Sterility testing explained (USP <71>)

A sterility test checks that a sample from a batch shows no microbial growth. That sounds simple, but the test has very little statistical power. If 0.1% of the units in a batch were contaminated and you tested 20 of them, you would miss the contamination about 98 times out of 100. So the sterility test does not prove a batch is sterile. It confirms that nothing went visibly wrong in a process that was already validated and controlled.

Keep that idea in mind through the rest of this article. Almost every rule around sterility testing, from where you test to when you may invalidate a result, follows from it.

What the pharmacopoeias say

USP <71>, Ph. Eur. 2.6.1, JP 4.06 and IP 2.2.11 are harmonised. The wording differs slightly, but the methods, media, quantities and interpretation are the same. If you write one SOP carefully against USP <71>, it will satisfy the others, provided you cite the edition you work to and check the IP for any local notes.

The two methods

Membrane filtration (preferred)

The product is filtered through a membrane with a nominal pore size of not more than 0.45 µm, usually 47 to 50 mm in diameter. Cellulose nitrate membranes suit aqueous, oily and weakly alcoholic solutions; cellulose acetate is used for strongly alcoholic solutions. Any micro-organisms stay on the membrane, while most of the product, including any preservative or antibiotic, passes through.

The membrane is then rinsed to wash away residual product. Use a validated rinse fluid such as Fluid A (peptone 1 g/L), Fluid D (Fluid A plus polysorbate 80, for products containing lecithin or oil) or Fluid K (for oily products and devices). The usual rule is not more than five rinses of 100 mL per membrane, unless method suitability shows that more is needed and does not harm recovery.

After rinsing, either cut the membrane in half aseptically and put one half in each medium, or use a closed canister system with two canisters and add the media directly. Closed canisters are now the standard in most Indian sterile sites because they reduce handling and the risk of false positives.

Use membrane filtration whenever the product can be filtered. It is the only sensible choice for antimicrobial products, because the rinse removes the activity that would otherwise kill any contaminant in the medium.

Direct inoculation

The product is added straight into the media. It is used for products that cannot be filtered: some suspensions, ointments and creams that will not dissolve, implants, and many medical devices. The volume of product must not be more than 10% of the volume of the medium, so that the product does not dilute or inhibit the medium.

If the product is antimicrobial, you must neutralise it (for example with a suitable inactivator for a preservative, or penicillinase for some beta-lactams) or dilute it enough that it stops being inhibitory. Method suitability tells you whether you succeeded.

How many units, and how much from each?

The pharmacopoeias set minimums. Your specification or the marketing authorisation can ask for more, never fewer.

Number of containers to test (injectables)

Batch size	Minimum number to test
Not more than 100 containers	10% or 4 containers, whichever is greater
More than 100 but not more than 500	10 containers
More than 500 containers	2% or 20 containers, whichever is less
Large-volume parenterals	2% or 10 containers, whichever is less

Quantity per medium from each container (liquids)

Quantity in container	Minimum quantity for each medium
Less than 1 mL	The whole contents
1 mL to 40 mL	Half the contents, but not less than 1 mL
More than 40 mL, up to 100 mL	20 mL
More than 100 mL	10% of the contents, but not less than 20 mL

For solids, the rules follow the same idea in grams, and for antibiotics in bulk there is a separate row. Always copy the current table from the edition you follow into your SOP annexure, so that the analyst never has to calculate from memory.

Media and incubation

Medium	Detects	Incubation
Fluid Thioglycollate Medium (FTM)	Anaerobic bacteria, and aerobic bacteria near the surface	30–35 °C
Soybean-Casein Digest Medium (SCDM)	Fungi and aerobic bacteria	20–25 °C

Incubate both media for not less than 14 days, whichever method you use. Examine them several times during incubation and at the end. FTM has a pink oxidised layer at the top because of the resazurin indicator. If that pink layer extends to more than one third of the medium before use, it has absorbed too much oxygen. You can restore it once by heating in a water bath, but do not reheat it a second time.

If the product makes the medium cloudy so that growth cannot be judged, transfer suitable portions (not less than 1 mL) to fresh vessels of the same medium between day 3 and day 7 and at day 14, then incubate the subcultures for not less than 4 more days.

Media quality checks

Every lot of medium, whether you prepare it or buy it ready to use, needs two checks before or in parallel with use:

• Sterility: incubate a portion of each lot, uninoculated, for 14 days. No growth is allowed.
• Growth promotion: inoculate with not more than 100 CFU of each test organism and incubate. Visible growth must appear within 3 days for bacteria and within 5 days for fungi.

Growth promotion for sterility media uses the same organisms as method suitability: Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 9027), Clostridium sporogenes (ATCC 19404 or 11437) and Bacillus subtilis (ATCC 6633) in FTM; Bacillus subtilis, Candida albicans (ATCC 10231) and Aspergillus brasiliensis (ATCC 16404) in SCDM. Do not let the culture go beyond five passages from the reference culture.

Method suitability

Before routine testing of a new product, show that the product does not inhibit growth under the test conditions. This is often called bacteriostasis and fungistasis (B&F) testing.

1. Run the test exactly as you will in routine use: same quantity of product, same membrane, same rinse fluid and volume.
2. For membrane filtration, add not more than 100 CFU of each test organism to the final rinse portion. For direct inoculation, add the organism to the medium containing the product.
3. Run a positive control at the same time without product.
4. Incubate for not more than 5 days.

Growth in the test containers must be visibly comparable to the control. If it is not, the product is still inhibitory. Increase the number of rinses, change the rinse fluid, add a neutraliser, or increase the volume of medium, and repeat until it passes. Record the final, validated conditions in a ready reference table that analysts can see at the bench.

Repeat method suitability whenever the formulation, the test procedure or the membrane or canister supplier changes.

Reading the result

At the end of incubation, examine every container. If there is no evidence of growth, the product complies. If growth is found, the product does not comply, unless the test can be clearly shown to be invalid for reasons unrelated to the product.

Never write off a turbid container as "product precipitate" without proof. Subculture it, stain it and identify any growth. If there is no growth on subculture and you can show that the same product causes the same turbidity in a sterile control, document that evidence.

When can a test be invalid?

Only when at least one of these is clearly shown:

• The microbiological monitoring of the sterility testing facility shows a fault.
• A review of the testing procedure used during the test shows a fault.
• Microbial growth is found in the negative controls.
• After identification, the growth can be unequivocally put down to faults in the material or technique used in the test.

If the test is declared invalid, repeat it with the same number of units as the original test. If no growth is found in the repeat, the product complies. If growth is found, the product does not comply.

Investigating a positive

A good sterility failure investigation runs in two directions at once: the laboratory and the manufacturing process.

In the laboratory, check:

• Environmental and personnel monitoring results for the session, including glove prints and sleeve plates
• Negative controls, media sterility and growth promotion records for the lots used
• Isolator or cabinet integrity: glove leak tests, VHP cycle records, HEPA filter certification and pressure trends
• Analyst training and the analyst's recent aseptic technique qualification
• Whether the same organism appears in the lab's EM isolate library

In manufacturing, check:

• Media fill history for the line and the shift
• Filter integrity test results before and after filtration
• Sterilisation records for components, equipment and the product
• Environmental monitoring of the filling room during that batch
• Interventions recorded during filling

Identify the organism to species level, preferably by a genotypic method, and compare it with isolates from both the lab and the production area. A match with the production environment points one way; a match only with the lab environment, together with a documented lab fault, points the other.

Good practice in the sterility suite

• Test in an isolator, or in a Grade A zone with a Grade B background.
• Decontaminate everything that enters the isolator, and record the cycle.
• Run negative controls with every session.
• Monitor the environment and the analyst's gloves during every test.
• Qualify analysts through aseptic technique training and periodic requalification, including gowning qualification.
• Identify every isolate from a positive test to species level, and compare it with your EM organism library.
• Trend everything: positives, invalid tests, glove leak failures and EM excursions in the sterility suite.

Rapid sterility methods

Rapid methods based on ATP bioluminescence, respiration or flow cytometry can give a result in days rather than two weeks. They are useful for products with a short shelf life, such as cell therapies and radiopharmaceuticals. They need full validation under USP <1223> or Ph. Eur. 5.1.6, including accuracy, specificity, limit of detection and equivalence with the compendial method, before they can replace it.

Common interview questions on this topic

• Why is membrane filtration preferred over direct inoculation?
• Why is FTM incubated at 30–35 °C and SCDM at 20–25 °C?
• How many vials would you test from a batch of 300 vials?
• What are the four conditions under which a sterility test can be invalidated?
• What is the difference between growth promotion and method suitability?

Go deeper

The Sterility Testing Masterclass walks through each step with real invalidation case studies, and the SOP Builder includes a full sterility test SOP with record formats that you can generate with your own company details.