Culture age and drying time as variables of the AOAC Sporicidal Test

In the United States, the AOAC Sporicidal Activity of Disinfectants Method 966.04 is the standard for identifying a liquid chemical germicide as a sterilant. Furthermore, the highest level of a disinfectant must also be a sterilant as defined by Method 966.04, when used in its sterilant mode for a longer exposure time. The AOAC Sporicidal Test is also used as a part of the standard test methods to define a sterilant for Australia and the European Union. Many laboratories have identified variables of this test that can affect the sterilization exposure time for sterilants, or even the ability to classify a chemical as a sterilant. Method 966.04 requires spore-labeled porcelain penicylinders (cylinders) and silk suture loops, collectively referred to as carriers, to be dried for 24 h, but allows these carriers to be used for at least 7 days, in effect allowing a drying time of 24 h to at least 7 days. We tested the resistance of cylinders that had been labeled with Bacillus subtilis spores cultured for 72, 96, and 120 h, and dried for 24, 48, and 72 h against a 60 min exposure to 2.0% alkaline glutaraldehyde, and 2, 5, 10, 15, and 20 min exposures to 2.5N HCl. All the culture incubation and drying times met the standard of resistance to 2.5N HCI for at least 2.0 min at 20 degrees C, and all carriers contained at least 10(5) colony-forming units (CFU) of B. subtilis per carrier. However, for 3 repeated tests, regardless of incubation time, an average of 96% of the carriers were sterilized by the 2.0% glutaraldehyde after drying for 24 h, and an average of 61 % were sterilized after drying for 48 or 72 h. We propose that the variable of drying time be eliminated from Method 966.04.