Abstract
A fast Endospore Germinability Assay (EGA) was validated with traditional plate counts to enumerate single endospore germination events for monitoring surface sterilization. The assay is based on a time-gated luminescence microscopy technique enabling visualization and enumeration of individual germinating endospores. Germinating endospores release calcium dipicolinate to form highly luminescent terbium dipicolinate complexes surrounding each germinating endospore. EGA and heterotrophic plate counting (HPC) were used to evaluate the swab/rinse recovery efficiency of endospores from stainless steel surfaces. EGA and HPC results were highly correlated for endospore recovery from stainless steel coupons inoculated with range of 1,000 endospores per coupon down to sterility. Dosage-dependent decrease of surface endospore germinability were observed in dry heat, UV irradiation, oxygen plasma and vaporized hydrogen peroxide treatments, measured with EGA and HPC. EGA is a fast and complementary method to traditional HPC for quantitative sterility assurance testing of surfaces. This work introduces and validates a 15-minute or faster assay for germinable endospores to complement the conventional lengthy, culture-based surface sterility validation, which is critical in hospitals, food and pharmaceutical industries to help minimize nosocomial infection, food spoilage, and pharmaceutical contamination.