Bioburden Testing: Definition, Procedure, USP Limits, and Medical Device Standards

Before a batch of syringes or a bottle of medicine can be sterilized, one number decides how that sterilization is designed and validated: how many living microorganisms are already on the product. Count too loosely and the sterilization may not be enough; count carefully and the process can be set correctly and proven. That count is the bioburden, and measuring it reliably is the job this test exists to do.

Bioburden is the number of viable (living) microorganisms on or in a product before it is sterilized. Bioburden testing is the laboratory method that counts them.

The microorganisms come from many sources: raw materials, water, the manufacturing environment, cleaning processes, equipment, and the people and components involved in assembly. Knowing how many are present, and what types, is essential before sterilization, because the sterilization process must be strong enough to deal with the actual starting load. Bioburden testing measures that starting load.

In short, bioburden testing answers two questions: how many viable microorganisms are on the product, and what kinds. The answers are used to design and validate the sterilization process and to monitor that manufacturing stays in control. It is used heavily in the pharmaceutical, medical device, and cosmetics industries.

Figure: Bioburden testing

Bioburden testing at a glance

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:

1. It helps to figure out the correct way of sterilizing the raw materials and water used to produce various products.
2. It also helps determine the number of viable microorganisms in the medical devices after production and before use.
3. 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.

Figure: Steps of Bioburden Testing

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.

1. For water samples, filtration followed by plating is preferred.
2. Sometimes materials are shaken ultrasonically, after which the substance is filtered and placed in an agar medium.
3. Sometimes stomaching, rinsing, and flushing are done, followed by filtration and plating on an agar medium.
4. If nothing of the above methods works, direct swabbing or contact plate technique is usable.
5. The agar plate is then incubated in two ways.

Two media methods: Fungal media such as SDA (Sabouraud dextrose agar) are incubated at 20 to 25°C for 2 to 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 such as 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:

1. The time of sampling should be at the end of processing and packaging.
2. The sample should be from homogenous bulk.
3. Aseptic handling and technique are required.
4. Clean and sterile media and containers should be used.
5. A correct method of sampling, selection, and identification is necessary.
6. Validation of the processes after every test is a must.
7. A proper method of circulating alert and action levels should be established.
8. The expiry time of the sample can influence the validity of the test.

Acceptable Level for Bioburden Testing

There is no single universal bioburden limit. Acceptable levels depend on the product, how it is used, and the stage of manufacturing, and they are set by the manufacturer against recognized standards.

For water and filtered solutions, a commonly applied in-process action level is not more than 10 CFU per 100 mL before final filtration, though companies set their own alert and action levels.

For nonsterile pharmaceutical products, the acceptance criteria are given in USP chapter 1111. For example, oral non-aqueous products commonly allow a total aerobic microbial count (TAMC) of up to 1,000 CFU per gram or mL and a total yeast and mold count (TYMC) of up to 100 CFU per gram or mL, with tighter limits for other routes of administration and a requirement that specified (objectionable) organisms be absent. Always apply the specific limit that matches the product and its route of use.

Common Microorganism Found

The microorganism commonly found during bioburden testing is bacterial species. Sometimes yeast and mold may also be recovered.

1. Staphylococcus species
2. Bacillus species
3. Coliform (in water sample): Escherichia coli is the most common coliform obtained as bioburden from water samples.
4. Yeast
5. 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 bio load. It is also common practice to perform bioburden testing to validate sterilization methods. Some of the fields that commonly apply bioburden testing are:

1. Microbiology laboratories: These laboratories constantly deal with microorganisms but also require a sterile working zone. So, bioburden testing is widely used to determine the total aerobic microbial count (TAMC).
2. Medical products and device manufacturing companies: These companies must produce materials and devices that contain very few or almost non-bio load 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.
3. 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.
4. 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 and microbial limit testing in pharmaceuticals follow USP chapters 61 (Microbial Enumeration Tests) and 62 (Tests for Specified Microorganisms), with acceptance criteria in chapter 1111. For medical devices, bioburden follows ISO 11737-1.

Difference Between Bioburden and Endotoxin Testing

Endotoxin testing detects bacterial endotoxin, which is lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria. Bioburden testing is different: it counts living bacteria and fungi, not the endotoxin molecule.

Bioburden testing measures the number of viable (living) microorganisms. Endotoxin testing measures the amount of the endotoxin (LPS) molecule itself, which can be present even when no living bacteria remain, because dead Gram-negative bacteria still release endotoxin. This is why a product can pass sterility yet still contain endotoxin, and why the two tests are done separately.

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 and microbial enumeration follow USP chapters 61 and 62. Endotoxin testing follows USP chapter 85 (Bacterial Endotoxins Test). Sterility testing follows USP chapter 71.

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.

Bioburden vs sterility testing

Bioburden testing and sterility testing are often confused, but they answer different questions at different stages.

The simple way to hold it: bioburden is a number measured before sterilization to set the process, and sterility is a pass/fail check after sterilization to confirm it worked. A low bioburden makes it easier to achieve and prove sterility.

Bioburden testing for medical devices (ISO 11737)

For medical devices, bioburden testing follows the international standard ISO 11737-1. This matters because the bioburden result is used to set and validate the sterilization dose, especially for radiation and ethylene oxide sterilization.

The device-specific points are:

1. Recovery from the whole device. The microorganisms must be removed from the device surface, often by shaking, sonication, or flushing, so they can be counted. Because no removal method is perfect, a recovery efficiency (correction factor) is applied, as described above.
2. Routine monitoring. Device manufacturers test bioburden regularly (for example on a set number of devices per batch or per period) to show the manufacturing process stays in control and the sterilization dose remains valid.
3. Choice of sterilization. A consistently low bioburden supports a lower, validated sterilization dose. A rising bioburden can signal a manufacturing problem and may require investigation before products are released.

This is why bioburden testing is a routine, ongoing part of medical device manufacturing, not a one-time check.

How to Remember

Bioburden is the before number. It counts living microorganisms before sterilization, to set and validate the process. Sterility is the after check.

Count, then characterize, then validate. Recover the organisms, count them (CFU), identify what they are, and validate the recovery with a correction factor.

61 and 62 count, 1111 sets the limit, 85 is endotoxin, 71 is sterility. The USP chapters: 61 enumerate, 62 specified organisms, 1111 acceptance criteria, 85 endotoxin, 71 sterility.

Devices follow ISO 11737. For medical devices, the bioburden standard is ISO 11737-1.

Recovery is never perfect, so correct for it. The correction factor (1 divided by recovery efficiency) adjusts the count for organisms the method missed.

Key exam facts

Where Students Get Confused

"Bioburden testing and sterility testing are the same." No. Bioburden is a count of microorganisms before sterilization, used to design the process. Sterility testing is a pass/fail check after sterilization to confirm the product is sterile.

"Bioburden testing detects endotoxin." No. Bioburden counts living microorganisms. Endotoxin testing measures the LPS molecule, which is a separate test (USP 85). A product can have low bioburden and still contain endotoxin.

"There is one universal bioburden limit." No. Limits depend on the product and its route of use. Pharmaceutical acceptance criteria are in USP 1111, and medical devices follow ISO 11737-1. Companies also set their own alert and action levels.

"Endotoxin is a phospholipid." No. Endotoxin is lipopolysaccharide (LPS). Its lipid A portion is a glycolipid, but the molecule as a whole is an LPS, not a phospholipid.

"The raw plate count is the bioburden." Not quite. Because recovery is never complete, the raw count is adjusted using a correction factor (1 divided by the recovery efficiency) to estimate the true bioburden.

References

• United States Pharmacopeia. General Chapters <61>, <62>, and <1111> (microbial examination and acceptance criteria for nonsterile products); <71> (sterility); <85> (bacterial endotoxins).
• International Organization for Standardization. (2018). ISO 11737-1: Sterilization of health care products, Microbiological methods, Part 1: Determination of a population of microorganisms on products.
• Sandle, T. (2016). Bioburden determination. In Pharmaceutical Microbiology (pp. 81–91). Woodhead Publishing.
• U.S. Food and Drug Administration. (2020). Pharmaceutical Microbiology Manual.
• Denyer, S. P., et al. (2011). Sterilization procedures and sterility assurance. In Hugo and Russell's Pharmaceutical Microbiology (8th ed.). Wiley-Blackwell.