Failed B&F test? Next steps for medical device manufacturers

Background

Sterility testing is one of the most critical steps in ensuring the safety and compliance of medical devices, pharmaceuticals, and other healthcare products. However, before sterility testing can be performed, regulatory guidelines such as USP <71> Sterility Tests and ISO 11737-2 require a preliminary check called the Sterility Method Suitability Test, commonly referred to as the Bacteriostasis and Fungistasis (B&F) Test.

Why is this step so important? Because certain substances in a product, such as residual disinfectants, preservatives, or antimicrobial agents, can inhibit microbial growth. These inhibitory effects, known as bacteriostatic (inhibiting bacteria growth) and fungistatic (inhibiting fungi growth) properties, can lead to false-negative sterility results. In other words, your product might appear sterile simply because the test organisms cannot grow, not because the product is truly free of contamination.

This is especially relevant for medical devices, where cleaning agents, surface treatments, or formulation components often include antimicrobial chemicals. If these inhibitors are not neutralized during testing, the sterility test becomes unreliable, and that can have serious implications for product release and regulatory compliance.

An analyst in a laboratory precisely pipettes a liquid into a test tube, showcasing meticulous laboratory techniques and focus.

The Challenge

Despite following standard sterility testing protocols, many medical device manufacturers are failing the Bacteriostasis and Fungistasis (B&F) Test. This trend is concerning because a failed B&F test means the sterility test results cannot be trusted, potentially delaying product release and increasing compliance risks.

Why does this happen? In most cases, the issue lies in the presence of residual antimicrobial substances in the product or packaging. In other cases, certain raw materials may react to the sterilization process, such as gamma irradiation, creating compounds that inhibit microbial growth. These substances, whether introduced during cleaning, disinfection, formulation, or formed during sterilization, can interfere with the test by preventing microorganisms from growing. When microorganisms cannot grow, the sterility test may falsely indicate that the product is sterile, creating a false sense of security.

Technical Note: Gamma radiation can cause chemical changes in polymers and additives, sometimes producing reactive by-products with antimicrobial properties. This is why products sterilized by irradiation often require additional validation for B&F compliance.

The effect of high energy (gamma) irradiation on polymers in different manner, Adapted from Tamada, M, 2010. Radiation Processing of Polymers and Its Applications. In H. Kudo (Ed.), Radiation applications (pp. 63–80). Springer. https://doi.org/10.1007/978-981-10-7350-2_8

The challenge is:

1. Identifying the root cause: Pinpointing which substances are responsible for the inhibitory effect can be complex, especially when multiple chemicals are involved.

2. Neutralizing the inhibitors effectively: Even after identification, finding the right neutralizer or media that works across all potential inhibitors requires technical expertise and careful validation.

For manufacturers, this problem is more than just a technical hurdle, it impacts timelines, regulatory compliance, and ultimately patient safety. Understanding why these failures occur and how to resolve them is critical for maintaining sterility assurance.

Investigation

When a B&F test fails, the first assumption should be that test controls are valid, meaning the positive and negative controls behaved as expected. If controls are valid, the next step is a systematic investigation to identify the root cause of microbial inhibition.

Here’s how to approach it:

1. Review the Manufacturing Process

Start by mapping out every step of the product’s lifecycle:

• Raw materials: Check if any components have inherent antimicrobial properties or if they can react during sterilization (e.g., gamma irradiation causing chemical changes).

• Cleaning and disinfection steps: Identify chemicals used for surface treatment or equipment cleaning.

• Packaging materials: Some packaging additives can leach into the product and inhibit microbial growth.

2. Identify Potential Inhibitors

Common inhibitors include:

• Alcohols (e.g., ethanol, isopropanol)

• Aldehydes (e.g., formaldehyde, glutaraldehyde)

• Bis-biguanides (e.g., chlorhexidine)

• Oxidizing agents (e.g., sodium hypochlorite)

• Phenolic compounds

• Quaternary ammonium compounds

• Mercurials

  
  

Also consider by-products from sterilization:

• Gamma irradiation can break down polymers such as latex or nitrile or additives, creating compounds with bacteriostasis or fungistasis properties.

• Ethylene oxide sterilization can leave residual chemicals such as Ethylene glycol which can inhibit microbial growth. Ethylene oxide sterilization could also react with the medical device to create a compound with bacteriostasis or fungistasis properties.

3. Analyze Product Formulation

If the product contains preservatives or stabilizers, these may have bacteriostatic or fungistatic effects. Even trace amounts can interfere with microbial growth in the test media.

4. Conduct Chemical Residue Testing

Perform analytical tests (e.g., GC-MS, HPLC) to detect residual disinfectants or sterilization by-products. This helps confirm whether inhibitors are present and in what concentration.

5. Evaluate Environmental Factors

Sometimes, the issue isn’t the product but the test environment:

• Was the media prepared correctly?

  • If yes, the positive control or the growth promotion test should show positive growth.

• Were neutralizers included in the media?

  • If no, maybe adding neutralizers might neutralize the bacteriostasis and fungistasis effect.

• Was the incubation temperature and time appropriate?

  • If yes, the positive control or the growth promotion test should show positive growth.

Solution

Once the investigation confirms the presence of microbial growth inhibitors, the next step is to neutralize their effects so that the sterility test can accurately detect contamination if present. Here’s how to approach it:

1. Select Appropriate Neutralizers

Neutralizers are substances added to the test media to counteract antimicrobial agents without harming the microorganisms being tested. The choice of neutralizer depends on the type of inhibitor identified:

Neutralizing Agents for Common Disinfectants/Antimicrobial Agents

Disinfectant/Antimicrobial Agent	Neutralizing Agent
Alcohols	Dilution or polysorbate 80
Glutaraldehyde	Glycine and sodium bisulfite
Sodium hypochlorite	Sodium thiosulfate
Chlorhexidine	Polysorbate 80 and lecithin
Mercuric chloride and other mercurials	Thioglycolic acid/Thioglycollate / sodium thiosulphate / thioglycollate with cysteine
Quaternary ammonium compounds	Polysorbate 80 and lecithin
Phenolic compounds	Dilution or polysorbate 80 and lecithin
Benzalkonium chloride 0.01%	0.5% Lechitin and 3% Polysorbate-80
Parabenz	5% Polysorbate-80 or 0.07% lecithin and 0.5% polysorbate-80
Azide	Azolectin
Sorbic acid	Dilution and polysorbate-80
Collagen implant	3% Polysorbate 80
Organic acids	Polysorbate-80
Penicillin / cephalosporins	Penicillinase (β-lactamase – volume determined from antibiotic assay). Considered less effective for cephalosporins – membrane filtration recommended
Chloraphenicol	Chloramphenicol acetyltransferase
Sulphonamide	P-aminobenzoic acid

Neutralizing Agents for Common Disinfectants/Antimicrobial Agents Adapted from Clontz, L. (2009). Microbial limit and bioburden tests: Validation approaches and global requirements (2nd ed.). CRC Press and Adapted from Sandle, T. (2022). Sterility testing – Overcoming difficult products [White paper]. Reading Scientific Services Ltd (RSSL)

Including the right neutralizer ensures that the antimicrobial effect is neutralized without compromising the growth of test organisms.

2. Use Broad-Spectrum Neutralizing Media

If multiple inhibitors are suspected or the exact inhibitor is unknown, consider using a broad-spectrum neutralizing medium such as Dey-Engley (DE) Neutralizing Broth that is designed to neutralize a wide range of disinfectants and antiseptics. This simplifies the process when dealing with complex formulations or sterilization by-products.

3. Validate Neutralization Effectiveness

After adding neutralizers or switching to a neutralizing medium:

• Repeat the B&F test to confirm that microorganisms can grow in the presence of the product.

• Ensure that the neutralizer itself does not inhibit microbial growth or interfere with the sterility test.

4. Adjust Sterilization or Cleaning Processes

If inhibitors originate from sterilization (e.g., gamma irradiation by-products) or cleaning chemicals:

• Review and optimize sterilization parameters to minimize chemical changes.

• Implement additional degassing or aeration steps for EO sterilization to reduce EO residual.

Outcome

By identifying and neutralizing microbial inhibitors, manufacturers will be able to comply with the B&F test and proceed with accurate and valid sterility testing. Thus, improving confidence in product safety.

Conclusion

Failing a B&F test doesn’t mean your product is unsafe, it means your testing method needs adjustment. With the right approach, you can troubleshoot effectively and ensure reliable sterility results.

Reference

Clontz, L. (2009). Microbial limit and bioburden tests: Validation approaches and global requirements (2nd ed.). CRC Press.

Duggan, K., Ijaz, M. K., McKinney, J., & Maillard, J.-Y. (2024). Reviewing the evidence of antimicrobial activity of glycols. Journal of Applied Microbiology, 135(4), lxae071. https://doi.org/10.1093/jambio/lxae071

International Organization for Standardization. (2008). ISO 10993-7:2008 Biological evaluation of medical devices — Part 7: Ethylene oxide sterilization residuals (2nd ed.). ISO.

International Organization for Standardization. (2014). ISO 11135:2014 Sterilization of health-care products — Ethylene oxide — Requirements for the development, validation and routine control of a sterilization process for medical devices (2nd ed.). ISO.

International Organization for Standardization. (2013). ISO 11137-2:2013 Sterilization of health care products — Radiation — Part 2: Establishing the sterilization dose (3rd ed.). ISO.

International Organization for Standardization. (2018). ISO 11737-1:2018 Sterilization of health care products — Microbiological methods — Part 1: Determination of a population of microorganisms on products (3rd ed.).

International Organization for Standardization. (2019). ISO 11737-2:2019 Sterilization of health care products — Microbiological methods — Part 2: Tests of sterility performed in the definition, validation and maintenance of a sterilization process (3rd ed.). ISO.

SaniChem Resources. (2024, February 12). Sterility validation: Bacteriostasis & fungistasis. SaniChem Resources. https://www.sanichem.my/post/bacteriostasis-and-fungistasis-sterility-validation

SpecialChem. (2025, September 10). Gamma radiation resistance. SpecialChem. https://www.specialchem.com/plastics/guide/gamma-radiation-resistance

Sandle, T. (2022). Sterility testing – Overcoming difficult products [White paper]. Reading Scientific Services Ltd (RSSL)

Tamada, M. (2018). Radiation processing of polymers and its applications. In H. Kudo (Ed.), Radiation applications (pp. 63–80). Springer. https://doi.org/10.1007/978-981-10-7350-2_8