Sterility Method Suitability: The B/F Test

Bacteriostasis and fungistasis testing or more commonly called the B/F test is a fundamental component of sterility method validation performed to ensure that a product does not interfere with the detection of viable microorganisms during sterility testing. Many sterile pharmaceutical products, biologics, and medical devices contain substances that can inhibit microbial growth, including preservatives, antibiotics, antiseptics, or active ingredients with intrinsic antimicrobial properties. In addition, certain physicochemical characteristics such as extreme pH, high osmolarity, elevated viscosity, or the presence of organic solvents may suppress microbial proliferation. While these properties may support product stability, they can compromise the reliability of sterility testing by preventing the growth of contaminating organisms in culture media. If such inhibition is not identified and neutralized, a contaminated product may incorrectly appear sterile, creating serious patient safety and regulatory risks.

For this reason, compendial standards such as the United States Pharmacopeia (USP <71>) and ISO 11737-2: 2019 require demonstration of method suitability prior to routine sterility testing. Bacteriostasis and fungistasis studies are typically conducted during initial validation of the sterility method, after formulation changes, when the testing method is modified, or when testing is transferred to a different laboratory. The objective is to confirm that the sterility test, when performed on a specific product, is capable of detecting low levels of viable microorganisms despite any inhibitory characteristics of the product matrix.

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The study involves inoculating a small, controlled population of generally in between 10 to 100 colony-forming units (CFU) of standardized compendial challenge microorganisms into sterility test media containing the product, alongside positive control media without the product. The commonly specified test strains include Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Clostridium sporogenes, Candida albicans, and Aspergillus brasiliensis. These organisms represent a broad spectrum of microbial types, including Gram-positive bacteria, Gram-negative bacteria, spore-forming aerobes, anaerobes, yeast, and mold, thereby providing a comprehensive challenge to the test system.

Test Strain	Media	Culture conditions
Staphylococcus aureus Pseudomonas aeruginosa Clostridium sporogenes	Fluid thioglycolate medium	30 - 35°C No more than 5 days
Aspergillus brasiliensis Bacillus subtilis Candida albicans	Soy-bean Casein Digest Medium	22.5 ± 2.5°C No more than 5 days

After inoculation, the media are incubated under prescribed temperature conditions appropriate for aerobic and anaerobic growth. The growth observed in the product-containing media is compared to that in the positive controls. The sterility test method is considered suitable when the challenged microorganisms exhibit visible growth comparable to that of the controls, without significant delay or suppression. If growth is absent or significantly reduced in the presence of the product, inhibitory activity is confirmed and corrective measures must be implemented.

Mitigation strategies may include dilution of the product to reduce antimicrobial concentration, membrane filtration followed by thorough rinsing to remove inhibitory substances, or the incorporation of validated neutralizing agents capable of inactivating preservatives or antimicrobial compounds. Any selected approach must be scientifically justified and shown not to be toxic to the challenge organisms. Refer to our article on Failed B&F test? Next steps for medical device manufacturers for more information.

In summary, the B/F test ensures that sterility testing methods remain capable of detecting viable contamination even in products with antimicrobial characteristics. By verifying method suitability using defined compendial test strains, laboratories strengthen sterility assurance, protect patient safety, and maintain regulatory compliance.

References

1. United States Pharmacopeia. General Chapter <71> Sterility Tests. United States Pharmacopeial Convention.

2. ISO 11737-2. Sterilization of medical devices — Microbiological methods — Part 2: Tests of sterility performed in the definition, validation and maintenance of a sterilization process.