Evaluation of Procedures for the Collection, Processing, and Analysis of Biomolecules from Low-Biomass Surfaces

Abstract

To comprehensively assess microbial diversity and abundance via molecular-analysis-based methods, procedures for sample collection, processing, and analysis were evaluated in depth. A model microbial community (MMC) of known composition, representative of a typical low-biomass surface sample, was used to examine the effects of variables in sampling matrices, target cell density/molecule concentration, and cryogenic storage on the overall efficacy of the sampling regimen. The MMC used in this study comprised 11 distinct species of bacterial, archaeal, and fungal lineages associated with either spacecraft or clean-room surfaces. A known cellular density of MMC was deposited onto stainless steel coupons, and after drying, a variety of sampling devices were used to recover cells and biomolecules. The biomolecules and cells/spores recovered from each collection device were assessed by cultivable and microscopic enumeration, and quantitative and speciesspecific PCR assays. rRNA gene-based quantitative PCR analysis showed that cotton swabs were superior to nylon-flocked swabs for sampling of small surface areas, and for larger surfaces, biological sampling kits significantly outperformed polyester wipes. Species-specific PCR revealed differential recovery of certain species dependent upon the sampling device employed. The results of this study empower current and future molecular-analysis-based microbial sampling and processing methodologies.

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

“…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).…”

“…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.…”

Microbial Community Structures of Novel Icelandic Hot Spring Systems Revealed by PhyloChip G3 Analysis

“…Standards were prepared from known concentrations of PCR amplicon of the 16S rRNA gene from Escherichia coli spanning 10 8 to 10 2 gene copies/ lL. Universal bacterial primers targeting the 16S rRNA gene, 1369F (5¢-CGG TGA ATACGT TCY CGG-3¢), and modified 1492R (5¢-GGW TAC CTTGTT ACG ACT T-3¢) were used for this analysis (Kwan et al, 2011).…”

Microbiology: lessons from a first attempt at Lake Ellsworth

“…During the manufacture and assembly of the lake measurement probes [47] (figure 2), swab samples were collected in order to assess the efficiency of the four-stage sterilization process undertaken (detergent wash, ethanol rinse, a commercially available biocide (Tenon Biocleanse) and hydrogen peroxide vapour exposure) as previously reported [48]. Randomly chosen titanium grade V surfaces of the probes were sampled with human DNA-free SK-1S DNA Buccal Swabs (Isohelix), which were wetted with PCR grade water (Sigma) in a sterile environment.…”