Purifying, Separating, and Concentrating Cells From a Sample Low in Biomass

Frequently there is an inability to process and analyze samples of low biomass due to limiting amounts of relevant biomaterial in the sample. Furthermore, molecular biological protocols geared towards increasing the density of recovered cells and biomolecules of interest, by their very nature, also concentrate unwanted inhibitory humic acids and other particulates that have an adversarial effect on downstream analysis. A novel and robust fluorescence-activated cell-sorting (FACS)-based technology has been developed for purifying (removing cells from sampling matrices), separating (based on size, density, morphology), and concentrating cells (spores, prokaryotic, eukaryotic) from a sample low in biomass. The technology capitalizes on fluorescent cell-sorting technologies to purify and concentrate bacterial cells from a low-biomass, high-volume sample. Over the past decade, cell-sorting detection systems have undergone enhancements and increased sensitivity, making bacterial cell sorting a feasible concept. Although there are many unknown limitations with regard to the applicability of this technology to environmental samples (smaller cells, few cells, mixed populations), dogmatic principles support the theoretical effectiveness of this technique upon thorough testing and proper optimization. Furthermore, the pilot study from which this report is based proved effective and demonstrated this technology capable of sorting and concentrating bacterial endospore and bacterial cells of varying size and morphology. Two commercial off-the-shelf bacterial counting kits were used to optimize a bacterial stain/dye FACS protocol. A LIVE/DEAD BacLight Viability and Counting Kit was used to distinguish between the live and dead cells. A Bacterial Counting Kit comprising SYTO BC (mixture of SYTO dyes) was employed as a broad-spectrum bacterial counting agent. Optimization using epifluorescence microscopy was performed with these two dye/stains. This refined protocol was further validated using varying ratios and mixtures of cells to ensure homogenous staining compared to that of individual cells, and were utilized for flow analyzer and FACS labeling. This technology focuses on the purification and concentration of cells from low-biomass spacecraft assembly facility samples. Currently, purification and concentration of low-biomass samples plague planetary protection downstream analyses. Having a capability to use flow cytometry to concentrate cells out of low-biomass, high-volume spacecraft/ facility sample extracts will be of extreme benefit to the fields of planetary protection and astrobiology. Successful research and development of this novel methodology will significantly increase the knowledge base for designing more effective cleaning protocols, and ultimately lead to a more empirical and true account of the microbial diversity present on spacecraft surfaces. Refined cleaning and an enhanced ability to resolve microbial diversity may decrease the overall cost of spacecraft assembly and/or provide a means to begin to assess challenging planetary protection missions

Phylogenetic Diversity of Microbial Isolates from the Mars Pathfinder

As spacecraft are sent to different planets, they take with them microscopic pieces of life from Earth. It is the task of the Biotechnology and Planetary Protection Group to keep as much of this life off other planets as possible as well as document any life that may have been sent. During the construction of the Mars Pathfinder, samples were collected from various locations on the spacecraft to test for contamination. These samples were then isolated, grown, documented, preserved and their 16S rRNA genes were sequenced for identification. The 16S rRNA gene sequence is utilized because it is a highly conserved portion of the transcriptional machinery of bacteria but also has known variable regions allowing it to be and amplified and used for distinguishing different genera and species of microbial life. All of the bacterial strains analyzed from this study were members of the genus Bacillus. Seventeen strains were sequenced and identified at greater than 98% homology to known type strains. After identifying the bacterial contaminant types, the National Aeronautics and Space Administration’s Jet Propulsion Laboratory will be able to better determine cleanliness protocols to maintain the international standard and protect Mars from Earth contamination on future missions

Development of a Centrifugal Technique for the Microbial Bioburden Analysis of Freon (CFC-11)

NASA Procedural Requirement 8020.12C entitled "Planetary Protection Provisions for Robotic Extraterrestrial Missions" states that the source-specific encapsulated microbial density for encapsulated organisms (div(0)) in nonmetallic materials ranges from 1-30 spores/cubic cm. The standard laboratory procedure, NASA Standard Procedures for the Microbial Examination of Space Hardware, NHB 5340.1B, does not provide any direction into the methodologies to understand the bioburden within such a fluid as CFC-11 (Freon). This general specification value for the Freon would be applicable to the Freon charged within the Mars Science Laboratory fs (MSL fs) Heat Rejection System. Due to the large volume required to fill this system, MSL could not afford to conservatively allocate 55.8% of the total spore budget of the entire laboratory system (rover, descent stage, cruise stage, and aeroshell) of 5.00 X 10(exp 5) spores at launch. A novel filtration approach was developed to analyze the Freon employing a 50 kDa molecular weight cutoff (MCO) filter, followed by 0.22-micron pore-size filter to establish a calculated microbial bioburden

Employing a Grinding Technology to Assess the Microbial Density for Encapsulated Organisms

Projects that utilize large volumes of nonmetallic materials of planetary protection concern pose a challenge to their bioburden budget, as the most conservative value of 30 spores/cubic cm is typically used. The standard laboratory procedures do not provide any direction into the methodologies to understand the embedded bioburden within such nonmetallic components such as adhesives, insulation, or paint. A tailored, novel, destructive hardware technology employing a household box grater was developed to assess the embedded bioburden within the adhesives, insulation, and paint for the Mars Science Laboratory (MSL) project

Using a Blender to Assess the Microbial Density of Encapsulated Organisms

There are specific NASA requirements for source-specific encapsulated microbial density for encapsulated organisms in non-metallic materials. Projects such as the Mars Science Laboratory (MSL) that use large volumes of non-metallic materials of planetary protection concern pose a challenge to their bioburden budget. An optimized and adapted destructive hardware technology employing a commercial blender was developed to assess the embedded bioburden of thermal paint for the MSL project. The main objective of this optimization was to blend the painted foil pieces in the smallest sizes possible without excessive heating. The small size increased the surface area of the paint and enabled the release of the maximum number of encapsulated microbes. During a trial run, a piece of foil was placed into a blender for 10 minutes. The outside of the blender was very hot to the touch. Thus, the grinding was reduced to five 2-minute periods with 2-minute cooling periods between cycles. However, almost 20% of the foil fraction was larger (>2 mm). Thus, the largest fractions were then put into the blender and reground, resulting in a 71% increase in particles less than 1 mm in size, and a 76% decrease in particles greater than 2 mm in size. Because a repeatable process had been developed, a painted sample was processed with over 80% of the particles being <2 mm. It was not perceived that the properties (i.e. weight and rubber-like nature) of the painted/foil pieces would allow for a finer size distribution. With these constraints, each section would be ground for a total of 10 minutes with five cycles of a 2-minute pulse followed by a 2-minute pause. It was observed on several occasions that a larger blade affected the recovery of seeded spores by approximately half an order of magnitude. In the standard approach, each piece of painted foil was aseptically removed from the bag and placed onto a sterile tray where they were sized, cut, and cleaned. Each section was then weighed and placed into a sterile Waring Laboratory Blender. Samples were processed on low speed. The ground-up samples were then transferred to a 500-mL bottle using a sterile 1-in. (.2.5-cm) trim brush. To each of the bottles sterile planetary protection rinse solution was added and a modified NASA Standard Assay (NASA HBK 6022) was performed. Both vegetative and spore plates were analyzed

Isolation of the Paenibacillus phoenicis, a Spore-Forming Bacterium

A microorganism was isolated from the surfaces of the cleanroom facility in which the Phoenix lander was assembled. The isolated bacterial strain was subjected to a comprehensive polyphasic analysis to characterize its taxonomic position. Both phenotypic and phylogenetic analyses clearly indicate that this isolate belongs to the genus Paenibacillus and represents a novel species. Bacillus spores have been utilized to assess the degree and level of microbiological contamination on spacecraft and their associated spacecraft assembly facilities. Spores of Bacillus species are of particular concern to planetary protection due to the extreme resistance of some members of the genus to space environmental conditions such as UV and gamma radiation, vacuum, oxidation, and temperature fluctuation. These resistive spore phenotypes have enhanced potential for transfer, and subsequent proliferation, of terrestrial microbes on another solar body. Due to decreased nutrient conditions within spacecraft assembly facility clean rooms, the vegetative cells of Bacillus species and other spore-forming Paenibacillus species are induced to sporulate, thereby enhancing their survivability of bioreductio

Adaptation of a Filter Assembly to Assess Microbial Bioburden of Pressurant Within a Propulsion System

A report describes an adaptation of a filter assembly to enable it to be used to filter out microorganisms from a propulsion system. The filter assembly has previously been used for particulates greater than 2 micrometers. Projects that utilize large volumes of nonmetallic materials of planetary protection concern pose a challenge to their bioburden budget, as a conservative specification value of 30 spores per cubic centimeter is typically used. Helium was collected utilizing an adapted filtration approach employing an existing Millipore filter assembly apparatus used by the propulsion team for particulate analysis. The filter holder on the assembly has a 47-mm diameter, and typically a 1.2-5 micrometer pore-size filter is used for particulate analysis making it compatible with commercially available sterilization filters (0.22 micrometers) that are necessary for biological sampling. This adaptation to an existing technology provides a proof-of-concept and a demonstration of successful use in a ground equipment system. This adaptation has demonstrated that the Millipore filter assembly can be utilized to filter out microorganisms from a propulsion system, whereas in previous uses the filter assembly was utilized for particulates greater than 2 micrometers

COSPAR Sample Safety Assessment Framework (SSAF)

The Committee on Space Research (COSPAR) Sample Safety Assessment Framework (SSAF) has been developed by a COSPAR appointed Working Group. The objective of the sample safety assessment would be to evaluate whether samples returned from Mars could be harmful for Earth's systems (e.g., environment, biosphere, geochemical cycles). During the Working Group's deliberations, it became clear that a comprehensive assessment to predict the effects of introducing life in new environments or ecologies is difficult and practically impossible, even for terrestrial life and certainly more so for unknown extraterrestrial life. To manage expectations, the scope of the SSAF was adjusted to evaluate only whether the presence of martian life can be excluded in samples returned from Mars. If the presence of martian life cannot be excluded, a Hold & Critical Review must be established to evaluate the risk management measures and decide on the next steps. The SSAF starts from a positive hypothesis (there is martian life in the samples), which is complementary to the null-hypothesis (there is no martian life in the samples) typically used for science. Testing the positive hypothesis includes four elements: (1) Bayesian statistics, (2) subsampling strategy, (3) test sequence, and (4) decision criteria. The test sequence capability covers self-replicating and non-self-replicating biology and biologically active molecules. Most of the investigations associated with the SSAF would need to be carried out within biological containment. The SSAF is described in sufficient detail to support planning activities for a Sample Receiving Facility (SRF) and for preparing science announcements, while at the same time acknowledging that further work is required before a detailed Sample Safety Assessment Protocol (SSAP) can be developed. The three major open issues to be addressed to optimize and implement the SSAF are (1) setting a value for the level of assurance to effectively exclude the presence of martian life in the samples, (2) carrying out an analogue test program, and (3) acquiring relevant contamination knowledge from all Mars Sample Return (MSR) flight and ground elements. Although the SSAF was developed specifically for assessing samples from Mars in the context of the currently planned NASA-ESA MSR Campaign, this framework and the basic safety approach are applicable to any other Mars sample return mission concept, with minor adjustments in the execution part related to the specific nature of the samples to be returned. The SSAF is also considered a sound basis for other COSPAR Planetary Protection Category V, restricted Earth return missions beyond Mars. It is anticipated that the SSAF will be subject to future review by the various MSR stakeholders

Draft Genome Sequence of Bacillus safensis JPL-MERTA-8-2, Isolated from a Mars-Bound Spacecraft.

Here, we present the draft genome of Bacillus safensis JPL-MERTA-8-2, a strain found in a spacecraft assembly cleanroom before launch of the Mars Exploration Rovers. The assembly contains 3,671,133 bp in 14 contigs