Sterility testing worksheet

How does the choice of nutrient media influence the result of sterility testing? Explain

The choice of nutrient media significantly influences the result of sterility testing because unsuitable media or inappropriate culture conditions may fail to detect viable organisms that are actually present. To be completely certain no organisms are present, one would need a universal culture medium and infinite variety of incubation conditions- which is not practically possible. Therefore, in actual practice, only media that support the growth of non-fastidious bacteria, yeasts, and moulds are used. This means that if a contaminant is fastidious (requiring special nutrients or conditions), it might not grow in the chosen media, leading to a false negative result.

Thus, the reliability of sterility testing is limited by the capabilities of the media used, and some microorganisms may remain undetected simply because the media cannot support their growth

Briefly describe the European pharmacopoeia recommended media for sterility testing

The European Pharmacopoeia recommends the use of two media for sterility testing by the direct inoculation method.

a. Fluid mercaptoacetate medium (also known as fluid thioglycolate medium), which contains glucose and sodium mercaptoacetate (sodium thioglycolate) 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).

Method of sterility testing of products containing no antimicrobial agent.

1. Direct inoculation

The direct inoculation method involves introducing test samples directly into nutrient media. The European Pharmacopoeia recommends two media:

• Fluid thioglycolate medium (with glucose and sodium thioglycolate) for anaerobic organisms, incubated at 30–35 °C.

• Tryptone soya broth (soybean casein digest medium) for aerobic bacteria (30–35 °C) and fungi (20–25 °C).

Limits are set on the sample-to-medium ratio to preserve the medium's nutrient properties.

2. Membrane filtration

This widely recommended pharmacopoeial method involves filtering fluids through a sterile membrane (pore size ≤ 0.45 μm) to trap microorganisms. After washing, the filter is aseptically divided and incubated in suitable media. Water-soluble solids are dissolved in a diluent, and oil-based products in solvents like isopropyl myristate, before filtration.

3. Test for low level of contamination:

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.

Why do we need some forms of modification of products containing antimicrobials for sterility testing?

Sterility testing for products containing antimicrobial agent requires certain modifications because these agents can inhibit the growth of microorganism, potentially leading to false negative results. In other words, the product may appear sterile not because it is free from contamination, but because the antimicrobial agents have prevented any viable organisms from growing in the culture media. To ensure accurate and reliable test results, it is necessary to remove or neutralize the antimicrobial activity before performing the test so that microorganisms can grow spontaneously. This allows any contaminating microorganisms to grow if present.

Thus, we can say that modifications are essential to prevent interference with microbial detection and to confirm the true sterility of the product

Method of sterility testing of products containing antimicrobials

1. 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.

2. 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.

3. 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.

What are the limitations of sterility testing?

• False-negative results:

  • The test may miss microorganisms if the culture media or incubation conditions are not appropriate.

• No universal culture medium:

  • There is no single medium that can grow all possible types of microorganisms.

• Limited incubation conditions:

  • It's not possible to recreate every environmental condition needed for all potential contaminants.

• Does not detect viruses:

  • The test cannot detect viruses, which are small enough to pass through sterilizing filters.

• Limited to non-fastidious organisms:

  • It mainly supports the growth of common bacteria, yeasts, and moulds, not more delicate (fastidious) microbes.

• Not a complete guarantee of sterility:

  • A negative result only means no microbial growth was observed, not that the product is 100% sterile.

• Destructive test:

  • The product being tested is destroyed during the sterility test and cannot be used afterwards.

Precautions of sterility testing

• Test the culture media first:

  • Media should be checked for nutrient content (if it can support microbial growth or not) and non-toxicity using specific microorganisms.

• Allow recovery of damaged microbes:

  • Some microorganisms might be damaged during sterilization, so they must be given optimal growth conditions.

• Strict aseptic conditions:

  • Testing must be done under aseptic conditions to avoid contamination.

  • Laminar airflow cabinets are used to create a clean environment.

• Use of isolators:

  • Isolators are used to keep the operator and environment physically separated from the test materials, reducing contamination risk.

• Environmental monitoring:

  • Air and surfaces in the testing area should be regularly sampled to ensure cleanliness.

• Use of negative controls:

  • Sterility testing should include samples known to be sterile to confirm that the environment is not introducing contamination.

  • These can be double-sterilized or sterilized by highly reliable methods like radiation

Why should we do positive and negative control test in sterility testing?

• To confirm the test conditions support microbial growth (positive control):

  • It is important to prove that microorganisms can grow under the test conditions.

  • Positive controls use small numbers of known microorganisms (like Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Clostridium sporogenes, Candida albicans, or Aspergillus niger).

  • If microbial growth is seen, it confirms the culture media is effective and the conditions are suitable.

• To ensure the test environment and method are not contaminated (negative control):

  • A negative control is a sterile sample without the product, processed alongside the test.

  • It should show no microbial growth, proving that the media, environment, and personnel did not introduce contamination.

• To validate the sterility test results:

  • Positive and negative controls help verify that any microbial growth observed in a test sample is due to the product, not due to external contamination or test failure.

• To justify or prevent unnecessary retesting:

  • If contamination is suspected (e.g. faulty air filtration, unsterile media, or handling errors), controls help identify the issue.

  • A sterility test can only be repeated if there is clear evidence the first test was invalid.

Under what circumstances a sterility testing can be repeated?

A sterility test may only be repeated if there is clear and documented evidence that the test was not valid. This ensures that retesting is not done just to avoid an inconvenient result. The following are acceptable reasons for repeating a sterility test:

1. Failure of the air filtration system:
If the filtration system in the testing area, such as the laminar air flow cabinet, was not working properly, it could have allowed airborne contaminants to enter the workspace during the test, leading to a false positive result.

2. Non-sterility of the culture media:
If the media used for the test was later found to be contaminated or not sterile, it can no longer be trusted to give an accurate result.

3. Contamination from personnel during testing:
If there is evidence that the contamination was introduced by the operator's poor aseptic technique, such as touching sterile parts or improper handling.