Rapid micro-detection system for the enumeration of bacterial endospores

NASA continuously monitors spacecraft surfaces to assure a very low presence of bacterial endospores upon landing on the surface of Mars. In order to meet the rigid schedules of spacecraft assembly, a more rapid, sensitive spore detection assay is being considered as an alternate method to the current three-day NASA standard culture-based assay. The millipore microstar rapid system (RMDS) has been used successfully for rapid bioburden enumeration in a wide range of applications. It combines membrane filtration, adenosine triphosphate (ATP) bioluminescence chemistry, and image analysis based on photon detection with a charge coupled device (CCD) camera. The RMDS is rapid and simple, shows high sensitivity (1 colony forming unit [CFU]/sample), and correlates excellently with traditional culture-based methods. In addition, by utilizing the milliflex filtration system, the RMDS is ideal for sampling both large sample volumes and those samples containing inhibitory substances. In this study, we have evaluated the use of the RMDS as a rapid spore detection tool for NASA planetary protection applications. This is accomplished by preceding the RMDS incubation protocol with a heat shock step, 15 minutes at 80degC, as a direct selection of spores. Different luciferase enzymes were tested in order to reduce the typical 18-24 hour incubation time to ~5 hours. Of the reagents evaluated, a formulation of a highly sensitive bioluminescence reagent was found to be more sensitive than the present millipore microstar reagents. Assay times of ~5 hours were repeatedly demonstrated along with low image background noise. In order to evaluate the applicability of this method, seven species of Bacillus (nine strains) that have been repeatedly isolated from clean room environments were assayed. All strains were detected in ~5 hours. The improved RMDS-based spore detection is under validation and is expected to achieve the goal of "same shift" measurement of spore bioburden during spacecraft- - assembly