USP <71> Sterility Tests

USP General Chapter <71> provides the harmonized procedures for sterility testing of articles required to be sterile. It is interchangeable with Ph. Eur. 2.6.1 and JP 4.06 under ICH Q4B Annex 8. The sterility test detects viable microorganisms by culture in defined media but does not itself ensure product sterility; sterility assurance is primarily achieved through validated manufacturing and aseptic/terminal sterilization controls.

Scope: Applies to substances, preparations, or articles designated sterile in USP–NF monographs. Testing per <71> is recognized as interchangeable with Ph. Eur. 2.6.1 and JP 4.06 when performed as written.

Objective: A qualitative test for absence of viable microorganisms, providing favorable recovery conditions by incubating test portions in specified media for not less than 14 days.

Aseptic Test Conditions

• Must be performed aseptically to prevent extraneous contamination.
• Typically conducted in a qualified isolator or laminar-airflow cabinet within a controlled cleanroom environment, per regional GMP expectations (e.g., EU GMP Annex 1 “Grade A in Grade B”).

Culture media and incubation. Two liquid media support a broad range of organisms:
• Fluid Thioglycollate Medium (FTM): incubate 30–35 °C (primarily for bacteria, including anaerobes).
• Soybean–Casein Digest Medium (SCDM/TSB): incubate 20–25 °C (for aerobic bacteria and fungi).
Incubate for 14 days (minimum). Growth-promotion and sterility checks for each medium lot are required.

Test methods (harmonized).
• Membrane filtration—used whenever the nature of the product permits (e.g., filterable aqueous, alcoholic, or oily preparations, or those miscible with suitable solvents). Use membrane filters with nominal pore size ≤ 0.45 µm whose microorganism-retaining effectiveness has been established; rinse suitably to reduce inhibitory residues before incubation.
• Direct inoculation—used when membrane filtration is not feasible (e.g., certain viscous or non-filterable articles); aseptically add specified volumes directly to the media.

Method suitability (product-specific recovery).

Verify that the test method does not inhibit microbial growth by inoculating ≤100 CFU of the following compendial organisms into the product preparation:
Staphylococcus aureus, Bacillus subtilis subsp. spizizenii, Pseudomonas aeruginosa, Clostridium sporogenes, Candida albicans, and Aspergillus brasiliensis.

Demonstrate recovery/detection comparable to control preparations. Reassess suitability when introducing a new product or changing test conditions.

Observation & Interpretation

Examine test vessels periodically and at the end of the 14-day incubation.

Complies (Pass): No evidence of microbial growth.

Does Not Comply (Fail): Growth observed, unless investigation clearly establishes test invalidity unrelated to the product (in which case the test may be repeated once with the same number of units).