Bioburden Testing Vs. Sterility Testing: What Is The Difference?

Bioburden and sterility testing are generally conducted in parallel because both represent key processes to ensure a sufficient sterility assurance for single-use medical devices. In principle, the respective aims are clear.

• Bioburden testing is a quantitative and qualitative assay, aiming to evaluate the number and the species of living microorganisms present on a medical device before sterilization.
• Sterility testing is a qualitative assay, aiming to ensure no living microorganisms are present on a medical device after sterilization.

Bioburden Testing Procedure

Unlike pharmaceuticals (liquid or powder), a medical device is not a product we can dissolve, dilute, or divide. To analyze bioburden on a medical device, the maximum quantity of germs needs to be removed from the medical device without killing them. To do so, a medical device needs to be immersed (or filled) in (with) a neutral eluent able to separate microorganisms from an inorganic surface without causing microbial lethality or promoting growth. When done, the medical devices are submitted to successive treatments (shaking, ultrasounds, etc.) to remove the microorganisms. Several methods can be used dependent on the type of tested product or the specified microorganisms. The eluent can be directly included in a solid growth medium, but the preferred method is to filter the eluent and incubate the filtration membrane on solid growth media, selected for the specified microorganisms.

When screening, different types of microorganism are required (aerobic, anaerobic, yeast/mold, etc.). There may also be a need to grow the microorganism in a different culture condition, where the eluent can be split, filtered, and incubated in different conditions. The obtained results need to be corrected to estimate the total number of germs initially present on the device.

Prior to performing bioburden routine testing, the extraction method must be validated, and consideration taken that it is never possible to remove 100% of microorganisms from a medical device, statistically speaking. There are several steps necessary to validate this method:

1. Evaluate the Efficacy of the Extraction: Several strategies could be used. The most classic and recommended is to inoculate sterile devices with a known amount of a germ (classically a spore suspension of Bacillus atrophaeus). Then the quantity of recovered germs is compared against the quantity enumerated in the inoculum to determine the percentage of recovery and calculate a correction factor to apply to bioburden routine testing results. The aim is to be as close as possible to the real quantity of the living germs present on a device. Other methods can be used as iterative extractions of non-sterile devices using the natural bioburden of the product to test as recommended in ISO 11737-1.
2. Evaluate the In Vitro Conditions Used in Routine Testing (Bacteriostasis/Fungistasis Testing): The presence of the device or the physical extraction of germs does not cause inhibition (or growth promotion) of germs during the treatment, leading to a potential underestimation of the real quantity of living microorganisms present on the tested product. A variety of representative vegetative microorganisms are used to inoculate the eluent. By adding these germs to the eluent, the method of extraction can be evaluated to see if it affects the number of germs recovered compared to the known inoculated quantity.
3. Screen Culture Conditions: This is required to perform bioburden routine testing, considering the type of germs recovered from the tested product (aerobes, anaerobes, yeast-mold, spores, etc.).

How a routine bioburden testing is performed and what needs to be validated is an important point to understand similarities and differences between bioburden and sterility and their respective application.

Sterility Testing Procedure

As with bioburden testing, several methods can be used to control the sterility of a product. The main recommended method is direct inoculation of the evaluated product with a liquid and adapted growth medium. This method is based on the property of each living microorganism to grow in an adapted growth medium and produce macroscopic signs of growth (turbidity, flocculation, etc.). This method is only qualitative.

As we discussed with the bioburden testing procedure, before any routine sterility testing a bacteriostasis/fungistasis assay is required. This addresses the absence of macroscopic signs of growth, due to the absence of living germ and growth inhibition in vitro testing conditions. This can be confusing for device manufacturers, but this test cannot be substituted by the bacteriostasis/fungistasis assay performed for bioburden. A variety of representative vegetative microorganisms are used, but the testing conditions are not the same and the inhibition is evaluated by comparing a positive control (medium inoculated without device) to the inoculated media containing the tested product. The presence of a potential inhibition is determined by a comparison of macroscopic signs of growth in a liquid medium.

A second method that can be used for sterility testing and is similar to the bioburden test method. When direct inoculation is not possible for technical reasons, it is possible to perform an extraction procedure of germs with membrane filtration. This is inoculation of the liquid medium by the filter, and not by the device directly. In these conditions, a method validation is required following a procedure similar to the bioburden process. However, this is not a preferred method considering that a mechanical extraction of germs cannot be guaranteed with 100% efficiency. The risk is that not all the living germs that are present on the device are extracted and false negative results for sterility may be obtained. It is the reason why ISO 11737-2 recommends the direct inoculation method.

Bioburden vs. Sterility Testing: When to Perform Each of These Tests?

The method to perform bioburden is described in ISO 11737-1. Bioburden analysis is conducted before sterilization for two reasons:

1. To ensure that the quantity and quality of microorganisms present on the device does not represent a challenge for sterilization
2. To monitor bioburden to ensure control and stability of production chains

The method to perform sterility testing is described in several standards, each with a specific scope. Sterility testing can be conducted for various reasons (on medical devices or biological indicators for sterilization validation, batch release testing, monitoring, etc.). ISO 11737-2 describes the methodology, but its scope is strictly limited to medical device testing and sterilization validation using a reduced or partial sterilization exposure. The main example is radiation validation and dose audit for which both bioburden and sterility testing are conducted together before and after sterilization to select a verification dose of radiation. Sterility testing is directly dependent of bioburden for two reasons:

1. The number of living microorganisms allows for empirical determination of the verification dose sufficient to sterilize the medical devices in a worst-case scenario. This ensures a sufficient sterility assurance level is maintained in routine conditions of sterilization.
2. The type of germs present on the medical device allows us to select the optimal growth conditions for cultivating surviving germs after sterilization at the verification dose. In this application, bioburden and sterility testing are complementary.

Additional standards for sterility testing that are not in the scope of ISO 11737-2 include the United States Pharmacopeia (USP) or European Pharmacopoeia. These can be used as standards for sterility testing employing a complete sterilization exposure or testing on biological indicators. However, USP and European Pharmacopoeia are specific to pharmaceuticals and not adapted for non-divisible products like medical devices.

Confusion often occurs when reviewing the differences between bioburden and sterility testing, particularly as the two tests are generally performed together for the same application and the two testing procedures have some method similarities. However, the purpose and the criticality of the results provided are not the same. Bioburden aims to evaluate the quantity and species of living microorganisms on a medical device before sterilization, whereas sterility testing aims to ensure that no more living microorganisms remain on a medical after sterilization. Although the reliability of the procedure for bioburden analysis has to be attested and validated, a variability of bioburden on a medical device could be easily managed, whereas a defect of the sterilization efficacy or false negative sterility results represent a real danger for patient safety.

NAMSA provides bioburden testing services.