Sterility Testing

What is sterilization?

Sterilization is defined as the process where all the living microorganisms, including bacterial spores are killed.

What is sterility test?

A sterility test is essentially a test which assesses whether  a sterilized pharmaceutical or medical product is free from contaminating microorganisms by incubation of  either the whole or a part of that product with a nutrient  medium. This is a destructive test.

Sterility test is applied to?

substance, preparations or articles which, according to the Pharmacopoeia, are required to be sterile.

Methods of sterility test

•Mainly three methods are available when conducting sterility tests:

1. Direct inoculation

2. Membrane filtration

3. Test for low level contamination

Direct inoculation method

The direct inoculation method involves introducing test samples directly into nutrient media

Direct inoculation medias

a. Fluid mercaptoacetate medium (also known as fluid thioglycolate medium), which contains glucose and sodium mercaptoacetate (sodium thioglycollate) and is particularly suitable for the cultivation of anaerobic organisms (incubation temperature 30 – 35 ° C)

b. Soyabean casein digest medium (also known as tryptone soya broth), which will support the growth of both aerobic bacteria (incubation temperature 30 – 35 ° C) and fungi (incubation temperature 20 – 25 ° C).

Membrane filtration

This technique is recommended by most pharmacopoeias and is the main method used to test most products. It involves filtering fluids through a sterile membrane filter (pore size ≤ 0.45 μm), which traps any microorganisms on the filter’s surface. After washing, the filter is aseptically divided, and portions are placed in culture media for incubation at the proper temperature and time. Water-soluble solids can be dissolved in a suitable diluent and tested this way, while oil-soluble products can be dissolved in a solvent like isopropyl myristate.

Test for low level of concentration

A sensitive method for detecting low levels of contamination in intravenous infusion fluids involves the addition of a concentrated culture medium to the fluid in its original container.

Limitations of sterility testing

• False-negative results – The test may fail to detect viable organisms due to unsuitable media or inappropriate cultural conditions.

• Lack of a universal medium – No single culture medium can support the growth of all possible contaminants.

• Limited incubation conditions – It is impossible to provide an infinite variety of incubation conditions to detect all potential microorganisms.

• Exclusion of viruses – Pharmacopoeial sterility tests do not detect viruses, which can pass through sterilizing filters due to their small size.

• Does not guarantee sterility – The test only provides a final check but does not ensure absolute sterility, as some microorganisms may remain undetected.

• Restricted microbial detection – The test mainly detects non-fastidious bacteria, yeasts, and molds, missing more demanding or unusual contaminants.

• Destructive test – Since the sterility test requires the use of the product itself, tested samples cannot be used or sold after testing.

Precautions in sterility testing

• Assessment of media – The culture media used must be tested for nutritive (growth-supporting) properties and lack of toxicity using specified organisms.

• Optimal conditions for damaged microorganisms – Any surviving microorganisms from the sterilization process may be weakened and should be given the best possible conditions for growth.

• Aseptic testing environment – Sterility testing must be performed under strict aseptic conditions, such as in a laminar airflow cabinet, to prevent accidental contamination.

• Facility control tests – As per the European Pharmacopoeia, air and surface sampling must be conducted to verify the adequacy of the testing environment.

• Use of negative controls – Tests should include known sterile samples (e.g., those sterilized by radiation or multiple sterilization cycles) to confirm the reliability of the sterility test.

• Minimizing contamination risk – Isolators are often used to physically separate the operator from the test materials, reducing the risk of introducing contaminants.

Sterility testing of products containing antimicrobial agents

1. Specific inactivation

2. Dilution

3. Membrane filtration

Specific inactivation

An appropriate inactivating (neutralizing) agent is incorporated into the culture media. The inactivating agent must be non - toxic to microorganisms, as must any product resulting from an interaction of the inactivator and the antimicrobial agent. Benzylpenicillin and Ampicillin are inactivated by β - lactamase (from B. cereus). Other inactivators are chloramphenicol acetyltransferase (inactivates chloramphenicol) and enzymes that modify aminoglycoside antibiotics.

Dilution

The antimicrobial agent is diluted in the culture medium to a level at which it ceases to have any activity, for example phenols, cresols and alcohols.

Membrane filtration

This method has traditionally been used to overcome the activity of antibiotics for which there are no inactivating agents, although it could be extended to cover other products if necessary, e.g. those containing preservatives for which no specific or effective inactivators are available. Basically, a solution of the product is filtered through a hydrophobic-edged membrane filter that will retain any contaminating microorganisms. The membrane is washed in situ to remove any traces of antibiotic adhering to the membrane and is then transferred to appropriate culture media.

Positive & Negative controls of sterility testing

• Positive Control:

  • Ensures that microorganisms can grow under test conditions.

  • Involves adding a small number of specific microorganisms to the culture medium with the sample to check for growth.

  • The European Pharmacopoeia recommends using:

    • Aerobic bacteria – Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa

    • Anaerobic bacteria – Clostridium sporogenes

    • Fungi – Candida albicans, Aspergillus niger

  • Microbial growth must be present to confirm that the culture medium supports proper growth.

• Negative Control:

  • Ensures that the test environment, media, and technique are free from contamination.

  • Uses sterile media without any product, processed alongside the test samples.

  • There should be no microbial growth in the negative control, confirming that any growth in the test sample is due to contamination in the product, not a lab error.

Sampling techniques

Sterility testing relies on proper sampling techniques to ensure accurate and reliable detection of viable microorganisms. Random sampling is used for aseptically processed and filled products to ensure fair representation, while for products sterilized in their final containers, samples should be taken from the coolest or least sterilant-accessible parts of the load. All sampling procedures must be conducted using aseptic techniques in a controlled environment, such as a laminar flow hood or cleanroom, with personnel wearing sterile gloves, gowns, and masks to prevent contamination.

Since sterility testing examines only a small portion of a batch, it is a statistical operation rather than a definitive confirmation of sterility. For example, let’s assume p represents the proportion of contaminated containers, and q represents the proportion of non-contaminated containers in a batch. If p = 0.1 (10% of containers are contaminated) and q = 1 − p = 0.9 (90% are non-contaminated), then:

(photo)

This example shows that the likelihood of obtaining one contaminated and one non-contaminated item in a sample is 18%. As the sample size increases, the probability of detecting contamination increases, reducing the chance of passing a contaminated batch as sterile. However, even with a satisfactory test result, it cannot be concluded that the entire batch is sterile—only that the tested sample has passed the sterility test.

Retest

A sterility test may be repeated only when there is clear evidence that the initial test was invalid. Justifiable circumstances for a retest include:

• Failure of the air filtration system: If the filtration system in the testing facility fails, airborne contaminants might enter the product or media during the test.

• Non-sterility of the media: If the culture media used for testing is found to be non-sterile, it may compromise the results.

• Contamination by personnel: If there is evidence that contamination occurred during testing due to errors or poor practices by the operating personnel.

In these cases, a retest is necessary to ensure the accuracy of the sterility test results.