A Rapid Single Spore Enumeration Assay

The Rapid Single Spore Enumeration Assay (RapidSSEA) is being developed for the Mars Technology Program to aid planetary protection personnel in their efforts to validate bioburden reduction on spacecraft surfaces prior to launch. RapidSSEA is based on imaging and counting individual bacterial spores in a microscope field-of-view, where the contrast is generated by a highly luminescent complex that forms when dipicolinic acid (DPA) is released from spores during germination and binds to terbium (Tb³⁺) ions in the surrounding medium. Under pulsed UV excitation, the long-lived (tau ~ 1 ms) DPA-triggered Tb luminescence is imaged by a lifetime-gated camera, essentially eliminating fluorescent interferents from the image, which enables counting of bacterial spores swabbed from environmental samples. Here we report results from the first side-by-side comparison of RapidSSEA and the NASA standard assay. Both assays were used to assess the spore population on stainless steel coupons inoculated with Bacillus atrophaeus spores. Using RapidSSEA, 38.0% plusmn 5.8% of the total inoculated spore population was recovered and counted within 30 minutes