Disinfectant Validation Procedure

1.0 Objective:

The objective of this protocol is to provide sufficient documented evidence in order to prove that the disinfectants employed for cleaning and sanitization are effective against wide range of microorganisms by employing Membrane Filtration method to establish the effective working concentration and contact time.

2.0 Scope:

The scope of this Validation protocol is to assure the efficacy of different disinfectant used for cleaning as well as sanitizing the production manufacturing, microbiology testing areas and rest of the company premises (general areas) which could be capable enough to kill wide range of microbial flora generally prevailing in those areas.

3.0 Pre –Validation Requirement:

3.1 Selection of Microorganisms:

The microorganisms selected are based on different classifications. The selected microorganisms include:

ü  Gram positive bacteria (Staphylococcus aureus ATCC No. 6538 or equivalent)

ü  Gram positive spore forming bacteria (Bacillus subtilis ATCC No. 6633 or equivalent)

ü  Gram negative bacteria (Pseudomonas aeruginosa  ATCC No. 9027 or equivalent)

ü  Yeast (Candida albicans ATCC No. 10231 or equivalent)

ü  Mold (Aspergillus brasiliensis ATCC No.16404 or equivalent)

ü  Environment Isolate if available

3.2 Selection of Disinfectants:

Disinfectants should be selected based on the following characteristics as mentioned below.

ü  Should be able to kill a wide range of microbial flora including pathogenic microbial strains.

ü  Should be non-toxic to humans on general application.

ü  Should not corrode the equipment surfaces on application.

ü  Should not possess any bad odour or residues on application.

3.3 Required Media, Diluents and other Accessories:

ü  Sterile molten Soyabean Casein Digest Agar (SCDA)

ü  Sterile molten Sabouraud dextrose agar (SDA)

ü  Sterile Normal saline (0.9 % of sodium chloride solution)

ü  Sterile Purified Water or water for injection

ü  Sterile Membrane Filtration assembly with vacuum pump

ü  Sterile Forceps

ü  Vortex Mixer.

ü  Sterilized graduated 1ml and 10ml glass pipette.

ü  Calibrated micropipettes and sterile micro tips

ü  Sterile 0.1% w/v peptone water.

ü  Measuring cylinder.

ü  Disinfectant of different concentration.

ü  Sterile 0.9 % w/v normal saline tubes (10 ml)

ü  Sterile cotton swabs.

ü  Contact Plates.

ü  Cyclomixer

ü  Poured sterile SCDA plates

4.0 Selection of Different Surfaces:

Different surface are selected for the validation of Disinfectants to provide the documented evidence that all the disinfectants with required concentration and contact time are effective on all the surfaces which we have included as per company premises product manufacturing and testing areas. 

4.1 List of different surfaces included for disinfectant validation studies are given below:

ü  Stainless Steel surface

ü  Glass surface

ü  GI doors surface

ü  Panel surface

ü  Granite surface

ü  Cota stone Surface

ü  PU coated surface

ü  Paint coated surface

ü  Epoxy coated surface

ü  Glove Surface

ü  Tiles Surface

5.0 Method for Disinfectant Validation:

Membrane Filtration technique is one of the simplest and effective analytical method for evaluation of sanitizer/disinfectant efficacy. This method involves the direct mixing of microbial cells or spores with the sanitizer/disinfectant and subsequent filtering of the solution at various intervals of contact time between sanitizer/disinfectant solution and microbial cultures. This method completely removes any the chances of bacteriostasis or Fungistasis.

6.0 Process for Validation of Efficacy of Disinfectant will be performed under following subheadings:

6.1 Preparation of challenge inoculums:

ü  Take required type of working culture (Spore culture in case of Bacillus subtilis).

ü  Take loopful of each working culture (Spore culture in case of Bacillus subtilis) and inoculate in to different tubes containing 10 ml Sterile saline and dilute the suspension serially using  10 fold dilution method from 10^-1 to 10^-8.

ü  Pipette out 0.1 ml of 10^-1 diluted culture on to each of two Sterile Petri dishes.

ü  Repeat this step for 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, 10^-7, and 10^-8. Repeat the above 4 steps for all other cultures.

ü  Add 20 ml of SCDA medium for bacteria, SDA for Fungi that is melted and cooled at approximately 45⁰ C.

ü  Cover the Petri dishes and mix the sample with Agar.

ü  Allow to solidify at room temperature. Invert the Petri dishes and incubate bacteria at 30-35⁰ C for 48 hours, Candida albicans and Aspergillus brasiliensis at 20-25⁰ C for 5 days.

ü  Preserve all the dilutions at 2-8⁰ C. Examine the plate count as the average of two plates in terms of CFU’s/0.1 ml. Select the dilutions containing 10,000 to 100000 CFU’s/0.1 ml, store at 2-8⁰ C and discard all other dilutions.

ü  Record the data in Attachment No-II.

6.2 Preparation of Bacillus spore culture:

ü  Take a pre-incubated sterile SCDA slant and a working culture of Bacillus.

ü  Pick a loopful of Bacillus culture and streak on surface of sterile SCDA slant.

ü  Incubate the slant at 30-35 ⁰C for 7 days.

ü  Perform spore staining of this incubated culture and observe for its purity and spore formation.

üIf the culture shows spore, preserve this culture at 2-8⁰C and use this spore culture for the preparation of inoculum of Bacillus as mentioned above.

ü  Record the data in Attachment No-I.

ü  Use the preserved dilution culture suspension within 7 days.

7.0 Disinfectants and Concentrations:

Prepare the disinfectants in sterile purified water or sterile water for injection and filter the disinfectants with 0.22 micron filters. The concentration and contact time of disinfectants should be based on the manufacturer recommendation. Prepare three different concentration of one disinfectants in which select one concentration which is recommended by manufacturer, one lower concentration and one higher concentration than the recommended concentration. Same with the contact time, use one contact time which is recommended by manufacturer, then choose one lesser contact time and one higher contact time. For example if recommended concentration by manufacturer is 1.0% for any disinfectant with 10 minutes of contact time then we can select 0.5% which is lower concentration with contact time of 5 minutes which is again less contact time than the recommended contact time. Select one higher concentration of 2.0% which is higher than the recommended and higher contact time of 15 minutes. By preparing three different concentrations and different contact time perform the validation study.

7.1 Use Dilution Method:

ü  Dilute disinfectant solution as per the selected concentration.

ü  Use sterile purified water or sterile water for injection as diluent.

ü  Arrange required number of sterile empty test tubes in a test tube rack.

ü  Use calibrated micropipettes or graduated pipettes for dispensing the inoculums and disinfectant solution.

ü  Add 10 ml of manufacturer recommendation concentration of disinfectant solution in a test tube.

ü  Add 10 ml of lower and higher concentrations of disinfectant in other test tubes.

ü  Arrange one sterile tube containing 10 ml of diluent (for initial count of challenge inoculum).

ü  Add 0.1 ml of prepared challenge inoculum to 10 ml of diluent.

ü  Dilute the solution using 10 fold dilution from 10^-1 to 10^-8 and plate from 10^-1 Dilution.

ü  Incubate these plates for initial count determination at appropriate temperatures.

ü  Arrange the sterile filter holder having 0.45µm membrane filter on manifold and assemble the manifold to vacuum source.

ü  Add 0.1 ml of challenge inoculum to all the tubes which contains disinfectant solution.

ü Contact time of all concentrations (0.5%, 1.0%, and 2.0%) should be done at room temperature first for 5 minutes. For positive controls, inoculation done in duplicate for each challenge microbial culture strain and Negative control should be kept un-inoculated. After the completion of ‘5’ minutes contact time, the solutions should be filtered using separate filter holder. Each membrane filter was rinsed with 3 X 100 ml sterile 0.1% peptone water to remove the traces of disinfectants.

ü  Neutralizers could be used if required to neutralize any traces of disinfectant on the filter or we may use media containing neutralizers.

ü  After rinsing, place each membrane filter on the surface of individual pre-incubated agar medium plates.

ü  Similarly challenge all the prepared concentration for contact time of 10 and 15 minutes intervals individually, mix and filter the solution of each tube using individual filter holder for each time.  Rinse each membrane filter with 3 X 100 ml sterile 0.1% peptone water.

ü  After rinsing place each membrane filter on the surface of individual pre-incubated agar medium plates.

ü  Incubate all plates at 30^oC – 35^oC for 48 hours for bacteria and the plates with the challenge inoculums of Candida albicans and Aspergillus niger at 20^oC – 25^oC for 5 days. Examine all plates each day for the presence of any microbial growth and compare with initial count.

ü  Similarly Challenge different concentrations of other disinfectants with 0.1 ml of the inoculums used in the previous test at different contact time individually and proceed as given above employing membrane filtration.

ü  Record the data in Attachment No-III.

7.2 Surface Challenge Test:

This method involves application of disinfectants to the different surfaces, challenged with standard test organisms, at the selected use concentration with a specified contact time and determining the log reduction of the challenge microorganisms.

7.3 Determination of efficacy of disinfectant:

ü  Perform all the activities under Laminar Air Flow.

ü  Prepare plates of Soyabean casein digest Agar (SCDA) and Sabouraud Dextrose Agar (SDA).

ü  Use pre-incubated plates for validation study.

ü Use the dilutions of the culture suspension and challenge the surfaces of the material to be evaluated with 0.1 ml of the inoculums.

ü  Spread the cell uniformly over the surface of 2 inch X 2 inch square with the help of sterile L shaped spreader.

ü  Allow the suspension to dry.

ü  After completely drying spray the recommended concentration of the chosen disinfectant on the surface where inoculums are spread and note the contact time.

ü  Take required number of readymade sterile swab for sampling and wet those swabs in sterile 0.9% saline solution under LAF.

ü  Take the swab from the surface of 2 inch X 2 inch square by following horizontal strokes first followed by vertical strokes.

ü  Replace the swab in to the tube with saline and mark the swab tube properly with date of sampling and name of organisms.

ü  Vortex the tube by cyclomixer to make the suspension uniform.

ü  Filter the content of the swab by 0.45µm pore size filter paper.

ü  Wash membrane with 3x100 ml of 0.1% sterile peptone water to remove residue of disinfectant.

ü  Aseptically transfer the membrane filter to the pre-incubated plate of Soyabean casein digest agar and Sabouraud dextrose agar. Avoid air trapping between the filter and agar surface.

ü  Incubate the plates of bacteria at 30-35°C for 48 hours and for fungus 5 days.

ü  Observe the plates after completion of incubation; express the results in cfu/plate.

ü  Record the data in Attachment No-IV.

ü  Fill the validated concentration and contact time for particular disinfectant as per Attachment No.-V.

8.0 Acceptance Criteria:

ü  The concentration of disinfectant which shows required reduction in microbial count at the least concentration and contact time shall be selected as effective concentration. 

ü  The disinfectant solution should bring a minimum of 3-log reduction for vegetative cells and minimum 2 log reduction for spore cells. 

9.0 Re-Validation:

The Disinfectant Efficacy validation shall be re-validated in one or more of following cases:

ü  Replacement of any disinfectant.

ü  Change in the composition of disinfectant by manufacturer.

ü  Change in cleaning procedure

10.0 Change Control System:

Any changes in the proposed equipment/system/procedure shall be carried out through the change control procedure. 

 11.0 Validation Report:

ü  The Validation report shall consist of a summary document, in narrative form, which shall briefly describe the activity performed along with the observations recorded.

ü  This report shall also include the related documents and attachments/annexure which completed at the time of validation activity.

Attachment No-I

Preparation of Bacillus subtilis Spores

Working Culture No		MTCC No.
Equivalent ATCC No.		Incubation Temperature
Agar medium used		Incubation Period
Medium Lot No.		Incubator ID No.

 

Staining Method used:

Result of Staining: ________________________________________________________________________________                                                                                ________________________________________________________________________________

Done By/Sign:                             Checked By/Sign:                  Reviewed By/Sign:                                   

Attachment No-II

Preparation of Challenge inoculum

Name of the Culture:                                                 Working culture tube No.Used:         

Strain No:                                                                   Equivalent ATCC No.:

Name of the Diluent used:                                         Dilution made:

Agar Medium used:                                                   Medium Lot No:

Incubation period and Temperature:                         Incubator I.D.No.:

Dilution Plated	Count/0.1ml/plate
	Plate-1	Plate-2	Average

Interpretation: As per the above Observation it is estimated that the________ dilution tube contain the culture concentration of ______________________ CFU’s/0.1 ml

  Done By/Sign:                             Checked By/Sign:                 Reviewed By/Sign:    

Attachment No-III

 Use Dilution Test

 Use Dilution Test

Name of Disinfectant:                                                     MTCC No.:

Name of the Organism:                                                   Equivalent ATCC No.:

Date of test:                                                                     Date of observation:                                                                      

Name of the Organism
Initial Count
Log of Initial Count
Concentration
Contact Time  _______________
Contact Time  _______________
Contact Time  _______________

 Log Reduction Calculation:

Concentration and Contact time	Log of CFU’s recovered in initial count – Log of CFU’s recovered in Test

Remarks:   Minimum concentration and Contact Time found to be effective_______________

Acceptance Criteria:The disinfectant should bring a minimum of 3-log reduction or more.

Done By/Sign:                             Checked By/Sign:                  Reviewed By/Sign:    

Attachment No-IV

Surface Challenge Test

  Surface Challenge Test

  Name of Disinfectant:                                                                 Name of Surface:

  Recommended Concentration:

  Date of test:                                                                                  Date of observation:

  Observations:

Cultures	Contact Time	Initial count	Log of Initial Count (a)	Count Observed in Test	Log of Observed Count (b)	Log Reduction (a-b)
Staphylococcus aureus
Bacillus
Pseudomonas aeruginosa
C.albicans
Aspergillus brasiliensis
Negative Control

Remarks: The disinfectant is able/ not able to bring _____ log reduction  for  the microorganism at _______% concentration and _______min and is  suitable  /not suitable for use.

  

   Done by/Date:                                 Checked By/Date:                             Approved by/Date:

Attachment No-V

List of Disinfectants with Validated concentration and Contact time

S.No	Name of Disinfectant	Contact Time	Effective Concentration (%)
1
2
3
4
5

Done by/Date:                                         Checked By/Date:             Approved by/Date:


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