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All sourcesNASA NTRSDOE OSTI
NASA NTRS2011

Low Fluid Shear Culture of Staphylococcus Aureus Represses hfq Expression and Induces an Attachment-Independent Biofilm Phenotype

Background: The opportunistic pathogen, Staphylococcus aureus, experiences fluctuations in fluid shear during infection and colonization of a human host. Colonization frequently occurs at mucus membrane sites such as in the gastrointestinal tract where the bacterium may experience low levels of fluid shear. The response of S. aureus to low fluid shear remains unclear. Methods: S. aureus was cultured to stationary phase using Rotating-Wall Vessel (RWV) bioreactors which produce a physiologically relevant low fluid shear environment. The bacterial aggregates that developed in the RWV were evaluated by electron microscopy as well as for antibiotic resistance and other virulence-associated stressors. Genetic expression profiles for the low-shear cultured S. aureus were determined by microarray analysis and quantitative real-time PCR. Results: Planktonic S. aureus cultures in the low-shear environment formed aggregates completely encased in high amounts of extracellular polymeric substances. In addition, these aggregates demonstrated increased antibiotic resistance indicating attachment-independent biofilm formation. Carotenoid production in the low-shear cultured S. aureus was significantly decreased, and these cultures displayed an increased susceptibility to oxidative stress and killing by whole blood. The hfq gene, associated with low-shear growth in Gram negative organisms, was also found to be down-regulated in S. aureus. Conclusions: Collectively, this data suggests that S. aureus decreases virulence characteristics in favor of a biofilm-dwelling colonization phenotype in response to a low fluid shear environment. Furthermore, the identification of an Hfq response to low-shear culture in S. aureus, in addition to the previously reported responses in Gram negative organisms, strongly suggests an evolutionarily conserved response to mechanical stimuli among structurally diverse prokaryotes.

Ott, C. Mark↗
NASA NTRS2011

Methods of Antimicrobial Coating of Diverse Materials

Methods of coating diverse substrate materials with antimicrobial agents have been developed. Originally intended to reduce health risks to astronauts posed by pathogenic microorganisms that can grow on surfaces in spacecraft, these methods could also be used on Earth for example, to ensure sterility of surgical inserts and other medical equipment. The methods involve, generally, chemical preparation of substrate surfaces to enable attachment of antimicrobial molecules to the substrate surfaces via covalent bonds. Substrate materials that have been treated successfully include aluminum, glass, a corrosion-resistant nickel alloy, stainless steel, titanium, and poly(tetrafluoroethylene). Antimicrobial agents that have been successfully immobilized include antibiotics, enzymes, bacteriocins, bactericides, and fungicides. A variety of linkage chem istries were employed. Activity of antimicrobial coatings against gram-positive bacteria, gram-negative bacteria, and fungi was demonstrated. Results of investigations indicate that the most suitable combination of antimicrobial agent, substrate, and coating method depends upon the intended application.

Akse, James R.↗
NASA NTRS2011

Climate Change, Extreme Weather Events, and Fungal Disease Emergence and Spread

Empirical evidence from multiple sources show the Earth has been warming since the late 19th century. More recently, evidence for this warming trend is strongly supported by satellite data since the late 1970s from the cryosphere, atmosphere, oceans, and land that confirms increasing temperature trends and their consequences (e.g., reduced Arctic sea ice, rising sea level, ice sheet mass loss, etc.). At the same time, satellite observations of the Sun show remarkably stable solar cycles since the late 1970s, when direct observations of the Sun's total solar irradiance began. Numerical simulation models, driven in part by assimilated satellite data, suggest that future-warming trends will lead to not only a warmer planet, but also a wetter and drier climate depending upon location in a fashion consistent with large-scale atmospheric processes. Continued global warming poses new opportunities for the emergence and spread of fungal disease, as climate systems change at regional and global scales, and as animal and plant species move into new niches. Our contribution to this proceedings is organized thus: First, we review empirical evidence for a warming Earth. Second, we show the Sun is not responsible for the observed warming. Third, we review numerical simulation modeling results that project these trends into the future, describing the projected abiotic environment of our planet in the next 40 to 50 years. Fourth, we illustrate how Rift Valley fever outbreaks have been linked to climate, enabling a better understanding of the dynamics of these diseases, and how this has led to the development of an operational predictive outbreak model for this disease in Africa. Fifth, We project how this experience may be applicable to predicting outbreaks of fungal pathogens in a warming world. Lastly, we describe an example of changing species ranges due to climate change, resulting from recent warming in the Andes and associated glacier melt that has enabled amphibians to colonize higher elevation lakes, only to be followed shortly by the emergence of fungal disease in the new habitats.

Tucker, Compton J.↗
NASA NTRS2011

Microbiological Lessons Learned from the Space Shuttle

After 30 years of being the centerpiece of NASA s human spacecraft, the Space Shuttle will retire. This highly successful program provided many valuable lessons for the International Space Station (ISS) and future spacecraft. Major microbiological risks to crewmembers include food, water, air, surfaces, payloads, animals, other crewmembers, and ground support personnel. Adverse effects of microorganisms are varied and can jeopardize crew health and safety, spacecraft systems, and mission objectives. Engineering practices and operational procedures can minimize the negative effects of microorganisms. To minimize problems associated with microorganisms, appropriate steps must begin in the design phase of new spacecraft or space habitats. Spacecraft design must include requirements to control accumulation of water including humidity, leaks, and condensate on surfaces. Materials used in habitable volumes must not contribute to microbial growth. Use of appropriate materials and the implementation of robust housekeeping that utilizes periodic cleaning and disinfection will prevent high levels of microbial growth on surfaces. Air filtration can ensure low levels of bioaerosols and particulates in the breathing air. The use of physical and chemical steps to disinfect drinking water coupled with filtration can provide safe drinking water. Thorough preflight examination of flight crews, consumables, and the environment can greatly reduce pathogens in spacecraft. The advances in knowledge of living and working onboard the Space Shuttle formed the foundation for environmental microbiology requirements and operations for the International Space Station (ISS) and future spacecraft. Research conducted during the Space Shuttle Program resulted in an improved understanding of the effects of spaceflight on human physiology, microbial properties, and specifically the host-microbe interactions. Host-microbe interactions are substantially affected by spaceflight. Astronaut immune functions were found to be altered. Selected microorganisms were found to become more virulent during spaceflight. The increased knowledge gained on the Space Shuttle resulted in further studies of the host-microbe interactions on the ISS to determine if countermeasures were necessary. Lessons learned from the Space Shuttle Program were integrated into the ISS resulting in the safest space habitat to date.

Pierson, Duane L.↗
NASA NTRS2006

Instrument Would Detect and Collect Biological Aerosols

A proposed compact, portable instrument would sample micron-sized airborne particles, would discriminate between biological ones (e.g., bacteria) and nonbiological ones (e.g., dust particles), and would collect the detected biological particles for further analysis. The instrument is intended to satisfy a growing need for means of rapid, inexpensive collection of bioaerosols in a variety of indoor and outdoor settings. Purposes that could be served by such collection include detecting airborne pathogens inside buildings and their ventilation systems, measuring concentrations of airborne biological contaminants around municipal waste-processing facilities, monitoring airborne effluents from suspected biowarfare facilities, and warning of the presence of airborne biowarfare agents

Savoy, Steve↗
NASA NTRS2011

Characterization of Volume F Trash from Four Recent STS Missions: Microbial Occurrence, Numbers, and Identifications

The fate of space-generated solid wastes, including trash, for future missions is under consideration by NASA. Several potential treatment options are under active technology development. Potential fates for space-generated solid wastes: Storage without treatment; storage after treatment(s) including volume reduction, water recovery, sterilization, and recovery plus recycling of waste materials. For this study, a microbial characterization was made on trash returned from four recent STS missions. The material analyzed were 'Volume F' trash and other bags of accompanying trash. This is the second of two submitted papers on these wastes. This first one covered trash content, weight and water content. Upon receipt, usually within 2 days of landing, trash contents were catalogued and placed into categories: drink containers, food waste, personal hygiene items, and packaging materials, i.e., plastic film and duct tape. Microbial counts were obtained with cultivatable counts on agar media and direct counts using Acridine Orange fluorescent stain (AODC). Trash bag surfaces, 25 square cm , were also sampled. Direct counts were approximately 1 x 10(exp 6) microbes/square cm and cultivatable counts ranged from 1 x 10 to 1 X 10(exp 4) microbes/ square cm-2. Aerobic microbes, aerobic sporeformers, and yeasts plus molds were common for all four missions. Waste items from each category were placed into sterile ziplock bags and 1.5 L sterile DI water added. These were then dispersed by hand shaking for 2 min. prior to inoculation of count media or determining AODC. In general, cultivatable microbes were found in drinks, food wastes, and personal hygiene items. Direct counts were usually higher than cultivatable counts. Some pathogens were found: Staphylococcus auerus, Escherichia coli (fecal wastes). Count ranges: drink pouches - AODC 2 x 10(exp 6) to 1 X 10(exp 8) g(sub fw) (exp -1); cultivatable counts variable between missions; food wastes: Direct counts were close to aerobic plate counts. Counts ranged from 10(exp 6) to 10(exp 9) per g(sub fw). Identities of isolates from cultivation media were obtained using a Biolog Microbial ID System or microSEQ molecular ID methodology using an ABI3130 gene analyzer.

Strayer, Richard F.↗
NASA NTRS2005

NASA Tech Briefs, May 2005

Topics covered include: Fastener Starter; Multifunctional Deployment Hinges Rigidified by Ultraviolet; Temperature-Controlled Clamping and Releasing Mechanism; Long-Range Emergency Preemption of Traffic Lights; High-Efficiency Microwave Power Amplifier; Improvements of ModalMax High-Fidelity Piezoelectric Audio Device; Alumina or Semiconductor Ribbon Waveguides at 30 to 1,000 GHz; HEMT Frequency Doubler with Output at 300 GHz; Single-Chip FPGA Azimuth Pre-Filter for SAR; Autonomous Navigation by a Mobile Robot; Software Would Largely Automate Design of Kalman Filter; Predicting Flows of Rarefied Gases; Centralized Planning for Multiple Exploratory Robots; Electronic Router; Piezo-Operated Shutter Mechanism Moves 1.5 cm; Two SMA-Actuated Miniature Mechanisms; Vortobots; Ultrasonic/Sonic Jackhammer; Removing Pathogens Using Nano-Ceramic-Fiber Filters; Satellite-Derived Management Zones; Digital Equivalent Data System for XRF Labeling of Objects; Identifying Objects via Encased X-Ray-Fluorescent Materials - the Bar Code Inside; Vacuum Attachment for XRF Scanner; Simultaneous Conoscopic Holography and Raman Spectroscopy; Adding GaAs Monolayers to InAs Quantum-Dot Lasers on (001) InP; Vibrating Optical Fibers to Make Laser Speckle Disappear; Adaptive Filtering Using Recurrent Neural Networks; and Applying Standard Interfaces to a Process-Control Language.

Source record↗
NASA NTRS2011

Altered Innate and Lymphocytic Immune Responses in Mouse Splenocytes Post-Flight

Space flight is known to affect immune responses of astronauts and animals, decreasing lymphocytic responses to mitogenic stimuli, delayed typed hypersensitivity reactions, and T-cell activation. Despite changes in immune suppression, there are no reports of consistent adverse clinical events post flight. To further investigate the spectrum of affected immune responses, murine splenocytes were stimulated immediately post-shuttle flight (14 days on STS-135) with T-cell stimulators or toll-like receptor agonists. Comparisons were made to ground control splenocytes from age-matched mice. Cell phenotypes were assessed, as well as activation markers and associated cytokine production. The CD4+ population decreased with no concurrent decrease in CD8+ cells from shuttle mice post flight compared to ground controls. Regarding antigen presenting cell populations, the number of CD11c+ cells were slightly elevated post flight, compared to ground controls, with increased MHC Class I expression (I-A(sup b)) and no change in Class II expression (H-2K(sup b)). CD86+ populations were also significantly diminished. However, the decreased markers did not correlate with activity. Stimulation of splenocytes post flight showed significant increase in bead uptake, increased Class I expression, increased TNF-alpha and IL-6 production in response to TLR-2 (zymosan) and TLR-4 (LPS) agonists. While most activated (ConA or anti-CD3/anti-CD28) CD4+ cells showed markedly diminished responses (reduced IL-2 production), non-specific T cell responses to superantigen (SEA/SEB) increased post flight as determined by expression of early activation markers. Production of additional cytokines was also dysregulated postflight. Overall, persistent immune changes during space flight could represent unique clinical risks for exploration class missions. The consequences of pathogenic encounter remain an important concern that should be addressed.

Hwang, ShenAn↗
NASA NTRS2005

Electroporation System for Sterilizing Water

A prototype of an electroporation system for sterilizing wastewater or drinking water has been developed. In electroporation, applied electric fields cause transient and/or permanent changes in the porosities of living cells. Electroporation at lower field strengths can be exploited to increase the efficiency of chemical disinfection (as in chlorination). Electroporation at higher field strengths is capable of inactivating and even killing bacteria and other pathogens, without use of chemicals. Hence, electroporation is at least a partial alternative to chlorination. The transient changes that occur in micro-organisms at lower electric-field strengths include significantly increased uptake of ions and molecules. Such increased uptake makes it possible to achieve disinfection at lower doses of chemicals (e.g., chlorine or ozone) than would otherwise be needed. Lower doses translate to lower costs and reduced concentrations of such carcinogenic chemical byproducts as trichloromethane. Higher electric fields cause cell membranes to lose semipermeability and thereby become unable to function as selective osmotic barriers between the cells and the environment. This loss of function is the cause of the cell death at higher electric-field intensities. Experimental evidence does not indicate cell lysis but, rather, combined leaking of cell proteins out of the cells as well as invasion of foreign chemical compounds into the cells. The concept of electroporation is not new: it has been applied in molecular biology and genetic engineering for decades. However, the laboratory-scale electroporators used heretofore have been built around small (400-microliter) cuvettes, partly because the smallness facilitates the generation of electric fields of sufficient magnitude to cause electroporation. Moreover, most laboratory- scale electroporators have been designed for testing static water. In contrast, the treatment cell in the present system is much larger and features a flow-through geometry, such that electric fields strong enough to effect 99.9- percent disinfection can be applied to water flowing in a pipe.

Schlager, Kenneth J.↗
NASA NTRS2004

Ultraviolet-Absorption Spectroscopic Biofilm Monitor

An ultraviolet-absorption spectrometer system has been developed as a prototype instrument to be used in continuous, real-time monitoring to detect the growth of biofilms. Such monitoring is desirable because biofilms are often harmful. For example, biofilms in potable-water and hydroponic systems act as both sources of pathogenic bacteria that resist biocides and as a mechanism for deterioration (including corrosion) of pipes. Biofilms formed from several types of hazardous bacteria can thrive in both plant-growth solutions and low-nutrient media like distilled water. Biofilms can also form in condensate tanks in air-conditioning systems and in industrial heat exchangers. At present, bacteria in potable-water and plant-growth systems aboard the space shuttle (and previously on the Mir space station) are monitored by culture-plate counting, which entails an incubation period of 24 to 48 hours for each sample. At present, there are no commercially available instruments for continuous monitoring of biofilms in terrestrial or spaceborne settings.

Micheels, Ronald H.↗
NASA NTRS2011

Development of a Novel Self-Enclosed Sample Preparation Device for DNA/RNA Isolation in Space

Modern biology techniques present potentials for a wide range of molecular, cellular, and biochemistry applications in space, including detection of infectious pathogens and environmental contaminations, monitoring of drug-resistant microbial and dangerous mutations, identification of new phenotypes of microbial and new life species. However, one of the major technological blockades in enabling these technologies in space is a lack of devices for sample preparation in the space environment. To overcome such an obstacle, we constructed a prototype of a DNA/RNA isolation device based on our novel designs documented in the NASA New Technology Reporting System (MSC-24811-1/3-1). This device is self-enclosed and pipette free, purposely designed for use in the absence of gravity. Our design can also be modified easily for preparing samples in space for other applications, such as flowcytometry, immunostaining, cell separation, sample purification and separation according to its size and charges, sample chemical labeling, and sample purification. The prototype of our DNA/RNA isolation device was tested for efficiencies of DNA and RNA isolation from various cell types for PCR analysis. The purity and integrity of purified DNA and RNA were determined as well. Results showed that our developed DNA/RNA isolation device offers similar efficiency and quality in comparison to the samples prepared using the standard protocol in the laboratory.

Zhang, Ye↗
NASA NTRS2011

Characterization of Volume F Trash from the Three FY11 STS Missions: Trash Weights and Categorization and Microbial Characterization

The project reported here provides microbial characterization support to the Waste Management Systems (WMS) element of NASA's Life Support and Habitation Systems (LSHS) program. Conventional microbiological methods were used to detect and enumerate microorganisms in STS Volume F Compartment trash for three shuttle missions: STS 133, 134, and 135. This trash was usually made available within 2 days of landing at KSC. The Volume F bag was weighed, opened and the contents were cataloged and placed into categories: personal hygiene items - inclUding EVA maximum absorbent garments (MAGs) and Elbow packs (daily toilet wipes, etc), drink containers, food waste (and containers), office waste (paper), and packaging materials - plastic film and duct tape. The average wet trash generation rate for the three STS missions was 0.362 % 0.157 kgwet crew 1 d-1 . This was considerably lower and more variable than the average rate for 4 STS missions reported for FY10. Trash subtotals by category: personal hygiene wastes, 56%; drink items, 11 %; food wastes, 18%; office waste, 3%; and plastic film, 12%. These wastes have an abundance of easily biodegraded compounds that can support the growth of microorganisms. Microbial characterization of trash showed that large numbers of bacteria and fungi have taken advantage of this readily available nutrient source to proliferate. Exterior and interior surfaces of plastic film bags containing trash were sampled and counts of cultivatable microbes were generally low and mostly occurred on trash bundles within the exterior trash bags. Personal hygiene wastes, drink containers, and food wastes and packaging all contained high levels of, mostly, aerobic heterotrophic bacteria and lower levels of yeasts and molds. Isolates from plate count media were obtained and identified .and were mostly aerobic heterotrophs with some facultative anaerobes. These are usually considered common environmental isolates on Earth. However, several pathogens were also isolated: Staphylococcus aureus and Escherichia coli.

Strayer, Richard F.↗
NASA NTRS2005

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.↗
NASA NTRS2011

Radiation-Resistant Hybrid Lotus Effect for Achieving Photoelectrocatalytic Self-Cleaning Anticontamination Coatings

An experiment involving radiation-resistant hydrophobic coatings is planned for space exposure and experimental testing on the International Space Station (ISS) in 2011. The Lotus biocide coatings are designed for supporting space exploration missions. This innovation is an antibacterial, anti-contamination, and self-cleaning coating that uses nano-sized semiconductor semimetal oxides to neutralize biological pathogens and toxic chemicals, as well as to mitigate dust accumulation (see figure). The Lotus biocide coating is thin (approximately microns thick), lightweight, and the biocide properties will not degrade with time or exposure to biological or chemical agents. The biocide is stimulated chemically (stoichiometric reaction) through exposure to light (photocatalysis), or by an applied electric field (electrocatalysis). The hydrophobic coating samples underwent preliminary high-energy proton and alpha-ray (helium ion) irradiations at the Lawrence Berkeley National Laboratory 88" cyclotron and demonstrated excellent radiation resistance for a portion of the Galactic Cosmic Ray (GRC) and Solar Proton spectrum. The samples will undergo additional post-flight studies when returned to Earth to affirm further the radiation resistance properties of the space exposed coatings.

Taylor, Edward W.↗
NASA NTRS2011

Cytometer on a Chip

A cytometer now under development exploits spatial sorting of sampled cells on a microarray chip followed by use of grating-coupled surface-plasmon-resonance imaging (GCSPRI) to detect the sorted cells. This cytometer on a chip is a prototype of contemplated future miniature cytometers that would be suitable for rapidly identifying pathogens and other cells of interest in both field and laboratory applications and that would be attractive as alternatives to conventional flow cytometers. The basic principle of operation of a conventional flow cytometer requires fluorescent labeling of sampled cells, stringent optical alignment of a laser beam with a narrow orifice, and flow of the cells through the orifice, which is subject to clogging. In contrast, the principle of operation of the present cytometer on a chip does not require fluorescent labeling of cells, stringent optical alignment, or flow through a narrow orifice. The basic principle of operation of the cytometer on a chip also reduces the complexity, mass, and power of the associated laser and detection systems, relative to those needed in conventional flow cytometry. Instead of making cells flow in single file through a narrow flow orifice for sequential interrogation as in conventional flow cytometry, a liquid containing suspended sampled cells is made to flow over the front surface of a microarray chip on which there are many capture spots. Each capture spot is coated with a thin (.50-nm) layer of gold that is, in turn, coated with antibodies that bind to cell-surface molecules characteristic of the cell species of interest. The multiplicity of capture spots makes it possible to perform rapid, massively parallel analysis of a large cell population. The binding of cells to each capture spot gives rise to a minute change in the index of refraction at the surface of the chip. This change in the index of refraction is what is sensed in GCSPRI, as described briefly below. The identities of the various species in a sample of cells is spatially encoded in the chip by the pattern of capture spots. The number of cells of a particular species is determined from the magnitude of the GCSPRI signal from that spot. GCSPRI as used here can be summarized as follows: The cytometer chip is fabricated with a diffraction grating on its front surface. The chip is illuminated with a light emitting diode (LED) from the front. By proper choice of grating parameters and of the wavelength and the angle of incidence of a laser beam, laser light can be made to be coupled into an electromagnetic mode that resonates with surface plasmons and thus couples light into surface plasmons. Coupling of light into a surface plasmon at a given location reduces the amount of incident light reflected from that location. A change in the index of refraction at the surface of a capture spot gives rise to a change in the resonance condition. Depending on the specific design, the change in the index of refraction could manifest itself as a brightening or darkening, a change in the wavelength needed to excite the plasmon at a given angle of incidence, or a change in the angle of incidence needed to excite the plasmon at a given wavelength. Whereas a multiwavelength laser system with multichannel detection would be needed to detect multiple species in conventional flow cytometry, it suffices to use an LED and a single detector channel in the GCSPRI approach: this contributes significantly to reductions in cost, complexity, size, mass, and power. GCSPRI cytometer chips could be made of plastic and could be mass-produced cheaply by use of molding and other methods adopted from the manufacture of digital video disks. These methods are amenable to a high degree of miniaturization: such additional features as fluidic channels, reaction chambers, and fluid-coupling ports could readily be incorporated into the chips, without incurring substantial additional costs.

Fernandez, Salvador M.↗
NASA NTRS2011

PMA-PhyloChip DNA Microarray to Elucidate Viable Microbial Community Structure

Since the Viking missions in the mid-1970s, traditional culture-based methods have been used for microbial enumeration by various NASA programs. Viable microbes are of particular concern for spacecraft cleanliness, for forward contamination of extraterrestrial bodies (proliferation of microbes), and for crew health/safety (viable pathogenic microbes). However, a "true" estimation of viable microbial population and differentiation from their dead cells using the most sensitive molecular methods is a challenge, because of the stability of DNA from dead cells. The goal of this research is to evaluate a rapid and sensitive microbial detection concept that will selectively estimate viable microbes. Nucleic acid amplification approaches such as the polymerase chain reaction (PCR) have shown promise for reducing time to detection for a wide range of applications. The proposed method is based on the use of a fluorescent DNA intercalating agent, propidium monoazide (PMA), which can only penetrate the membrane of dead cells. The PMA-quenched reaction mixtures can be screened, where only the DNA from live cells will be available for subsequent PCR reaction and microarray detection, and be identified as part of the viable microbial community. An additional advantage of the proposed rapid method is that it will detect viable microbes and differentiate from dead cells in only a few hours, as opposed to less comprehensive culture-based assays, which take days to complete. This novel combination approach is called the PMA-Microarray method. DNA intercalating agents such as PMA have previously been used to selectively distinguish between viable and dead bacterial cells. Once in the cell, the dye intercalates with the DNA and, upon photolysis under visible light, produces stable DNA adducts. DNA cross-linked in this way is unavailable for PCR. Environmental samples suspected of containing a mixture of live and dead microbial cells/spores will be treated with PMA, and then incubated in the dark. Thereafter, the sample is exposed to visible light for five minutes, so that the DNA from dead cells will be cross-linked. Following this PMA treatment step, the sample is concentrated by centrifugation and washed (to remove excessive PMA) before DNA is extracted. The 16S rRNA gene fragments will be amplified by PCR to screen the total microbial community using PhyloChip DNA microarray analysis. This approach will detect only the viable microbial community since the PMA intercalated DNA from dead cells would be unavailable for PCR amplification. The total detection time including PCR reaction for low biomass samples will be a few hours. Numerous markets may use this technology. The food industry uses spore detection to validate new alternative food processing technologies, sterility, and quality. Pharmaceutical and medical equipment companies also detect spores as a marker for sterility. This system can be used for validating sterilization processes, water treatment systems, and in various public health and homeland security applications.

Venkateswaran, Kasthuri J.↗
NASA NTRS2011

Microbial Characterization of Solid-Wastes Treated with Heat Melt Compaction Technology

The research purpose of the project was to determine the fate of microorganisms in space-generated solid wastes after processing by a Heat Melt Compactor (HMC), which is a candidate solid waste treatment technology. Five HMC product disks were generated at Ames Research Center (ARC), Waste Management Systems element. The feed for two was simulated space-generated trash and feed for three was Volume F compartment wet waste returned on STS 130. Conventional microbiological methods were used to detect and enumerate microorganisms in HMC disks and in surface swab samples of HMC hardware before and after operation. Also, biological indicator test strips were added to the STS trash prior to compaction to test if HMC processing conditions, 150 C for approx 3 hr and dehydration, were sufficient to eliminate the test bacteria on the strips. During sample acquisition at KSC, the HMC disk surfaces were sanitized with 70% alcohol to prevent contamination of disk interiors. Results from microbiological assays indicated that numbers of microbes were greatly reduced but not eliminated by the 70% alcohol. Ten 1.25 cm diameter cores were aseptically cut from each disk to sample the disk interior. The core material was run through the microbial characterization analyses after dispersal in sterile diluent. Low counts of viable bacteria (5 to 50 per core) were found but total direct counts were 6 to 8 orders of magnitude greater. These results indicate that the HMC operating conditions might not be sufficient for complete waste sterilization, but the vast majority of microbes present in the wastes were dead or non-cultivable after HMC treatment. The results obtained from analyses of the commercial spore test strips that had been added fo the wastes prior to HMC operation further indicated that the HMC was sterilizing the wastes. Nearly all strips were recovered from the HMC disks and all of these were negative for spore growth when run through the manufacturer's protocol. The 10(exp 6) or so spores impregnated into the strips were no longer viable. Control test strips, i.e., not exposed to the HMC conditions, were all strongly positive. All isolates from the cultivable counts were identified, leading to one concern: several were identified as Staphylococcus aureus, a human pathogen. The project reported here provides microbial characterization support to the Waste Management Systems element of the Life Support and Habitation Systems program.

Strayer, Richard F.↗
NASA NTRS2011

Cytometer on a Chip

A cytometer now under development exploits spatial sorting of sampled cells on a microarray chip followed by use of grating-coupled surface-plasmon-resonance imaging (GCSPRI) to detect the sorted cells. This cytometer on a chip is a prototype of contemplated future miniature cytometers that would be suitable for rapidly identifying pathogens and other cells of interest in both field and laboratory applications and that would be attractive as alternatives to conventional flow cytometers. The basic principle of operation of a conventional flow cytometer requires fluorescent labeling of sampled cells, stringent optical alignment of a laser beam with a narrow orifice, and flow of the cells through the orifice, which is subject to clogging. In contrast, the principle of operation of the present cytometer on a chip does not require fluorescent labeling of cells, stringent optical alignment, or flow through a narrow orifice. The basic principle of operation of the cytometer on a chip also reduces the complexity, mass, and power of the associated laser and detection systems, relative to those needed in conventional flow cytometry. Instead of making cells flow in single file through a narrow flow orifice for sequential interrogation as in conventional flow cytometry, a liquid containing suspended sampled cells is made to flow over the front surface of a microarray chip on which there are many capture spots. Each capture spot is coated with a thin (approximately 50-nm) layer of gold that is, in turn, coated with antibodies that bind to cell-surface molecules characteristic of one the cell species of interest. The multiplicity of capture spots makes it possible to perform rapid, massively parallel analysis of a large cell population. The binding of cells to each capture spot gives rise to a minute change in the index of refraction at the surface of the chip. This change in the index of refraction is what is sensed in GCSPRI, as described briefly below. The identities of the various species in a sample of cells is spatially encoded in the chip by the pattern of capture spots. The number of cells of a particular species is determined from the magnitude of the GCSPRI signal from that spot. GCSPRI as used here can be summarized as follows: The cytometer chip is fabricated with a diffraction grating on its front surface. The chip is illuminated with a light emitting diode (LED) from the front. By proper choice of grating parameters and of the wavelength and the angle of incidence of a laser beam, laser light can be made to be coupled into an electromagnetic mode that resonates with surface plasmons and thus couples light into surface plasmons. Coupling of light into a surface plasmon at a given location reduces the amount of incident light reflected from that location. A change in the index of refraction at the surface of a capture spot gives rise to a change in the resonance condition. Depending on the specific design, the change in the index of refraction could manifest itself as a brightening or darkening, a change in the wavelength needed to excite the plasmon at a given angle of incidence, or a change in the angle of incidence needed to excite the plasmon at a given wavelength. Whereas a multiwavelength laser system with multichannel detection would be needed to detect multiple species in conventional flow cytometry, it suffices to use an LED and a single detector channel in the GCSPRI approach: this contributes significantly to reductions in cost, complexity, size, mass, and power. GCSPRI cytometer chips could be made of plastic and could be mass-produced cheaply by use of molding and other methods adopted from the manufacture of digital video disks. These methods are amenable to a high degree of miniaturization: such additional features as fluidic channels, reaction chambers, and fluid-coupling ports could readily be incorporated into the chips, without incurring substantial additional costs.

Fernandez, Salvador M.↗