Protocol No. / xxxxxxxxxxxxx
Disinfectant Validation Protocol / Page No. / 1 of 7
Area: Q.C - MICROBIOLOGY

DEPARTMENT

/

Microbiology

EFFECTIVE DATE

CHANGE HISTORY
Date / Supersede / CCR No. /

Changes Made

/

Revision No.

Nil / NA / Original Issue / 00

TABLE OF CONTENTS

S. No. / Description / Page No.
1.0 / Protocol approval / 2
2.0 / Objective / 3
3.0 / Scope / 3
4.0 / Validation Approach / 3
5.0 / Responsibilities and identification of execution team / 3
6.0 / Test procedures / 3
7.0 / Recording of observations / 7
8.0 / Discrepancy and corrective action report / 7
9.0 / Compilation, reviewand summary report / 7
10.0 / Appendix / 8
11.0 / Requalification Criteria / 8

1.0PROTOCOL APPROVAL

Signing of this approval page of protocol indicates agreement with the validation approach described in this document. If any modification in the validation approach becomes necessary, a revision through change control shall be prepared, checked and approved. This protocol cannot be executed until approved by following personnel.

Department / Name /

Designation

/ Signature /Date

Prepared by

Quality Control
Microbiology

Reviewed by

Quality Control
Microbiology

Approved by

Quality Assurance

Authorised by

HOD - QA

2.0OBJECTIVE

To establish documented evidence which will provide a High degree of assurance and reliability about the efficacy of the Disinfectants.

3.0SCOPE

The scope of this protocol is to outline procedure for efficacy of disinfectant/s used in the surface and area sanitization of controlled and critical clean rooms. To be performed at the time of revalidation.

4.0VALIDATION APPROACH

Disinfectants are tested for their efficacy at the different concentration and different intervals of contact time. The disinfectants when used as per the recommendations of the manufacture and above concentrations and the contact time should be able to inhibit the growth of the test microorganisms.

5.0RESPONSIBILITIES AND IDENTIFICATION OF EXECUTION TEAM

5.1Responsibilities: The group comprising of representatives from each of the following departments and they shall be responsible for the overall compliance with this protocol.

Department /

Responsibility

Quality Control
Microbiology / Execute, Participate and provide necessary support for the validation activity. Preparation and review of the Qualification Report of the qualification documents and its compliance to meet the acceptance criteria of the protocol.
Quality Assurance / Monitoring the qualification activities, compilation, review and authorisation of the Installation Qualification report and its compliance to meet the acceptance criteria of the protocol.

5.2Identification of Executors: All the identified executors involved with this Protocol are to Record Name, Designation, Signature and Date.

6.0TEST PROCEDURES

6.1Requirements

Before proceeding for validation following materials are required.

6.1.1Cultures

  • Staphylococcus aureus.
  • B.Subtilis.
  • E.coli.
  • Candida albicans
  • Apergillus niger.
  • Environmental Isolate (if available).
  • Disinfectants for Validation.
  • Sterile distilled water.
  • Poured SCDA plates and slants.
  • Poured PDA plates and slant.
  • Sterile forceps.
  • Sterile membrane filtration units.
  • Sterile membranes.
  • Vortex Mixer.
  1. Preparation of Spore forming Culture
  2. Prepare SCDA slants as per the SOP QC232.
  3. Incubate slant for 48 hrs at 30 – 35  C as preincubation to cross check the contamination.
  4. From the working culture streak a loopful of Bacillus subtilis culture on to the newly prepared slant.
  5. Incubate the slants at 30 – 35  C for 48 hrs.
  6. After 48 hrs of incubation cross check the culture for any cross contamination by simple gram staining technique SLT No 104.
  7. During staining also check for the presence of vegetative or spore cells.
  8. After Gram Staining preserve the cultures for further 7 days.
  9. After 7 days cross check the culture for any spore formation by negative/spore Staining technique.
  10. Enter the details in annexure no 1.
  11. Preparation of Challenge Inoculam
  12. Prepare fresh SCDA /PDA slants as per the SOP QC232.
  13. Incubate those SCDA slants at 30–35C for 48 hrs and PDA slants at 20 - 25C for 3 days to cross check any type of contamination.
  14. From the working culture streak a loopful of culture into the freshly prepared slants.
  15. Incubate those inoculated slants at 30–35C for 48 Hours for bacterial cultures and 20–25C for 5 days for fungal cultures.
  16. Add 5.0 ml of sterile saline into the slants aseptically taking care not to contaminate the slants.
  17. With the help of a inoculating loopstreak the lawn of organisms.
  18. Take 1.0 ml of the culture suspension and make serial dilutions ranging from 1:10 to 1: 1000.
  19. Plate the serial dilutions from 1:10 to 1:1000 by taking 1.0 ml of the culture in sterile petri dishes.
  20. Pour sterile molten SCDA (for bacteria) and PDA (for fungi) on to the inoculated plates.
  21. Perform the entire analysis in duplicate for each dilution.
  22. Incubate the plates at the required specified temperature.
  23. After plating the required dilutions do not discard the dilutions and preserve the samples at a temperature of 2 – 8C till the incubation period.
  24. After the incubation count the number of colonies.
  25. Select the dilution which is having 10000 to 100000 cells / ml for validation study.
  26. Preserve the culture suspension. Record the data in Annexure no. 1.
  27. Preparation of Disinfectant solution
  28. Prepare different concentrations of disinfectant/s as per the manufacturer recommended concentration and above the recommendation to establish the efficacy of the disinfectant.
  29. Enter the details in the annexure –1.
  30. Determining the Efficacy of the Disinfectant by Use Dilution Method
  31. Prepare test tubes having 9.0 ml of sterile distilled water.
  32. Add 1.0 ml of the use dilution for one disinfectant.
  33. Vortex the tube for 5.0 minutes.
  34. Add 0.1 ml of any one culture into the test sample.
  35. Make the sample dilution in such a way that each contact time has two sets of samples.
  36. Give a contact time of 0 hrs, 2 min, 5.0 min, 10 min.
  37. After the specified contact time, filter the samples through a 0.45  membrane.
  38. Give three washings of 100 ml each with 0.1 % sterile peptone water.
  39. After filtration with the help of a sterile forceps take the membrane and place it on a SCDA or m (HPC) agar. Incubate the plates.
  40. After incubation count the number of colonies present on the membrane.
  41. Note down the number of colonies.
  42. This will be the final count of the exposed culture.
  43. Select the plates, which have least to nil counts.
  44. Proceed in the same manner taking all the cultures to be tested.
  45. Contact time for the usage of the disinfectant will be set on the basis of the results, which will have least counts.
  46. Note down the results in Annexure –1.
  47. Determining the Efficacy of the Disinfectant by Surface Method.
  48. To get a practical approach for the efficacy apply the disinfectants on to any of the surface which is present in that particular area, which will be decontaminated by spraying the disinfectant.
  49. Take S.S strips and Epoxy coated material having a surface area of 25 cm2.
  50. Wrap the strips with an aluminium foil and sterilize it in a Dry heat Sterilizer at 2000 C for 2 hrs. For epoxy coated strips, disinfect as per the manufacturer recommendation.
  51. From the previously determined suspension having 10000 – 100000 cells per ml inoculate one culture on to four different S. S surface and Epoxy coated surface.
  52. With the help of a sterile spatula spread the culture on the surface.
  53. Perform the entire practise in Culture handling area.
  54. Keep it on the LAF bench for 30 minutes for drying.
  55. After the exposed duration for drying, spray the disinfectant on to any one surface of the recommended used dilution.
  56. Allow the surface to be with the sanitizer for 0hrs, 2min, 5.0 min, and 10 min.
  57. With the help of a sterile moistened swab, swab the surface gently covering all the area of the surface.
  58. Use different swabs for all the strips.
  59. Place the swab sticks in a test tube having sterile solution of fluid casein digest Soya broth.
  60. Vortex the test tube gently for 5. 0 minutes.
  61. Aseptically filter the samples through a 0.45  membrane.
  62. Give three washings of 100 ml each with 0.1 % sterile peptone water.
  63. After the filtration with the help of a sterile forceps take the membrane and place it on a SCDA or m (HPC) agar.
  64. Incubate the plates at 30 – 35  C for 48 Hours for bacterial cultures and 20 – 25  C for 5 days for fungal cultures.
  65. After incubation count the number of colonies present on the membrane.
  66. Note down the number of colonies in both the test sample and that with the unexposed strip.
  67. Proceed in the same manner taking all the cultures to be tested and the various sanitizers.
  68. Acceptance Criteria: The decrease in the bacterial load to the exposed disinfectant indicates that the disinfectant is capable of reducing the contaminant when used in the area.

7.0RECORDING OF OBSERVATIONS

Record the observations after the execution of each test procedures, in the annexure–1.

8.0DISCREPANCY AND CORRECTIVE ACTION REPORT

Document any discrepancies observed during the validation in annexure -1. Include the corrective actions of the same. When all the discrepancies are satisfactorily resolved or an approved action plan is developed which ensures that the discrepancy will be resolved.

9.0COMPILATION, REVIEW AND SUMMARY REPORT

Compile and review that all test functions have been completed, reconciled and attached to this protocol. Verify that the approvals for deviations have been taken and are resolved appropriately to the satisfaction.

Performance Qualification shall be considered acceptable when all the conditions specified in the test procedures have been met.

Prepare the summary report in the annexure –2 (Performance Qualification Report) and submit this for review, approval and authorisation to Validation Core Team.

10.0APPENDIX

10.1Abbreviations and definitions

Abbreviation / Definitions
VPQC / Validation protocol quality control.
SOP / Standard Operating Procedure.
QA / Quality Assurance.
Cfu / Colony Forming Unit.
°C / Degree Celsius.
NA / Not Applicable.
SCDA / Soyabean casein digest agar.
M (HPC) agar / Membrane hetero tropic count agar.
S.S / Stainless steel.
PDA / Potato dextrose agar.

10.2References

10.1.1Validation Master Plan.

10.1.2Preparation Of Culture Suspensions (SOPQC231).

10.1.3Preparation And Qualification Of Media (SOPQC232).

10.1.4Preparation of dilute disinfectants (SOPQC215).

10.3Annexure

10.3.1Annexure – 1 (Recording Of Observations For Disinfectant Validation).

10.3.2Annexure – 2 (Validation Report).

11.0REVALIDATION CRITERIA

Revalidation shall be carried out in case of

A new disinfectant is received.

If the manufacturer revise the concentration of ingredients.

If a new microorganism is isolated from that particular area.