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Indicators are routinely used to check the effectiveness of various cleaning, disinfection, and sterilization processes. They include biological, chemical, and other (e.g., mechanical) indicators.

Biological indicators (Fig. 1.23) consist of a standardized population of microorganisms inoculated onto a carrier material. They are particularly widely used in the monitoring and validation of sterilization processes. Bacterial endospores are commonly used as the test microorganisms, as they are generally nonpathogenic and stable and demonstrate high resistance to various sterilization processes (Table 1.21). Defined bacterial strains, obtained from standard culture collections (e.g., the American Type Culture Collection), are used. The intrinsic resistance of the inoculated spore population can vary depending on the culturing methods used and other variables. Therefore, to standardize the use of biological indicators, manufacturers test each batch of indicators to determine the population and the relative resistance to a given sterilization process (e.g., D-value determination at 121’C with saturated steam and at 55^°C, 800 mg of ethylene oxide/liter, and 70% relative humidity for ethylene oxide). Depending on the application, other microorganisms may be used, but to a much lesser extent.

FIGURE 1.23 Example of a self-contained biological indicator. The 3M Attest 1292 Rapid Readout Biological Indicator is used to monitor steam sterilization cycles. Reproduced with permission of 3M Health Care.

The carrier can consist of any material, with typical examples being paper, stainless steel, glass, and plastics. In its true definition, a biological indicator consists of the inoculated coupon placed into a “primary pack,” which can be a protective envelope or pouch, or within an assembled vial or ampoule. In their simplest form, biological indicators are present within a protective envelope, and following exposure to a given sterilization process, the inoculated coupon is aseptically removed from its pack and incubated in a specified growth medium to determine the presence or absence of spore viability. Due to the release of dipicolinic acid (see section 8.3.11) upon germination and outgrowth of the spores, pH indicator dyes can be used in the growth media to indicate the presence of viability before visible growth (turbidity) is observed. To minimize aseptic handling, self-contained biological indicators that include the inoculated carrier within a vial containing a sealed ampoule of growth medium have also been developed (Fig. 1.23). Following exposure, the medium ampoule is broken to allow incubation without handling of the coupon. Further “rapid-review” biological indicators are available that detect the presence of certain endospore enzymes (e.g., (α-D-glucosidase) whose destruction by heat correlates with the loss of viability of the spore; the presence of enzyme activity can be detected fluorimetrically and can give a rapid indication of spore viability (usually within 1 to 4 h). In addition, these indicators are further incubated to demonstrate the presence or absence of growth, as for traditional biological indicators. Various standards that define the requirements for and use of biological indicators are given in Table 1.22.

TABLE 1.21 Bacterial-endospore species used to monitor and validate sterilization processes

Sterilization process	Biological indicator
Moist heat	Geobacillus stearothermophilus
Dry heat	Bacillus atrophaeus
Irradiation	Bacillus pumilus
Ethylene oxide	Bacillus atrophaeus
Low-temperature steam formaldehyde	G. stearothermophilus
Hydrogen peroxide vapor	G. stearothermophilus

Chemical indicators change color (or provide another visible change) on exposure to a given disinfectant or sterilization process (Fig. 1.24). They can range from simple process indicators that indicate exposure to a given process parameter (e.g., exposure to heat, but not necessarily at the right temperature and for the right amount of time) to more specific integrator indicators, which change color only on exposure to multiple variables (e.g., temperature and time for steam sterilization and concentration or time, temperature, and humidity for ethylene oxide sterilization). They can be classified in various ways, and an example is given in Table 1.23.

Chemical indicators are widely used, as they give an instant result and in some cases (as with some integrators) can be correlated to a biological indicator result. Applications include specific direct parameters that are required for disinfection or sterilization (e.g., verification of the presence of a minimal concentration of a biocidal formulation prior to use or that a range of conditions have been met in a sterilizer), but also other, indirect variables that are important to the efficacy of the process (e.g., Bowie-Dick tests are used to confirm the adequate removal of air in prevacuum-type steam sterilizers [see section 5.2]). Examples of various standards that define the requirements for and use of chemical indicators are given in Table 1.24.

TABLE 1.22 Examples of biological-indicator standards

Referencea	Title	Summary
ISO 11138-1	Sterilization of Health Care Products—Biological Indicators—Part 1: General Requirements	General requirements for production, labeling, test methods, and performance characteristics of biological indicator systems to be used in the validation and routine monitoring of sterilization processes
ISO 11138-2	Sterilization of Health Care Products—Biological—Part 2: Biological Indicators for Ethylene Oxide Sterilization Processes	Specific requirements for biological indicators used for ethylene oxide sterilization, including test organism and performance criteria
ISO 11138-3	Sterilization of Health Care Products—Biological Indicators—Part 3: Biological Indicators for Moist Heat Sterilization Processes	Specific requirements for biological indicators used for moist-heat (steam) sterilization, including test organisms and performance criteria
ISO 14161	Sterilization of Health Care Products—Biological Indicators—Guidance for the Selection, Use and Interpretation of Results	Guidance for the selection, use, and interpretation of results of biological indicators used in the development, validation, and routine monitoring of sterilization processes
EN 866-1	Biological Systems for Testing Sterilizers and Sterilization Processes. Part 1—General Requirements	General requirements for production, labeling, test methods, and performance characteristics of biological indicator systems to be used in the validation and routine monitoring of sterilization processes
USP XXIII	Biological Indicators—Resistance Performance Tests	Testing of the resistances and population of biological indicators
EP 5.1.2	Biological Indicators of Sterilization	Requirements for biological indicators, including population and resistance

^aISO, International Standards Organization; EN, European Norm, from the CEN (European Committee for Standardization); USP, United States Pharmacopeia; EP, European Pharmacopoeia.

Other, miscellaneous indicators include mechanical indicators, such as gauges or sensors that measure temperature, concentration, pressure, time, etc., that are used to monitor various physical parameters during a given process and cleaning indicators that use artificial test soils inoculated onto a surface to test (generally by visual inspection) physical removal during a cleaning process or cycle. Mechanical indicators play an important role in the parametric release of a product or process as an alternative to the use of chemical and biological indicators for routine monitoring of sterilization processes (see section 1.4.2.4).

TABLE 1.23 A typical classification of chemical indicators

Class	Type	Description
1	Process indicators	Indicate exposure to minimal process conditions; used to differentiate exposed from unexposed items (e.g., autoclave tape)
2	Indicators for use in specific tests	Indicate that a specific process is obtained, which is linked to the sterilization process (e.g., a Bowie-Dick test indicates the adequate removal of air from a prevacuum steam sterilizer)
3	Single-parameter indicators	Indicate a change on exposure to one parameter (e.g., concentration of a biocide or temperature).
4	Multiparameter indicators	Indicate a change on exposure to at least two parameters
5	Integrating indicators	Indicate a change on exposure to all the critical parameters of a given process (e.g., ethylene oxide sterilization with temperature, biocide concentration, relative humidity, and time)

FIGURE 1.24 Example of a chemical-indicator color change.