Sterility Test, how to perform Sterility Test in the best way?

Sterility Test Sterility test is the basic requirements for the products claim it is sterile for its intended use. This is the core requirements to ensure the sterile status of the products which never contain the viable microorganism at the time of use to the patients or must confirm before released for sales. Sterility testing […]

Sterility Test

Sterility test is the basic requirements for the products claim it is sterile for its intended use. This is the core requirements to ensure the sterile status of the products which never contain the viable microorganism at the time of use to the patients or must confirm before released for sales.

Sterility testing need to be as precise as possible due to its critical point of use such as pharmaceutical products, tissue materials, blood products, serum preparations, vaccine preparation, Insulin preparations, powder for injections etc. and the other products which are claim to be sterile or free from viable microorganisms.

Various types of firm, food factory, pharmaceutical industry, beverage manufacturers, and medical device manufacturer’s etc. company use Sterility testing procedures which company deal with the sterile products and this is mandatory for them. Generally, a microbiologist or a group of microbiologists are involve to perform the sterility test based on company work flow.

A Consistent sterility testing is the core to the develop or validate a specific product or procedure. Continuous robust test method is mandatory get the accurate test result repeatedly. A robust quality infrastructure is required to support the biopharmaceutical, pharmaceutical, and medical device industries.

Direct Inoculation and Membrane Filtration Methods

Sterility testing is required to ensure viable contaminating microorganisms are not evident in a product. This testing is conducted by direct inoculation or membrane filtration methods and can be performed in an isolator or cleanroom environment.

Sterility Testing Techniques

There are several sterility Testing are available-

Recommended Sterility Test: Two Types

  • Direct inoculation
  • Membrane filtration

Additional Test: Two Types

  • Bacteriostasis/fungistasis testing–b/f testing
  • Vaporized Hydrogen Peroxide (VHP) ingress testing

Direct Inoculation

Here two types of media are used to directly inoculate the test article for the determination of the both aerobic and anaerobic microorganisms. The both media are for 14 day from the start of the test day and sporadic observations as well as final/end day observations are done to check the any type of evidence of microbial contamination.

A suitable volume of growth media is used to inoculate small volume of sample which is directly collect from sample container by applying aseptic technique then it incubate for 14 days. Direct Inoculation Sterility Testing has some significant limitations. The sensitivity is low for the test as small volume of a full container is inoculate to the respective culture media. At the starting of inoculation, if the sample appears cloudy or turbid then this very challenging to detect the turbidity and the end of the test period for the microbial growth.

Membrane Filtration Sterility Testing

Here the simultaneous filtration of test sample and standard preparation perform through two membrane filters and the samples are subsequently incubated for 14 days from the start of the test day, and finally check/determine the visibility of the microorganisms both aerobic and anaerobic.

The filterable pharmaceuticals product are subject to Membrane Filtration Sterility Testing which have been described in EU Pharmacopoeia < 2.6.1>, USP <71> & JP Pharmacopoeia <4.06>. To perform the test 0.45 µm membrane filter is used to pass the sample, then culture medium is added for incubation. The sensitivity of the test is more precise as the whole or composite sample is passed through the filter. Another best opportunity of the Membrane Filtration Sterility Testing is, its rinse away components present in the sample which may cause the turbidity or inhibit growth as for example preservatives or antibiotics.

Bacteriostasis/Fungistasis Testing–B/F Testing

Bacteriostasis/fungistasis testing is perform in conjunction with the sterility test evaluate whether or not the test article is inhibitory to the growth of the different microorganisms. To evaluate the sterility result Bacteriostasis/fungistasis test is essential to ensure that test article don’t contain any antimicrobial properties and it don’t inhibit the detection of microorganism at sterility test.

Vaporized Hydrogen Peroxide (VHP) Ingress Testing

An isolator required to perform the Vaporized Hydrogen Peroxide (VHP) Ingress Testing which undergo undergoes VHP decontamination. This assay assesses if VHP enter to the test article is apparent that may affect the validity of the result.

What is Sterility Test USP <71>?

Sterility test USP <71> is the chapter of USP[United States Pharmacopeia] which represents how the sterility test to be perform, and the detail description, methodology and how the product to be tested based on the fill volume and sample size.

Media use in sterility testing

The sterility testing of the all product subject to sterile require two types of media which to be cultured in separate two media. In sterility testing, two types of culture media are used to promote the growth of residual anaerobes, as well as aerobes and fungi.

FTM[Fluid Thioglycolate Medium] and SCDM[Soybean Casein Digest Medium], these two type of media are used use to culture anaerobic and some aerobic bacteria and fungi.

Generally FTM is use for culture of anaerobic and some aerobic bacteria on the other hand, SCDM is use for fungi and aerobic bacteria. Before examination the samples are incubated for 14 days at 32.5°C and 22.5°C. Media must be turbidity free. Presence of turbidity in the respective culture media subject to growth of microorganism and it must be investigate.

Sterility testing methods for medical devices

For medical devices testing, direct transfer sterility testing is recommended. The respective devices are tested is in direct contact with the designated test media during the incubation period where microorganism is growing on or in the device to be check. Transfusion and infusion assemblies related products which contain fluid pathway declared sterile then product flush sterility testing is preferred for this type of product. Here a rinsing fluid is used to flush the product lumen then the elute is passed through the membrane filter and after that it place on the suitable media for incubation for 14 days.

sterility test

Sterility Testing Procedure


Be assured of strict devotion with aseptic technique and no occurrence of secondary contamination in every step of the test. Traffic in the LF workbench should be reduce and well-ordered. Use cellulose acetate membrane filters when strongly alcoholic solutions are subject to filter. If the solution being tested has antimicrobial properties, rinse the membrane at least three times with sterile dilution fluid.

Avoid piercing/ splitting of HEPA filter with liquid and spraying of solutions on the workbench. Use single syringe for single batch or test and avoid touch contamination of the needle and plunger of the syringes. All rubber stoppers of vials and neck of ampoules should be out of hand touch as hands are clean but not sterile. Check all the media for clarity as well as sterility before use. Perform positive control/growth promotion test in another separate area from sterility test area under Bio-safety Cabinet.

Media and Diluents:

Tryptone Soya Broth (TSB) 

Fluid Thioglycollate Medium (FTM). [When medium is stored, store at a temperature between 2ºC and 25 ºC in a sterile, air tight container. If more than upper one third of the medium has acquired a pink color, then to remove the pink color, the medium may be restored once by heating the containers in a water bath until the pink color vanishes and by cooling quickly.] USP Diluting Fluid A/rinse solution (1g/L peptone water/Sterile water for injection)

USP Diluting Fluid D (To each Liter of Fluid A/rinse solution add 1 mL of polysorbate 80, adjust to a pH of 7.1± 0.2). For Cephalosporin/Penicillin, add a quantity of sterile β lactamase, adequate to deactivate any residual antibiotic activity on the membranes after the solution of the test specimen has been filtered. Filtered 70% IPA (Isopropyl Alcohol).

 Prepare these dehydrated mediums and ensure effectiveness of the media before, or in parallel, with the sterility test on the product subject to be examined. Growth promotion test of medium should be justified.

Method of Testing:

  • Membrane filtration
  • Direct inoculation

Criteria of membrane filter:

  • Filter I: Cellulose Nitrate Filter[CNF] for aqueous, oily and weakly alcoholic solutions and Filter II: Cellulose Acetate Filter[CAF] for strongly alcoholic solutions.
  • Diameter of membrane filter: 47 mm
  • Pore size of the membrane filter: Not greater than 0.45 µm.

Test sample:

Sterility test is a destructive test [You can’t return the test sample] and it is impossible to test every single item for sterility. Sample for testing should be representative of the batch, which ensures that the results of the tests are substantial. Arbitrary samples are optimally selected every Lth unit, where L = the total units in the batch per number of sample required.

The following number of samples should be used in sterility test described in Table – 1 and Table -2.When the test samples are turbid and is impossible to filter the samples follow the direct transfer method unless in other case follow membrane filtration method.

Sample preparation:

Excessive care must be exercised when opening an article so that the sample to be tested for sterility is not contaminated by Microorganisms present on the exterior part of the container. The exterior surfaces of ampoules and closures of vials must be cleansed with 70% IPA or Hydrogen peroxide. Allow for 10/15 minutes and assemble the sampling unit’s previously sterile tray at inverted position.

Transfer the samples through pass box into the sterility testing area. FTM [Fluid Thioglycollate Medium] and SCDM[Soybean-Casein Digest Medium] or TSB[Tryptone Soya Broth] is used for the sterility test. Prepare these dehydrated mediums.

Before use, each batch of medium should justify for sterility by incubating portions of the medium for not less than 7 days. Growth promotion test [Nutritive properties] should be justified.

Test for Sterility of the product/material [Membrane filtration method]:

Prepare required amount of FTM, TSB, and USP Diluting Fluid–A and sterilized it. After completion of sterilization transfer all testing materials and accessories into sterility testing area by opening sterile side door of the autoclave.

Enter into the sterility testing area through change room. Clean & sanitize the working place. Accumulate the Sterilized filtration unit cautiously, using not more than 0.45µm cellulose nitrate filters (47mm dia.). Convey samples and other equipment into testing area from pass box after cleaning the surface    with 70% IPA.

For Raw Materials transfer aseptically 5 -10 gm of tested sample into 500 ml screw caped  conical flask containing 200 ml of USP diluting fluid-A and shake gently when it completely   dissolve. For Finished Product collect required amount of test sample (see step Table) & transfer aseptically 300 mg of solids into a 500 ml screw caped conical flask containing 200 ml of USP Diluting Fluid-A and mix or constitute, as directed in the labeling, the containers and transfer a quantity of liquid equivalent to about 300 mg into a 500 ml screw caped conical flask containing 200 ml of USP diluting fluid-A.

Transfer the whole content to the filter cup assembly under strict aseptic condition. Filter the whole content with aid of vacuum pump [negative pressure] from the filter cup. If the tested sample under test has inherent Bacteriostatic & Fungistatic properties or contains preservative, rinse the filter paper using USP Diluting Fluid A.

After completing the previous step, cut the filter paper into two equal sections with sterile scissor and aseptically transfer into 100 ml bottle containing TSB & FTM media separately. Mark 1×100 mL of sterile FTM and 1×100 mL of sterile TSB medium without any sample and inoculum, as negative control.

After finishing of the test transfer out all the materials through pass box, clean the working place thoroughly and sanitize with approved disinfecting solution before leaving the area.

Direct Transfer Method:

Prepare required amount of FTM, TSB, (distribute it in screw cap 100 ml bottle) and USP diluting Fluid-A and sterilized it. After completion of sterilization transfer all testing materials and accessories into sterility testing by opening sterile side door of the autoclave.

Enter into the sterility testing area through change room. Clean & sanitize the working place. Prepare required amount of test sample (as per mention Table) and aseptically transfer it into 500 ml screw   caped conical flask containing 200 ml of USP Diluting Fluid-A, and shake it gently.

Agitate the flask and aseptically withdraw 5 ml of test specimen into both of sterile TSB & FTM medium. Mix each test specimen with the appropriate medium, but do not aerate excessively. Incubate the test mixture and both negative controls as directed in the Incubation condition. Examine the media visually for growth.

Where the material being tested renders the medium turbid, so that the presence or absence of microbial growth cannot be determined by visual examination, transfer suitable portions of the medium to fresh containers of the same medium at least once during the period from the third to the seventh day after the test is started.

Continue incubation of the original and of the transfer containers for a total of not less than 14 days from the original inoculation.

Sterility Test of Syringes ( 5 mL, 10 mL & 20 mL)

Take the required quantity of the material followed by previously mentioned table. Aseptically disassemble the syringes into the components like barrel, plungers, needle and needle shield.

Transfer half of the syringe components into one media bottle having sufficient quantity of the FTM and another media bottle having TSB so that components can submerge completely within media and if require pour additional media.

Select the bottle for sterility test on the basis of the size of the components of syringes so that upon addition of media sufficient air space will be available.

Incubation Conditions:

All the test containers, incubated for not less than 14 days at 32.5 ± 2.50C for the Fluid Thioglycollate Medium and at 22.5 ± 2.50C for the Soybean-Casein Digest Medium or Tryptone Soya Broth Medium regardless of the method used for sterility testing.

Observe the media bottle on a periodic basis over Digest Medium regardless of the method used for sterility testing. Observe the tested as well as negative control incubated media bottles on each working day for any kind of macroscopic evidence of microbial growth and record the results in the report sheet with sign and date of the observer.

Interpretations of the Results:

At intervals during the incubation period and the completion of the prescribed incubation time,   examine the media for any visible growth (Turbidity). If confusion arises, make subculture on TSA medium and/or justify evidence of growth.

If no evidence of growth is found, the preparation being examined, pass the sterility test and issue report form (Annexure I). If evidence of growth is found, isolate and identify the organism and make a full case investigation.

If the cause of microbial growth failed to reveal, perform repeat test with same number of test sample. Take double number the sample in case of high risk product.

Download : Appendix-I-Sterility Test Report

Download : Appendix-II-Sterility Test observation Register

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