Types of Biological Indicators

General Requirements

A biological indicator is a microbiological test system consisting of a standardized viable population of specific microorganisms (usually bacterial spores) inoculated on a carrier contained within its primary pack ready for use and providing a defined resistance to a specified sterilization process.

Biological indicators provide means to directly assess the microbial lethality of a sterilization process. When used in conjunction with physical and/or chemical process monitors, biological indicators provide an indication of the effectiveness of a given sterilization process.

A sterilization process should be considered as satisfactory only when the desired microbiological results, as determined by an appropriate sterilization cycle development, validation and routine monitoring programme, have been realized failure to achieve acceptable microbial challenge results forms the basis for declaring a failure in the sterilization process. Biological indicators may also be used to evaluate the capability of processes used to decontaminate isolators or aseptic cleanroom environments.

Types of Biological Indicators

Biological indicators come in various forms designed for specific types of sterilization each type incorporates a viable culture of a known species of microorganisms. There are at least three different types of biological indicators. Some biological indicators may also contain two different species and concentrations of spores.

In one form, the spores are inoculated on a carrier of convenience, such as filter paper placed within a primary pack that protects the carrier from damage or contamination while allowing the sterilant to contact the test organisms. Aseptic technique must be employed when transferring the inoculated carrier to the growth medium in order to avoid accidental contamination. The carrier and primary packaging is not degraded by the specific sterilization process and is designed to minimize any loss of the original inoculum during transport, handling and shelf-life storage.

In another form, called the self-contained biological indicator, the unit consists of both an ampoule of growth medium and an inoculated carrier with the desired population of test organisms, in another vial. The sterilant enters and exits the outer vial through a filter in the cap. This form of biological indicator together with the self-contained growth medium can be considered a system and the entire system provides resistance to the sterilization process.

To allow for the time lag that may occur while the sterilizing agent reaches the contained microorganisms in the system, the D-value (Decimal Reduction Value), process end-point, kill time and the survival time should be characterized for the system and not solely for the paper carrier of the self-contained unit. Following processing, the ampoule of growth media is crushed and brought into contact with the inoculated carrier, thereby eliminating the need to aseptically transfer the carrier to a separate vial of growth media.
Related: New Genetically Engineered Biological Indicators

Other self-contained biological indicators consist of hermetically sealed ampoules containing the test organisms suspended in growth media. The growth medium often also contains a dye, which indicates positive or negative growth following incubation. Due to their construction, this type of biological indicator is sensitive to temperature only and is employed primarily for monitoring sterilization of liquids. These biological indicators may be incubated directly following exposure to the sterilization process under the specified conditions. Growth or no growth of the treated spores is determined visually (either by observing a specified color change of an indicator incorporated in the medium or by turbidity) or by microscopic examination of the inoculated medium.

The design of the self-contained system is such that there is minimal loss of the original inoculum during transport and handling and the resistance characteristics comply with the labeling of the self-contained system. During or after the sterilization process, the materials used in the self-contained system do not retain or release any substance that can inhibit the growth of low numbers of surviving indicator microorganism under culture conditions. Adequate steps must be taken to demonstrate that the recovery medium has retained its growth support characteristics after exposure to the sterilization process.

Custom biological indicators for use in sterilization in-place (SIP), or wherever necessary, may be prepared by adding an aliquot of spore suspension to representative units of the lot to be sterilized (inoculated product). If it is not practical to add the spores to the actual product units, spores can be added to a simulated product, which has been validated to demonstrate a similar resistance to the actual product.

Spore suspensions with a known D-value should be used to inoculate the actual or simulated inoculated product. The population, D-value (the time required at a temperature T to reduce a specific microbial population by 90 percent, or by a factor of 10), Z-value (the number of degrees of temperature change necessary to change the D-value by a factor of 10), where applicable, and end-point kill time of the inoculated actual or simulated product should be determined.

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