PROTOCOL APPROVAL

Signing of this approval page of protocol indicates agreement with the Testing approach described in this document. If any process, procedure changes protocol shall be prepared and approved. This protocol cannot be executed until signed and approved by following personnel

Quality Control--Microbiology Execute, Participate and provide necessary support for the validation activity. 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.

1.PURPOSE

To establish a protocol test for the Validation of Sterility testing by membrane filtration method will produce the consistent results analyzed as per the standard operating procedure.

2. SCOPE

The scope of the test extends to the Validation of Sterility testing by membrane filteration method

3. RESPONSIBILITY

The responsibility for executing the protocol shall be with the technical director under whose guidance the analysis shall be carried out.  The following persons shall be involved in the execution of the protocol.

Head Quality Control:   

Microbiologist  1    :  

Microbiologist  2    : 

4. DOCUMENT REFERENCE:

1. Sterility test validation USP

5.PROCEDURE FOR ANALYSIS:

Equipment:

*  Analytical balance, able to determine 0.1 mg

*  Conical flasks, Volumetric flasks, 100ml, 50 ml and 20 ml

*  Magnetic stirrer or ultrasonic bath

*  Micropipette

*  Petriplates

*  Test tubes

*  LAF

*  Autoclave

*  Dry heat sterilizer

Reagents & Materials:

*  Sodium chloride

* Soyabean Casein Digest broth

*  Soyabean digest agar

*  Sabouraud Dextrose Agar

*  Fluid thioglycollate medium

Organisms:     

Pseudomonas aeruginosa-ATCC 9027;

Staphylococcus aureus-ATCC 6538;

Escherichia coli-ATCC 8739

Candida albicans-ATCC 10231;

Aspergillus niger-ATCC 16404;     

Pseudomonas aeruginosa-ATCC 9027;

Staphylococcus aureus-ATCC 6538;

Escherichia coli-ATCC 8739

Candida albicans-ATCC 10231;

Aspergillus niger-ATCC 16404;

Preparation of Inoculum: Prepare the Slants and incubate for 24 hours for pre incubation.

Take 24 hours active culture slant transfer all five cultures and incubate respective temperatures mentioned in the table.

 GROWTH PROMOTION TEST: Media tested for growth promotion

Validation of Sterility Test by Membrane Filtration method is done by following procedures

A.  Test for Residual Antimicrobial Activity

The Test for Residual Antimicrobial Activity is carried out the test procedure as described in general sterility test, up to the final wash procedure. To the final wash add an inoculum of viable cells of the specific bacteria and fungi. After the final wash with the added microorganisms has been passed through the filter, inoculate filter paper in FTM & incubate at 30 to 35ºC or in SCDM and incubate at 20 to 25ºC as per table 1.

Growth of each of the added microorganisms should be apparent within 48 hrs. If conspicuous growth does not occur within 3 days for bacteria and 5 days for fungi, the

Test  procedure is not valid and must be modified.

B.  Test for Antimicrobial Activity

To demonstrate that the mixture does not manifest antimicrobial activity, carry out the test as described in sterility test procedure, up to the incubation step and add an inoculum of viable cells of the specific bacteria and fungi respectively to FTM and SCDM and incubate at 30 to 35ºC and 20 to 25ºC respectively.

Growth of each of each of the added microorganisms should be apparent within 48 hrs. If conspicuous growth does not occur within 3 days for bacteria and 5 days for fungi, the test procedure is not valid and must be modified

C.  Stasis Test (Efficacy of the test media at the end of incubation period)

The Stasis Test is designed to demonstrate that the media (i.e. FTM and SCDM) inoculated with the test preparations will support growth for full incubation period. After incubation of the media has been completed in accordance with the instruction given in the sterility test for negative control, add to representative tube containing FTM that has been incubated at 30-35ºC, an inoculum of viable cells of specific bacteria. To the next tube containing SCDM that has been incubated at 20-25ºC, add an inoculum of viable cells of specific fungi. Return all the inoculated tubes to their previous temperature and incubation continued.

All the tubes should show growth of added microorganisms within 48 hours. If conspicuous growth does not occur within 3 days for bacteria and 5 days for fungi, the test is considered invalid.

A.  TEST FOR RESIDUAL ANTIMICROBIAL ACTIVITY)

Objectives:

      The test is performed to ensure that; any residual of Antimicrobial Activity is satisfactory eliminated by using the steps mentioned in this protocol.

Procedure: