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At least 55 records · Page 3

Tissuelike 3D Assemblies of Human Broncho-Epithelial Cells

Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that showed signs of damage by viruses and virus titers (see figure) that indicated large increases in the populations of viruses during the days following inoculation.

Goodwin, Thomas J.↗

The Influence of Ecological Isolation on the Structural and Functional Stability of Complex Microbial Communities

To help understand how the behavior of microorganisms and microbial communities in insular space habitats may differ from the behavior of these groups on Earth, long-term incubations (100+ days) were conducting using wastewater bioreactors (batch fed) designed to mimic "closed" and "open" ecological systems. The issue of immigration was considered, and the goal of the research was to determine whether the stability of microbial communities in space is reduced due to their prolonged isolation. Bioreactors were established by inoculating flasks of sterile synthetic wastewater with the microbial community obtained from a local treatment facility; each day, one-third of the medium in the flask was replaced with an equal volume of sterile artificial wastewater. Flasks were divided into two treatments: "closed" and "open" to recruitment of additional microorganisms. "Closed" flasks were maintained as described above, while the medium used to feed the "open" flasks was supplemented daily with a small amount of raw sewage (which provided a continuous source of new potential community members). Significant differences in microbial community structure and function developed in the two sets of communities, and the results suggest that the open community was more stable and better able to adjust to changing environmental conditions. Each community's resistance to environmental (temperature fluctuations) and biological stresses (starvation and invasion by an opportunistic pathogen Pseudomonas aeruginosa) was monitored. Experiments were also conducted to determine whether the effect of isolation changes depending on the microbial communities' initial diversity or composition; communities with a low(er) initial diversity were less stable. Overall, the results indicate that isolation will be an important factor influencing the activity of microbial communities on board spacecraft. A possible way of mitigating these effects would be to include communities with high initial diversity or to periodically re-inoculate the systems using diverse inocula transported from Earth.

Franklin, R. B.↗

Assessment of Bacterial Spores in Solid Materials: Curriculum Improvements Partnership Award for the Integration of Research (CIPAIR)

This summer, we quantified the release, by cryogenic grinding at liquid nitrogen temperatures, of microbes present in 4 different spacecraft solids: epoxy 9309, epoxy 9394, epoxy 9396, and a silicone coating. Three different samples of each material were prepared: aseptically prepared solid material, powdered material inoculated with a known spore count of Bacillus atrophaeus, and solid material artificially embedded with a known spore count of Bacillus atrophaeus. Samples were cryogenically ground as needed, and the powders were directly cultured to determine the number of microbial survivors per gram of material. Recovery rates were found to be highly material-dependent, varying from 0.2 to 50% for inoculated material surfaces and 0.002 to 0.5% for embedded spores. A study of the spore survival rate versus total grinding time was also performed, with results indicating that longer grinding time decreases recovery rates of viable spores.

embedded bioburden↗

Mass culture of photobacteria to obtain luciferase

Inoculating preheated trays containing nutrient agar with photobacteria provides a means for mass culture of aerobic microorganisms in order to obtain large quantities of luciferase. To determine optimum harvest time, growth can be monitored by automated light-detection instrumentation.

Chappelle, E. W.↗

Enhanced degradation of aluminum metal in the presence of selected microorganisms

Experiments were conducted to determine the effects of microorganisms, substrates, pressures, humidities, and oxygen concentrations upon aluminum corrosion. In addition, the effects of microbes upon coated and treated aluminum were examined and an attempt to correlate aluminum in solution with degradation of the samples was undertaken. The organisms, humidities, oxygen levels, and substrates all played a major role in the corrosion of aluminum. Quantitation of aluminum losses indicated that the total metal losses from inoculated samples were significantly greater than those of the uninoculated samples.

Tennyson, J. M.↗

Dry-heat Resistance of Bacillus Subtilis Var. Niger Spores on Mated Surfaces

Bacillus subtilis var. niger spores were placed on the surfaces of test coupons manufactured from typical spacecraft materials including stainless steel, magnesium, titanium, and aluminum. These coupons were then juxtaposed at the inoculated surfaces and subjected to test pressures of 0, 1000, 5000, and 10,000 psi. Tests were conducted in ambient, nitrogen, and helium atmospheres. While under the test pressure condition, the spores were exposed to 125 C for intervals of 5, 10, 20, 50, or 80 min. Survivor data were subjected to a linear regression analysis that calculated decimal reduction times.

Simko, G. J.↗

The effects of various cure cycles upon the viability of Bacillus subtillis var. niger spores within solid propellant.

Saturethane solid propellant was used in all tests. The spore inoculum was evenly distributed in the propellant. Samples weighing approximately 5 g were aseptically removed, placed into curing ovens, and exposed to cure temperatures. Initial tests were conducted at 82 and 93 C. Analysis of the obtained data indicated that the survivor curves were not linear. Exposure of the inoculated propellant samples to 93 C reduced the initial population to less than 0.01% in about 20 hours. At 82 C, approximately 168 hours were required for a similar reduction. Tests involving curing temperatures of 105 and 115 C were also conducted. It is pointed out that changes in the mechanism of spore inactivation due to chemical and physical changes in the propellant could account for the nonlinear survivor curves obtained.

Brewer, W. A.↗

A study of psychrophilic organisms isolated from the manufacture and assembly areas of spacecraft to be used in the Viking mission

The effect of storage of dry heat treated Teflon ribbons under nitrogen gas followed by high vacuum on the recovery of hardy organisms from the ribbons was studied. A similar experiment was performed on spore crops of hardy organisms recovered previously from Cape Canaveral. Hardy organisms have been inoculated onto slides and subjected to an artificial Martian environment in an attempt to demonstrate their growth in this environment. Additional experiments using the artificial Martian environment include response of soil samples from the VAB with both constant temperature and freeze-thaw cycles. These experiments were performed with dried soil and soil containing added water. Other investigations included the effect of heatshock on soil samples, psychrophilic counts of new soil samples from the manufacture area of the Viking spacecraft, effect of pour plate versus spread plate on psychrophilic counts, and preparation of spore crops of hardy organisms from Cape Canaveral.

Foster, T. L.↗

Evaluations of lunar samples for the presence of viable organisms

Samples from the six successful Apollo lunar exploration missions were examined for the presence of biological formed elements and were used to inoculate a variety of culture media designed to promote growth of a broad spectrum of microorganisms. No evidence of viable organisms was obtained from any of these analyses. Following incubation of the lunar material-culture medium complexes, microbial growth dynamics studies were conducted with known test species to evaluate the possible presence of toxic factors. Only extracts of culture media which had been in contact with a mixture of lunar material from both Apollo 11 core tubes proved to be toxic to all species tested. Attempts to reproduce this toxic effect with individual Apollo 11 core samples obtained at other parts of the core and analyzed under somewhat different conditions were unsuccessful. In all, 48 different lunar samples were examined. These samples were collected at the lunar surface, in trenches, and in core samples to a depth of 297 cm.

Taylor, G. R.↗

Plating isolation of various catalase-negative microorganisms from soil

A unique plating procedure was developed that allows isolation, but not enumeration, of representatives of the catalase-negative soil microflora. The numbers recovered, however, are low as compared to the numbers recovered when the modified dilution-to-extinction isolation procedure is used. The latter procedure provides prolonged inoculation in sealed tubes containing a nutritionally rich broth medium over small submerged agar slants. In contrast, the plating procedure utilizes nutritionally minimal media and the shorter incubations mandated by the inherent problems associated with plating.

Labeda, D. P.↗

Oxygen effects on mortality of mice infected with Diplococcus pneumoniae

Mice infected by intraperitoneal injection of Diplococcus pneumoniae were held at 1 atm in either hypoxic (12%), hyperoxic (75%), or a normal (21%) oxygen environment. Mortality rates indicated prolongation of survival in hypoxia and shortened survival in hyperoxia. Exposure of mice to the experimental gas mixtures prior to inoculation did not alter the results.

Angrick, E. J.↗

Hazard analysis of Clostridium perfringens in the Skylab Food System

The Skylab Food System presented unique microbiological problems because food was warmed in null-gravity and because the heat source was limited to 69.4 C (to prevent boiling in null-gravity). For these reasons, the foods were manufactured using critical control point techniques of quality control coupled with appropriate hazard analyses. One of these hazard analyses evaluated the threat from Clostridium perfringens. Samples of food were inoculated with C. perfringens and incubated for 2 h at temperatures ranging from 25 to 55 C. Generation times were determined for the foods at various temperatures. Results of these tests were evaluated taking into consideration: food-borne disease epidemiology, the Skylab food manufacturing procedures, and the performance requirements of the Skylab Food System. Based on this hazard analysis, a limit for C. perfringens of 100/g was established for Skylab foods.

Bourland, C. T.↗

Electrochemical detection device

A standard pH reference electrode and a platinum cathodic electrode are positioned in a container with suitable nutrient medium for microbial growth plus the sample to be tested. The two electrodes are connected to electronic circuitry including an up/down counter whicn counts up for the first 80 minutes after a test has initiated. Then the potential between the two electrodes is tracked by the electronic circuitry and after there is a change of 10 mv a signal is sent to the up/down counter to cause it to reverse its count. When there is a additional 20 mv change in the potential between the two electrodes another signal is sent to the up/down counter, signalling it to stop. The resulting count on the counter is equal to the length of time for the inoculum to begin the production of measurable amounts of H2 after inoculation.

Young, R. N.↗

Bacterial degradation of polychlorinted biphenyls in sludge from an industrial sewer lagoon

A laboratory experiment was conducted to determine if polychlorinated biphenyls (PCB's) found in an industrial sewer sludge can be effectively degraded by mutant bacteria. The aerated sludge was inoculated daily with mutant bacteria in order to augment the existing bacteria with bacteria that were considered to be capable of degrading PCB's. The pH, nitrogen, and phosphorus levels were monitored daily to maintain an optimum growing medium for the bacteria. A gas chromatographic method was used to determine the PCB concentrations of the sludge initially and also throughout the experiment. Results and discussion of the bacterial treatment of polychlorinated biphenyls are presented.

Kim, W. S.↗

Characterization of neurospora circadian rhythms in space

To determine whether the circadian rhythm of conidiation in neurospora crassa is endogenously derived or is driven by some geophysical time cue, an experiment was conducted on space shuttle flight STS-9, where inoculated race tubes were exposed to the microgravity environment of space. The results demonstated that the rhythm can persist in space. However, there were several minor alterations noted; an increase in the period of the oscillation and the variability of the growth rate and a diminished rhythm amplitude, which eventually damped out in 25% of the flight tubes. On day seven of the flight, the tubes were exposed to light while their growth fronts were marked. It appears that some aspects of this marking process reinstated a robust rhythm in all the tubes which continued throughout the remainder of the flight. It was hypothesized that the damping found prior to the marking procedure on STS-9 may have been a result of the hypergravity pulse of launch and not due to the microgravity of the orbital lab; furthermore, that the marking procedure, by exposing the samples to light, had reinstated rhythmicity. To test this, an investigation was conducted into the effects of acute and chronic exposure to hypergravity.

Ferraro, James S.↗

Autonomous support for microorganism research in space

A preliminary design for performing on orbit, autonomous research on microorganisms and cultured cells/tissues is presented. An understanding of gravity and its effects on cells is crucial for space exploration as well as for terrestrial applications. The payload is designed to be compatible with the Commercial Experiment Transporter (COMET) launch vehicle, an orbiter middeck locker interface, and with Space Station Freedom. Uplink/downlink capabilities and sample return through controlled reentry are available for all carriers. Autonomous testing activities are preprogrammed with in-flight reprogrammability. Sensors for monitoring temperature, pH, light, gravity levels, vibrations, and radiation are provided for environmental regulation and experimental data collection. Additional experimental data acquisition includes optical density measurement, microscopy, video, and film photography. On-board full data storage capabilities are provided. A fluid transfer mechanism is utilized for inoculation, sampling, and nutrient replenishment of experiment cultures. In addition to payload design, representative experiments were developed to ensure scientific objectives remained compatible with hardware capabilities. The project is defined to provide biological data pertinent to extended duration crewed space flight including crew health issues and development of a Controlled Ecological Life Support System (CELSS). In addition, opportunities are opened for investigations leading to commercial applications of space, such as pharmaceutical development, modeling of terrestrial diseases, and material processing.

Fleet, Mary L.↗

Microbiological test results using three urine pretreatment regimes with 316L stainless steel

Three urine pretreatments, (1) Oxone (Dupont) and sulfuric acid, (2) sodium hypochlorite and sulfuric acid, (3) and ozone, were studied for their ability to reduce microbial levels in urine and minimize surface attachment to 316L stainless steel coupons. Urine samples inoculated with Bacillus insolitus and a filamentous mold, organisms previously recovered from the vapor compression distillation subsystem of NASA Space Station Freedom water recovery test were tested in glass corrosion cells containing base or weld metal coupons. Microbial levels, changes in pH, color, turbidity, and odor of the fluid were monitored over the course of the 21-day test. Specimen surfaces were examined by scanning electron microscopy at completion of the test for microbial attachment. Ozonated urine samples were less turbid and had lower microbial levels than controls or samples receiving other pretreatments. Base metal coupons receiving pretreatment were relatively free of attached bacteria. However, well-developed biofilms were found in the heat-affected regions of welded coupons receiving Oxone and hypochlorite pretreatments. Few bacteria were observed in the same regions of the ozone pretreatment sample.

Huff, Timothy L.↗