Bioburden describes the number of viable microorganisms present in a product or on a sterile barrier system. The bioburden may be introduced by various sources like raw materials, environment, cleaning processes, and manufacturing and assembling components.
Bioburden testing is a recommended method for determining the total quantity and types of viable microorganisms in unsterilized material or products before performing sterilization. It is a crucial method for selecting the correct process of sterilizing, finalizing, and distributing materials and products.
The vital step in bioburden testing is the collection of samples or recovery methods of microorganisms from products and medical devices. Bioburden testing is highly used in pharmaceutical industries, manufacturers of medical devices, and cosmetics industries.
Table of Contents
Purpose of Bioburden Testing
The bioburden testing methods isolate and enumerate viable microorganisms before sterilizing any materials and products. These testing methods have many different purposes in different laboratories, which are as follows:
- It helps to figure out the correct way of sterilizing the raw materials and water used to produce various products.
- It also helps determine the number of viable microorganisms in the medical devices after production and before use.
- Bioburden testing helps maintain laboratory standards as per the rules or protocols followed.
Procedure of Bioburden Testing
Bioburden testing is the method of determining total viable count (TVC). Bioburden testing can be carried out as TAMC (total aerobic microbial count) or a combination of TAMC and TYMC (total yeast and mold count). For TAMC microbial pour plate and spread plate method are used.
The procedure of bioburden testing for manufacturing (raw materials and packaging practices) in or on a medical device has the following four distinct stages: Microorganism recovery, Enumeration of microorganisms, Bioburden characterization, Validating the method.
Microorganism Recovery
There are many ways of recovering microbial samples based on the material types to be tested.
- For water samples, filtration followed by plating is preferred.
- Sometimes materials are shaken ultrasonically, after which the substance is filtered and placed in an agar medium.
- Sometimes stomaching, rinsing, and flushing are done, followed by filtration and plating on an agar medium.
- If nothing of the above methods works, direct swabbing or contact plate technique is usable.
- The agar plate is then incubated in two ways.
- Two media methods: Fungal-specific media like SDA (Sarbouraud dextrose agar) is incubated at 25 ℃ for 2-7 days. Another TAMC-specific media like TSA (trypticase soy agar) is incubated at 37 ℃ for 18-48 hours.
- One media dual temperature method: All-purpose media like SCD (soybean casein digest) medium is used and incubated firstly at 30-35 ℃ for 2-3 days and then at 20-25 ℃ for 5-7 days. It is a preferable method in areas with low bioburden.
Enumeration of the Microorganisms
The cultured microorganisms are then enumerated by using either MPN (most probable number), membrane filtration, or aerobic plate count method. The microbial load is expressed in CFU (colony forming unit), which helps to develop a standard level.
Bioburden Characterization
The identification of microorganisms is made using various methods. For the identification of bacteria, observation of colony morphology, Gram staining, and other differential staining for studying cell morphology and performing different biochemical tests is usually done. Observing colony morphology and different types of fungal staining methods is used to identify yeast and molds.
Besides these methods, molecular methods like PCR and blotting techniques are also performed to characterize microorganisms.
Validate the Method
Using calculated correction factor(s) during the recovery of bioburden helps calculate the bioburden estimate from the raw sterilization count. The correction factor helps to validate the recovery method.
A correction factor is a numerical value provided by recovery efficiency (RE). The RE helps in assessing the efficiency of the extraction method. According to ISO 11737-1, the RE is measured in two ways; inoculated recovery and repetitive extraction (exhaustive recovery).
In the inoculated method, the materials have low bioburden or are pre-sterilized, which reduces the effect of other competitive microorganisms in order to obtain an accurate count of the inoculated organism. The RE is the ratio of the colony count of the target microorganism recovered to the positive control. It is the preferred method. Usually, a spore-forming microorganism like Bacillus is applied to the material and allowed to dry for this method.
Recovery Efficiency (RE)= CFU recovered in one rinse/Inoculation Population
In the repetitive method, product samples are rinsed (three to five times). The viable bioburden is measured after every extraction. The RE is calculated by comparing the total number of colonies recovered (from all the rinses) to the first rinse. This method is unsuitable for powders, gels, or any other dissolving material.
Recovery Efficiency (RE)= Total rinse CFU/First Rinse CFU
Correction factor= 1/RE
Things to consider
While performing a bioburden, the following factors need to be considered:
- The time of sampling should be at the end of processing and packaging.
- The sample should be from homogenous bulk.
- Aseptic handling and technique are required.
- Clean and sterile media and containers should be used.
- A correct method of sampling, selection, and identification is necessary.
- Validation of the processes after every test is a must.
- A proper method of circulating alert and action levels should be established.
- The expiry time of the sample can influence the validity of the test.
Acceptable Level for Bioburden Testing
According to EMA (European medicine association), not more than 10 CFU per 100 ml is an acceptable level for bioburden testing in the samples obtained through filtration. However, these levels vary based on region, company, and industry.
Common Microorganism Found
The microorganism commonly found during bioburden testing is bacterial species. Sometimes yeast and mold may also be recovered.
- Staphylococcus species
- Bacillus species
- Coliform (in water sample): Escherichia coli is the most common coliform obtained as bioburden from water samples.
- Yeast
- Mold
Application of Bioburden Testing
Bioburden testing is the estimation of microorganisms in samples like containers, medical devices, water, in-process samples, final bulk products before sterilization, product contact surfaces, or materials that require frequent assessment of the bioload. It is also common practice to perform bioburden testing to validate sterilization methods. Some of the fields that commonly apply bioburden testing are:
- Microbiology laboratories: These laboratories constantly deals with microorganism but also require sterile zone for working. So, bioburden testing is widely used to determine the total aerobic microbial count (TAMC).
- Medical products and device manufacturing companies: These companies must produce materials and devices that contain very few or almost non-bioload because these come in constant contact with immunocompromised persons. So, bioburden testing of these products pre-sterilization is preferred and recommended. It is essential to validate the sterilization method.
- Pharmaceutical industries: Final bulk of oral drugs before sterilization, water used for producing drugs, containers, and surfaces are constantly used as samples for bioburden testing in pharmaceutical industries.
- Cosmetic Industries: The raw materials for cosmetic and bulk products before sterilization are subjected to bioburden testing. It is also performed on the equipment used to produce those cosmetic products.
Difference Between Microbial Limit Test and Bioburden Testing
Bioburden testing is a quantitative method that is performed in non-sterile, pre-sterile, unfiltered and filtered samples. Microbial limit test (MLT) is a qualitative and quantitative method performed for non-sterile materials.
Usually, MLT is the method of determining the total aerobic microbial count (TAMC) and total yeast and mold count (TYMC) separately. In bioburden testing, the total viable count (TVC) is calculated as a whole.
Bioburden testing follows guidelines from USP (United States Pharmacopeia) 60, 61, and 62. In contrast, MLT follows the guidelines of USP 61 and 62.
Difference Between Bioburden and Endotoxin Testing
Endotoxin testing determines bacterial endotoxin, a phospholipid found in the outer membrane of gram-negative bacteria. In contrast, bioburden is not limited to determining endotoxin but expands to counting bacteria and fungi.
Bioburden testing determines the total number of viable (living) microorganisms. Whereas endotoxin testing detects and quantifies non-viable bacteria as dead gram-negative bacteria release endotoxin.
Endotoxin is performed in sterile and nonpyrogenic devices or equipment, usually, that come in direct or indirect contact with cardiovascular, lymphatic system, or cerebrospinal fluids. At the same time, bioburden testing helps validate the sterilization method and monitor raw materials, packaging, and the environment.
Bioburden testing follows USP (United States Pharmacopeia) 60, 61, and 62. But endotoxin testing follows the guidelines of the USP 161.
Endotoxin tests do not require culturing, so a report is generated within a day. At the same time, bioburden testing requires culturing and can take up to 7 to 10 days for reporting.
References
- Bioburden – Eurofins Scientific. (n.d.). Retrieved November 16, 2022, from https://www.eurofins.it/media/91471/40024_eurofins_biolab_ctp_laboratories_test_bioburden.pdf
- Sandle, Tim. (2013). A new standard for bioburden testing: USP chapter in development. GMP review. 12. 10-12. https://www.researchgate.net/publication/258074441_A_new_standard_for_bioburden_testing_USP_chapter_in_development
- Pharmaceutical Microbiology Manual. FOOD AND DRUG ADMINISTRATION OFFICE OF REGULATORY AFFAIRS Office of Regulatory Science. (2020, August 25). Retrieved November 16, 2022, from https://www.fda.gov/media/88801/download
- Sandle, T. (2016). Bioburden determination. Pharmaceutical Microbiology, 81–91. https://doi.org/10.1016/b978-0-08-100022-9.00007-4
- Clontz, L. (2008, October 14). Microbial limit and Bioburden tests: Validation Approaches and Global Requirements. Google Books. Retrieved November 16, 2022, from https://books.google.com/books/about/Microbial_Limit_and_Bioburden_Tests.html?id=tNltHmj7-7EC
- Yang, H., Li, N., & Chang, S. (2013). A risk-based approach to setting sterile filtration bioburden limits. PDA Journal of Pharmaceutical Science and Technology, 67(6), 601–609. https://doi.org/10.5731/pdajpst.2013.00942
- Denyer, S. P., Hodges, N., Gorman, S. P., Gilmore, B. F., Hugo, W. B., & Russell, A. D. (2011). Sterilization procedures and sterility assurance. In Hugo and Russell’s Pharmaceutical Microbiology (eighth, pp. 370–371). essay, Wiley-Blackwell.
