Validation of aClostridiumEndospore Viability Assay and Analysis of Greenland Ices and Atacama Desert Soils

Abstract

ABSTRACTA microscopy-based endospore viability assay (micro-EVA) capable of enumerating germinableClostridiumendospores (GCEs) in less than 30 min has been validated and employed to determine GCE concentrations in Greenland ices and Atacama Desert soils. Inoculation onto agarose doped with Tb3+andd-alanine triggersClostridiumspore germination and the concomitant release of ∼108molecules of dipicolinic acid (DPA) per endospore, which, under pulsed UV excitation, enables enumeration of resultant green Tb3+-DPA luminescent spots as GCEs with time-gated luminescence microscopy. The intensity time courses of the luminescent spots were characteristic of stage IClostridiumspore germination dynamics. Micro-EVA was validated against traditional CFU cultivation from 0 to 1,000 total endospores/ml (i.e., phase-bright bodies/ml), yielding 56.4% ± 1.5% GCEs and 43.0% ± 1.0% CFU. We also show thatd-alanine serves as aClostridium-specific germinant (three species tested) that inhibitsBacillusgermination of spores (five species tested) in that endospore concentration regime. Finally, GCE concentrations in Greenland ice cores and Atacama Desert soils were determined with micro-EVA, yielding 1 to 2 GCEs/ml of Greenland ice (versus <1 CFU/ml after 6 months of incubation) and 66 to 157 GCEs/g of Atacama Desert soil (versus 40 CFU/g soil).