Rapid endospore viability assay of Clostridium sporogenes spores

A rapid Endospore Viability Assay (EVA), previously developed for Bacillus spores, was modified for enumeration of germinable Clostridium sporogenes spores. The EVA is based on the detection of dipicolinic acid (DPA), which is released during stage I germination and quantified by terbium (III) ion Tb-DPA luminescence. Germination of C. sporogenes spores in aqueous suspension was induced by L-alanine and NaHCO3 addition, and germinable endospore numbers were determined by reference to a standard curve. Determination of the fractions of germinable C. sporogenes spores by EVA and phase-contrast microscopy yielded comparable results of 54.0% ± 2.9% and 59.3% ± 2.6%, respectively, while only 32.3% ± 5.3% of spores produced colonies on reinforced clostridial medium (RCM). Rates of germination were measured as a function of temperature (30 °C–60 °C) using EVA, yielding a linear relationship between the square root of the rate constant and inverse temperature.