Development and testing of a high throughput dry-heat microbial reduction assay for bacterial isolates from spacecraft assembly facilities

The Jet Propulsion Laboratory (JPL) microbial archive holds several thousand organisms and some of those are known to exhibit high dry-heat microbial resistance (DHMR). A collection of 237 putative dry-heat resistant organisms, known as the Teflon Ribbon Collection (TRC), was screened for dry-heat resistance. Using a NASA standard dry-heat organism, Bacillus atrophaeus (ATCC 9732, Bacillus subtilis var. niger), it was found that starting concentrations (10e7 to 10e3) and refrigeration storage times (up to 3 weeks post-heat exposure) were negligible for dry-heat screening purposes. Using 16S rRNA analysis, the collection of 237 isolates was divided into clusters based on 97% sequence similarity, resulting in 78 representative isolates. Of these 78 isolates, 29 were selected for dry-heat testing based on relative abundance and uniqueness. To advance dry-heat resistance screening times, a new thermal insert with twelve times the capacity to test more isolates per heat-exposure was designed and fabricated for use with a heritage dry-heat test system. The 29 representative organisms from each cluster were then subjected to the new high-throughput, dry-heat screening process. Based on clustering, known dry-heat resistant isolates such as V3-13 were useful in finding other unknown high dry-heat resistant isolates, such as M8-28. Additionally, more information on the collection's dry-heat hardness was attained as previously untested isolates were subjected to the screening processes. A majority of the tested representative isolates were found to be more dry-heat resistant than ATCC 9372, B. atrophaeus.