Sterility Testing

What information is given by a sterility test, and what are its limitations?

Sterility tests can show that organisms capable of growing in the test media under the selected conditions are absent from the fraction of the batch that has been tested. It should not be used as the sole means of controlling sterile processing.

What are the two methods of sterility testing?

• Membrane filtration method

• Direct inoculation method

Describe the processes involved in the membrane filtration method of sterility testing.

1. Sterilise both the filtration system and the membrane.

2. Pass the product through the membrane filter.

3. Transfer the whole membrane to the culture medium.

4. Incubate the media for not less than 14 days.

What method of sterility testing is preferred, where possible?

Membrane filtration method

What is the required pore size for the membrane filtration method of sterility testing?

Not greater than 0.45 microns.

What types of solutions are cellulose nitrate filters able to retain microorganisms from?

Aqueous, oily, or weakly alcoholic solutions.

What types of solutions are cellulose acetate filters able to retain microorganisms from?

Strongly alcoholic solutions.

What types of solutions are specially adapted filters able to retain microorganisms from?

Solutions containing antibiotics.

What should be done if a product has antimicrobial properties, and we want to conduct sterility testing using the membrane filtration method?

Wash the membrane not less than 3 times by filtering through it each time the volume of the chosen sterile diluent used in the method suitability test.

What is the process in the direct inoculation method of sterility testing?

1. Transfer the required quantity of the product directly into the culture medium, so that the volume of the product is not more than 10% of the total volume of the medium.

2. Incubate for not less than 14 days. Observe the culture several times during the incubation period.

How does the direct inoculation method need to be modified for oily products?

Shake cultures containing oily products gently each day of incubation.

How does the direct inoculation method need to be modified for products with antimicrobial properties?

Neutralisation of the antimicrobial excipient, or dilution of the product.

How does the direct inoculation method need to be modified for large volume products?

Use a concentrated culture medium, so that it is still able to support microbial growth despite dilution with the large volume of product.

How many containers need to undergo sterility testing out of a batch of less than 100 parenteral preparations?

10% or 4 containers, whichever is more.

What are the two types of culture media commonly used in sterility testing?

• Fluid thioglycolate medium (FTM)

• Soya-bean casein digest medium (SCDM)

What types of microorganisms is FTM a suitable culture media for, and what is the usual incubation temperature?

FTM can support the culture of aerobic and anaerobic bacteria. The usual incubation temperature is 30-35C.

What types of microorganisms is SCDM a suitable culture media for, and what is the usual incubation temperature?

SCDM can support the culture of aerobic bacteria and fungi. The usual incubation temperature is 30-35C for aerobic bacteria, and 20-25C for fungi.

What are the three control tests that are used in any sterility testing procedure?

• Sterility of procedure test (negative control)

• Growth promotion test (positive control)

• Validation test (main positive control)

What is the purpose of the sterility of procedure test?

To determine whether the culture media used is sterile, and that the operational procedure was sterile.

What is the purpose of the growth promotion test?

To confirm that the media culture is able to support microbial growth under the test conditions.

What is the purpose of the validation test?

To determine that there is no growth inhibitory effect from the product, such as the presence of an antimicrobial preservative.

When should the sterility of procedure test be performed in relation to the sterility of product test?

Parallel

When should the growth promotion test be performed in relation to the sterility of product test?

Usually prior to the sterility of product test.

When should the validation test be performed in relation to the sterility of product test?

Parallel

What is the procedure for conducting a growth promotion test?

Inoculate portions of FTM and SCDM with separate species of microorganism. Incubate for not more than 3 days in the case of bacteria, and not more than 5 days in the case of fungi.

When can a sterility test be considered invalid?

• Fault in the data of the microbiological monitoring

• Testing procedure review reveals a fault

• Microbial growth is found in the negative controls

• Determined the identity of the microorganisms and confirmed that the growth was unrelated to the material and/or technique used in conducting the procedure

What is the procedure after a sterility test is determined to be invalid?

The test is repeated with the same number of units as in the original test.