Sterility testing explained: USP <71> compared with rapid microbiological methods

The test uses two media so that both oxygen-loving and oxygen-avoiding organisms have a chance to grow: fluid thioglycollate medium, incubated at 30–35 °C, mainly for anaerobic and aerobic bacteria, and soybean-casein digest medium, incubated at 20–25 °C, for fungi and aerobic bacteria. Containers are examined during and at the end of an incubation of not less than 14 days.

Membrane filtration is preferred when the material can be filtered: the sample passes through a 0.45 µm membrane that is rinsed and then placed in the media, which also washes away anything that might suppress growth. Direct inoculation, where sample is added straight to the media, is used when filtration is not practical.

Before results count, the laboratory must show the sample does not stop microorganisms from growing, by spiking low numbers of test organisms in the presence of the sample (bacteriostasis and fungistasis testing). A report that does not mention method suitability leaves that question open.

Sterility testing is a sampling test. Because only a limited number of units are tested, it can miss low-level contamination. That is why regulated sterile manufacturing relies on validated aseptic processing or terminal sterilization, with the sterility test as one check among many rather than proof on its own.

Rapid methods replace waiting for visible growth with a faster signal: ATP bioluminescence, automated detection of CO2 or pressure changes from growing organisms, flow or solid-phase cytometry, or nucleic-acid amplification. They can shorten the wait from two weeks to a few days.

Because they detect differently, they must be validated before replacing the compendial test, covering accuracy, precision, specificity, limit of detection, robustness, and equivalence or non-inferiority. USP <1223> and Ph. Eur. 5.1.6 describe that validation. For licensed biologics in the US, 21 CFR 610.12 was revised to allow validated alternative sterility methods.

Lyophilized research materials are not sold as sterile products, and a sterility claim needs a lot-specific report naming the method, media, incubation period, and method-suitability result. Our currently published reports cover HPLC purity, measured quantity, and on some lots LC-MS identity. They do not include sterility results. If a lot needs sterility data, ask through the testing page and we will tell you whether a report exists.