Sterilization deals with the destruction of microorganisms by exposing materials to conditions that are rapidly lethal to
them. The validation of these processes generally relies on the use of resistant microorganisms termed biological indicators (BIs) that are positioned within the sterilizer. The destruction of these microorganisms during validation confirms the
lethality of the process for the routine use of the sterilizer. Routine sterilization must be sufficiently lethal to the bioburden
expected on or in the materials being processed. Information about the process resistance of both the BI and bioburden is
essential to understanding process effectiveness. Over- or under-estimating the resistance of either the indicator or the
indigenous bioburden can lead to failed validation studies, potentially contaminated batches, sterility failure investigations,
and other serious situations.
Determination of microbial resistance
Determining microbial resistance once was only the concern of BI manufacturers and firms producing terminally sterilized products.
There are several instances in which the firm using BIs could benefit by making its own resistance determination. These situations
arise when either spore strips or suspension indicators are used as follows:
- Spore strips are labeled by their manufacturer, with resistance based on their test methods and laboratory media. Differences
in the methods and media at the end user's site can lead to altered resistance.
- Suspensions of BIs come with a labeled resistance as well. That value is for the specific lot when tested as a spore strip.
BI manufacturers caution their customers that these values may change when the indicator is inoculated onto a different substrate.
- Elastomeric closures have been shown to alter the resistance of BI organisms dried on their surface as is currently expected
in regulatory submissions
- Resistance determination and screening of bioburden organisms can be detected in presterilization sampling
- BI resistance is determined in support of cycle development, initial or revalidation of terminal sterilization processes
- Sterility-test and media-fill failure investigations sometimes incorporate concerns regarding the resistance of presterilization
bioburden organisms.
BIER vessels
BI evaluation resistometer (BIER) vessels have been used for more than 50 years to measure the resistance of bacterial spores
when monitoring steam-sterilization processes. The earliest designs of steam-BIER vessels were developed by food-processing
technologists, and were based on the concept of retorts used in the food-processing industry. These steam retorts maintained
a large volume and constant flow of steam under pressure in an environment in which BI units were quickly inserted and removed.
This process provided a nearly instantaneous come-up and cool-down condition for the exposed samples.
Medical and pharmaceutical technologists developed BIER vessels around conventional autoclave technology. The autoclave is
a steam-pressure vessel in which materials are placed into a chamber at ambient pressure. The door is closed, steam is admitted
into the chamber, and steady-state conditions are achieved. With specially engineered systems using very small chambers, the
system can achieve nearly instantaneous come-up and cool-down conditions.
These special sterilizers were almost exclusively custom designed. Today, commercial units are offered by a limited number
of manufacturers. The Fedegari system (model FOB 3, Fedegari Autoclavi SpA, Povia, Italy) tested in this study is a 140-L
laboratory steam autoclave with an accessory attachment that allows rapid sample insertion into a chamber at steady-state
conditions and rapid removal of the sample. The chamber's internal sample guide can be removed from the interior of the unit,
thus allowing the unit to be used as a standard laboratory autoclave. This BIER vessel design can be used not only for BIs,
but also for chemical indicators and process-challenge devices.
Figure 1: The BIER vessel's square-wave system. Samples placed
in the BIER vessel are taken from ambient conditions, brought to the
sterilizing condition, and returned to the ambient conditions.
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The BIER vessel establishes a steady-state condition within a small sterilization chamber and relies on the rapid insertion
and removal of samples for the conduct of the D-value determination. In its purest form, the BIER operates as a square wave system, in which the samples are instantaneously
taken from ambient conditions, brought to the sterilizing condition, and returned to ambient conditions (see Figure 1). BIER-vessel
standards for saturated steam mandate a maximum of 10 s for the come-up and come-down steps.
