DISINFECTANT VALIDATION
TABLE OF CONTENTS
Section No | Topics | Page No |
1.0 | Protocol Approval | |
2.0 | Objective | |
3.0 | Acceptance Criteria | |
4.0 | Responsibilities | |
5.0 | Validation Methodologies | |
6.0 | Evaluation of test Result | |
7.0 | Re-validation Frequency | |
8.0 | Documentation | |
9.0 | Conclusion |
- PROTOCOL APPROVAL
Name / Designation | Signature | Date |
Prepared By | ||
Microbiologist | ||
Checked By | ||
Microbiology | ||
Manager. Quality Control | ||
Approved By | ||
Plant Manager | ||
Manager. Quality Assurance |
2.0 OBJECTIVE:
To evaluate the efficiency of disinfectants which are being used for the surface and area sanitization of controlled and critical clean rooms.
3.0. ACCEPTANCE CRITERIA :
- The disinfectants when used as per the recommendations of the manufacture for dilution and the contact time should be able to inhibit the growth of the test microorganisms .
4.0 RESPONSIBILITIES :
Validation Group consists of following members :
- QA/QC Department Representative
Manager, QA
Manager QC
Manager, Microbiology
Microbiologists
- Specific Responsibilities
- Microbiologist will write the protocol in consultation with QC Manager .
- Manager, QA will check the protocol for its completeness, accuracy, technical excellence and applicability.
- Head of the Department QA will be responsible for final approval of protocol.
- Microbiologist will be responsible for preparation of slants , sub culturing of organisms , streaking on the slants .
- Microbiologist will also be responsible for preparing the used dilution as per the manufacturers recommendation.
5.0 VALIDATION TEST PROCEDURE:
Before proceeding for validation following materials are required.
Staphylococcus aureus
B.Subtilis
E.coli.
Candida albicans
Apergillus niger.
Micrococcus species Environmental Isolate
- Disinfectants for Validation.
- Sterile distilled water.
- Sterile Molten Soyabean Caesin Digest Agar
- Sterile Molten Potato Dextrose Agar
- Poured SCDA plates.
- Poured PDA plates
- Sterile forceps
- Sterile membrane filtration units.
- Sterile membranes
- Vortex Mixer.
I – Preparation of Spore forming Culture
- Prepare SCDA slants as per the SOP.
- Incubate slant for 48 hrs at 30 – 35 ° C as pre-incubation to cross check the contamination.
- from the working culture streak a loopful of Bacillus subtilis culture on to the newly prepared slant.
- Incubate the slants at 30 – 35 ° C for 48 hrs.
- After 48 hrs of incubation cross check the culture for any cross contamination by simple gram staining technique.
- During Gram Staining also check for the presence of vegetative or spore cells.
- After Gram Staining preserve the cultures for further 7 days.
- After 7 days cross check the culture for any spore formation by simple Negative Staining technique.
II – Negative staining.
- Prepare a thin smear on a clean slide.
- Place the slide on a staining rack above boiling water.over the smear with small pieces of tissue paper.
- Pour malachite green and continue heating till boiling for 5 minutes.
- Do not allow the stain to dry.
- Gently wash the slide with water.
III- Preparation of Challenge Inoculam.
- Prepare fresh SCDA /PDA slants as per the SOP 12043.
- Incubate those slants at 30 – 35 ° C for 48 hrs to cross check any type of contamination.
- From the working culture streak a loopful of culture into the freshly prepared slants as per the SOP.
- Incubate those inoculated slants at 30 – 35 ° C for 48 hrs for bacterial cultures and 20 – 25 ° C for 5 days for fungal cultures.
- Add 5.0 ml of sterile saline into the slants aseptically taking care not to contaminate the slants .
- With the help of a sterile spatula streak the lawn of organisms.
- Take 1.0 ml of the culture suspension and make serial dilutions ranging from 1:10 to 1: 100 .
- Plate the serial dilutions from 1:10 to 1:100 taking 1.0 ml of the culture in sterile petri-dishes.
- Pour sterile molten SCDA and PDA on to the inoculated plates.
- Incubate the plates at the required specified temperature.
- 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 .
- After the incubation count the number of colonies .
- Select the dilution which is having 10000 to 100000 cells / ml for validation study .
- Preserve the culture suspension.Record the data in Annexure.
IV- Preparation of Disinfectant solution
- Take the concentrated solution as received from the supplier and dilute the disinfectant to prepare a stock solution, which is twice that of the recommended concentration.
- This will be the used dilution.
V – Determining the initial microbial count.
- With the help of an calibrated micropipette pipette out 1.0 ml of any of the culture and inoculate into 9.0 ml of the 0.9 % sterile saline solution.
- Vortex it for 5.0 minutes.
- Filter the sample through a 0.45 m membrane.
- Give three washings of 100 ml each with .1 % sterile peptone water.
- After filtration , with the help of a sterile forcep take the membrane and place the membrane on a SCDA or m (HPC) agar plate.
- Incubate the plate .
- After incubation count the number of colonies present on the membrane.
- Note down the number of colonies.
- This will be the initial counts of the culture.
- In the same manner proceed for all the other cultures which are going to be tested.
- Record the initial counts in the Annexure and report as Counts / 0.1 ml
IV – Determining the Efficacy of the Disinfectant by Use Dilution Method.
- Prepare test tubes having 9.0 ml of sterile distilled water.
- Add 1.0 ml of the use dilution for one disinfectant.
- Vortex the tube for 5.0 minutes.
- Add 0.1 ml of any one culture into the test sample.
- Make the sample dilution in such a way that each contact time have two sets of samples.
- Give a contact time of 0 hrs, 5.0 min , 10 min .
- After the specified contact time, filter the samples through a 0.45 m membrane.
- Give three washings of 100 ml each with .1 % sterile peptone water.
- After filtration with the help of a sterile forcep take the membrane and place it on a SCDA or m (HPC) agar.
- Incubate the plates .
- After incubation count the number of colonies present on the membrane.
- Note down the number of colonies.
- This will be the final counts of the exposed culture .
- Select the plates which have least to Nil counts.
- Proceed in the same manner taking all the cultures to be tested .
- Contact time for the usage of the disinfectant will be set on the basis of the results which will have least counts.
V- Determining the Efficacy of the Disinfectant by Surface Method
- 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.
- Take S.S strips and Epoxy coated material having a surface area of 25 cm2.
- Wrap the strips and the epoxy coated surface with a aluminium foil and sterilize it in an Dry heat Steriliser at 200 0 C for 2 hrs.
- From the previously determined suspension having 10000 – 100000 cells per ml inoculate one culture on to three different S. S surface and Epoxy coated surface.
- With the help of a sterile spatula spread the culture on the surface.
- Keep it on the LAF bench for 30 minutes for drying.
- After the exposed duration for drying spray the disinfectant on to any one surface of the recommended used dilution .
- Allow the surface to be with the sanitiser for 0hrs, 5.0 min , 10 min.
- With the help of a sterile moistened swab , swab the surface gently covering all the area of the surface.
- Use different swabs for all the strips.
- Place the two swab sticks in a test tube having sterile solution of fluid caesin digest Soya broth .
- Vortex the test tube gently for 5. 0 minutes .
- Aseptically filter the samples through a 0.45 m membrane.
- Give three washings of 100 ml each with .1 % sterile peptone water.
- After the filtration with the help of a sterile forcep take the membrane 5.52 and place it on a SCDA or m (HPC) agar.
- Incubate the plates .
- After incubation count the number of colonies present on the membrane.
- Note down the number of colonies in both the test sample and that with the unexposed strip.
- Proceed in the same manner taking all the cultures to be tested and the various sanitisers.
6.0 EVALUATION OF RESULTS :
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.
7.0 Schedule Revalidation:
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.
8.0 DOCUMENTATION:
Validation Report contains the following documents.
- Approved Validation protocol
- Media Preparation as per SOP.
- Gram Staining GTP.
- Test report for different disinfectants
- List of Approved Disinfectants.
- Summary Conclusion .
9.0 CONCLUSION :
Summary report will contain discussion and conclusion which clearly state the successful achievement of objective of validation studies.