Bioburden is the term for the number of bacteria living on a surface that has not been sterilised. Bioburden testing, also known as microbial limit or microbial cleanliness testing, is performed on pharmaceutical products and medical products for quality control purposes. This test focuses on bacteria rather than viruses because viruses cannot survive outside a host long enough to be present on a medical product. Essentially, it tests how clean the mask is when it is packaged and delivered to the buyer. The lower the bioburden, the cleaner the mask is. The PRO mask has an average bioburden of 2.06 CFU/g.

The bioburden is calculated by counting the number of colony-forming units (CFUs), groups of two or more bacterium, on a newly opened mask and dividing the number of CFUs by the weight of the mask in grams. CFUs are counted rather than individual bacterium due to their small size and their tendency to group together. The FDA requires that the bioburden of medical masks is less than 30 CFUs per gram.

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