A biological indicator is defined in the International Organization for Standardization (ISO) 11139 terminology document as: "a test system containing viable microorganisms providing a defined resistance to a specified sterilization process" (1). Biological indicators (BIs) are used by medical-device manufacturers, pharmaceutical manufacturers, and healthcare institutions to provide measured response of the efficacy of sterilization processes; the sterility of medical items cannot be inspected or tested in a practical manner. BIs use known quantities of micro-organisms with a high degree of resistance to specific sterilization processes to generate quantitative information regarding efficacy of the processes.
The presence of viable micro-organisms in or on a BI makes this form of sterilization indicator an appropriate tool to evaluate the sterilization process. BIs are unique in comparison with chemical indicators and physical monitors, which are also valuable and provide immediate or real-time information respectively. The latter monitors, however, do not directly provide a biological result.That said, the use of viable micro-organisms requires a post-exposure incubation period to allow for growth of any test organisms that may have survived the sterilization process. This time delay in obtaining feedback from the sterilization cycle is often viewed as a major limitation to the use of BIs, particularly in applications for routine monitoring.
Viable micro-organisms, by nature, require time under suitable conditions of temperature, oxygen tension, nutrients, water, and so forth, to multiply and subsequently create a visual indication of their viability. The result of viability is typically demonstrated by turbidity in a broth culture medium, colonies on an agar surface, or a pH indicator or other metabolic-derived color changes that signify microbial metabolism and growth. A BI that is positive at any time during the predetermined incubation period that growth is detected is a straightforward result (i.e. a positive is a positive regardless of the incubation time). However, a negative BI result is ambiguous relative to the length of incubation. Because today's BIs use well-characterized bacterial endospores, all of the necessary conditions for germination and growth are established. Additionally, the performance requirements of BIs have been well characterized in the ISO 11138 BI Standards series (2). The only BI parameter that is not universally accepted to date is the length of the incubation time necessary to establish viability of BI organisms.
The release of medical items as "sterile" in both industry and in healthcare facilities is commonly based on negative BI monitoring results. The time from completion of the sterilization process to release of goods for distribution or patient use can have major economic and patient care implications. The incubation time at which a BI can be declared negative is therefore a significant issue and is to some extent complicated by the fact that any scientific conclusion based on a negative result is always open to debate.
The current ISO 14161:2009 standard, which provides guidance for users of BIs, recommends an incubation period of 7 days for established sterilization processes, such as ethylene oxide and moist heat, and 14 days for nonstandard or new sterilization processes (3). This guidance is consistent with the requirements of the BI manufacturer's standard ISO 11138-1:2006, but the 11138-1 document also specifies that the BI incubation time and temperature be validated (4). Additional text in ISO 14161:2009 regarding BI incubation time (12.3.3) states that:
Neither ISO BI document provides information on test methodology for validating BI incubation time. This lack of detail has led to confusing and nonuniform requirements for RIT documentation that have historically been applied to medical device and pharmaceutical manufacturers throughout the world.