Fluorescent antibody detection of microorganisms
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Experiments indicate that hardy organisms will likely grow in the Martian environment if moisture is available, and that these organisms definitely present a threat to contamination of the biopackage if they are transported to the surface of Mars.
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Firefly luciferase ATP assay is used in clinical and industrial applications, such as determination of urinary infection levels, microbial susceptibility testing, and monitoring of yeast levels in beverages. Three categories of extractants were investigated for their extracting efficiency. They were ionizing organic solvents, nonionizing organic solvents, and inorganic acids. Dimethylsulfoxide and formamide represented the ionizing organic solvents, while n-butanol, chloroform, ethanol, acetone, and methylene chloride were used for the nonionizing organic solvents. Nitric acid and perchloric acid were chosen for the inorganic acids category. Pathogens were tested with each solvent. They included: Saccharomyces carlsbergensis, E. coli, Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter species, Proteus mirabilis, Proteus vulgaris, Staphylococcus epidermidis, Streptococcus faecalis, Pseudomonas aeruginosa, and Candida albicans. These results are shown in graphic representations.
The anaerobic utilization of phosphite or phosphine and the significance of this conversion to potential contamination of Jupiter were investigated. A sporeforming organism was isolated from Cape Canaveral soil which anaerobically converts hypophosphite to phosphate. This conversion coincides with an increase in turbidity of the culture and with phosphate accumulation in the medium. Investigations of omnitherms (organisms which grow over a broad temperature range, i.e. 3 -55 C were also conducted. The cellular morphology of 28 of these isolates was investigated, and all were demonstrated to be sporeformers. Biochemical characterizations are also presented. Procedures for replicate plating were evaluated, and those results are also presented. The procedures for different replicate-plating techniques are presented, and these are evaluated on the basis of reproducibility, percentage of viable transfer, and ease of use. Standardized procedures for the enumeration of microbial populations from ocean-dredge samples from Cape Canaveral are also presented.
Results are presented on the anaerobic conversion of phosphite to phosphate. It is demonstrated that in the presence of both phosphite and hypophosphite, the phosphite is the preferred phosphorous source. An investigation in which P-32 labeled hypophosphite was added to the basal medium demonstrates that the labeled hypophosphite was incorporated into the metabolic reactions of the cell. Other data show that as cell growth occurs, the phosphite and/or hypophosphite levels decrease. The Bacillus sp. capable of anaerobic utilization of phosphite was isolated from Cape Canaveral soil samples, and it is partially characterized. Also included are continued investigations of omnitherms. The data presented show that some of these possess significant resistance to the Viking dry-heat cycle, and that they retain their omnithermic characteristic after recovery from the heat cycle. Other physiological characteristics of these isolates are also presented. It is demonstrated that omnitherms can be isolated from Cape Canaveral soil.
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Results of studies in anaerobic phosphorus metabolism are presented. Specific topics discussed include: (1) anaerobic utilization of PH3; (2) reduction of phosphate or phosphite; (3) isolation of organisms which utilize phosphite or phosphate anaerobically as a final hydrogen acceptor; and (4) the toxicity of PH3 to the organisms. Techniques of anaerobic microbiology associated with space hardware were also studied. These include: (1) the Brewer anaerobe jar/GasPak system; (2) a new procedure to grow aerobes and anaerobes simultaneously; (3) a culture medium to differentiate oblagate from facultative anaerobes; and (4) a procedure to quantitate O2 sensitivity of anaerobes.
Soil samples from Cape Canaveral were subjected to a simulated Martian environment and assayed periodically over 45 days to determine the effect of various environmental parameters on bacterial populations. The simulated environment was based on the most recent available data, prior to the Viking spacecraft, describing Martian conditions and consisted of a pressure of 7 millibars, an atmosphere of 99.9% CO2 and 0.1% O2, a freeze-thaw cycle of -65 C for 16 h and 24 C for 8 h, and variable moisture and nutrients. Reduced pressure had a significant effect, reducing growth under these conditions. Slight variations in gaseous composition of the simulated atmosphere had negligible effect on growth. The freeze-thaw cycle did not inhibit growth, but did result in a slower rate of decline after growth had occurred. Dry samples exhibited no change during the 45-day experiment, indicating that the simulated Martian environment was not toxic to bacterial populations. Psychrotrophic organisms responded more favorably to this environment than mesophiles, although both types exhibited increases of approximately 3 logs in 7 to 14 days when moisture and nutrients were available.
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Experiments to identify free living organisms in soils that may be substantially simpler in genetic content, and mirroring a more primitive stage of evolution than the species with which we are familiar to date, were designed. Organic chemical studies on the composition and disposition of elementary carbon leave nothing wanting as an aboriginal substrate for the original of life and early chemical evolution. Such studies were missed when it came to the interpretation of the Viking lander data, and needed for conceptual planning of future planetary missions.
Anaerobic and aerobic sporeformers and non-sporeformers were cultivated anaerobically in nutrient media under various pressures (up to 1800 psi) of pure H2, CH4, NH3, and H2S. Viability assays were performed periodically to determine growth, survival, or spore survival. Hydrogen up to 1800 psi demonstrated little or no suppression of growth with the possible exception of Bacillus coagulans at 1800 psi. The obligate anaerobes grew very well. Under CH4 the obligate anaerobes again exhibited the most prolific growth, whereas the facultative anaerobes grew well except under higher pressures. Ammonia at low pressure was extremely toxic to all test organisms. At 100 psi all populations were killed within 24 hours except Staphylococcus aureus which survived for 72 hours and the Bacillus spp. which produced a surviving population of approximately 10,000 spores/ml. All populations in H2S were killed within 24 to 48 hours except Proteus mirabilis which decreased to 100 cells/ml and the Bacillus spp. Spore survival studies of two months duration demonstrated that B. coagulans and B. pumilus survived under all experimental conditions. Clostridium novyi type B and C. sporogenes were killed rapidly in NH3 and H2S and demonstrated no sporulation.
Approximately 46% of the lunar sample (10084,151), 125.42 mg, was solubilized in 680 ml 0.01 M salicylic acid. Atomic absorption spectroscopic analysis of the solubilized lunar sample showed the following amount of metal ions: Ca, 3.1; Mg, 4.0; K, 0.09; Na, 0.67; Fe, 7.3; Mn, 1.6; Cu, Ni, Cr, less than 0.1 each. All are in ppm. Salicylic acid used to solubilize the lunar sample was highly inhibitory to the growth of mixed soil microbes. However, the mineral part of the lunar extract stimulated the growth. For optimal growth of the soil microbes the following nutrients must be added to the moon extract; sources of carbon, nitrogen, sulfur, phosphorus, and magnesium in addition to water.
The survival of Baccillus subtilis, Azotobacter chroococcum, and the enteric bacteriophage MS2 has been examined in clays representing terrestrial (Wyoming type montmorillonite) and Martian (Fe3+ montmorillonite) soils exposed to terrestrial and Martian environmental conditions of temperature and atmospheric composition and pressure. An important finding is that MS2 survived simulated Mars conditions better than the terrestrial environment, probably owing to stabilization of the virus caused by the cold and dry conditions of the simulated Mars environment. This finding, the first published indication that viruses may be able to survive in Mars-type soils, may have important implications for future missions to Mars.
The work described in this research program originated with the choice by NASA of an ion mobility spectrometer for air quality monitoring on-board the international spacestation. Though the gas chromatograph-ion mobility spectrometer analyzer known as VOA met or exceeded expectations, limitations in the basic understanding of response and the utilization of foundational principles into usable technology was considered unacceptable. In this research program, a comprehensive model for the origins of mobility spectra was proposed, tested and verified. The principles considered responsible for the appearance of mobility spectra have now been elucidated through this project. This understanding has been applied in automated identification of mobility spectra using neural networks and routine procedures for this now exist. Finally, the limitation on linear range has been shown to be a technical limitation and not a fundamental limitation so that a hardware component was crafted to extend the linear range of a mobility spectrometer by 10X. This project has led to one Ph.D. dissertation and one MS thesis. In addition, over ten public presentations at professional meetings and six journal publications have resulted from this program of research. The findings are so plentiful that total analysis of the findings may require four to six years or more. The findings confirm that the decision to use VOA was sound and that the chemical and physical principles of mobility spectrometry are both understandable and predictable.
A photographic atlas of scanning electron microscope (SEM) images of Cambrian (Tommotian) microfossils from the phosphorites of Khubsugul Mongolia is presented. SEM images of modern cyanobacteria and bacteria are provided for comparison. The importance of bacterial fossils and morphological biomarkers to astrobiology and the understanding of the origin of phosphorites is considered.
New spectral measurements of bacteria taken at cryogenic temperatures provide insights on the surface composition of Europa as well as the detectability of microbes on the surface. Additional information is contained in the original extended abstract.