Objectives: Dental instrument sterilization with steam autoclaves is critical to maintaining infection control standards in dental practice, and preventing patient-to-patient transmission of pathogenic bacteria and viruses. The American Dental Association and the United States Centers for Disease Control and Prevention recommend, and many state dental laws require, weekly use of biological spore tests to verify dental instrument sterilization outcomes. However, the most widely used biological spore test needs microbial culture incubation for 2 days after autoclave exposure, which limits swift identification of sterilization failure. To address this issue, this study evaluated the reliability of a new rapid biological spore test for determining the sterilization efficacy of dental steam autoclaves within 20 minutes. Methods: Two commercial biological spore tests were evaluated in Temple University dental school steam autoclaves, 1.) the Steris Celerity 20 Steam Biologic Indicator with a 20-minute outcome time requirement, and 2.) the 3M Attest 1262 Biological Indicator with a 48-hour outcome time requirement. Both biological spore tests employed live thermoresistant Geobacillus stearothermophilus spores as an indicator of whether sterilization conditions in steam autoclaves were met or not. To compare their efficacy, a total of 157 pairs of the two biological spore tests were placed into dental steam autoclaves with dental instrument cassettes, and subjected to manufacturer-recommended steam autoclave temperature and air pressure operating conditions for an adequate sterilization time of 15 minutes. Two additional groups of 10 pairs each of the two biological indicators were subjected to appropriate steam autoclave temperature and air pressure settings, but only for aborted non-sterilizing time periods of 10 and 5 minutes, respectively. Subsequent aseptic processing and laboratory incubation of both biological indicators was initiated within 2-24 hours, and followed manufacturer recommendations. After autoclave exposure, Steris Celerity 20 Steam Biologic Indicator test ampoules were incubated in a specialized instrument for 20 minutes at 57 °C, which also spectrophotometrically evaluated the microbial culture medium for fluorescent α- glucosidase enzyme signal changes. No change in fluorescent intensity represented successful sterilization, whereas increased fluorescence indicated survival of viable G. stearothermophilus spores germinating into vegetative bacterial cells after failed sterilization. 3M Attest 1262 Biological Indicator ampoules were incubated for 48 hours in a laboratory heating block at 57 °C, after which a pH-based color change in the microbial culture broth was visually assessed. No change in the color of the culture broth (purple color remains) indicated successful sterilization, whereas development of a yellow color in the culture broth, as a result of viable G. stearothermophilus spore germination into vegetative bacterial cells, denoted failed sterilization. Results: A total of 354 biological indicators were exposed to dental steam autoclaves sterilization cycles, incubated for either 20 minutes or 48 hours, and evaluated for G. stearothermophilus spore growth. The Steris Celerity and 3M Attest biological spore tests both uniformly detected successful sterilization, with no G. stearothermophilus spore growth, after 15 minutes of steam autoclave exposure at manufacturer recommended steam autoclave temperature and air pressure operating conditions. This provided 100% agreement, and no statistically significant difference in the prevalence of successful sterilization outcomes, between 157 pairs of both biological indicator types after 15 minutes of steam autoclave exposure. Similarly, both biological spore test systems were also in complete agreement after only 5 minutes of steam autoclave exposure, with 100% of both biological indicators positive for G. stearothermophilus spore growth, indicating failed sterilization. In contrast, after 10 minutes of steam autoclave exposure, there was a complete lack of agreement between the two types of biological indicators. All 10 Steris Celerity spore tests were positive, whereas all 10 3M Attest ampoules were negative, for G. stearothermophilus spore growth after 10 minutes of steam autoclave exposure. Relative to this disagreement, a non-biological chemical indicator strip that was part of the Steris biological indicator test system failed to have a darkened bar develop and extend into the “Accept (OK)” portion of the strip for all Steris Celerity spore tests exposed to either 5 minutes or 10 minutes of steam autoclave exposure, indicating that adequate autoclave steam, temperature and/or time parameters had not been attained for sterilization. Conclusions: The Steris Celerity biological spore test was successful in rapidly determining the sterilization efficacy of dental steam autoclaves within only a 20-minute incubation time period, as compared to 48 hours of incubation required by the widely-used 3M Attest biological spore test. As a result, this rapid assay offers earlier detection of steam autoclave sterilization failure before potentially contaminated dental instruments are used in clinical patient care. The alarming failure of 3M Attest biological spores to grow after a non-sterilizing 10-minute steam autoclave exposure time warrants further product evaluation. / Oral Biology