Rapid culture-independent microbial analysis aboard the international space station (ISS) stage two: quantifying three microbial biomarkers.

Abstract A portable, rapid, microbial detection unit, the Lab-On-a-Chip Application Development Portable Test System (LOCAD-PTS), was launched to the International Space Station (ISS) as a technology demonstration unit in December 2006. Results from the first series of experiments designed to detect Gram-negative bacteria on ISS surfaces by quantifying a single microbial biomarker lipopolysaccharide (LPS) were reported in a previous article. Herein, we report additional technology demonstration experiments expanding the on-orbit capabilities of the LOCAD-PTS to detecting three different microbial biomarkers on ISS surfaces. Six different astronauts on more than 20 occasions participated in these experiments, which were designed to test the new beta-glucan (fungal cell wall molecule) and lipoteichoic acid (LTA; Gram-positive bacterial cell wall component) cartridges individually and in tandem with the existing Limulus Amebocyte Lysate (LAL; Gram-negative bacterial LPS detection) cartridges. Additionally, we conducted the sampling side by side with the standard culture-based detection method currently used on the ISS. Therefore, we present data on the distribution of three microbial biomarkers collected from various surfaces in every module present on the ISS at the time of sampling. In accordance with our previous experiments, we determined that spacecraft surfaces known to be frequently in contact with crew members demonstrated higher values of all three microbial molecules. Key Words: Planetary protection-Spaceflight-Microbiology-Biosensor. Astrobiology 12, 830-840.

Setting a standard: the limulus amebocyte lysate assay and the assessment of microbial contamination on spacecraft surfaces.

Historically, colony-forming units as determined by plate cultures have been the standard unit for microbiological analysis of environmental samples, medical diagnostics, and products for human use. However, the time and materials required make plate cultures expensive and potentially hazardous in the closed environments of future NASA missions aboard the International Space Station and missions to other Solar System targets. The Limulus Amebocyte Lysate (LAL) assay is an established method for ensuring the sterility and cleanliness of samples in the meat-packing and pharmaceutical industries. Each of these industries has verified numerical requirements for the correct interpretation of results from this assay. The LAL assay is a rapid, point-of-use, verified assay that has already been approved by NASA Planetary Protection as an alternate, molecular method for the examination of outbound spacecraft. We hypothesize that standards for molecular techniques, similar to those used by the pharmaceutical and meat-packing industries, need to be set by space agencies to ensure accurate data interpretation and subsequent decision making. In support of this idea, we present research that has been conducted to relate the LAL assay to plate cultures, and we recommend values obtained from these investigations that could assist in interpretation and analysis of data obtained from the LAL assay.

Rapid culture-independent microbial analysis aboard the International Space Station (ISS).

A new culture-independent system for microbial monitoring, called the Lab-On-a-Chip Application Development Portable Test System (LOCAD-PTS), was operated aboard the International Space Station (ISS). LOCAD-PTS was launched to the ISS aboard Space Shuttle STS-116 on December 9, 2006, and has since been used by ISS crews to monitor endotoxin on cabin surfaces. Quantitative analysis was performed within 15 minutes, and sample return to Earth was not required. Endotoxin (a marker of Gram-negative bacteria) was distributed throughout the ISS, despite previous indications that mostbacteria on ISS surfaces were Gram-positive [corrected].Endotoxin was detected at 24 out of 42 surface areas tested and at every surface site where colony-forming units (cfu) were observed, even at levels of 4-120 bacterial cfu per 100 cm(2), which is below NASA in-flight requirements (<10,000 bacterial cfu per 100 cm(2)). Absent to low levels of endotoxin (<0.24 to 1.0 EU per 100 cm(2); defined in endotoxin units, or EU) were found on 31 surface areas, including on most panels in Node 1 and the US Lab. High to moderate levels (1.01 to 14.7 EU per 100 cm(2)) were found on 11 surface areas, including at exercise, hygiene, sleeping, and dining facilities. Endotoxin was absent from airlock surfaces, except the Extravehicular Hatch Handle (>3.78 EU per 100 cm(2)). Based upon data collected from the ISS so far, new culture-independent requirements (defined in EU) are suggested, which are verifiable in flight with LOCAD-PTS yet high enough to avoid false alarms. The suggested requirements are intended to supplement current ISS requirements (defined in cfu) and would serve a dual purpose of safeguarding crew health (internal spacecraft surfaces <20 EU per 100 cm(2)) and monitoring forward contamination during Constellation missions (surfaces periodically exposed to the external environment, including the airlock and space suits, <0.24 EU per 100 cm(2)).

A field-based cleaning protocol for sampling devices used in life-detection studies.

Analytical approaches to extant and extinct life detection involve molecular detection often at trace levels. Thus, removal of biological materials and other organic molecules from the surfaces of devices used for sampling is essential for ascertaining meaningful results. Organic decontamination to levels consistent with null values on life-detection instruments is particularly challenging at remote field locations where Mars analog field investigations are carried out. Here, we present a seven-step, multi-reagent decontamination method that can be applied to sampling devices while in the field. In situ lipopolysaccharide detection via low-level endotoxin assays and molecular detection via gas chromatography-mass spectrometry were used to test the effectiveness of the decontamination protocol for sampling of glacial ice with a coring device and for sampling of sediments with a rover scoop during deployment at Arctic Mars-analog sites in Svalbard, Norway. Our results indicate that the protocols and detection technique sufficiently remove and detect low levels of molecular constituents necessary for life-detection tests.