Bioburden Testing: Which Method is the Best?

Bioburden, the population of viable microorganisms found on a medical device, pharmaceutical raw material, or product prior to sterilization; is a critical parameter in medical device manufacturing. Determining the precise bioburden is essential for validating sterilization processes, ensuring product safety, and complying with stringent regulatory standards.

The "best" testing method is not a one-size-fits-all solution; rather, it is a strategic choice based on the sample’s characteristics, the expected level of contamination, and efficiency requirements.

Regardless of the device being tested, the bioburden testing process follows four fundamental steps:

1. Selection & Preparation: Choosing a representative sample and the appropriate extraction fluid.

2. Extraction: Physically removing microorganisms from the device into the fluid.

3. Enumeration: Filtering or plating the fluid to grow and count microbial colonies (CFUs).

4. Validation: Applying a "Correction Factor" to account for microbes that were not successfully removed during extraction.

Curating the best bioburden method requires matching these steps, specifically extraction and enumeration, to the unique physical properties and the expected microbial load of each device.

3 Basic Steps of performing Bioburden Testing

Matching Device Types to Bioburden Extraction Methods

The extraction phase aims to dislodge microbes into a liquid medium for counting. The choice of method depends entirely on the device's geometry and material:

Medical Device Type	Recommended Extraction Method	Why It Is Suitable
Complex Internal Lumens (e.g., Catheters, Tubing, Endoscopes)	Immersion and Rinsing	Fluids must be flushed through internal channels to ensure microbes aren't trapped in "dead spaces" or narrow bores.
Intricate, Hard Surfaces (e.g., Orthopedic Screws, Dental Implants, Surgical Tools)	Sonication	Ultrasonic waves provide the energy needed to "shake off" highly adherent microbes from microscopic crevices and threads.
Absorbent or Flexible Materials (e.g., Gauze, Textiles, Wound Dressings)	Stomaching / Masticating	Mechanically compressing and "massaging" the material in a sterile bag to release microbes trapped within fibrous matrices.
Large, Non-Submersible Equipment (e.g., Imaging Plates, Large Hospital Hardware)	Swabbing / Agar Overlay	When a device is too large to fit in a container or cannot be submerged, swabbing or agar overlaying provides a localized estimate of surface contamination.
Simple, Solid Devices (e.g., Tongue Depressors, Plastic Handles)	Orbital Shaking / Agitation	Sufficient for devices with accessible surfaces where microbes are easily reached by moving fluid.

Enumeration: Choosing the Right Counting Method

Once extracted, the enumeration method must be chosen based on the nature of the device and the expected microbial load:

• Membrane Filtration: This is the preferred choice for most devices such as standard surgical instruments. It allows for the testing of large fluid volumes, which is essential for detecting low levels of contamination on high-quality surgical tools.

• Pour Plate: If a device extraction (like from gauze) results in a cloudy fluid that would clog a filter, the Pour Plate method is used. It is reliable for direct analysis where filtration is impossible such as particulate-heavy samples. Since only a fraction of the extract will be used for this method, it is suitable for devices with higher microbial load.

• Most Probable Number (MPN): For devices coated in thick gels such as viscous product or those expected to be nearly sterile (low bioburden/CFU count or low-bioburden products), the Most Probable Number (MPN) method provides a statistical estimate where traditional plating may fail.

Microorganisms on agar plate

The Role of Chemistry and Neutralization

In searching for the best bioburden method for your device, the device's material also dictates the fluid chemistry. Devices with hydrophobic (water-repellent) surfaces require surfactants (like Polysorbate 80) to lift microbes. Crucially, if a device has an antimicrobial coating (such as silver-ion or antibiotic-impregnated catheters), the fluid must contain neutralizers to prevent these chemicals from killing the microbes before they can be counted.

Validation: The Final Essential Step

Regardless of the device type, standards such as ISO 11737-1 and USP <61> mandate validation. Manufacturers must determine a Correction Factor through Recovery Efficiency studies, either by seeding the specific device with known organisms (Inoculated Product Method) or by extracting it multiple times (Repetitive Recovery).

Conclusion

The "best" bioburden method is a validated combination of extraction and enumeration tailored to the device's shape and material. This customized approach remains the only way to ensure that sterilization processes are based on an accurate understanding of a device's true microbial challenge, ultimately ensuring patient safety.

References:

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

2. 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.).

3. USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests.

4. USP <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms.