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Health significance and occurrence of injured bacteria in drinking water

Enteropathogenic and indicator bacteria become injured in drinking water with exposure to sublethal levels of various biological, chemical and physical factors. One manifestation of this injury is the inability to grow and form colonies on selective media containing surfactants. The resulting underestimation of indicator bacteria can lead to a false estimation of water potability. m-T7 medium was developed specifically for the recovery of injured coliforms (both "total" and fecal) in drinking water. The m-T7 method was used to survey operating drinking water treatment and distribution systems for the presence of injured coliforms that were undetected with currently used media. The mean recovery with m-Endo LES medium was less than 1/100 ml while it ranged between 6 and 68/100ml with m-T7 agar. The majority of samples giving positive results with m-T7 medium yielded no detectable coliforms with m-Endo LES agar. Over 95% of the coliform bacteria in these samples were injured. Laboratory experiments were also done to ascribe the virulence of injured waterborne pathogens. Enteropathogens including Salmonella typhimurium, Yersinia enterocolitica and Shigella spp. required up to 20 times the chlorine levels to produce the same injury in enterotoxigenic Escherichia coli (ETEC) and nonpathogenic coliforms. Similar results were seen with Y. enterocolitica exposed to copper. The recovery of ETEC was followed by delayed enterotoxin production, both in vitro and in the gut of experimental animals. This indicates that injured waterborne enteropathogenic bacteria can be virulent.

NASA Discipline Number 13-40

Enhancement of anion-exchange chromatography of DNA using compaction agents

The use of adsorptive chromatography for preparative nucleic acid separations is often limited by low capacity. The possibility that the adsorbent surface area sterically accessible to nucleic acid molecules could be increased by reducing their radius of gyration with compaction agents has been investigated. The equilibrium adsorption capacity of Q Sepharose anion-exchange matrix for plasmid DNA at 600 mM NaCl was enhanced by up to ca. 40% in the presence of 2.5 mM spermine. In addition, compaction agent selectivity has been demonstrated. Spermine, for example, enhances the adsorption of both plasmid and genomic DNA, spermidine enhances binding only of plasmid, and hexamine cobalt enhances only the binding of genomic DNA. Compaction may be generally useful for enhancing adsorptive separations of nucleic acids.

NASA Discipline Environmental Health

Gender not a factor for altitude decompression sickness risk

INTRODUCTION: Early, retrospective reports of the incidence of altitude decompression sickness (DCS) during altitude chamber training exposures indicated that women were more susceptible than men. We hypothesized that a controlled, prospective study would show no significant difference. METHODS: We conducted 25 altitude chamber decompression exposure profiles. A total of 291 human subjects, 197 men and 94 women, underwent 961 exposures to simulated altitude for up to 8 h, using zero to 4 h of preoxygenation. Throughout the exposures, subjects breathed 100% oxygen, rested or performed mild or strenuous exercise, and were monitored for precordial venous gas emboli (VGE) and DCS symptoms. RESULTS: No significant differences in DCS incidence were observed between men (49.5%) and women (45.3%). However, VGE occurred at significantly higher rates among men than women under the same exposure conditions, 69.3% and 55.0% respectively. Women using hormonal contraception showed significantly greater susceptibility to DCS than those not using hormonal contraception during the latter two weeks of the menstrual cycle. Significantly higher DCS incidence was observed in the heaviest men, in women with the highest body fat, and in subjects with the highest body mass indices and lowest levels of fitness. CONCLUSION: No differences in altitude DCS incidence were observed between the sexes under our test conditions, although men developed VGE more often than women. Age and height showed no significant influence on DCS incidence, but persons of either sex with higher body mass index and lower physical fitness developed DCS more frequently.

NASA Discipline Environmental Health

ATP as a biomarker of viable microorganisms in clean-room facilities

A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.

NASA Discipline Environmental Health

The effect of simulated weightlessness on hypobaric decompression sickness

BACKGROUND: A discrepancy exists between the incidence of ground-based decompression sickness (DCS) during simulated extravehicular activity (EVA) at hypobaric space suit pressure (20-40%) and crewmember reports during actual EVA (zero reports). This could be due to the effect of gravity during ground-based DCS studies. HYPOTHESIS: At EVA suit pressures of 29.6 kPa (4.3 psia), there is no difference in the incidence of hypobaric DCS between a control group and group exposed to simulated weightlessness (supine body position). METHODS: Male subjects were exposed to a hypobaric pressure of 29.6 kPa (4.3 psi) for up to 4 h. The control group (n = 26) pre-oxygenated for 60 min (first 10 min exercising) before hypobaric exposure and walking around in the altitude chamber. The test group (n = 39) remained supine for a 3 h prior to and during the 60-min pre-oxygenation (also including exercise) and at hypobaric pressure. DCS symptoms and venous gas emboli (VGE) at hypobaric pressure were registered. RESULTS: DCS occurred in 42% in the control and in 44% in simulated weightlessness group (n.s.). The mean time for DCS to develop was 112 min (SD +/- 61) and 123 min (+/- 67), respectively. VGE occurred in 81% of the control group subjects and in 51% of the simulated weightlessness subjects (p = 0.02), while severe VGE occurred in 58% and 33%, respectively (p = 0.08). VGE started after 113 min (+/- 43) in the control and after 76 min (+/- 64) in the simulated weightlessness group. CONCLUSIONS: No difference in incidence of DCS was shown between control and simulated weightlessness conditions. VGE occurred more frequently during the control condition with bubble-releasing arm and leg movements.

NASA Discipline Environmental Health

Mathematical model of diffusion-limited gas bubble dynamics in unstirred tissue with finite volume

Models of gas bubble dynamics for studying decompression sickness have been developed by considering the bubble to be immersed in an extravascular tissue with diffusion-limited gas exchange between the bubble and the surrounding unstirred tissue. In previous versions of this two-region model, the tissue volume must be theoretically infinite, which renders the model inapplicable to analysis of bubble growth in a finite-sized tissue. We herein present a new two-region model that is applicable to problems involving finite tissue volumes. By introducing radial deviations to gas tension in the diffusion region surrounding the bubble, the concentration gradient can be zero at a finite distance from the bubble, thus limiting the tissue volume that participates in bubble–tissue gas exchange. It is shown that these deviations account for the effects of heterogeneous perfusion on gas bubble dynamics, and are required for the tissue volume to be finite. The bubble growth results from a difference between the bubble gas pressure and an average gas tension in the surrounding diffusion region that explicitly depends on gas uptake and release by the bubble. For any given decompression, the diffusion region volume must stay above a certain minimum in order to sustain bubble growth.

NASA Discipline Environmental Health

Spectroscopic detection of biological NO with a quantum cascade laser

Two configurations of a continuous wave quantum cascade distributed feedback laser-based gas sensor for the detection of NO at a parts per billion (ppb) concentration level, typical of biomedical applications, have been investigated. The laser was operated at liquid nitrogen temperature near lambda = 5.2 microns. In the first configuration, a 100 m optical path length multi-pass cell was employed to enhance the NO absorption. In the second configuration, a technique based on cavity-enhanced spectroscopy (CES) was utilized, with an effective path length of 670 m. Both sensors enabled simultaneous analysis of NO and CO2 concentrations in exhaled air. The minimum detectable NO concentration was found to be 3 ppb with a multi-pass cell and 16 ppb when using CES. The two techniques are compared, and potential future developments are discussed.

NASA Discipline Environmental Health

Yb fiber laser pumped mid-IR source based on difference frequency generation and its application to ammonia detection

A Yb fiber laser pumped cw narrow-linewidth tunable mid-IR source based on a difference frequency generation (DFG) in a periodically poled LiNbO3 (PPLN) crystal for trace gas detection was demonstrated. A high power Yb fiber laser and a distributed feedback (DFB) laser diode were used as DFG pump sources. This source generated mid-IR at 3 microns with a powers of ~2.5 microW and a spectral linewidth of less than 30 MHz. A frequency tuning range of 300 GHz (10 cm-1) was obtained by varying the current and temperature of the DFB laser diode. A high-resolution NH3 absorption Doppler-broadened spectrum at 3295.4 cm-1 (3.0345 microns) was obtained at a cell pressure of 27 Pa from which a detection sensitivity of 24 ppm m was estimated.

NASA Discipline Environmental Health

Three-dimensional tissue assemblies: novel models for the study of Salmonella enterica serovar Typhimurium pathogenesis

The lack of readily available experimental systems has limited knowledge pertaining to the development of Salmonella-induced gastroenteritis and diarrheal disease in humans. We used a novel low-shear stress cell culture system developed at the National Aeronautics and Space Administration in conjunction with cultivation of three-dimensional (3-D) aggregates of human intestinal tissue to study the infectivity of Salmonella enterica serovar Typhimurium for human intestinal epithelium. Immunohistochemical characterization and microscopic analysis of 3-D aggregates of the human intestinal epithelial cell line Int-407 revealed that the 3-D cells more accurately modeled human in vivo differentiated tissues than did conventional monolayer cultures of the same cells. Results from infectivity studies showed that Salmonella established infection of the 3-D cells in a much different manner than that observed for monolayers. Following the same time course of infection with Salmonella, 3-D Int-407 cells displayed minimal loss of structural integrity compared to that of Int-407 monolayers. Furthermore, Salmonella exhibited significantly lower abilities to adhere to, invade, and induce apoptosis of 3-D Int-407 cells than it did for infected Int-407 monolayers. Analysis of cytokine expression profiles of 3-D Int-407 cells and monolayers following infection with Salmonella revealed significant differences in expression of interleukin 1alpha (IL-1alpha), IL-1beta, IL-6, IL-1Ra, and tumor necrosis factor alpha mRNAs between the two cultures. In addition, uninfected 3-D Int-407 cells constitutively expressed higher levels of transforming growth factor beta1 mRNA and prostaglandin E2 than did uninfected Int-407 monolayers. By more accurately modeling many aspects of human in vivo tissues, the 3-D intestinal cell model generated in this study offers a novel approach for studying microbial infectivity from the perspective of the host-pathogen interaction.

NASA Discipline Environmental Health

Acridine orange staining reaction as an index of physiological activity in Escherichia coli

The assumption that the acridine orange (AO) color reaction may be used as an index of physiological activity was investigated in laboratory grown Escherichia coli. Spectrofluorometric observations of purified nucleic acids, ribosomes and the microscopic color of bacteriophage-infected cells stained with AO confirmed the theory that single-stranded nucleic acids emit orange to red fluorescence while those that are double-stranded fluoresce green in vivo. Bacteria growing actively in a rich medium could be distinguished from cells in stationary phase by the AO reaction. Cells from log phase appeared red, whereas those in stationary phase were green. However, this differentiation was not seen when the bacteria were grown in a minimal medium or when a variation of the staining method was used. Also, shifting bacteria in stationary phase to starvation conditions rapidly changed their AO staining reaction. Boiling and exposure to lethal concentrations of azide and formalin resulted in stationary-phase cells that appeared red after staining but bacteria killed with chlorine remained green. These findings indicate that the AO staining reaction may be suggestive of physiological activity under defined conditions. However, variables in staining and fixation procedures as well as uncertainties associated with mixed bacterial populations in environmental samples may produce results that are not consistent with the classical interpretation of this reaction. The importance of validating the putative physiological implications of this staining reaction is stressed.

NASA Discipline Number 04-10

Probing porous media with gas diffusion NMR

We show that gas diffusion nuclear magnetic resonance (GD-NMR) provides a powerful technique for probing the structure of porous media. In random packs of glass beads, using both laser-polarized and thermally polarized xenon gas, we find that GD-NMR can accurately measure the pore space surface-area-to-volume ratio, S/V rho, and the tortuosity, alpha (the latter quantity being directly related to the system's transport properties). We also show that GD-NMR provides a good measure of the tortuosity of sandstone and complex carbonate rocks.

NASA Discipline Environmental Health

Rapid direct methods for enumeration of specific, active bacteria in water and biofilms

Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScan(R), a new instrument that is very sensitive and rapid. The ChemScan(R) laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between results obtained with the ChemScan and traditional plate counts of mixed natural bacterial populations in water. The continuing evolution of these methods will be valuable in the rapid and accurate analysis of environmental samples.

Review, Tutorial

Fourier-transform optical microsystems

The design, fabrication, and initial characterization of a miniature single-pass Fourier-transform spectrometer (FTS) that has an optical bench that measures 1 cm x 5 cm x 10 cm is presented. The FTS is predicated on the classic Michelson interferometer design with a moving mirror. Precision translation of the mirror is accomplished by microfabrication of dovetailed bearing surfaces along single-crystal planes in silicon. Although it is miniaturized, the FTS maintains a relatively high spectral resolution, 0.1 cm-1, with adequate optical throughput.

NASA Discipline Environmental Health

Development of an automated diode-laser-based multicomponent gas sensor

The implementation and application of a portable fiber-coupled trace-gas sensor for the detection of several trace gases, including CO2, CH4, and H2CO, are reported. This particular sensor is based on a cw fiber-amplified near-infrared (distributed Bragg reflector) diode laser and an external cavity diode laser that are frequency converted in a periodically poled lithium niobate crystal to the mid-IR spectroscopic fingerprint region (3.3-4.4 micrometers). A continuous absorption spectrum of CH4 and H2CO from 3.37 to 3.10 micrometers with a spectral resolution of 40 MHz (approximately 0.0013 cm-1) demonstrated the spectral performance that can be achieved by means of automated wavelength tuning and phase matching with stepper motor control. Autonomous long-term detection of ambient CO2 and CH4 over a 3- and 7-day period was also demonstrated.

NASA Discipline Environmental Health

Effective utilization of quantum-cascade distributed-feedback lasers in absorption spectroscopy

A variable duty cycle quasi-cw frequency scanning technique was applied to reduce thermal effects resulting from the high heat dissipation of type I quantum-cascade lasers. This technique was combined with a 100-m path-length multipass cell and a zero-air background-subtraction technique to enhance detection sensitivity to a parts-in-10(9) (ppb) concentration level for spectroscopic trace-gas detection of CH4, N2O, H2O, and C2H5OH in ambient air at 7.9 micrometers. A new technique for analysis of dense high resolution absorption spectra was applied to detection of ethanol in ambient air, yielding a 125-ppb detection limit.

NASA Discipline Environmental Health

Compact CH4 sensor based on difference frequency mixing of diode lasers in quasi-phasematched LiNbO3

A compact, portable and robust room temperature CH4 sensor is reported. By difference frequency mixing a 500 mW alpha-DFB diode laser at 1066 nm and an erbium-doped fiber amplified 1574 nm DFB diode laser in periodically poled lithium niobate up to 7 (mu)W of narrowband radiation at 3.3 microns is generated. Real-time monitoring of CH4 over a 7 day period using direct absorption in an open-path multipass cell (L = 36 m) demonstrates a detection precision of +/- 14 ppb.

NASA Discipline Environmental Health

Measurement of heat conduction through stacked screens

This paper describes the experimental apparatus for the measurement of heat conduction through stacked screens as well as some experimental results taken with the apparatus. Screens are stacked in a fiberglass-epoxy cylinder, which is 24.4 mm in diameter and 55 mm in length. The cold end of the stacked screens is cooled by a Gifford-McMahon (GM) cryocooler at cryogenic temperature, and the hot end is maintained at room temperature. Heat conduction through the screens is determined from the temperature gradient in a calibrated heat flow sensor mounted between the cold end of the stacked screens and the GM cryocooler. The samples used for these experiments consisted of 400-mesh stainless steel screens, 400-mesh phosphor bronze screens, and two different porosities of 325-mesh stainless steel screens. The wire diameter of the 400-mesh stainless steel and phosphor bronze screens was 25.4 micrometers and the 325-mesh stainless steel screen wire diameters were 22.9 micrometers and 27.9 micrometers. Standard porosity values were used for the experimental data with additional porosity values used on selected experiments. The experimental results showed that the helium gas between each screen enhanced the heat conduction through the stacked screens by several orders of magnitude compared to that in vacuum. The conduction degradation factor is the ratio of actual heat conduction to the heat conduction where the regenerator material is assumed to be a solid rod of the same cross sectional area as the metal fraction of the screen. This factor was about 0.1 for the stainless steel and 0.022 for the phosphor bronze, and almost constant for the temperature range of 40 to 80 K at the cold end.

NASA Discipline Environmental Health