Comparison of Innovative Molecular Approaches and Standard Spore Assays for Assessment of Surface Cleanliness

Abstract

A bacterial spore assay and a molecular DNA microarray method were compared for their ability to assess relative cleanliness in the context of bacterial abundance and diversity on spacecraft surfaces. Colony counts derived from the NASA standard spore assay were extremely low for spacecraft surfaces. However, the PhyloChip generation 3 (G3) DNA microarray resolved the genetic signatures of a highly diverse suite of microorganisms in the very same sample set. Samples completely devoid of cultivable spores were shown to harbor the DNA of more than 100 distinct microbial phylotypes. Furthermore, samples with higher numbers of cultivable spores did not necessarily give rise to a greater microbial diversity upon analysis with the DNA microarray. The findings of this study clearly demonstrated that there is not a statistically significant correlation between the cultivable spore counts obtained from a sample and the degree of bacterial diversity present. Based on these results, it can be stated that validated state-of-the-art molecular techniques, such as DNA microarrays, can be utilized in parallel with classical culture-based methods to further describe the cleanliness of spacecraft surfaces.

Pyrosequencing-Derived Bacterial, Archaeal, and Fungal Diversity of Spacecraft Hardware Destined for Mars

“…Coupling the innovative pyrosequencing techniques employed in this study with other emerging molecular methodologies, such as generation 3 PhyloChip DNA microarrays (4,28), microscopy-based viability assays (33), etc., could lead to significantly improved approaches for monitoring and mitigating the diverse microbial populations associated with ultraclean environments, without the biases that plague culture-dependent techniques. Ultimately, pyrosequencing analyses supported, and accentuated, the general trends observed with regard to environmental clustering where the greatest microbial diversity was encountered in ordinary room facilities, followed next by cleanroom floors, and, finally, spacecraft components (Fig.…”

“…Surface sampling of spacecraft assembly cleanroom (SAC) floors and ground support equipment (GSE) was performed using either premoistened polyester wipes or biological sampling kits (BiSKit; QuickSilver Analytics, Abingdon, MD) as previously described (4,24). The two cleanroom facilities examined during this study were both certified as class 8 per ISO 14644-1.…”

“…Only the group IV wipes collected at Kennedy Space Center (group IV samples) were stored at Ϫ20°C (14 days) prior to being transported on dry ice to JPL for further processing and analysis. To facilitate recovery of microorganisms and biomolecules from the polyester matrix, 200 ml of sterile rinse solution (85 mg of potassium dihydrogen phosphate, 200 mg of Tween 80 [per liter of water], pH 7.2) was added to each polyester wipe-containing Corning bottle prior to vigorous shaking and ultrasonic treatment, as previously described (4,24). Previous studies have demonstrated that SAC samples contain extremely low biomass and seldom yield detectable PCR amplification products (34,56).…”

“…Group III samples (15 sets; 110 m 2 ) were analyzed together to evaluate the extent of correlation between the endospore burden associated with spacecraft hardware surfaces and the resulting operational taxonomic unit (OTU) abundance. This group comprised spacecraft hardware surface samples whose bacterial endospore burden had been previously determined (4). Group IV samples (4 sets; 52 m 2 ) were analyzed together to investigate how pyrosequencing results differed across various mission subsystem components (e.g., cruise stage, rover).…”

Implementing Planetary Protection on the Atlas V Fairing and Ground Systems Used to Launch the Mars Science Laboratory

“…Individual components of the MSL flight system, and Atlas V flight hardware and ground support systems alike, were sampled and examined via standard NASA spore assays, as previously described (La Duc et al, 2004;Osman et al, 2008;Cooper et al, 2011;Kwan et al, 2011). After having been sampled, hardware was stored in clean, sealed antistatic bags until installment and/or deployment on the spacecraft.…”