Outer Space and the Quest for Cleanliness

Sources: Yvette Piceno and Dan Hawkes

Bacterial spores are resistant to many things (e.g., heat, desiccation) and are thought to be the most likely way our spacecraft could introduce life to (or “contaminate”) places we visit beyond Earth.  As such, a great deal of effort has gone into cleaning spacecraft surfaces and using spore counts as a means of documenting cleanliness. The NASA spore assay was developed in the 1960s and depends on culturing bacterial spores. Given the knowledge gained in the intervening decades, though, the assay is outdated. While it still serves as a proxy for “cleanliness” in one sense (if there are many cultivable spores in a sample, then the surface is not clean), there are many conditions under which spores and non-spore cells (or vegetative cells) likely could grow. Testing all the possible conditions would be time-consuming and costly. 

Molecular methods offer a faster, more comprehensive means of assaying the microbial “bio-burden.” Molecular methods—such as using DNA to identify bacteria present in a sample—provide a more comprehensive means of determining the extent of bacterial bio-burden, as DNA can be extracted from live and dormant/dead vegetative cells and spores. In a recent study (Cooper et al., 2011), an investigative team led by Jet Propulsion Lab at Cal Tech, and  including ESD’s Yvette Piceno and Gary Andersen, extracted DNA from sample wipes and analyzed that DNA using PhyloChip technology, to assess the breadth of bacteria recovered from clean spacecraft surfaces. The information was compared to spore counts of the same surfaces to determine if there were a relationship between the number of spores cultured (using NASA standard aerobic, mesophilic, heterotrophic conditions) and the number of different bacterial taxa found by PhyloChip. A separate quantitative method of DNA amplification for the 16S rRNA gene (qPCR) was also used to provide another measure of bio-burden.

While more than a third of the samples (including handling and negative controls) showed good agreement among the three methods, a third of the samples showed amplifiable DNA from samples with no cultivable spores, and in one case, there were cultivable spores but no amplifiable DNA by either molecular method. This last sample may be an example of spores not being lysed during DNA extraction, making it impossible to detect them by molecular means. By and large, however, molecular methods were able to detect the presence of bacterial DNA even when spores could not be cultured from actual samples, and there was no direct relationship between the number of cultured spores and the diversity of bacteria assessed by PhyloChip (underscoring that knowing the number of spores in a sample does not provide information about they types of bacteria present).

(a) Comparison of subfamily richness and spore counts grouped by the spore abundance categories and based on the NASA standard spore assay. The results show there is no strong correlation between sample category and subfamily count. Category A, 0 spores; category B, 1 to 50 spores; and category C, >50 spores. The following samples were some form of negative control: GI-b, GI-18b, GI-19b, GI-20b: (b) Calculated cell density based on 16S rRNA gene copies compared to spore counts and total hybridization intensity. Average 16S rRNA gene copies are 3.6 per cell. Gene copies correlate similarly to hybridization intensities, but show no correlation to spore counts.

These findings lead to the conclusion that it is time to update the methods used to determine cleanliness of very-low-biomass surfaces. Making sure cleanroom surfaces are “clean” is important to many industries, including (but not limited to) food production and the medical industry. The findings of this study indicate that newer assays may be in order for these facilities as well, as science continually pushes the envelope in defining what “clean” is.

For more information on microbiome profiling technology, visit the Second Genome website.