period count the plates with 30 – 300 colonies per plate.
  • Average the duplicate plates and multiply by the dilution factor. (Reciprocal of the dilution).
  • Repeat the exercise from step 6.1.5.2.1 to 6.1.5.2.10 with all the challenge organisms.

 

Negative Control –

  • Plate out 1.0 mL aliquot from the each test tube containing 10 mL 0.9% Normal Saline in to sterile petriplates in duplicate.
  • Pour sterile molten Soyabean Casein Digest Agar Medium and Sabourauds Dextrose Agar Medium into the plates respectively.
  • Incubate all the plates at 32.5 ± 2.5°C for 48 – 72 hrs in case of Soyabean Casein Digest Agar Medium and 22.5 ± 2.5°C for 72 – 120 hrs in case of Sabourauds Dextrose Agar Medium.
  • After completion of incubation period count the plates for any colonies.

Positive Controls –

  • For each challenge organisms, place an inoculated carrier (Disc) into each test tube containing 10 mL Sterile 0.9% Normal Saline for one minute (1 minute).
  • After one minutes (1 minutes), macerate inoculated carrier using glass beads and vortex mixture.
  • These tubes are considered the 10 1
  • Transfer 1.0 mL from the above dilution to another tube containing 9.0 mL of the 9% Normal Saline.
  • Perform the serial dilutions in the same manner ranging from 1:10 to1: 100000000.
  • After serial dilution plate out in duplicate 1.0 mL aliquot from each tube.
  • Pour sterile molten Soyabean Casein Digest Agar Medium for bacterial cultures and Sabourauds Dextrose Agar Medium for all the yeast & mold cultures into the plates.
  • Incubate all the plates at 32.5 ± 2.5°C for 48 – 72 hrs for bacterial cultures and 22.5 ± 2.5°C for 72 – 120 hrs for yeast & mold.
  • After completion of incubation period count the plates with 30-300 colonies per plate.
  • Average the duplicate plates and multiply by the dilution factor (reciprocal of the dilution).
  • Repeat the exercise from step 6.1.5.4.1 to 6.1.5.4.10 with all the challenge organisms.

Acceptance Criteria –

  • Compare the counts of the test control and the positive controls.
  • There should not be more than a half log difference between the test control and the positive controls for the neutralizing agent to be acceptable.
  • Calculate the log reduction for each challenge organism using the following formula:

Log Reduction = log No – Log N, . Where No = The average count (Positive Control), N = The average               count (Test Control)

  • No colonies should be observed in the negative control.

 Disinfectant Solution Validation –

All the disinfectants, which are being used for the surface and area sanitization of controlled and critical clean rooms, must be initially validated using the following procedures.

  • Prepare the disinfectant solution according to Section 6.1.2.
  • Prepare neutralizing agent (Dey /Engley (D/E) broth according to Section 6.1.3.
  • Prepare discs (Inoculated carriers) according to Section 6.1.4.

Disinfectant Exposure (Contact Period) –

One minute (1 minute), Two minutes (2 minutes), Five minutes (5 minutes), Ten minutes (10 minutes) and Thirty minutes (30 minutes).

Test Control –

  • Place an inoculated carrier (Disc) into 1.0 mL filtered disinfectant solution for recommended contact period.
  • Follow the contact period and after contact period immediately add 9.0 mL Dey /Engley (D/E) broth which contains neutralizing agent and vortex until well mixed.
  • Periodically macerate inoculated carrier (Disc) using glass beads and vortex mixture.
  • These tubes are considered the 10 1
  • Transfer 1.0 mL from the above dilution to another tube containing 9.0 mL of 9% Normal Saline.
  • Perform the serial dilutions in the same manner ranging from 1:10 to1: 100000.
  • After serial dilution plate out in duplicate 1.0 mL aliquot from each tube.
  • Pour sterile molten Soyabean Casein Digest Agar Medium for bacterial cultures and Sabourauds Dextrose Agar Medium for all the yeast and mold cultures into the plates.
  • Incubate all the plates at 32.5 ± 2.5°C for 48 – 72 hrs for bacterial cultures and 22.5 ± 2.5°C for 72 – 120 hrs for yeast & mold.
  • After completion of incubation period count the plates with 30-300 colonies per plate.
  • Average the duplicate plates and multiply by the dilution factor (reciprocal of the dilution).
  • Repeat the exercise from step 6.1.6.5.1 to 6.1.6.5.11 with all the challenge organisms, all the concentration of the disinfectant used and for all the contact period.

Positive Control –

  • For each challenge organism, place an inoculated carrier (Disc) into each test tube containing the 10 mL 0.9% Normal Saline for one minute (1 minute).
  • After one minute (1 minute), macerate inoculated carrier using glass beads and vortex mixture.
  • These tubes are considered the 10 1
  • Transfer 1.0 mL from the above dilution to another tube containing 9.0 mL of 9% Normal Saline.
  • Perform the serial dilutions in the same manner ranging from 1:10 to1: 100000000.
  • After serial dilution plate out in duplicate 1.0 mL aliquot from each tube.
  • Pour sterile molten Soyabean Casein Digest Agar Medium for bacterial cultures and Sabourauds Dextrose Agar Medium for all the yeast and mold cultures into the plates.
  • Incubate all the plates at 32.5 ± 2.5°C for 48 – 72 hrs for bacterial cultures and 22.5 ± 2.5°C for 72 – 120 hrs for yeast & mold.
  • After completion of incubation period count the plates with 30-300 colonies per plate.
  • Average the duplicate plates and multiply by the dilution factor (reciprocal of the dilution).
  • Repeat the exercise from step 6.1.6.6.1 to 6.1.6.6.10 with all the challenge organisms.

Negative Control –

  • Place an uninoculated carrier (Disc) into each test tube containing the 1.0 mL 0.9% Normal Saline for one minute (1 minute), add 9.0 mL Dey/Engley (D/E) broth, which contains neutralizing agent.
  • After one minute (1 minute), macerate inoculated carrier using glass beads and vortex mixture.
  • Plate out 1.0 mL aliquot from the each test tube in to sterile petriplates in duplicate.
  • Pour sterile molten Soyabean Casein Digest Agar Medium and Sabourauds Dextrose Agar Medium into the plates respectively.
  • Incubate all the plates at 32.5 ± 2.5°C for 48 – 72 hrs in case of Soyabean casein digest agar and 22.5 ± 2.5°C for 72 – 120 hrs in case of Sabourauds Dextrose Agar Medium.
  • After completion of incubation period count the plates for any colonies.

 Interpretation of result –

  • No colonies should be observed in the negative control.
  • 3 Log reduction or greater should be achieve in the test.
  • Calculate the log reduction for each contact period, each disinfectant concentration for each challenge organism using the following formula:

Log Reduction = log No – Log N.

Where No = The average count (Positive Control)

N = The average count (Test Control)

Determine the contact period where a 3 Log reduction or greater is achieved.  This time period will serve as contact period for the disinfectants solution.

  • Determine the concentration (use dilution) and contact period at which the disinfectant solution show no growth.

 

Acceptance Criteria –

  • No colonies should be observed in the negative controls.
  • 3 Log reduction or greater should be obtained in the disinfectant solution tested at a particular concentration and contact period. If 3 Log reduction is not achieve then section 6.1.6.5 to be repeated to determine a new concentration and contact period for the disinfectant solution.
  • Determine the concentration (use dilution) and contact period at which the disinfectant solution show 3 Log reduction or greater.

 Surface Challenge Test  –

   Preparatory Activities

  • Prepare and filter the disinfectant solution as per recommended used dilution.
  • Take S.S strips and Epoxy coated material having a surface area of 25 cm2.
  • Wrap the strips with an aluminium foil and sterilize it in autoclave as per SOP No. – N/QC/093.
  • For Epoxy coated surface, disinfect the area as per the recommended concentration.
  • Prepare the suspension of challenge organisms having minimum population of 104 to 105 CFU/mL as per step 6.1.1.
  • Prepare the swabs as per SOP No. – N/QC/068.

 

 Test Control

  • Inoculate 1 mL of culture suspension on the six different S. S strips and Epoxy coated surface.
  • With the help of a sterile spatula spread the culture suspension on the surface of the S. S strips and Epoxy coated surface.
  • Perform the entire practice under Laminar airflow unit.
  • After spreading keep the S. S strips and Epoxy coated surface under Laminar airflow unit for 30 minute for drying.
  • After drying the S. S strips and Epoxy coated surface, spray the disinfectant on one S. S strips and one epoxy coated surface with the recommended used dilution.
  • After recommended contact period wipe the surface with the help of a sterile swab, swab the surface gently covering all the area of the surface..
  • Repeat the step from 6.2.2.1 to 6.2.2.6 with all the challenge organisms.
  • Use different swabs for all the strips and Epoxy surface.
  • Analyze the swab as per the SOP – N/QC/065.
  • After incubation count the number of colonies as CFU / 25 cm2.

    Positive Control

  • Inoculate 1 mL of culture suspension on the one S. S strips and one Epoxy coated surface.
  • With the help of a sterile spatula spread the culture suspension on the surface.
  • Perform the entire practice under Laminar airflow unit.
  • After spreading keep the S. S strips and Epoxy coated surface under Laminar airflow unit for 30 minute for drying.
  • After drying wipe the surface with the help of a sterile swab, swab the surface gently covering all the area of the surface.
  • Repeat the step from 6.2.3.1 to 6.2.3.5 with all the challenge organisms.
  • Analyse the swab as per the SOP – N/QC/065.
  • After incubation count the number of colonies as CFU / 25 cm2.

 Negative Control

  • Wipe the surface of the one uninoculated S. S strips and one Epoxy coated surface with the help of a sterile swab, swab the surface gently covering all the area of the surface.
  • Analyse the swab as per the SOP – N/QC/065.
  • After incubation count the number of colonies as CFU / 25 cm2.

Acceptance Criteria

  • No colonies should be observed in the negative control.
  • There should be 3 – log reduction or greater after exposure to recommended disinfectant concentration (use dilution) and recommended contact period.
  • The decrease in the microbial load to the exposed disinfectant indicates that the disinfectant is capable of reducing the contaminant when used in the area.

 

FREQUENCY OF VALIDATION:

 Whenever a new disinfectant is received.

 If the manufacturer revises the concentration of ingredients.

REFERENCES Source:

Chapter 1072 – Disinfectants and Antiseptics (United State Pharmacopoeia  – 30 NF 25 2007)

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