Sterility Test: Meaning, Methods, Media and Limits (USP <71>)

In short: A sterility test checks whether a sample of a product shows any growth of viable micro-organisms in culture media over 14 days. It is a required batch test for sterile products, but it only examines a small sample, so a pass does not prove the whole batch is sterile. Sterility is assured by a validated manufacturing process. This article is for QC microbiology staff and students.

What is a sterility test?

Sterility means the absence of viable micro-organisms. A sterility test is a pharmacopoeial method in which a representative sample of a product is placed in suitable culture media and incubated, and the media are examined for growth. If no growth is seen (and the controls are valid), the sample passes the test.

In the United States, 21 CFR 211.167(a) requires appropriate laboratory testing of each batch of a drug product that is purported to be sterile and/or pyrogen-free, with written test procedures that are followed. The pharmacopoeial tests are USP <71> Sterility Tests and Ph. Eur. general chapter 2.6.1 Sterility.

Test conditions and precautions

The test is done under aseptic conditions, normally in a grade A zone (laminar airflow cabinet or isolator) within a suitable background cleanroom. The precautions used to avoid contamination must not affect any micro-organisms that the test is meant to detect. The working area is monitored regularly by sampling (settle plates, contact plates, air and personnel monitoring).

Media

Two media are used (USP <71> / Ph. Eur. 2.6.1, harmonised):

• Fluid thioglycollate medium (FTM), incubated at 30 to 35 °C. It supports anaerobes and also aerobic bacteria.
• Soybean-casein digest medium (SCDM, also called tryptic soy broth or TSB), incubated at 20 to 25 °C. It supports aerobic bacteria and fungi.

Each batch of medium must pass a growth promotion test and be sterility-checked before use. The pharmacopoeias list test strains for the growth promotion and method suitability tests: Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Clostridium sporogenes, Candida albicans and Aspergillus brasiliensis.

A method suitability test (also called the bacteriostasis and fungistasis test) shows that the product itself does not inhibit growth in the test.

Two methods

The number of containers to be tested and the minimum quantity per medium depend on the batch size, dosage form and container volume. Take these from the tables in the pharmacopoeia chapter and your approved specification, not from older summaries.

Incubation and reading the results

Inoculated media are incubated for 14 days and inspected at intervals, at least at the end, for turbidity or other visible growth. If the product makes the medium cloudy so that growth cannot be judged, a portion is transferred to fresh medium and incubated again, as the pharmacopoeia describes.

• No growth, and valid controls: the product complies with the test.
• Growth seen: the product does not comply, unless the test is shown to be invalid for reasons unrelated to the product, such as growth in the negative control, a fault in the facility monitoring data, an error in the procedure, or identification of the isolate that points clearly to a lab or material fault.
• A retest is allowed only after such an invalidity is documented, using the same number of units. Growth in the retest means failure.

Example procedure (illustrative template)

The steps below are an illustrative example only. Adapt them to your own quality system, validated method and the current pharmacopoeia.

Objective: To check that a sterile product is free from viable micro-organisms by membrane filtration.

Materials: sterile closed filtration unit with 0.45 µm, 47 mm membrane filters; FTM and SCDM, 100 ml per vessel (already growth-promotion tested); sterile 0.1% peptone water as rinse fluid; filtered 70% isopropyl alcohol; sterile forceps, scissors and gloves; laminar airflow cabinet or isolator.

Procedure:

1. Sterilise filtration units, forceps and accessories by a validated cycle (the original SOP used an autoclave at 121 °C for 30 minutes).
2. Disinfect the outside of the samples with filtered 70% IPA and pass them into the test room through the pass box.
3. Assemble the filtration unit in the LAF, place the membrane and wet it with sterile 0.1% peptone water.
4. Filter the product contents (for example, the contents of the required number of ampoules). Powders are first dissolved in sterile diluent.
5. Rinse the membrane with the validated volume of rinse fluid (the original SOP used 3 x 100 ml).
6. Cut the membrane aseptically into two halves; place one half in FTM and the other in SCDM.
7. Run a negative control for each medium by repeating the process with sterile diluent, and a positive control with a known organism in a separate area.
8. Incubate FTM at 30 to 35 °C and SCDM at 20 to 25 °C for 14 days and record observations on each working day.

Frequency: every manufacturing batch of a product labelled sterile.

Limits of the sterility test

• The test examines only a limited number of units from the batch, so it can detect only gross contamination. A low level of contamination in the batch can pass undetected.
• It cannot prove that a batch is sterile. It can only show that no contamination was found in the sample tested under the test conditions.
• Culture media and incubation conditions cannot grow every organism, so some organisms may be missed.
• False positives from the testing environment are possible if aseptic technique or isolator control is weak.

For these reasons, the assurance of sterility rests on the validated sterilisation or aseptic process, environmental and process monitoring, and the contamination control strategy. Parametric release, which replaces the sterility test with process data, is permitted only in narrow cases (for example, FDA CPG 490.200 for moist-heat terminally sterilised parenterals).

EU GMP Annex 1 context

The revised EU GMP Annex 1 (Manufacture of Sterile Medicinal Products, published 25 August 2022) came into operation on 25 August 2023, with one point (8.123) extended to 25 August 2024. Section 10.5 states that the sterility test of the finished product is only the last of a series of control measures by which sterility is assured, and that it cannot be used to assure the sterility of a product that does not meet its design, procedural or validation parameters. Annex 1 also requires the test to be validated for the product concerned.

Related reading

See also: Sterility testing in pharmaceutical product as per USFDA guideline.

Frequently asked questions

Why are two media used?

FTM and SCDM are chosen because between them they support anaerobes, aerobes and fungi at their usual incubation temperatures.

How long is a sterility test incubated?

14 days, with observation during the incubation period.

Can a failed sterility test be repeated?

Only if an investigation shows the test was invalid for a cause unrelated to the product. Retesting to get a pass is not acceptable.

Does a pass mean the batch is sterile?

No. It shows that no growth was found in the tested sample. Sterility depends on a validated process.

References

1. United States Pharmacopeia, General Chapter <71> Sterility Tests (current USP–NF; harmonised with Ph. Eur. 2.6.1 and JP 4.06).
2. European Pharmacopoeia, general chapter 2.6.1 Sterility (current edition; harmonised with USP <71>).
3. US FDA, 21 CFR 211.167(a): Special testing requirements (eCFR/FDA).
4. EU GMP (EudraLex Volume 4), Annex 1: Manufacture of Sterile Medicinal Products, C(2022) 5938, 22 August 2022; in operation 25 August 2023 (health.ec.europa.eu).
5. US FDA CPG Sec. 490.200: Parametric Release of Parenteral Drug Products Terminally Sterilized by Moist Heat.