Biological Indicators

Biological indicators (BIs), or spore tests, assess directly the killing of known highly resistant, nonpathogenic bacterial spores. Geobacillus stearothermophilus (G. stearothermophilus) spores test steam and unsaturated chemical vapor sterilizers. Bacillus atrophaeus (B. atrophaeus) spores test dry heat sterilizers. Bacterial spores in the test products are more resistant and are present in greater numbers than common microbial contaminants found on patient-care items.

In oral healthcare facilities, BIs should be processed with a load in all sterilizers at least weekly. However, a BI must be processed in every load containing an implant and the implant should be quarantined until the result of the spore test is known. BIs should be placed in the sterilizer in accordance with the manufacturer’s directions. BIs come in three forms: spore strips, self-contained spore vials, and spore ampules.

Spore strips, small strips of thick filter paper covered with spores, are enclosed in glassine pouches. The pouches are placed into packages and processed. After processing, using an aseptic technique, the pouches are opened, and the strips are placed into a tube of sterile culture media for incubation (at a specified temperature for up to 7 days). No growth (a clear tube) indicates the test is negative. Growth (a cloudy tube) indicates the test is positive (i.e., sterilization failure).

Self-contained spore vials include a glass ampule of sterile media surrounded by a plastic vial with a spore strip inside. After processing, the BI is activated by crushing the vial (manually or with a device) to allow the culture media to come in contact with the spore strip. The vials are then incubated at the appropriate temperature for a maximum of 48 hours. Significant failures could be detected in ≤24 hours.

Some self-contained spore vials may include a pH indicator system in the glass ampule of the sterile media. The pH indicator in the growth media changes from purple to yellow with the generation of acid. Some spore vials can also be placed into a special incubator/reader, which delivers rapid results (in one hour). The readers displays a “red light” for positive results or a “green light” for negative results.

Glass ampules are available with G. stearothermophilus spores for use with steam processes. The ampules contain spores suspended in a culture broth solution. Since the spores and the media are mixed together, no vial crushing or activation is required after processing. A 48-hour incubation period is recommended, but growth usually occurs within 8-12 hours. Spore growth is evidenced by a yellow color change in the media along with visible turbidity at the bottom of the vial.

Biological testing may be accomplished in-office. Following the manufacturer’s recommendations, the test BI (e.g., ConFirm^® 10 In-Office Biological Monitoring System) is placed in a pack, pouch, tray, or cassette or in the sterilizer with unpackaged instruments. Test BIs from multiple sterilizers must be clearly identified. After processing, the test BI must be incubated (along with a positive control from the same lot but not processed through the sterilizer) within two hours (or refrigerated). Accurate records must be maintained (see Sterilization Monitoring Record).

Biological testing may also be accomplished by an independent entity. Following the manufacturer’s recommendations, the test BI (e.g., ConFirm^® Mail-in Biological Monitoring) is placed in a pack, pouch, tray, or cassette or in the sterilizer with unpackaged instruments. Test BIs from multiple sterilizers must be clearly identified. After processing, the test BI (along with a control BI from the same lot but not processed through the sterilizer) must be mailed within 24 hours to the testing agency. Documentation of test results must be provided by the testing agency.^2,3