Applications of a Rapid Endospore Viability Assay for Monitoring UV Inactivation and Characterizing Arctic Ice Cores†

We have developed a rapid endospore viability assay (EVA) in which endospore germination serves as an indicator for viability and applied it to (i) monitor UV inactivation of endospores as a function of dose and (ii) determine the proportion of viable endospores in arctic ice cores (Greenland Ice Sheet Project 2 [GISP2] cores; 94 m). EVA is based on the detection of dipicolinic acid (DPA), which is released from endospores during germination. DPA concentrations were determined using the terbium ion (Tb3)-DPA luminescence assay, and germination was induced by L-alanine addition. The concentrations of germinable endospores were determined by comparison to a standard curve. Parallel EVA and phase-contrast microscopy experiments to determine the percentage of germinable spores yielded comparable results (54.3 % 3.8 % and 48.9% 4.5%, respectively), while only 27.8 % 7.6 % of spores produced CFU. EVA was applied to monitor the inactivation of spore suspensions as a function of UV dose, yielding reproducible correlations between EVA and CFU inactivation data. The 90 % inactivation doses were 2,773 J/m2, 3,947 J/m2, and 1,322 J/m2 for EVA, phase-contrast microscopy, and CFU reduction, respectively. Finally, EVA was applied to quantify germinable and total endospore concentrations in two GISP2 ice cores. The first ice core contained 295 19 germinable spores/ml and 369 36 total spores/ml (i.e., the percentage of germinable endospores was 79.9% 9.3%), and the second core contained 131 4 germinable spores/ml and 162 17 total spores/ml (i.e., the percentage of germinable endospores was 80.9 % 8.8%), whereas only 2 CFU/ml were detected by culturing. Bacterial spores (i.e., endospores) are dormant structures that exhibit remarkable longevity, with reports ranging fro