THE EFFECT OF CO SUB 2 ON PH IN CULTURE MEDIA FOR ALGAE
Effect of carbon dioxide on ph of algal culture media
ALGAE↗
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Microfluidic droplets with amended culture media cultivate a greater diversity of soil microorganisms
ABSTRACT Uncultivated but abundant soil microorganisms have untapped potential for producing broad ranges of natural products, as well as for bioremediation. However, cultivating soil microorganisms while maintaining a broad microorganism diversity to enable phenotyping and functional analysis of as diverse individual isolates as possible remains challenging. In this study, we developed and tested the ability of several culture media formulations that contain defined soil metabolites or soil extracts to maintain microorganism diversity during culture. We also assessed their performance in microfluidic droplet cultivation where single-soil microorganism isolates were encapsulated and cultivated in picoliter-volume water-in-oil emulsion droplets to enable clonal growth needed for downstream functional analyses. Our results show that droplet cultivation with media supplemented by soil extract or soil metabolites enables the recovery of soil microorganisms with higher diversity (up to 1.5-fold higher richness) compared to bulk cultivation methods. Importantly, 1.7-fold more of less abundant (<1%) phyla and 11-fold more of unique genera were recovered, demonstrating the utility of this method for interrogating highly diverse soil microorganisms for broad ranges of applications. IMPORTANCE Although soil microorganisms hold a significant value in bioproduction and bioremediation, only a small fraction—less than 1%—can be cultured under specific media and cultivation conditions. This indicates that there are ample opportunities in harvesting the diverse environmental microorganisms if isolating and recovering these uncultured microorganisms are possible. This paper presents a new cultivation technique composed of isolating single-soil microorganism cell from anin situsoil microorganism community in microfluidic droplets and conducting in-droplet cultivation in media supplemented by soil extract or soil metabolites. This method enables the recovery of a broader diversity of the original microorganism community, laying the groundwork for a high-throughput phenotyping of these diverse microorganisms from their natural habitats.
Biotechnology & Applied Microbiology↗
Influence of culture media on the radiation resistance of Micrococcus radiodurans.
Culture medium effect on radiation resistance of microorganism Micrococcus radiodurans
Anderson, A. W.↗
Pathogen Propagation in Cultured Three-Dimensional Tissue Mass
A process for propagating a pathogen in a three-dimensional tissue mass cultured at microgravity conditions in a culture vessel containing culture media and a culture matrix is provided. The three-dimensional tissue mass is inoculated with a pathogen and pathogen replication in the cells of the tissue mass achieved.
Thomas J Goodwin↗
A Comprehensive Characterization of Microorganisms and Allergens in Spacecraft Environment
The determination of risk from infectious disease during long-duration missions is composed of several factors including (1) the host#s susceptibility, (2) the host#s exposure to the infectious disease agent, and (3) the concentration of the infectious agent, and (4) the characteristics of the infectious agent. While stringent steps are taken to minimize the transfer of potential pathogens to spacecraft, several medically significant organisms have been isolated from both the Mir and International Space Station (ISS). Historically, the method for isolation and identification of microorganisms from spacecraft environmental samples depended upon their growth on culture media. Unfortunately, only a fraction of the organisms may grow on a culture medium, potentially omitting those microorganisms whose nutritional and physical requirements for growth are not met. Thus, several pathogens may not have been detected, such as Legionella pneumophila, the etiological agent of Legionnaire#s disease. We hypothesize that environmental analysis using non-culture-based technologies will reveal microorganisms, allergens, and microbial toxins not previously reported in spacecraft, allowing for a more complete health assessment. The development of techniques for this flight experiment, operationally named SWAB, has already provided advances in NASA laboratory processes and beneficial information toward human health risk assessment. The first accomplishment of the SWAB experiment was the incorporation of 16S ribosomal DNA sequencing for the identification of bacteria. The use of this molecular technique has increased bacterial speciation of environmental isolates from previous flights three fold compared to conventional methodology. This increased efficiency in bacterial speciation provides a better understanding of the microbial ecology and the potential risk to the crew. Additional SWAB studies focused on the use of molecular-based DNA fingerprinting using repetitive sequencebased polymerase chain reaction (rep-PCR). This technology has allowed contamination tracking of microorganisms between crewmembers and their environment. This study not only demonstrated that ISS has a greater diversity of organisms than originally expected, but also provided insight into possible routes of infection to the crew. Additional ground-based studies used rep-PCR and protein based assays to determine the potential of methicillin resistant Staphylococcus aureus (MRSA) aboard ISS. MRSA has become increasingly common on Earth and pose a treatment problem for infections during flight. While no MRSA have been isolated from ISS to date, the mecA gene product that is responsible for methicillin resistance was isolated in other Staphylococcus species aboard ISS suggesting a potential of MRSA through gene transfer. Using improved sample collection technologies, flight sampling for SWAB was initiated in August 2006 and should continue through spring of 2007. The focus of these flight samples is the collection of DNA for evaluation by Denaturing Gradient Gel Electrophoresis (DGGE). Unlike other techniques, DGGE does not depend on any microbial growth on culture media allowing a more comprehensive assessment of the spacecraft interior. This study should provide insight into the true microbial ecology that is experienced by the crew during flight. This information will lead toward an accurate microbial risk assessment to help set flight requirements to protect the safety, health, and performance of the crew.
Ott, C. M.↗
Pathogen propagation in cultured three-dimensional tissue mass
A process for propagating a pathogen in a three-dimensional tissue mass cultured at microgravity conditions in a culture vessel containing culture media and a culture matrix is provided. The three-dimensional tissue mass is inoculated with a pathogen and pathogen replication in the cells of the tissue mass achieved.
Goodwin, Thomas J.↗
A Comprehensive Characterization of Microorganisms and Allergens in Spacecraft Environment
The determination of risk from infectious disease during long-duration missions is composed of several factors including the concentration and the characteristics of the infectious agent. Thus, a thorough knowledge of the microorganisms aboard spacecraft is essential in mitigating infectious disease risk to the crew. While stringent steps are taken to minimize the transfer of potential pathogens to spacecraft, several medically significant organisms have been isolated from both the Mir and International Space Station (ISS). Historically, the method for isolation and identification of microorganisms from spacecraft environmental samples depended upon their growth on culture media. Unfortunately, only a fraction of the organisms may grow on a culture medium, potentially omitting those microorganisms whose nutritional and physical requirements for growth are not met. Thus, several pathogens may not have been detected, such as Legionella pneumophila, the etiological agent of Legionnaire s disease. We hypothesize that environmental analysis using non-culture-based technologies will reveal microorganisms, allergens, and microbial toxins not previously reported in spacecraft, allowing for a more complete health assessment. The development of techniques for this flight experiment, operationally named SWAB, has already provided advances in NASA laboratory processes and beneficial information toward human health risk assessment. The translation of 16S ribosomal DNA sequencing for the identification of bacteria from the SWAB experiment to nominal operations has increased bacterial speciation of environmental isolates from previous flights three fold compared to previous conventional methodology. The incorporation of molecular-based DNA fingerprinting using repetitive sequence-based polymerase chain reaction (rep-PCR) into the capabilities of the laboratory has provided a methodology to track microorganisms between crewmembers and their environment. Both 16S ribosomal DNA identification and bacterial fingerprinting have improved NASA s capability to better understand spacecraft environments and determine the source of contamination events. Preflight sampling has been completed for air, surface, and water samples. In-flight sample collection has been completed for a total of 8 air and surface sample collection sessions. In-flight hardware has performed well and the surface sampling device received positive feedback from the crew for its ease of use. While processing and analysis continue for these samples, early results have begun to provide information on the spacecraft environment. Using a method called Denaturing Gradient Gel Electrophoresis (DGGE), several air and samples were evaluated to determine the types of organisms that were present. Using only molecular techniques, DGGE does not depend on any microbial growth on culture media, allowing a more comprehensive assessment of the spacecraft interior. Preliminary results have identified several microorganisms that would not have been isolated using current technology, though none of these organisms would be considered medically significant. Interestingly, the isolation of Gram negative organisms is greater using DGGE than conventional media based isolation. The cause of this finding is unclear, though it may be the result of the technique s ability to isolate both viable and non-viable bacteria. The next phase of the SWAB sample analysis is the use of quantitative polymerase chain reaction (QPCR) to look for specific medically significant organisms. While not as broad as DGGE, QPCR is much more sensitive and may reveal findings that were not seen during the initial evaluation. Together, this information will lead toward an accurate microbial risk assessment to help set flight requirements to protect the safety, health, and performance of the crew.
Castro, V.A.↗
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.↗
Analysis of gravity-induced particle motion and fluid perfusion flow in the NASA-designed rotating zero-head-space tissue culture vessel
The gravity induced motions, through the culture media, is calculated of living tissue segments cultured in the NASA rotating zero head space culture vessels. This is then compared with the media perfusion speed which is independent of gravity. The results may be interpreted as a change in the physical environment which will occur by operating the NASA tissue culture systems in actual microgravity (versus unit gravity). The equations governing particle motions which induce flows at the surface of tissues contain g terms. This allows calculation of the fluid flow speed, with respect to a cultured particle, as a function of the external gravitational field strength. The analysis is approached from a flow field perspective. Flow is proportional to the shear exerted on a structure which maintains position within the field. The equations are solved for the deviation of a particle from its original position in a circular streamline as a function of time. The radial deviation is important for defining the operating limits and dimensions of the vessel because of the finite radius at which particles necessarily intercept the wall. This analysis uses a rotating reference frame concept.
Wolf, David A.↗
Effect of Serum from Chickens Treated with Clenbuterol on Myosin Accumulation, Beta-Adrenergic Receptor Population, and Cyclic AM Synthesis in Embryonic Chicken Skeletal Muscle Cell Cultures
Broiler chickens at 35 days of age were fed 1 ppm clenbuterol for 14 days. This level of dietary clenbuterol led to 5-7% increases in weights of leg and breast muscle tissue. At the end of the 14-day period, serum was prepared from both control and clenbuterol-treated chickens and was then employed as a component of cell culture media at a final concentration of 20% (v/v). Muscle cell cultures were prepared from both the leg and breast muscle groups of twelve-day chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 micron clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 days beginning on the seventh day in culture. Neither the percent fusion nor the number of nuclei in myotubes were significantly affected by any of the treatments. The quantity of MHC was not increased by serum from clenbuterol-treated chickens in either breast and leg muscle cultures; however, MHC quantity was 50- 100% higher in cultures grown in control chicken serum to which 10 nM and 50 nM clenbuterol had also been added. The Beta-AR population was 4,000-7,000 Beta-AR per cell in cultures grown in chicken serum, with leg muscle cultures having approximately 25-30% more receptors than breast muscle cultures. Receptor population was not significantly affected by the presence of clenbuterol or by the presence of serum from clenbuterol-treated chickens. In contrast, the Beta-AR population in leg and breast muscle cultures grown in the presence of 10% horse serum was 18,000-20,000 Beta-AR per cell. Basal concentration of cAMP was not significantly affected by any of the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 micron isoproterenol, limited increases of 12-20% in cAMP concentration above basal levels were observed. However, when cultures grown in the presence of horse serum were stimulated with 1 micron isoproterenol, increases of 600-800 % in cAMP concentration above basal levels were observed. Thus, not only did cells grown in horse serum have a higher Beta-AR population, each receptor had a higher capacity for cAMP synthesis following isoproterenol stimulation. Finally, the hypothesis was tested that clenbuterol exerts its action on muscle protein content by changes in cAMP concentration. No correlation was apparent between basal cAMP concentration and MHC content.
Young, R. B.↗
Effect of Serum from Chickens Treated with Clenbuterol on Myosin Accumulation, Beta-Adrenergic Receptor Population, and Cyclic AMP Synthesis in Embryonic Chicken Skeletal Muscle Cell Cultures
Broiler chickens at 35 d of age were fed 1 ppm clenbuterol for 14 d. This level of dietary clenbuterol led to 5-7% increases in the weights of leg and breast muscle tissue. At the end of the 14-d period, serum was prepared from both control and clenbuterol-treated chickens, and was then employed as a component of cell culture media at a final concentration of 20% (v/v). Muscle cell cultures were prepared from both the leg and the breast muscle groups of 12-d chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 uM clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 d, beginning on the seventh d in culture. Neither the percent fusion nor the number of nuclei in myotubes was significantly affected by any of the treatments. The quantity of myosin heavy chains (MHCs) was not increased by serum from clenbuterol-treated chickens in either breast or leg muscle cultures; however, the MHC quantity was 50-150% higher in cultures grown in control chicken serum to which 10 and 50 nM clenbuterol had also been added. The B-adrenergic receptor (betaAR) population was 4000-7000 betaARs per cell in cultures grown in chicken serum with leg muscle cultures having approximately 25-30% more receptors than breast muscle Culture. Receptor population was not significantly affected by the presence of clenbuterol or by the presence of serum from clenbuterol-treated chickens. In contrast, the betaAR Population in leg and breast muscle cultures grown in the presence of 10% horse serum was 16,000-18,000 betaARs per cell. Basal concentration of cyclic adenosine 3':5'monophosphate (cAMP) was not significantly affected by the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 uM isoproterenol, limited increases of 12-20% in cAMP Concentration above the. basal levels were observed. However, when cultures grown in the presence of horse serum were stimulated with 1 uM isoproterenol, cAMP concentration was stimulated 5- to 9-fold above the basal levels. Thus, not only did cells, grown in horse serum have a higher PAR population, but also each receptor had a higher capacity for cAMP synthesis following isoproterenol stimulation. Finally, the hypothesis that clenbuterol exerts its action on muscle protein content by changes in cAMP concentration was tested. No correlation was apparent between basal cAMP concentration and MHC content.
Young, Ronald B.↗
Biona-C Cell Culture pH Monitoring System
Sensors 2000! is developing a system to demonstrate the ability to perform accurate, real-time measurements of pH and CO2 in a cell culture media in Space. The BIONA-C Cell Culture pH Monitoring System consists of S2K! developed ion selective sensors and control electronics integrated with the fluidics of a cell culture system. The integrated system comprises a "rail" in the Cell Culture Module (CCM) of WRAIR (Space Biosciences of Walter Read Army Institute of Research). The CCM is a Space Shuttle mid-deck locker experiment payload. The BIONA-C is displayed along with associated graphics and text explanations. The presentation will stimulate interest in development of sensor technology for real-time cell culture measurements. The transfer of this technology to other applications will also be of interest. Additional information is contained in the original document.
Friedericks, C.↗
Effects of light on protein patterns in gravitropically stimulated root caps of corn
In certain cultivars of corn (Zea mays var. Merit), light stimulates gravitropic bending of the root by influencing events in the root cap. In this paper, we report on changes in root cap proteins which occur as a result of the light treatment and single out specific proteins as potentially having a role in mediating the gravitropic response. For this work, we have used root caps maintained aseptically in culture media supplemented with auxin. If auxin is deleted from the culture medium, the protein profiles observed following illumination differ from that seen in caps provided light while in auxin-supplemented media. We also report that several of the proteins for which synthesis is stimulated by light appear to turn over rapidly, usually within 0.5 hour of formation.
Gravitropism/physiology/radiation effects↗
Surface, Water, and Air Biocharacterization (SWAB) Flight Experiment
The determination of risk from infectious disease during spaceflight missions is composed of several factors including both the concentration and characteristics of the microorganisms to which the crew are exposed. Thus, having a good understanding of the microbial ecology aboard spacecraft provides the necessary information to mitigate health risks to the crew. While preventive measures are taken to minimize the presence of pathogens on spacecraft, medically significant organisms have been isolated from both the Mir and International Space Station (ISS). Historically, the method for isolation and identification of microorganisms from spacecraft environmental samples depended upon their growth on culture media. Unfortunately, only a fraction of the organisms may grow on a specific culture medium, potentially omitting those microorganisms whose nutritional and physical requirements for growth are not met. To address this bias in our understanding of the ISS environment, the Surface, Water, and Air Biocharacterization (SWAB) Flight Experiment was designed to investigate and develop monitoring technology to provide better microbial characterization. For the SWAB flight experiment, we hypothesized that environmental analysis using non-culture-based technologies would reveal microorganisms, allergens, and microbial toxins not previously reported in spacecraft, allowing for a more complete health assessment. Key findings during this experiment included: a) Generally, advanced molecular techniques were able to reveal a few organisms not recovered using culture-based methods; however, there is no indication that current monitoring is "missing" any medically significant bacteria or fungi. b) Molecular techniques have tremendous potential for microbial monitoring, however, sample preparation and data analysis present challenges for spaceflight hardware. c) Analytical results indicate that some molecular techniques, such as denaturing gradient gel electrophoresis (DGGE), can be much less sensitive than culture-based methods. d) More sensitive molecular techniques, such as quantitative polymerase chain reaction (QPCR), were able to identify viral DNA from ISS environments, suggesting potential transfer of the organism between crewmembers. In addition, the hardware selected for this experiment represented advances for next-generation sample collection. The advanced nature of this collection hardware was noted, when the Sartorius MD8 Air Port air sampler from the SWAB experiment remained on board ISS at the request of JAXA investigators, who intend to use it in completion of their microbial ecology experiment.
Castro, V. A.↗
Ecology and life history of an amoebomastigote, Paratetramitus jugosus, from a microbial mat: new evidence for multiple fission
Five microbial habitats (gypsum crust, gypsum photosynthetic community, Microcoleus mat, Thiocapsa scum, and black mud) were sampled for the presence of the euryhaline, rapidly growing amoebomastigote, Paratetramitus jugosus. Field investigations of microbial mats from Baja California Norte, Mexico, and Salina Bido near Matanzas, Cuba, reveal that P. jugosus is most frequently found in the Thiocapsa layer of microbial mats. Various stages of the life history were studied using phase-contrast, differential-interference, and transmission electron microscopy. Mastigote stages were induced and studied by electron microscopy; mastigotes that actively feed on bacteria bear two or more undulipodia. A three-dimensional drawing of the kinetid ("basal apparatus") based on electron micrographs is presented. Although promitoses were occasionally observed, it is unlikely that they can account for the rapid growth of P. jugosus populations on culture media. Dense, refractile, spherical, and irregular-shaped bodies were seen at all times in all cultures along with small mononucleate (approximately 2-7 micrometers diameter) amoebae. Cytochemical studies employing two different fluorescent stains for DNA (DAPI, mithramycin) verified the presence of DNA in these small bodies. Chromatin-like material seen in electron micrographs within the cytoplasm and blebbing off nuclei were interpreted to the chromatin bodies. Our interpretation, consistent with the data but not proven, is that propagation by multiple fission of released chromatin bodies that become small amoebae may occur in Paratetramitus jugosus. These observations are consistent with descriptions of amoeba propagules in the early literature (Hogue, 1914).
Non-NASA Center↗
Improved Whole-Blood-Staining Device
Dramatic improvements have been made in NASA s Whole Blood Staining Device (WBSD) since it was last described in "Whole-Blood-Staining Device," NASA Tech Briefs, Vol. 23, No. 10 (October 1999), page 64. The new system has a longer shelf life, a simpler and more effective operational procedure, improved interface with instrumentation, and shorter processing time. More specifically, the improvements have targeted bag and locking clip materials, sampling ports, and air pocket prevention. The WBSD stains whole blood collected during spaceflight for subsequent flow cytometric analysis. In short, the main device stains white blood cells by use of monoclonal antibodies conjugated to various fluorochromes, followed by lysing and fixing of the cells by use of a commercial reagent that has been diluted according to NASA safety standards. This system is compact, robust, and does not require electric power, precise mixing, or precise incubation times. Figure 1 depicts the present improved version for staining applications, which is a poly(tetrafluoroethylene) bag with a Luer-lock port and plastic locking clips. An InterLink (or equivalent) intravenous- injection port screws into the Luer-lock port. The inflatable/collapsible nature of the bag facilitates loading and helps to minimize the amount of air trapped in the fully loaded bag. Some additional uses have been identified for the device beyond whole blood staining. The WBSD has been configured for functional assays that require culture of live cells by housing sterile culture media, mitogens, and fixatives prior to use [Figure 2(a)]. Simple injection of whole blood allows cell-stimulation culture to be performed in reduced gravity conditions, and product stabilization prior to storage, while protecting astronauts from liquid biohazardous materials. Also, the improved WBSD has reconstituted powdered injectable antibiotics by mixing them with diluent liquids [Figure 2(b)]. Although such mixing can readily be performed on Earth by shaking in glass vials, it cannot readily be performed this way in outer space without entraining air bubbles. The present device can be preloaded with the powder and diluent(s) in separate compartments. The powder and diluent( s) can be mixed, without introducing air bubbles, by removing the clip(s), then shaking. This use of the device could also be advantageous in terrestrial applications because it maintains the isolation of the constituents until the time of use.
Sams, Clarence F.↗
Reduced-Gravity Experiments Conducted to Help Bioreactor Development
The NASA Glenn Research Center and the NASA Johnson Space Center are collaborating on fluid dynamic investigations for a future cell science bioreactor to fly on the International Space Station (ISS). Project Manager Steven Gonda from the Cellular Biotechnology Program at Johnson is leading the development of the Hydrodynamic Focusing Bioreactor--Space (HFB-S) for use on the ISS to study tissue growth in microgravity. Glenn is providing microgravity fluid physics expertise to help with the design and evaluation of the HFB-S. These bioreactors are used for three-dimensional tissue culture, which cannot be done in ground-based labs in normal gravity. The bioreactors provide a continual supply of oxygen for cell growth, as well as periodic replacement of cell culture media with nutrients. The bioreactor must provide a uniform distribution of oxygen and nutrients while minimizing the shear stresses on the tissue culture.
Niederhaus, Charles E.↗
Hollow-Fiber Clinostat
Hollow-fiber clinostat, is bioreactor used to study growth and other behavior of cells in simulated microgravity. Cells under study contained in porous hollow fiber immersed in culture medium inside vessel. Bores in hollow fiber allow exchange of gases, nutrients, and metabolic waste products between living cells and external culture media. Hollow fiber lies on axis of vessel, rotated by motor equipped with torque and speed controls. Desired temperature maintained by operating clinostat in standard tissue-culture incubator. Axis of rotation made horizontal or vertical. Designed for use with conventional methods of sterilization and sanitation to prevent contamination of specimen. Also designed for asepsis in assembly, injection of specimen, and exchange of medium.
Rhodes, Percy H.↗