Sterility Testing (USP 71): Principle, Methods, Media, and Interpretation

A batch of injectable medicine is ready for release, and it must be sterile, because a single surviving microorganism injected into a patient can cause serious harm. But how do you prove that something contains no living microorganisms at all? You cannot test every unit, and you cannot see the absence of life directly. Sterility testing is the carefully designed answer to that hard question, and understanding both what it can and cannot tell you is the key to using it correctly.

Sterility testing is the laboratory method that checks whether a pharmaceutical or medical product is free from viable (living) microorganisms. Sterile means completely free of all living microorganisms, including highly resistant bacterial endospores. Products such as injectables, implants, and eye preparations must be sterile, because even a single surviving microorganism can cause serious infection.

Principle

The principle is simple: a sample of the product is added to nutrient culture media that support a wide range of microorganisms, and the media are incubated. If any viable microorganism is present, it multiplies and makes the medium turbid (cloudy). No growth means no detectable microorganisms. Because different organisms need different conditions, two media are used together: one for bacteria (including anaerobes) and one for fungi and aerobes.

Sterility testing is governed by pharmacopeia standards. The key one is USP chapter 71 (Sterility Tests), which is harmonized with the European Pharmacopoeia (EP 2.6.1) and the Japanese Pharmacopoeia, so the method is essentially the same worldwide.

USP 71, the standard for sterility testing

Sterility testing is defined by USP General Chapter 71 (Sterility Tests). This is the compendial standard used to confirm that sterile pharmaceutical products, biologics, and certain medical devices are free from viable microorganisms. Since 2002 it has been harmonized with the European Pharmacopoeia and the Japanese Pharmacopoeia, so the core requirements are the same across these pharmacopeias.

USP 71 specifies:

1. Two methods: membrane filtration (the preferred method when the product can be filtered) and direct inoculation (used when filtration is not feasible).
2. Two media: fluid thioglycollate medium (FTM) for bacteria, including anaerobes, and soybean casein digest medium (SCDM, also called tryptic soy broth) for fungi and aerobes.
3. A 14-day incubation of both media.
4. A qualitative pass or fail result: no growth in either medium is a pass; growth in either medium is a fail.

A method suitability test (also called bacteriostasis and fungistasis testing) that must be done before routine testing, to prove the product itself does not stop microorganisms from growing.

Media for Sterility Testing

Figure: Sterility test media

Sterility testing uses two culture media together, because no single medium grows every type of microorganism. One is chosen to grow bacteria, including anaerobes, and the other to grow fungi and aerobes. Both are incubated for 14 days.

Fluid thioglycollate medium (FTM)

FTM supports aerobes, anaerobes, and microaerophiles. Its reducing agents (sodium thioglycollate and L-cystine) create an oxygen gradient down the tube: aerobes grow near the oxygen-rich top, strict anaerobes grow in the oxygen-poor bottom, and microaerophiles grow in between. This is why FTM is the medium relied on to detect anaerobic contaminants. It is incubated at 30 to 35°C. For the full composition and preparation, see the thioglycollate broth article.

A variant, alternative thioglycollate medium (ATM), has the same base without the agar and resazurin and is read anaerobically. It is used for turbid or viscous products and for devices with narrow lumens.

Soybean casein digest medium (SCDM)

SCDM, also called tryptic soy broth (TSB), supports fungi (yeasts and molds) and aerobic bacteria. It is incubated at 20 to 25°C, the cooler range that favors fungal growth. SCDM is the broth form of the same soybean-casein digest base used in tryptic soy agar. For the full composition and preparation, see the tryptic soy agar article.

How to choose the method

USP 71 allows two methods, and the choice depends on the product.

Membrane filtration is generally preferred, because filtering concentrates any microorganisms onto the membrane and lets inhibitory substances (like preservatives or antibiotics) be rinsed away before culturing. Direct inoculation is used when the product cannot be filtered.

Methods of Sterility Testing

Sterility testing is done by membrane filtration and direct inoculation method.

Membrane Filtration

Figure: Sterility Test Unit

• Membrane filtration sterility testing for pharmaceutical products is recommended by United States Pharmacopeia (USP), European Pharmacopoeia (Ph. Eur), and Japanese (JP) Pharmacopoeia.
• Membrane filtration is the method of the filtration of fluids through the sterile membrane filter having a pore size ≤ 0.45 μm and a diameter of approximately 50mm.
• The product is filtered through a sterile membrane filter with a pore size of 0.45 μm or smaller, which retains any microorganisms present. Cellulose nitrate filters are used for aqueous, oily, and weak alcoholic solutions, and cellulose acetate filters for strong alcoholic solutions.
• If there is the presence of any microorganisms, then it is retained in the filter. Under the aseptic condition, filter through the membrane. Then aseptically remove the membrane and cut it into two halves.
• Then immerse one-half of the membrane in 100 ml of soybean casein digest medium and incubate at 20-25 ℃ for 14 days. Then immerse the other half in 100 ml of fluid thioglycollate medium and incubate at 30-35 ℃ for 14 days.

The samples whose quality needs to be checked can be any of the forms. It needs to be dissolved in a suitable diluent if it’s water-soluble. If it is oil soluble, then it is dissolved in a suitable solvent. Membrane filtration is used to test the following substances:

• Oil or oily preparation
• Ointments in solutions
• Soluble powder or bacteriostatic, fungistatic liquid
• Solid without antimicrobial properties and not soluble in the medium
• Nonbacteriostatic solids are not readily soluble in culture medium

Direct Inoculation

In the direct inoculation method, a test sample is directly inoculated in the culture media using a sterile pipette or syringe. If the test sample is an antimicrobial agent, it is neutralized by adding suitable inactivating agents to the medium. Then its incubated at 20-25 ℃ (soybean casein digest) and 30-35 ℃ (fluid thioglycollate) for 14 days.

Method suitability (bacteriostasis and fungistasis)

Before a product can be routinely tested, the laboratory must prove that the test itself works for that product. This is the method suitability test, also called the bacteriostasis and fungistasis (B&F) test.

The concern is that some products contain preservatives or antibiotics that could stop microorganisms from growing in the medium, which would hide a contaminated product and give a false pass. To rule this out, a small, known number of standard test organisms (not more than 100 CFU) is added to the product in the media, and the media are checked for growth. The compendial organisms used include Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Clostridium sporogenes, Candida albicans, and Aspergillus brasiliensis, covering aerobes, anaerobes, and fungi.

If these organisms grow normally, the method is suitable. If they are inhibited, the product's antimicrobial activity must be neutralized or removed (by adding an inactivating agent, by dilution, or by using membrane filtration to wash it away) before the real test.

Inactivation of Antimicrobial Agents in Sterility Testing

If any preservative is present in the product or the test sample is bacteriostatic or fungistatic, use the suitable sterile neutralizing agent. Then its action will be nullified so that it won’t prevent the growth of the contaminating microorganisms. To inactivate the antimicrobial agent, dilution can also be done. When the antimicrobial agent is diluted in the culture medium, it reaches the level at which it ceases to have any activity. An appropriate neutralizing or inactivating agent is incorporated into the culture media.

Results and Interpretation

Image source: Courseware

• The sample passes the sterility test if there is no growth of microbes after the incubation.
• If growth is observed and turbidity is seen, then a re-test is done.
• If growth is observed again in the second test and cannot be distinguished from the second test, it fails.
• But if it can be distinguished from the first test, then the second re-test is done using twice the number of samples.
• If growth is not observed in the second re-test, it passes the test. If growth is observed, then it fails the test.

What a sterility test can and cannot tell you

This is the most important idea in sterility testing, and it is often misunderstood.

Sterility is binary. A product is either sterile or it is not. A sterility test result is a pass or fail, not a number.

But absence cannot be proven directly. You can never test the entire batch, because testing destroys the sample. Only a small number of units are tested. So a pass does not prove that every single unit in the batch is sterile. It means that no viable microorganisms were detected in the specific samples tested, under the test conditions.

This has a practical consequence. A passing sterility test supports the sterility of a batch, but it does not guarantee it on its own. This is exactly why sterility is built in during manufacturing (through validated sterilization and low bioburden) rather than tested in at the end. The sterility test is the final confirmation, not the only safeguard. This is also why a low bioburden before sterilization matters so much: the fewer organisms present to begin with, the more confident the whole system can be.

This connects to bioburden testing, which measures the microbial load before sterilization. See the article on bioburden testing for how the two fit together.

How to Remember

Sterility testing is USP 71. The chapter number to remember. 71 is sterility, and it is harmonized worldwide.

Two methods, two media, 14 days. Membrane filtration or direct inoculation. FTM and SCDM. Incubated for 14 days.

FTM warm for bacteria, SCDM cool for fungi. FTM at 30 to 35°C for bacteria including anaerobes. SCDM at 20 to 25°C for fungi and aerobes.

Filter first, inoculate if you cannot. Membrane filtration is preferred; direct inoculation is for products that cannot be filtered.

A pass is "no growth in what we tested," not "the whole batch is sterile." Sterility testing cannot prove absolute sterility, because only a sample is tested.

Prove the test works first. Method suitability (bacteriostasis and fungistasis) confirms the product does not stop microorganisms from growing.

Key exam facts

Where Students Get Confused

"A passing sterility test proves the whole batch is sterile." No. Only a sample is tested, and testing destroys it. A pass means no microorganisms were detected in the units tested. It supports, but does not absolutely prove, the sterility of the whole batch.

"Sterility testing and bioburden testing are the same." No. Bioburden counts the microorganisms present before sterilization to design the process. Sterility testing is a pass/fail check after sterilization to confirm no viable microorganisms remain.

"One medium is enough." No. Two media are used because different organisms need different conditions. FTM grows bacteria including anaerobes, and SCDM grows fungi and aerobes.

"If a product has preservatives, you can test it directly." Not safely. Preservatives can stop microorganisms from growing and hide contamination. Their activity must be neutralized, diluted, or filtered away first, and the method suitability test confirms this.

"Membrane filtration and direct inoculation are interchangeable." They are alternatives, but membrane filtration is preferred when the product can be filtered, because it concentrates microorganisms and removes inhibitory substances. Direct inoculation is for non-filterable products.

References

• United States Pharmacopeia. General Chapter <71> Sterility Tests.
• Denyer, S. P., Hodges, N., Gorman, S. P., and Gilmore, B. F. (2011). Hugo and Russell's Pharmaceutical Microbiology (8th ed.). Wiley-Blackwell.
• Sandle, T. (2020). Sterility testing: overcoming difficult products.
• Bugno, A., et al. (2018). Performance survey and comparison between rapid sterility testing method and pharmacopoeia sterility test. Journal of Pharmaceutical Innovation, 13(1), 27–35.