Sterility test method | membrane filtration method Ultimate Guide

Scope & Purpose   : The purpose of this procedure is to provide written guidelines for performing the Sterility test method | membrane filtration method.

Requirements:

• Sterile Sterility test filter holder unit.
• Sterile membrane filters – 0.45 m. Pore size, 47 mm. Diameter.
• Sterile Soyabean Casein Digest Medium (SCDM), 100 ml. in each tube.
• Sterile Fluid Thioglycollate Medium (FTM), 100 ml. in each tube.
• Sterile scissors.
• Sterile duster.
• Sterile forceps.
• Sterile gloves.
• 70% v/v Isopropyl Alcohol.
• Laminar Air Flow cabinet (LAF).
• Test samples.
• Sterile gown, mask. And Booties.
• Sterile Bacteriological peptone (0.1 % w/v)

Implementation Steps:

• Keep on U.V. lamp of laminar air flow bench before one hour of sterility testing.
• Enter into the sterility testing room
• Switch “OFF” UV light & switch “ON” Laminar Air Flow. Wipe the working bench of LAF with 70% v/v IPA.
• All the sterilized materials required for sterility test is taken from the buffer room of autoclave unloading side in sterility test room.
• Samples for sterility test is transfer from pass box provided in material air lock to sterility test room.
• Assemble the filtration cones on sterility test filter holder unit which is attached to the source of vacuum.
• Before enter into the sterility room ensure vacuum pump on.
• Containers (bottles) for testing are taken in LAF workbench. The surface of the containers is decontaminated with disinfectant ( 70% v/v IPA).
• Transfer the volume from each container (as specified in IP/BP/USP Refer attached
• Open the knob of  the filtration assembly & allow to suck the solution through exhaust pipe Repeat the procedure from step No. 7 to 9 till all the samples are tested lot wise.
• After completion of filtration, close the knob of each filtration assembly and the top of the assembly is carefully removed, filters are cut into two halves with the help of sterile forceps & each half is transferred to sterile Soyabean Casein Digest Medium & Fluid Thioglycollate Medium for one sterility test lot.
• Repeat the same procedure for every lot.
• In case of samples containing antimicrobial / inhibitory properties rinse every lot by 300 ml of sterile Bacteriological peptone (0.1% w/v). Also filter 100 ml of sterile Bacteriological peptone (0.1% w/v) as a control. Filters are cut into two halves with the help of sterile forceps & each half is transferred to sterile Soyabean Casein Digest Medium & Fluid Thioglycollate Medium.
• After completion of filtration, close the knob  of each filtration assembly and the top of the assembly is carefully removed, filters are cut into two halves with the help of sterile forceps & each half is transferred to sterile Soyabean Casein Digest Medium & Fluid Thioglycollate Medium for one sterility test lot.
• Repeat the same procedure for every lot.
• The inoculated sterility test tubes of Soyabean Casein Digest Medium are incubated at 20 – 25OC & Fluid Thioglycollate Medium are incubated at 30 to 35OC for 7 days for the test as per IP.  And in case of BP and USP test incubate the tubes for 14 days.
• Put ‘OFF’ the vacuum pump after completion of testing.
• Positive controls  - Inoculate 1 tube each of sterile Soyabean Casein Digest medium with 10 to 100 cfu of the following organisms:

            i) B. subtilis (ATCC6633)

            ii) C.albicans (ATCC10231)

            iii) A. niger ( ATCC 16404)

• Inoculate 1 tube each of sterile Fluid Thioglycollate medium with 10 to100cfu of the following organisms:

            i) B. subtilis (ATCC6633)

            ii) S. aureus(ATCC6538p)

            iii) P. aeruginosa ( ATCC 9027)

            iv) C. albicans ( ATCC 10231)

            v) Cl. sporogenes ( ATCC 11437)

 Negative controls:

• Incubate 1 tube each of sterile soyabean casein digest medium and Sterile              Fluid Thioglycollate medium at 20 – 25 Degree C and 30 to 35 Degree C respectively
• OBSERVATION & INTERPRETATION OF RESULTS:
• Sterility test tubes are observed daily for macroscopic evidence of microbial growth:
• If no evidence of growth is found, the preparation being examined passes the test for sterility.
• evidence of microbial growth is found; the preparation being examined does not comply with the test for sterility. Do not repeat the test unless it can be clearly shown that the test was invalid for causes unrelated to the preparation being examined. The test may be considered invalid only when one or more of the following conditions are fulfilled:
• Microbial growth is found in negative control.
• Data on microbial monitoring of the sterility testing facility shows a fault,
• A review of the testing procedure used for the test in question reveals a fault.
• After identifying the microorganisms isolated from the containers showing microbial growth the growth may be ascribed without any doubt to faults with respect to the materials and/or the technique used in conducting the test procedure.
• If the test is declared to be invalided it is repeated with the same number of units as in the original test.
• If no evidence of microbial growth is found in the repeat test the product examined complies with the test for sterility. If microbial growth is found in the repeat test the product examined does not comply with the test for sterility & the product is rejected.       

Interpretation of sterility test results as per BP       

• Clarify the conditions under which you would invalidate a sterility test, thus permitting a retest to be conducted,
• Provide a statement of the circumstances under which you would invalidate a positive sterility test, thus permitting a retest to be conducted.
• Provide a statement of the circumstances in which you would use condition (d) of the BP “Observation and Interpretation of Results” to invalidate the initial sterility test result, and a description of the test methods used conform that isolates are identical.

Stasis Test   :

• Stasis test also referred to as an inhibition test, which is performed to ensure the following two points.
• To ensure that there are no inhibitory substances remaining in the product.
• And that the media is still capable of supporting the growth of micro-organisms at the end of the sterility test    incubation period.  It is useful to confirm the inactivation of antimicrobial substances in products.

Test Procedure:

• The stasis test should be done only for antibiotics. Like Fluconazole Injection, Ciprofloxacin Injection, Metronidazole Injection & Ofloxacin Injection.
• After successful incubation of 14 days of above maintained products sterility test media tubes further incubate 5 days for fungi with addition of Candida & Aspergillus niger (10 – 100 CFU) for 20 to 25 ºC  and 3 days for bacteria with addition of Clostridium sporogenes, Bacillus subtilis, Staphylococcus aureus, & Pseudomonasa aeruginosa (10 – 100 CFU) for 30 to 35 ºC
• Observation and Interpretation of the results for sterility testing :
• Clarify the conditions under which you would invalidate a positive sterility test, thus permitting a retest to be conducted.
• Provide a statement of the circumstances under which you would invalidate a positive sterility test, thus permitting a retest to be conducted.
• Provide a statement of the circumstances in which you would use condition (d) of the BP “Observation and Interpretation of Results” to invalidate the initial sterility   test result, and a description of the test methods used to conform that isolates are identical.
• If conspicuous growth is not apparent within the above specified incubated period the test is considered invalid and invalid stasis tests may be repeated once. If conspicuous growth is not obtained at the second attempt the test method should be modified and revalidated.
• If the media are found to support growth of the test micro-organisms then this test need not be applied to every sample.

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