This document discusses two methods for validating the extraction efficiency of a bioburden test method: repetitive recovery and product inoculation. The repetitive recovery method involves repeatedly extracting microorganisms from a sample until no new organisms are recovered to determine the overall recovery rate. The product inoculation method involves inoculating a product with known microbes and calculating the recovery rate based on the inoculum amount and recovered amount. While product inoculation establishes a correction factor, the drying process can impact microbe viability, so repetitive recovery is generally preferred for determining bioburden extraction efficiency.

1. Bioburden Method Validation
Extraction Efficiency Test
An Extraction Efficiency test will provide a validated Bioburden recovery test method for a particular product. This is
necessary to determine accurate product bioburden levels. It will show the efficiency of recovering microorganisms
that are present on the product. The data collected by performing this Extraction Efficiency test is used to determine
if a recovery factor needs to be applied to the results of the Bioburden portion of the validation. Insufficient recovery
during bioburden tests would result in an underestimation of the true bioburden of the product and an inadequate
sterilization dose could be applied to the product. If any changes are made to the product, such as the method of
production or the raw materials used, then the bioburden method must be re-validated. The two most commonly
used Extraction Efficiency methods to validate the Bioburden test method are Repetitive (Exhaustive) Recovery and
Product Inoculation.
Repetitive (Exhaustive) Recovery Method
The idea behind this method is that the extraction procedure on a single sample product is to be repeated until there
is no significant increase in the number of recovered microorganisms. The goal is to recover all viable
microorganisms by washing the sample product repeatedly. Using this method determines the efficiency of
recovering the naturally occurring bioburden from a product. The counts in colony forming units (CFU) that are
recovered from the first extraction are compared to the total counts recovered from all the washes to calculate a
percent recovery when doing just one extraction. The percent recovery is used to calculate a correction factor which
is then applied to the Bioburden test numbers for the product. In this way routine bioburden tests only require one
extraction. The CFU recovered from one extraction are then simply multiplied by the correction factor to determine
the total bioburden of a product. This represents the true bioburden value which is what is used to determine the
needed gamma radiation sterilization dose for the particular product. These doses are determined by following the
information in Table B.1 of the ANSI/AAMI/ISO 11137 guidelines.
Product Inoculation Method
In this method the product is inoculated with a known number of microorganisms in order to create an artificial
bioburden and thus establish recovery efficiencies and a correction factor. The product is inoculated and then the
inoculum is allowed to dry. Once the inoculum has dried, the chosen method for removing microorganisms from the
particular product is applied. A ratio of the recovered titer to initial inoculum count establishes the recovery efficiency
and correction factor for the product. We do not recommend The Product Inoculation Method because in many cases
it is impossible to guarantee the viability of the organisms during the drying process.