Bioburden is the population of viable microorganisms on or in a product, a component or a bulk solution immediately before it is sterilized. It is expressed as colony-forming units (CFU) per unit, per gram or per volume. Sterilization does not start from zero; it starts from the bioburden, and the whole sterility calculation depends on knowing and controlling it.

How it is measured

For a liquid such as bulk bacteriostatic water before filtration, a measured volume is passed through a membrane filter, the membrane is placed on a growth medium, incubated, and the colonies counted; alternatively a pour-plate or spread-plate count is used. The methods are those of USP <61> for microbial enumeration. Because bioburden organisms are those that survived the water system and any pretreatment, the identity of what grows is examined as well as the count: a Gram-negative bacterium in a water system is a warning about endotoxin, whatever its number.

Typical limits

• For a bulk solution immediately before sterile filtration, current good manufacturing practice guidance uses a limit of 10 CFU per 100 mL. A higher count means either the water system or the process upstream needs attention, and it also loads the sterilizing filter.
• For terminally sterilized products the bioburden of the filled units before the cycle is measured routinely, because it is an input to the sterility assurance level. A cycle validated for a bioburden of 100 CFU per unit is not proven for units carrying 10,000.

Why it matters even when the product is sterilized

• Sterility assurance. The 10⁻⁶ SAL is calculated from the starting population. See sterility assurance level explained.
• Endotoxin. Killing Gram-negative bacteria leaves their endotoxin behind, and sterilization does not remove it. Low bioburden before sterilization is the main way the 0.5 EU/mL endotoxin limit is met. See what is an endotoxin unit?
• Filter performance. A sterilizing filter has a finite capacity; a high bioburden shortens its life and increases the chance of an integrity failure. See sterile filtration explained.

Bioburden in the laboratory

The term also applies to anything a laboratory prepares. Solutions made with bacteriostatic water start with essentially zero bioburden from the diluent, and the preservative is there to keep the bioburden introduced during use from growing. Every entry into a vial adds to it, which is the reason for clean technique and the 28-day limit. See how many times can a vial be punctured? and antimicrobial effectiveness testing.

Related

How bacteriostatic water is manufactured · Glossary

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