Protocol No.
Sterility Test
Validation Protocol Page No. 1 of 6
AREA: Q.C - MICROBIOLOGY
DEPARTMENT QC -MICROBIOLOGY
PRODUCT NAME
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 description 3
5.0 Responsibilities and identification of execution team 3
6.0 Test procedures 3
7.0 Recording of observations 5
8.0 Discrepancy and corrective action report 5
9.0 Compilation, review and summary report 6
10.0 Appendix 6
11.0 Revalidation Criteria 6
Protocol No.
Sterility Test
Validation Protocol Page No. 2 of 6
AREA: Q.C - MICROBIOLOGY
1.0 PROTOCOL APPROVAL
Signing of this approval page of protocol indicates agreement with the method validation approach described in
this document. If any modification in the method 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
Protocol No.
Sterility Test
Validation Protocol Page No. 3 of 6
AREA: Q.C - MICROBIOLOGY
2.0 OBJECTIVE:
The protocol is designed so as to validate that any Bacteriostatic and Fungistatic activity inherent in the products
to be tested does not adversely affect the reliability of the test and the test procedure to be followed is suitable for
testing the products as per the pharmacoepial methods.
3.0 SCOPE
This protocol is applicable to sterile raw material and finished products, which are tested for sterility.
4.0 VALIDATION DESCRIPTION
The purpose of this protocol is to ensure that any Bacteriostatic and fungistatic activity inherent in the Product
does not adversely affects the reliability of the test procedure.
5.0 RESPONSIBILITIES AND IDENTIFICATION OF EXECUTION TEAM
5.1 Responsibilities: 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 Execute, Participate and provide necessary support for the validation activity.
Microbiology Preparation and review of the validation Report of the documents and its compliance to
meet the acceptance criteria of the protocol.
Quality Assurance Monitoring the validation activities, compilation, review and authorization of the
method validation report and its compliance to meet the acceptance criteria of the
protocol.
5.2 Identification of Executors: All the identified executors involved with this Protocol are to Record Name,
Designation, Signature and Date.
6.0 TEST PROCEDURES
Before proceeding for validations following materials are required.
Sterile product sample.
Sterile distilled water.
Sterile Molten Soyabean Casein Digest Agar.
Poured SCDA plates.
Sterile forceps.
Sterile membrane filtration units.
Sterile membranes.
Vortex Mixer.
0.1 % peptone water.
Sterile Fluid Thioglycollate Medium.
Sterile Soyabean Casein Digest Medium.
Cultures required suspensions of Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium
sporogenes, Bacillus subtilis, Candida albicans, Aspergillus Niger.
Protocol No.
Sterility Test
Validation Protocol Page No. 4 of 6
AREA: Q.C - MICROBIOLOGY
The validation will be performed under the following subheadings.
6.1 Preparation of 10 –100 cells / ml: Prepare the 10 – 100 cells / ml solution as per the SOP XXX.
6.2 To Determine Sterility of the Product.
[Link] After sampling, the sterile product sample will bring to microbiology lab.
[Link] Microbiologist will place the sterile product sample within the pass box after decontaminating the
outer surface of the pouch with disinfectant solution.
[Link] Enter the sterility testing room as per the SOP for sterile area entry and exit procedure for sterility
testing lab.
[Link] After entering in the sterility testing room, clean the LAF surface with 70 % filtered IPA
[Link] Place the Sterile product Samples within the LAF.
[Link] Perform the sterility test of the sample by following procedure described in XXX.
[Link] Operate the sterility test apparatus Equinox as per XXXXX.
[Link] After performance Incubate Soyabean Casein Digest Medium at 20 – 25oC for 14 days and Fluid
Thioglycollate Medium at 30 – 35oC for 14 days.
[Link] Observe the medium tubes/canisters daily for any growth.
[Link] Record the observations daily in the format given as annexure-1.
6.3 To Determine the Bacteriostasis and Fungistasis nature of the Product.
[Link] Perform the whole exercise under the LAF in Sterility testing area.
[Link] Take the sterile sample as per the XXXXX and dissolve the sample in 200 ml of 0.1 % peptone water.
[Link] Perform the sterility test for the above sample by following procedure described in SLT112.
[Link] Rinse the membrane with 2 X 100 ml portions of the sterile 0.1 % peptone water.
[Link] Inoculate the final rinse with 10 - 100 CFU/ mL of the test microorganisms.
[Link] After filtration aseptically transfer the membrane on to the SCDM / FTGM depending on the
organism being tested.
[Link] Perform the whole exercise with all the organisms listed below.
[Link] Note down the results in Annexure-1.
6.4 Positive Control:
[Link] Filter 2 X 100 ml portions of 0.1 % peptone water through 0.45 filter membrane.
[Link] Finally rinse the membrane with the third 100 ml 0.1 % sterile peptone water inoculated with 10 –
100 cells / ml of any culture listed above.
[Link] Repeat the above exercise with all the other organisms.
[Link] Inoculate the membrane into medium.
[Link]. Medium Organisms Strain Number
Staphylococcus aureus
01 Fluid Thioglycollate Pseudomonas aeruginosa
Clostridium sporogenes
Bacillus subtilis
02 Soyabean Casein Digest Candida albicans
Aspergillus niger
Protocol No.
Sterility Test
Validation Protocol Page No. 5 of 6
AREA: Q.C - MICROBIOLOGY
6.5 Negative Control:
[Link] Filter 3 X 100 ml portions of 0.1 % peptone water through 0.45 filter membrane.
[Link] Cut the membranes into two halves and inoculate each half separately into specified medium.
6.6 Incubation of Tubes:
[Link] Incubate test for sterility sample tubes of Fluid Thioglycollate Medium at 32.5 2.5°C and for
Soyabean Caesin digest Medium incubate at 22.5 2.5°C for 14 days.
[Link] The negative control should be incubated for 14 days, observed daily and recorded.
[Link] Incubate the Positive control tubes for 3 days in case of bacteria and 5 days incase of fungi.
6.7 Acceptance Criteria:
[Link] The growth of each test organisms in the test sample (with the product) is visually comparable to the
growth of the positive control.
[Link] The negative control should not show any growth during the 14 days of incubation.
7.0 RECORDING OF OBSERVATIONS
Record the observations after the execution of each test procedures, in the annexure–1.
8.0 DISCREPANCY 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.
9.0 COMPILATION, REVIEW AND SUMMARY REPORT
Compile and review that all test procedures 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.
Testing method validation shall be considered acceptable when all the conditions specified in the test procedures
have been met.
Prepare the summary report in the annexure –2 and submit this for review, approval and authorization to
Validation Core Team.
10.0 APPENDIX
10.1 Abbreviations and definitions
Abbreviation Definitions
10.2 References.
Protocol No.
Sterility Test
Validation Protocol Page No. 6 of 6
AREA: Q.C - MICROBIOLOGY
10.2.2 Preparation Of Culture Suspensions.
10.2.3 Sterility Testing.
10.2.4 Growth Promotion test.
10.2.5 Sterility Testing Area, Entry And Exit Procedure SOP.
10.2.6 Operation Of Equinox; Make: Millipore. SOP.
10.3 Annexure
[Link] Annexure – 1 (Recording Of Observations For Validation).
[Link] Annexure – 2 (Sterility Test Validation Report).
11.0 REVALIDATION CRITERIA:
Revalidation shall be carried out in case of
11.1 If the manufacturing process is changed.
11.2 If there is any change in testing procedure which is being used for sterility testing.
