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Bioburden Testing-Medical Device Testing

Updated: Oct 23, 2023

What Is Bioburden Testing

Bioburden is the population of viable microorganisms in or on a product, water, or raw material. Bioburden test determines the number of these microorganisms present, reported as colony forming units (CFU).

Why Test For Bioburden?

A knowledge of bioburden can be used in a number of situations as a part of ;

  • Validation and re-validation of sterilization processes

  • Routine monitoring for control of manufacturing processes

  • Monitoring of raw materials, components or packaging

  • Assessment of the efficiency of cleaning processes

  • An overall environmental monitoring programs

Bioburden Method

ISO 11737-1 specifies the requirement and provides guidance for the enumeration and microbial characterization of the population of viable microorganism on medical devices.

It also describes detailed procedure in conducting bioburden test on medical devices from selection of product samples and appropriate method for bioburden determination to microbial characterization of bioburden.

In addition, the validation of the bioburden method emphasizing on the extraction efficiency (correction factor determination) either by repetitive extraction or inoculation procedures can also be found in this document.

Membrane Filtration Method

The most common technique is membrane filtration where the solution of microbes is filtered through a sterile filter paper and the filter paper is transferred onto a suitable agar plate and incubated.

The procedure for bioburden test using the membrane filtration method is as follows:

  1. Extraction solvent is sterilized in a suitable size bottle and autoclaved just before use

  2. The sample is immersed aseptically into the solvent once cooled

  3. Extraction is carried out using ultra-sonication

  4. The extract is then passed through a membrane filter to collect any microorganism removed

  5. The membrane filter is then placed onto an agar plate and incubated

  6. Aerobic & anaerobic bacteria and also yeast & molds colonies are counted and recorded

  7. The total number of colonies in the test sample is calculated as the total cfu observed

Pour Plate Method

​Another technique is pour plate method where a small portion (1 mL) of the extract is mixed with molten agar medium and incubated under suitable conditions. The choice of growth media and incubation conditions will depend on the type of microorganisms to be enumerated.

For aerobic bioburden, both bacteria and fungi (yeast & molds) colonies are enumerated while for anaerobic bioburden, anaerobic bacteria colony counts are determined. The media, incubation condition and incubation period are summarized below.

 The media, incubation condition and incubation period for bioburden
Culturing Technique For Bioburden Testing

Most Probable Number (MPN) Method

The determination of bacterial quantity in a sample is a basic part of microbiology research. The most probable number (MPN) method is a popular quantitative technique for dividing a sample into culture broth and determining the presence or absence of microorganisms. This method yields an approximate bacterial concentration, but narrowing the confidence intervals needs a large number of repeats at acceptable dilutions. Furthermore, it should be noted that the MPN approach is limited to bacterial inspection and is not a reliable instrument for fungus enumeration.

How Many Samples Are Needed For Bioburden Testing?

Samples of product to be tested should be representative of the production batches. The number of samples required to give a reliable bioburden estimate (BE) will depend on the purpose of the bioburden enumeration.

Generally, for routine bioburden test for QC monitoring requires between 3-10 samples selected at random.

For sterilization validation, gamma sterilization require 10 samples from 3 different batches for a total of 30 samples, while for EO sterilization, 10 samples are tested.

For large samples, sample item portion (SIP) may be used where representative parts of the device are taken in portions and the SIP ratio is calculated and used in the final calculation of average BE.

What Extraction Technique to Use in the Removal of Viable Microorganisms For Bioburden Testing?

Various techniques of dislodging the viable microorganisms from the samples can be used including stomaching, ultrasonication, mechanical shaking, vortex mixing, flushing and even blending if possible.

The choices of extracting fluids may range from sterile water to sterilized solution of surfactants such as polysorbates.

Types of solvents used for the extraction of viable microorganisms for bioburden testing
Bioburden Extraction Solvent

There should be a balance between extracting the microorganisms aggressively without killing the viable microbes.

The efficiency of extracting the microbes from the medical device may be determined as part of the validation protocol in bioburden test. Repetitive recovery or repetitive extraction should be conducted to obtain the correction factor which will be used to compensate for the extraction efficiency.

Alternatively if the bioburden count is known or expected to be less than 10 CFU/device, the inoculation method should be applied to estimate the extraction efficiency and thus the correction factor.

Bioburden Enumeration

Once the incubation period is complete, the number of colony forming units (CFU) is counted either manually or with assistance by appropriate software.

Accuracy in counting the CFU is improved with the use of colony counters and microscopes. Counting small or spreading colonies is improved through experience. Using two or more personnel to do the counting improves the accuracy tremendously.

How to Report Bioburden Count?

The final bioburden count is determined by multiplying the observed CFU with the pre-determined correction factor (extraction efficiency) and SIP (sample in proportion) ratio if SIP sample was done. Bioburden may be reported as total bioburden, aerobic bioburden, fungi (yeast & molds) and anaerobic bioburden. If more than one sample per product is tested, an average bioburden estimate (Average BE) may be reported.

Bioburden Recovery Efficiency

In practice, it is impossible to enumerate every microorganism that is present on a product due to the nature of the product, the extraction method and culturing limitations.

Some method might be more effective at removing the microorganisms but might cause cell death, make them unculturable and thus not enumerable. Whereas some methods might be gentle to the microorganisms but are less effective at removal.

Therefore, care should be taken when choosing the most suitable and effective method to estimate the bioburden of a product.

Since complete removal of bioburden is not possible, bioburden recovery efficiency tests are performed to establish a bioburden correction factor. It is a measure of the ability of a specific method to remove, collect and/or culture microorganisms from a product.

The bioburden correction factor will “correct” the bioburden data so it will “include” or account for microorganisms that are not removed or cultured. Bioburden recovery efficiency tests can also be done to compare the effectiveness between test methods.

Bioburden Testing for Medical Devices at Sanichem Resources.

SaniChem offers a few variations of bioburden test methods to suit customers' requirements. These follow the accredited in-house method MB-BIOBURDEN-1 which is based on ISO 11737-1:2018. We conduct medical device tests for customers from all over the world such as Malaysia, Singapore, Thailand, Vietnam, Philippines, Saudi Arabia, United Arab Emirates, Pakistan, Egypt, Kenya and more.

Bioburden and Related Tests.

  1. MB102a - Determination of bioburden by membrane filtration (aerobic bacteria + yeast & molds) - 3 parameters

  2. MB102b - Total aerobic and anaerobic bioburden by membrane filtration method - 5 parameters

  3. MB102c - Total anaerobic bioburden by membrane filtration method

  4. MB102d - Determination of bioburden of liquid samples by pour plate method (aerobic bacteria + yeast & mold) - 3 parameters

  5. MB102e - Total aerobic and anaerobic bioburden by pour plate method

  6. M102f - Correction Factor determination for bioburden - repetitive extraction

  7. MB102g - Correction Factor determination for bioburden - inoculation method

In SaniChem Resources, we could help you to:

  • Understand Bioburden test for medical device.

  • Perform analysis on Bioburden testing.

  • Fulfill regulatory requirements of ISO 11135

Our test methods are based on requirements of:

  1. ISO 11737-2

  2. USP 61 & USP 62

Click the button below to contact us on any questions you have about bioburden testing

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