Each biological indicator (BI) that is manufactured contains a known number of microorganisms (also referred to as spores). In order to confirm that the BI actually contains the expected number of spores, an individual will need to perform a population assay. In USP <55>, this is referred to as Total Viable Spore Count. This particular chapter provides a detailed description of the steps to take when performing a population assay – from sample size (at least four test samples) to creating a dilution series, heat shock time and temps, and incubation time and temps.
For each dilution series, calculate the yield to be between 30-300 colonies on a plate. 1.0 mL of each dilution is then placed into two petri dishes, and swirled with 20 mL of agar. These plates are then incubated at a species-appropriate temperature for at least 48 hours. From there, a calculation is performed to figure out the average number of spores per unit.