Method of determining efficacy of a sterilization process

A method of determining the efficacy of a sterilization process, comprising:

• providing a biological sterilization indicator comprising;
• a housing;
• a container containing an aqueous liquid;
• a source of predetermined enzyme activity that is inactivated by a sterilant;
• a first indicator reagent with a first absorbance spectrum;
• a second indicator reagent that is converted by the source of predetermined enzyme activity to a biological derivative with a second fluorescence emission spectrum;
• wherein the first absorbance spectrum includes detectable absorbance in at least a portion of wavelengths of the second fluorescence emission spectrum;
• a substrate that receives and concentrates the first indicator reagent from the aqueous liquid;
• providing an instrument that detects the biological derivative, wherein the instrument comprises an optical path;
• exposing the predetermined enzyme activity to the sterilant;
• bringing the predetermined enzyme activity, the first indicator reagent, the second indicator reagent, and the substrate into fluid communication with the aqueous liquid;
• and
• detecting a quantity of fluorescene from the biological derivative in the aqueous liquid;
• wherein detecting the quantity of fluorescence from the biological derivative comprises using the instrument;
• wherein detecting the quantity of fluorescence comprises positioning the housing in the optical path passes through the aqueous liquid but does not intersect the substrate;
• wherein the quantity of fluorescence is an indication of the efficacy of the sterilization process.