Bioburden testing involves the detection of microorganisms that can be present in products or the environment. The methods vary, however, from method to method, and the results are not always 100 percent accurate. The enumeration process can vary depending on the technician performing the bioburden testing, which can lead to data that is less than 100 percent accurate. Fortunately, specific bioburden procedures are available that can detect both naturally occurring and artificially introduced microorganisms in products.

Inoculation method

Bioburden testing is a standard laboratory procedure which evaluates a product’s biological hazards. Performing bioburden tests requires inoculating the product with a Bacillus spore suspension. It is critical to repeat the bioburden procedure for each sample to ensure that the results are valid. Bioburden data should include recovery tests. Bioburden test procedures should be consistent with international standards such as ANSI/AMMI/ISO 11737-1 and EN 1174-3, which apply to sterilization of medical devices.

A more labor-intensive method is the repetitive method. The repetitive method requires multiple bioburden tests on a sample. Unlike the inoculation method, the repetitive method is not as consistent as a single extraction. The results are also inconsistent because the samples have different bioburden populations from the beginning. Thus, it is critical to choose the parameters and the number of repetitions that best reflect the nature of the product. The repetitions may range from three to ten on a sample depending on the bioburden levels.

Pour plate

In order to assess the level of microbial contamination in a drug, pharmaceutical companies must determine its microbial load. During different stages of the drug-making process, samples are taken from the drug and plated on agar. By measuring the number of contaminating microbes, pharmaceutical companies can devise precautions to limit microbial contamination. Using agar with a high melting point, pouring samples on agar can destroy large numbers of bacterial cells.

Bioburden characterization, including pour plating and spread plating, is only valuable if a baseline is established. If the results are aberrant, then it’s of no use. The microorganisms should be separated from new ones so that the results can be compared. This is usually done during more frequent monitoring in earlier stages of manufacturing and processing. To do this, the sample must be thoroughly sterilized and the medium must be boiled before being added to the petri dish.

Spread plate

The method of bioburden testing using spread plates is also known as the Copacabana Method. The test involves plating a mixture of E. coli cells and supplementing them with X-Gal. Blue colonies result from cells expressing a functional b-galactosidase enzyme while white ones result from cells with a mutation in the lacZ gene. A blue/white screen is used to differentiate between the two.

The technique used for this testing involves placing the tissue in stomacher bags containing extraction fluid and vigorously stirring it for one minute. Then, multiple aliquots of the extraction fluid were taken from the stomacher bags and pour-plated onto multiple plates. The growth of each strain on each plate was monitored over a seven-day period. Pre-processed allograft tissue contained 37 different microorganisms. The most common Gram-positive organisms were Staphylococcus epidermidis and Staphylococcus capitis.

Limit of detection for bioburden testing

The limits of detection for bioburden testing are a critical consideration in the development of a bioburden monitoring program. A bioburden test must be specific to the products and raw materials used in its production. Non-sterile products are generally tested for viable microorganisms and pathogens, as well as total viable counts. Although these products do not require sterilization, the presence of microbial organisms may adversely affect the therapeutic characteristics of the products. Therefore, it is essential to understand the limits of detection and the associated bioburden levels for non-sterile products.

The limits of detection for bioburden testing are different for different medical devices. The method used to determine the bioburden of medical devices varies depending on the type of material. Unlike other tests, ISO 11737-1:2018 does not define a single method for determining bioburden, and therefore, the manufacturer must determine which is appropriate for the product. The limits of detection for bioburden testing will be more accurate if the manufacturer understands the materials and the properties of the products.

Sterility validations for bioburden testing

In-process bioburden testing can determine the presence of microorganisms in a product. To accurately determine if a product contains bioburden, the method used for this testing must be validated. The data obtained from in-process bioburden testing may also be used to monitor seasonal trends and adverse trends. In addition to determining the presence of bioburden, in-process bioburden testing may also provide evidence of undetected contamination and biofilms.

Bioburden is the amount of live microorganisms present in a sample. This amount is measured in colony-forming units, which represent the number of live microorganisms present in the product. Various factors contribute to bioburden contamination, including personnel, raw materials, and inadequate environmental controls. In addition, water used during the manufacturing process may contain microbial contaminants. Performing bioburden testing is important for the manufacturing process.