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Sample records for single isolated microorganisms

  1. Development of a facile droplet-based single-cell isolation platform for cultivation and genomic analysis in microorganisms

    PubMed Central

    Zhang, Qiang; Wang, Tingting; Zhou, Qian; Zhang, Peng; Gong, Yanhai; Gou, Honglei; Xu, Jian; Ma, Bo

    2017-01-01

    Wider application of single-cell analysis has been limited by the lack of an easy-to-use and low-cost strategy for single-cell isolation that can be directly coupled to single-cell sequencing and single-cell cultivation, especially for small-size microbes. Herein, a facile droplet microfluidic platform was developed to dispense individual microbial cells into conventional standard containers for downstream analysis. Functional parts for cell encapsulation, droplet inspection and sorting, as well as a chip-to-tube capillary interface were integrated on one single chip with simple architecture, and control of the droplet sorting was achieved by a low-cost solenoid microvalve. Using microalgal and yeast cells as models, single-cell isolation success rate of over 90% and single-cell cultivation success rate of 80% were demonstrated. We further showed that the individual cells isolated can be used in high-quality DNA and RNA analyses at both gene-specific and whole-genome levels (i.e. real-time quantitative PCR and genome sequencing). The simplicity and reliability of the method should improve accessibility of single-cell analysis and facilitate its wider application in microbiology researches. PMID:28112223

  2. Diffusion-controlled metabolism for long-term survival of single isolated microorganisms trapped within ice crystals.

    PubMed

    Rohde, Robert A; Price, P Buford

    2007-10-16

    Two known habitats for microbial metabolism in ice are surfaces of mineral grains and liquid veins along three-grain boundaries. We propose a third, more general, habitat in which a microbe frozen in ice can metabolize by redox reactions with dissolved small molecules such as CO(2), O(2), N(2), CO, and CH(4) diffusing through the ice lattice. We show that there is an adequate supply of diffusing molecules throughout deep glacial ice to sustain metabolism for >10(5) yr. Using scanning fluorimetry to map proteins (a proxy for cells) and F420 (a proxy for methanogens) in ice cores, we find isolated spikes of fluorescence with intensity consistent with as few as one microbial cell in a volume of 0.16 microl with the protein mapper and in 1.9 microl with the methanogen mapper. With such precise localization, it should be possible to extract single cells for molecular identification.

  3. Single cell genomics of subsurface microorganisms

    NASA Astrophysics Data System (ADS)

    Stepanauskas, R.; Onstott, T. C.; Lau, C.; Kieft, T. L.; Woyke, T.; Rinke, C.; Sczyrba, A.; van Heerden, E.

    2012-12-01

    Recent studies have revealed unexpected abundance and diversity of microorganisms in terrestrial and marine subsurface, providing new perspectives over their biogeochemical significance, evolution, and the limits of life. The now commonly used research tools, such as metagenomics and PCR-based gene surveys enabled cultivation-unbiased analysis of genes encoded by natural microbial communities. However, these methods seldom provide direct evidence for how the discovered genes are organized inside genomes and from which organisms do they come from. Here we evaluated the feasibility of an alternative, single cell genomics approach, in the analysis of subsurface microbial community composition, metabolic potential and microevolution at the Sanford Underground Research Facility (SURF), South Dakota, and the Witwaterstrand Basin, South Africa. We successfully recovered genomic DNA from individual microbial cells from multiple locations, including ultra-deep (down to 3,500 m) and low-biomass (down to 10^3 cells mL^-1) fracture water. The obtained single amplified genomes (SAGs) from SURF contained multiple representatives of the candidate divisions OP3, OP11, OD1 and uncharacterized archaea. By sequencing eight of these SAGs, we obtained the first genome content information for these phylum-level lineages that do not contain a single cultured representative. The Witwaterstrand samples were collected from deep fractures, biogeochemical dating of which suggests isolation from tens of thousands to tens of millions of years. Thus, these fractures may be viewed as "underground Galapagos", a natural, long-term experiment of microbial evolution within well-defined temporal and spatial boundaries. We are analyzing multiple SAGs from these environments, which will provide detailed information about adaptations to life in deep subsurface, mutation rates, selective pressures and gene flux within and across microbial populations.

  4. Isolation and characterization of Chilembwe and Sinda Rock Phosphate solubilizing soil microorganisms

    USDA-ARS?s Scientific Manuscript database

    This study was conducted to isolate and characterize soil microorganisms capable of solubilizing Chilembwe and Sinda rock phosphates readily available in Zambia. Single isolates were obtained by direct plating and enrichment cultures with succinate, cellulose and glucose as the carbon sources. Isola...

  5. Isolation and characterization of Arctic microorganisms decomposing bioplastics.

    PubMed

    Urbanek, Aneta K; Rymowicz, Waldemar; Strzelecki, Mateusz C; Kociuba, Waldemar; Franczak, Łukasz; Mirończuk, Aleksandra M

    2017-12-01

    The increasing amount of plastic waste causes significant environmental pollution. In this study, screening of Arctic microorganisms which are able to degrade bioplastics was performed. In total, 313 microorganisms were isolated from 52 soil samples from the Arctic region (Spitsbergen). Among the isolated microorganisms, 121 (38.66%) showed biodegradation activity. The ability of clear zone formation on emulsified poly(butylene succinate-co-adipate) (PBSA) was observed for 116 microorganisms (95.87%), on poly(butylene succinate) (PBS) for 73 microorganisms (60.33%), and on poly(ɛ-caprolactone) (PCL) for 102 microorganisms (84.3%). Moreover, the growth of microorganisms on poly(lactic acid) (PLA) agar plates was observed for 56 microorganisms (46.28%). Based on the 16S rRNA sequence, 10 bacterial strains which showed the highest ability for biodegradation were identified as species belonging to Pseudomonas sp. and Rhodococcus sp. The isolated fungal strains were tested for polycaprolactone films and commercial corn and potato starch bags degradation under laboratory conditions. Strains 16G (based on the analysis of a partial 18S rRNA sequence, identified as Clonostachys rosea) and 16H (identified as Trichoderma sp.) showed the highest capability for biodegradation. A particularly high capability for biodegradation was observed for the strain Clonostachys rosea, which showed 100% degradation of starch films and 52.91% degradation of PCL films in a 30-day shake flask experiment. The main advantage of the microorganisms isolated from Arctic environment is the ability to grow at low temperature and efficient biodegradation under this condition. The data suggest that C. rosea can be used in natural and laboratory conditions for degradations of bioplastics.

  6. Characterization of microorganisms isolated from counterfeit toothpaste.

    PubMed

    Brzezinski, Jennifer L; Craft, David L

    2012-09-01

    The appearance of potentially counterfeit "Colgate" toothpaste on the American market prompted a criminal investigation by the United States Food and Drug Administration (FDA), including the collection of c. 60,000 tubes of toothpaste from retail outlets and product distributors. Microbiological testing was performed based on the FDA Bacteriological Analytical Manual, which determined the presence and number of bacteria present in the products. Bacteria were isolated from each "Colgate" variety; up to 2 × 10(6) cfu/g were isolated from some of the product units. Using conventional microscopic and biochemical bacterial identification methods, most of the bacteria isolated from these samples were Gram-negative rods of several genera, including Pseudomonas, Serratia, and Klebsiella. Most of the organisms isolated represent opportunistic pathogens, and therefore, counterfeit "Colgate" toothpaste containing high levels of bacteria pose a human health hazard. © 2012 American Academy of Forensic Sciences.

  7. Isolation of porphyran-degrading marine microorganisms from the surface of red alga, Porphyra yezoensis.

    PubMed

    Yoshimura, Takashi; Tsuge, Keisuke; Sumi, Toshihisa; Yoshiki, Masahiro; Tsuruta, Yumi; Abe, Shin-ichi; Nishino, Shiduo; Sanematsu, Seigo; Koganemaru, Kazuyoshi

    2006-04-01

    Marine microorganisms degrading porphyran (POR) were found on the surface of thalli of Porphyra yezoensis. Fifteen crude microorganism groups softened and liquefied the surface of agar-rich plate medium. Among these, 11 microorganism groups degraded porphyran that consisted of sulfated polysaccharide in Porphyra yezoensis. Following isolation, 7 POR-degradable microorganisms were isolated from the 11 POR-degradable microorganism groups.

  8. Complete nitrification by a single microorganism

    PubMed Central

    van Kessel, Maartje A.H.J.; Speth, Daan R.; Albertsen, Mads; Nielsen, Per H.; Op den Camp, Huub J.M.; Kartal, Boran; Jetten, Mike S.M.; Lücker, Sebastian

    2016-01-01

    Summary Nitrification is a two-step process where ammonia is considered to first be oxidized to nitrite by ammonia-oxidizing bacteria (AOB) and/or archaea (AOA), and subsequently to nitrate by nitrite-oxidizing bacteria (NOB). Described by Winogradsky already in 18901, this division of labour between the two functional groups is a generally accepted characteristic of the biogeochemical nitrogen cycle2. Complete oxidation of ammonia to nitrate in one organism (complete ammonia oxidation; comammox) is energetically feasible and it was postulated that this process could occur under conditions selecting for species with lower growth-rates but higher growth-yields than canonical ammonia-oxidizing microorganisms3. Still, organisms catalysing this process have not yet been discovered. Here, we report the enrichment and initial characterization of two Nitrospira species that encode all enzymes necessary for ammonia oxidation via nitrite to nitrate in their genomes, and indeed completely oxidize ammonium to nitrate to conserve energy. Their ammonia monooxygenase (AMO) enzymes are phylogenetically distinct from currently identified AMOs, rendering recent acquisition by horizontal gene transfer from known ammonia-oxidizing microorganisms unlikely. We also found highly similar amoA sequences (encoding the AMO subunit A) in public sequence databases, which were apparently misclassified as methane monooxygenases. This recognition of a novel amoA sequence group will lead to an improved understanding on the environmental abundance and distribution of ammonia-oxidizing microorganisms. Furthermore, the discovery of the long-sought-after comammox process will change our perception of the nitrogen cycle. PMID:26610025

  9. Isolation of Microorganisms Able To Metabolize Purified Natural Rubber

    PubMed Central

    Heisey, R. M.; Papadatos, S.

    1995-01-01

    Bacteria able to grow on purified natural rubber in the absence of other organic carbon sources were isolated from soil. Ten isolates reduced the weight of vulcanized rubber from latex gloves by >10% in 6 weeks. Scanning electron microscopy clearly revealed the ability of the microorganisms to colonize, penetrate, and dramatically alter the physical structure of the rubber. The rubber-metabolizing bacteria were identified on the basis of fatty acid profiles and cell wall characteristics. Seven isolates were strains of Streptomyces, two were strains of Amycolatopsis, and one was a strain of Nocardia. PMID:16535106

  10. Isolation of microorganisms for biological detoxification of lignocellulosic hydrolysates.

    PubMed

    López, M J; Nichols, N N; Dien, B S; Moreno, J; Bothast, R J

    2004-03-01

    Acid pretreatment of lignocellulosic biomass releases furan and phenolic compounds, which are toxic to microorganisms used for subsequent fermentation. In this study, we isolated new microorganisms for depletion of inhibitors in lignocellulosic acid hydrolysates. A sequential enrichment strategy was used to isolate microorganisms from soil. Selection was carried out in a defined mineral medium containing a mixture of ferulic acid (5 mM), 5-hydroxymethylfurfural (5-HMF, 15 mM), and furfural (20 mM) as the carbon and energy sources, followed by an additional transfer into a corn stover hydrolysate (CSH) prepared using dilute acid. Subsequently, based on stable growth on these substrates, six isolates--including five bacteria related to Methylobacterium extorquens, Pseudomonas sp, Flavobacterium indologenes, Acinetobacter sp., Arthrobacter aurescens, and one fungus, Coniochaeta ligniaria--were chosen. All six isolates depleted toxic compounds from defined medium, but only C. ligniaria C8 (NRRL 30616) was effective at eliminating furfural and 5-HMF from CSH. C. ligniaria NRRL 30616 may be useful in developing a bioprocess for inhibitor abatement in the conversion of lignocellulosic biomass to fuels and chemicals.

  11. Extracellular enzymes produced by microorganisms isolated from maritime Antarctica.

    PubMed

    Loperena, Lyliam; Soria, Verónica; Varela, Hermosinda; Lupo, Sandra; Bergalli, Alejandro; Guigou, Mairan; Pellegrino, Andrés; Bernardo, Angela; Calviño, Ana; Rivas, Federico; Batista, Silvia

    2012-05-01

    Antarctic environments can sustain a great diversity of well-adapted microorganisms known as psychrophiles or psychrotrophs. The potential of these microorganisms as a resource of enzymes able to maintain their activity and stability at low temperature for technological applications has stimulated interest in exploration and isolation of microbes from this extreme environment. Enzymes produced by these organisms have a considerable potential for technological applications because they are known to have higher enzymatic activities at lower temperatures than their mesophilic and thermophilic counterparts. A total of 518 Antarctic microorganisms, were isolated during Antarctic expeditions organized by the Instituto Antártico Uruguayo. Samples of particules suspended in air, ice, sea and freshwater, soil, sediment, bird and marine animal faeces, dead animals, algae, plants, rocks and microbial mats were collected from different sites in maritime Antarctica. We report enzymatic activities present in 161 microorganisms (120 bacteria, 31 yeasts and 10 filamentous fungi) isolated from these locations. Enzymatic performance was evaluated at 4 and 20°C. Most of yeasts and bacteria grew better at 20°C than at 4°C, however the opposite was observed with the fungi. Amylase, lipase and protease activities were frequently found in bacterial strains. Yeasts and fungal isolates typically exhibited lipase, celullase and gelatinase activities. Bacterial isolates with highest enzymatic activities were identified by 16S rDNA sequence analysis as Pseudomonas spp., Psychrobacter sp., Arthrobacter spp., Bacillus sp. and Carnobacterium sp. Yeasts and fungal strains, with multiple enzymatic activities, belonged to Cryptococcus victoriae, Trichosporon pullulans and Geomyces pannorum.

  12. Antibacterial Effect of Copper on Microorganisms Isolated from Bovine Mastitis.

    PubMed

    Reyes-Jara, Angelica; Cordero, Ninoska; Aguirre, Juan; Troncoso, Miriam; Figueroa, Guillermo

    2016-01-01

    The antimicrobial properties of copper have been recognized for several years; applying these properties to the prevention of diseases such as bovine mastitis is a new area of research. The aim of the present study was to evaluate in vitro the antimicrobial activity of copper on bacteria isolated from subclinical and clinical mastitis milk samples from two regions in Chile. A total of 327 microorganisms were recovered between March and September 2013, with different prevalence by sample origin (25 and 75% from the central and southern regions of Chile, respectively). In the central region, Escherichia coli and coagulase negative Staphylococci (CNS) were the most frequently detected in clinical mastitis cases (33%), while in the southern region S. uberis, S. aureus, and CNS were detected with frequencies of 22, 21, and 18%, respectively. Antibiotic susceptibility studies revealed that 34% of isolates were resistant to one or more antibiotics and the resistance profile was different between bacterial species and origins of isolation of the bacteria. The minimum inhibitory concentration of copper (MIC-Cu) was evaluated in all the isolates; results revealed that a concentration as low as 250 ppm copper was able to inhibit the great majority of microorganisms analyzed (65% of isolates). The remaining isolates showed a MIC-Cu between 375 and 700 ppm copper, and no growth was observed at 1000 ppm. A linear relationship was found between the logarithm of viable bacteria number and time of contact with copper. With the application of the same concentration of copper (250 ppm), CNS showed the highest tolerance to copper, followed by S. uberis and S. aureus; the least resistant was E. coli. Based on these in vitro results, copper preparations could represent a good alternative to dipping solutions, aimed at preventing the presence and multiplication of potentially pathogenic microorganisms involved in bovine mastitis disease.

  13. Antibacterial Effect of Copper on Microorganisms Isolated from Bovine Mastitis

    PubMed Central

    Reyes-Jara, Angelica; Cordero, Ninoska; Aguirre, Juan; Troncoso, Miriam; Figueroa, Guillermo

    2016-01-01

    The antimicrobial properties of copper have been recognized for several years; applying these properties to the prevention of diseases such as bovine mastitis is a new area of research. The aim of the present study was to evaluate in vitro the antimicrobial activity of copper on bacteria isolated from subclinical and clinical mastitis milk samples from two regions in Chile. A total of 327 microorganisms were recovered between March and September 2013, with different prevalence by sample origin (25 and 75% from the central and southern regions of Chile, respectively). In the central region, Escherichia coli and coagulase negative Staphylococci (CNS) were the most frequently detected in clinical mastitis cases (33%), while in the southern region S. uberis, S. aureus, and CNS were detected with frequencies of 22, 21, and 18%, respectively. Antibiotic susceptibility studies revealed that 34% of isolates were resistant to one or more antibiotics and the resistance profile was different between bacterial species and origins of isolation of the bacteria. The minimum inhibitory concentration of copper (MIC-Cu) was evaluated in all the isolates; results revealed that a concentration as low as 250 ppm copper was able to inhibit the great majority of microorganisms analyzed (65% of isolates). The remaining isolates showed a MIC-Cu between 375 and 700 ppm copper, and no growth was observed at 1000 ppm. A linear relationship was found between the logarithm of viable bacteria number and time of contact with copper. With the application of the same concentration of copper (250 ppm), CNS showed the highest tolerance to copper, followed by S. uberis and S. aureus; the least resistant was E. coli. Based on these in vitro results, copper preparations could represent a good alternative to dipping solutions, aimed at preventing the presence and multiplication of potentially pathogenic microorganisms involved in bovine mastitis disease. PMID:27199953

  14. Time evolution of trapped single cell microorganism

    NASA Astrophysics Data System (ADS)

    Bernatova, Silvie; Samek, Ota; Obruca, Stanislav; Sery, Mojmir; Zemanek, Pavel; Marova, Ivana

    2016-04-01

    The combination of optical tweezers and Raman micro-spectroscopy is frequently referred as Raman tweezers. A single focused laser beam is utilized here both as a source of Raman scattering and a source forming an optical trap. Raman tweezers have been recently used in variety of applications in cell biology as a useful tool for non-contact and non-destructive determination of living cells properties. Here we use Raman tweezers to follow response of cells on the length of their cultivation in mineral oil. Analyses of obtained Raman spectra are based on 2D correlation analysis and allow us to determine the chemical background of the cell response in a gentle way.

  15. Isolation of cultivable microorganisms from Polish notes and coins.

    PubMed

    Kalita, Michal; Palusińska-Szysz, Marta; Turska-Szewczuk, Anna; Wdowiak-Wróbel, Sylwia; Urbanik-Sypniewska, Teresa

    2013-01-01

    The potential role of currency in the spread of pathogenic microflora has been evaluated in many countries. In this study Polish paper notes and the coins in general circulation were assayed for the presence of cultivable bacteria and fungi. Bacterial isolates identification was based on cultural and biochemical characters and by comparison of the 16S rRNA gene sequence. Fungal isolates were recognized with biochemical and morphological criteria. Coagulase-negative staphylococci, (43.6% of the total bacterial count) including Staphylococcus saprophyticus, S. epidermidis, and S. hominis, and Enteroccus spp. (30.8% of the total bacterial count), i.e. E.faecalis, E.faecium and E. durans, were the most numerous bacterial contamination. Penicillium spp., and Aspergillus spp. were the most frequently detected moulds whereas Candida spp. was the most frequent yeast isolated from currency. A visible dependence between the banknote denomination, the physical condition of paper currency, and the number of bacteria and fungi was found. The overall count of bacteria isolated from currency was thousand-fold higher than that of fungal isolates. The total amount of bacteria and fungi recovered from the coins was approximately 2.7-fold lower than that isolated from the notes. In summary, the Polish currency notes were found to be contaminated mainly with commensal bacteria and fungi while the opportunistic pathogenic microorganisms Escherichia coli, Pseudomonas stutzeri and C. albicans were detected at a low frequency.

  16. Isolation of microorganisms involved in reduction of crystalline iron(III) oxides in natural environments.

    PubMed

    Hori, Tomoyuki; Aoyagi, Tomo; Itoh, Hideomi; Narihiro, Takashi; Oikawa, Azusa; Suzuki, Kiyofumi; Ogata, Atsushi; Friedrich, Michael W; Conrad, Ralf; Kamagata, Yoichi

    2015-01-01

    Reduction of crystalline Fe(III) oxides is one of the most important electron sinks for organic compound oxidation in natural environments. Yet the limited number of isolates makes it difficult to understand the physiology and ecological impact of the microorganisms involved. Here, two-stage cultivation was implemented to selectively enrich and isolate crystalline iron(III) oxide reducing microorganisms in soils and sediments. Firstly, iron reducers were enriched and other untargeted eutrophs were depleted by 2-years successive culture on a crystalline ferric iron oxide (i.e., goethite, lepidocrocite, hematite, or magnetite) as electron acceptor. Fifty-eight out of 136 incubation conditions allowed the continued existence of microorganisms as confirmed by PCR amplification. High-throughput Illumina sequencing and clone library analysis based on 16S rRNA genes revealed that the enrichment cultures on each of the ferric iron oxides contained bacteria belonging to the Deltaproteobacteria (mainly Geobacteraceae), followed by Firmicutes and Chloroflexi, which also comprised most of the operational taxonomic units (OTUs) identified. Venn diagrams indicated that the core OTUs enriched with all of the iron oxides were dominant in the Geobacteraceae while each type of iron oxides supplemented selectively enriched specific OTUs in the other phylogenetic groups. Secondly, 38 enrichment cultures including novel microorganisms were transferred to soluble-iron(III) containing media in order to stimulate the proliferation of the enriched iron reducers. Through extinction dilution-culture and single colony isolation, six strains within the Deltaproteobacteria were finally obtained; five strains belonged to the genus Geobacter and one strain to Pelobacter. The 16S rRNA genes of these isolates were 94.8-98.1% identical in sequence to cultured relatives. All the isolates were able to grow on acetate and ferric iron but their physiological characteristics differed considerably in

  17. identification of Pseudomonas spp. as amoeba-resistant microorganisms in isolates of Acanthamoeba.

    PubMed

    José Maschio, Vinicius; Corção, Gertrudes; Rott, Marilise Brittes

    2015-01-01

    Acanthamoeba is a "Trojan horse" of the microbial world. The aim of this study was to identify the presence of Pseudomonas as an amoeba-resistant microorganism in 12 isolates of Acanthamoeba. All isolates showed the genus Pseudomonas spp. as amoeba-resistant microorganisms. Thus, one can see that the Acanthamoeba isolates studied are hosts of Pseudomonas.

  18. Radiotolerance of microorganisms isolated from radiation fields on a university campus: implications for shallow subsurface growth of microorganisms on Mars

    NASA Astrophysics Data System (ADS)

    Mormile, Melanie R.; Elmer, Jacob J.; Spychala, Scott J.

    2007-09-01

    The surface of Mars is exposed to higher levels of solar and galactic cosmic ray irradiation than Earth due to its very weak magnetic field. Thus, microorganisms that could possibly survive in the shallow subsurface of Mars would likely be radiotolerant. To better understand microorganisms that might reside in this environment of Mars, a number of isolates were obtained from the area around a gamma-radiation source, 137Cs, located on the UMR campus. Radiation sensitivity assays were performed on the isolates as well as on the common bacterium, E. coli. All the organisms tested were able to withstand exposures up to 20 Gy. The E. coli control did not survive exposures of 200 Gy, while the isolate designated 1B-1 could. Another isolate, Cont-1, also withstood this exposure. Each of the isolates produced white growth on solid medium and their cells are rod-shaped. The study of these isolates and similar organisms could enhance our knowledge of these unique extremophilic bacteria and might provide insight into the microorganisms that could be present in the shallow subsurface of Mars.

  19. Isolation of microorganisms involved in reduction of crystalline iron(III) oxides in natural environments

    PubMed Central

    Hori, Tomoyuki; Aoyagi, Tomo; Itoh, Hideomi; Narihiro, Takashi; Oikawa, Azusa; Suzuki, Kiyofumi; Ogata, Atsushi; Friedrich, Michael W.; Conrad, Ralf; Kamagata, Yoichi

    2015-01-01

    Reduction of crystalline Fe(III) oxides is one of the most important electron sinks for organic compound oxidation in natural environments. Yet the limited number of isolates makes it difficult to understand the physiology and ecological impact of the microorganisms involved. Here, two-stage cultivation was implemented to selectively enrich and isolate crystalline iron(III) oxide reducing microorganisms in soils and sediments. Firstly, iron reducers were enriched and other untargeted eutrophs were depleted by 2-years successive culture on a crystalline ferric iron oxide (i.e., goethite, lepidocrocite, hematite, or magnetite) as electron acceptor. Fifty-eight out of 136 incubation conditions allowed the continued existence of microorganisms as confirmed by PCR amplification. High-throughput Illumina sequencing and clone library analysis based on 16S rRNA genes revealed that the enrichment cultures on each of the ferric iron oxides contained bacteria belonging to the Deltaproteobacteria (mainly Geobacteraceae), followed by Firmicutes and Chloroflexi, which also comprised most of the operational taxonomic units (OTUs) identified. Venn diagrams indicated that the core OTUs enriched with all of the iron oxides were dominant in the Geobacteraceae while each type of iron oxides supplemented selectively enriched specific OTUs in the other phylogenetic groups. Secondly, 38 enrichment cultures including novel microorganisms were transferred to soluble-iron(III) containing media in order to stimulate the proliferation of the enriched iron reducers. Through extinction dilution-culture and single colony isolation, six strains within the Deltaproteobacteria were finally obtained; five strains belonged to the genus Geobacter and one strain to Pelobacter. The 16S rRNA genes of these isolates were 94.8–98.1% identical in sequence to cultured relatives. All the isolates were able to grow on acetate and ferric iron but their physiological characteristics differed considerably in

  20. Antimicrobial Susceptibility of Microorganisms Isolated from Periapical Periodontitis Lesions.

    PubMed

    Narita, Masato; Shibahara, Takahiko; Takano, Nobuo; Fujii, Rie; Okuda, Katsuji; Ishihara, Kazuyuki

    2016-01-01

    Periapical periodontitis usually results from microbial infection, with these microorganisms occasionally migrating to the root canal, which can lead to further, potentially life-threatening, complications. Here, the susceptibility of 27 bacterial strains to various antimicrobial agents was evaluated. These strains comprised 13 species; 16 of the strains were clinical isolates from periapical lesions. Each strain was inoculated onto blood agar plates containing one of the antimicrobial agents. The plates were incubated anaerobically at 37°C for 96 hr and the minimal inhibitory concentrations (MICs) determined. Ten strains required an MIC of 32 μg/ml or greater for amoxicillin, 6 for cefmetazole, and 5 for cefcapene among β-lactam antibiotics; 8 strains required an MIC of 32 μg/ml or greater for clindamycin, 4 for azithromycin, and 11 for clarithromycin among macrolide antibiotics; 3 strains required an MIC of 32 μg/ml or greater for ciprofloxacin and 2 for ofloxacin among fluoroquinolones. The effect of cefcapene on 5 strains was evaluated after biofilm formation to investigate the relationship between biofilm formation and susceptibility. All strains showed a decrease in susceptibility after biofilm formation. The results revealed that several antimicrobial agents commonly used in a clinical setting, including amoxicillin, cefmetazole, and clindamycin, are potentially effective in the treatment of orofacial odontogenic infections. The development of resistant strains, however, means that this can no longer be guaranteed. In addition, azithromycin, ciprofloxacin, and ofloxacin were more effective than the 3 β-lactam antibiotics tested. These results suggest that sensitivity testing is needed if odontogenic infections are to be treated safely and effectively.

  1. Detection of variable DNA repeats in diverse eukaryotic microorganisms by a single set of polymerase chain reaction primers.

    PubMed Central

    Riley, D E; Samadpour, M; Krieger, J N

    1991-01-01

    We cloned and sequenced a variable DNA repeat from Trichomonas vaginalis, a flagellated protozoan parasite. Targeting of this repeat in the polymerase chain reaction resulted in complex and intense product patterns for a wide variety of eukaryotic microorganisms, including the pathogenic protozoan parasites T. vaginalis, Giardia lamblia, Leishmania donovani, three species of Trypanosoma, and four species of Acanthamoeba; the nonpathogenic protozoans, Paramecium tetraurelia and Tetrahymena thermophilia; and a yeast, Saccharomyces cerevisiae. Each microorganism exhibited a distinctive pattern of repeats. For example, a characteristic pattern was exhibited by six clinical T. vaginalis isolates. Eight G. lamblia isolates exhibited either one of two characteristic pattern types. There was no reaction with human DNA or DNA from the prokaryotes Ureaplasma urealyticum and Mycoplasma hominis. This approach may facilitate detection of a wide variety of eukaryotic microorganisms by use of a single primer set and holds promise for the development of typing schemes for both T. vaginalis and G. lamblia. Images PMID:1757544

  2. Single Cell Isolation and Analysis

    PubMed Central

    Hu, Ping; Zhang, Wenhua; Xin, Hongbo; Deng, Glenn

    2016-01-01

    Individual cell heterogeneity within a population can be critical to its peculiar function and fate. Subpopulations studies with mixed mutants and wild types may not be as informative regarding which cell responds to which drugs or clinical treatments. Cell to cell differences in RNA transcripts and protein expression can be key to answering questions in cancer, neurobiology, stem cell biology, immunology, and developmental biology. Conventional cell-based assays mainly analyze the average responses from a population of cells, without regarding individual cell phenotypes. To better understand the variations from cell to cell, scientists need to use single cell analyses to provide more detailed information for therapeutic decision making in precision medicine. In this review, we focus on the recent developments in single cell isolation and analysis, which include technologies, analyses and main applications. Here, we summarize the historical background, limitations, applications, and potential of single cell isolation technologies. PMID:27826548

  3. Effects of single-walled carbon nanotubes on soil microorganisms

    NASA Astrophysics Data System (ADS)

    Jin, L.; Chung, H.; Son, Y.

    2011-12-01

    Single-walled carbon nanotubes (SWCNTs) are novel materials that have the potential to be used in various commercial fields due to their unique physicochemical properties. As a result of commercial development of nanotechnology, SWCNTs may be discharged to the soil environment with unknown consequences. However, there are as yet no data in the scientific literature that demonstrate the effects of SWCNTs on microbial function in soils. Therefore, we aimed to determine the effects of SWCNTs on soil microbial activity through a 2-week incubation study on urban soils supplemented with different concentrations of SWCNTs ranging from 0 to 1000 μg CNT/g soil. Fluorometric test using fluorogenic substrates were employed for the measurement of several enzyme activities in soil samples. More specifically, we determined the changes in the activities of cellobiohydrolase, β-1,4-glucosidase, β-1,4-xylosidase, β-1,4-N-acetylglucosaminidase, L-leucine aminopeptidase and acid phosphatase which play important roles in the carbon, nitrogen, and phosphorus cycles in response to the addition of SWCNTs. We found that microbial enzyme activities decreased as the concentrations of SWCNT added increased. The lowest enzyme activities were observed under 1000 μg CNT/g soil. The overall pattern shows that enzyme activities decreased slightly in the first 2-3 days and increased in the later stage of the incubation. Our results suggest that relatively high concentrations of SWCNTs can inhibit microbial activities, and this may be due to microbial cell membrane damage caused by SWCNTs. However, further study needs to be conducted to determine the mechanism responsible for inhibitory effect of SWCNTs on soil microbial activity. It can be concluded that changes in the activities of extracellular enzymes can indicate the effect of SWCNTs on soil microorganisms and nutrient cycling.

  4. Obtaining genomes from uncultivated environmental microorganisms using FACS-based single-cell genomics.

    PubMed

    Rinke, Christian; Lee, Janey; Nath, Nandita; Goudeau, Danielle; Thompson, Brian; Poulton, Nicole; Dmitrieff, Elizabeth; Malmstrom, Rex; Stepanauskas, Ramunas; Woyke, Tanja

    2014-05-01

    Single-cell genomics is a powerful tool for exploring the genetic makeup of environmental microorganisms, the vast majority of which are difficult, if not impossible, to cultivate with current approaches. Here we present a comprehensive protocol for obtaining genomes from uncultivated environmental microbes via high-throughput single-cell isolation by FACS. The protocol encompasses the preservation and pretreatment of differing environmental samples, followed by the physical separation, lysis, whole-genome amplification and 16S rRNA-based identification of individual bacterial and archaeal cells. The described procedure can be performed with standard molecular biology equipment and a FACS machine. It takes <12 h of bench time over a 4-d time period, and it generates up to 1 μg of genomic DNA from an individual microbial cell, which is suitable for downstream applications such as PCR amplification and shotgun sequencing. The completeness of the recovered genomes varies, with an average of ∼50%.

  5. Microbial quality and molecular identification of cultivable microorganisms isolated from an urban drinking water distribution system (Limassol, Cyprus).

    PubMed

    Botsaris, George; Kanetis, Loukas; Slaný, Michal; Parpouna, Christiana; Makris, Konstantinos C

    2015-12-01

    Microorganisms can survive and multiply in aged urban drinking water distribution systems, leading to potential health risks. The objective of this work was to investigate the microbial quality of tap water and molecularly identify its predominant cultivable microorganisms. Tap water samples collected from 24 different households scattered in the urban area of Limassol, Cyprus, were microbiologically tested following standard protocols for coliforms, E. coli, Pseudomonas spp., Enterococcus spp., and total viable count at 22 and 37 °C. Molecular identification was performed on isolated predominant single colonies using 16SrRNA sequencing. Approximately 85% of the household water samples were contaminated with one or more microorganisms belonging to the genera of Pseudomonas, Corynebacterium, Agrobacterium, Staphylococcus, Bacillus, Delftia, Acinetobacter, Enterococcus, Enterobacter, and Aeromonas. However, all samples tested were free from E. coli. This is the first report in Cyprus molecularly confirming specific genera of relevant microbial communities in tap water.

  6. In vitro degradation of linamarin by microorganisms isolated from cassava wastewater treatment lagoons

    PubMed Central

    Vasconcellos, S. P; Cereda, M. P.; Cagnon, J. R.; Foglio, M.A.; Rodrigues, R.A.; Manfio, G. P.; Oliveira, V. M.

    2009-01-01

    This study aimed at isolating and characterizing of microorganisms able to use linamarin as sole carbon source. Thirty one microbial strains were isolated from manipueira, a liquid effluent of cassava processing factories. Among these strains, Bacillus licheniformis (isolate 2_2) and Rhodotorulla glutinis (isolate L1) were able to degrade 71% and 95% of added linamarin, respectively, within 7 days, showing high biodegradation activity and great potential for detoxification of cassava processing wastewaters. PMID:24031436

  7. Methods for observing microbial biofilms directly on leaf surfaces and recovering them for isolation of culturable microorganisms.

    PubMed

    Morris, C E; Monier, J; Jacques, M

    1997-04-01

    Epifluorescence microscopy, scanning electron microscopy, and confocal laser scanning microscopy were used to observe microbial biofilms directly on leaf surfaces. Biofilms were observed on leaves of all species sampled (spinach, lettuce, Chinese cabbage, celery, leeks, basil, parsley, and broad-leaved endive), although the epifluorescent images were clearest when pale green tissue or cuticle pieces were used. With these techniques, biofilms were observed that were about 20 (mu)m in depth and up to 1 mm in length and that contained copious exopolymeric matrices, diverse morphotypes of microorganisms, and debris. The epifluorescence techniques described here can be used to rapidly determine the abundance and localization of biofilms on leaves. An additional technique was developed to recover individual biofilms or portions of single biofilms from leaves and to disintegrate them for isolation of the culturable microorganisms they contained. Nineteen biofilms from broad-leaved endive, spinach, parsley, and olive leaves were thus isolated and characterized to illustrate the applications of this technique.

  8. Molecular epidemiology of microorganisms isolated from food workers and enteral feeding of public hospitals.

    PubMed

    Borges, Liana J; Campos, Maria Raquel H; Cardoso, Juliana L; André, Maria Cláudia D P B; Serafini, Álvaro B

    2010-09-01

    This study aimed to compare strains of Staphylococcus aureus and E. coli isolated from food workers and enteral diet samples obtained from 2 public hospitals (H1/H2) in Goiania, Goias, Brazil, by the means of antibiogram and pulsed field gel electrophoresis (PFGE). In the H1, strains of S. aureus were present in 2 enteral diet samples and in 13 food worker swabs. Strains of E. coli were found in an enteral diet sample from H1 and in 2 enteral diet samples from H2 and in 6 food worker swabs in the H1 and in 12 food worker swabs from H2. According to the antibiogram, the 6 susceptibility profiles (A to F) of 15 S. aureus strains colonizing personnel and enteral feeding did not allow the identification of the probable source of diet contamination. All 20 E. coli strains isolated from the H1 and H2 were grouped in 4 phenotypic profiles (A to D). The phenotypes A (H1) and C (H2) showed the same profile for microorganisms isolated from handlers and diets, suggesting more phenotypic similarity among these samples. PFGE genotyping showed that S. aureus isolates from diets were related to a single strain isolated from a food worker suggesting that in this case the reason for the diet contamination may be a result of food handling. The food worker appears to be the most probable source of E. coli contamination for enteral feeding from H2. This fact emphasizes on the food workers as a risk of bacterial transmission for the diets and that the diet chain production must be controlled. The study emphasizes the importance of monitoring the enteral diet microbiological quality and the factors associated to its contamination. The study highlights the use of molecular biology as an instrument to correlate strains to determine the origin of the final product contamination.

  9. Plating isolation of various catalase-negative microorganisms from soil

    NASA Technical Reports Server (NTRS)

    Labeda, D. P.; Hunt, C. M.; Casida, L. E., Jr.

    1974-01-01

    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.

  10. Plating isolation of various catalase-negative microorganisms from soil

    NASA Technical Reports Server (NTRS)

    Labeda, D. P.; Hunt, C. M.; Casida, L. E., Jr.

    1974-01-01

    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.

  11. On the isolation of halophilic microorganisms from salt deposits of great geological age

    NASA Technical Reports Server (NTRS)

    Stan-Lotter, Helga; Denner, Ewald

    1993-01-01

    From salt sediments of Triassic or Permian age from various locations in the world halophilic microorganisms were isolated. Molecular characteristics of several of the isolates suggested they belong to the archaebacteria. One group appears to represent novel strains; several properties of one such isolate, strain BIp, are described here. The existence of viable microorganisms in ancient sediment would have great implications with respect to our notions on evolution, the research for life in extraterrestrial environments, and the longterm survival of functional biological structures. Of crucial importance is thus the question if these microorganisms existed in the salt since the time of deposition or invaded at some later date. Some suggestions to address these issues experimentally are discussed.

  12. On the Isolation of Halophilic Microorganisms from Salt Deposits of Great Geological Age

    NASA Technical Reports Server (NTRS)

    Stan-Lotter, Helga; Denner, Ewald; Orans, Robin (Editor)

    1993-01-01

    From salt sediments of Triassic or Permian ace from various locations in the world halophilic microorganisms were isolated. Molecular characteristics of several of the isolates suggested they belong to the archaebacteriae. One group appears to represent novel strains; several properties or one such isolate, strain BIp, are described here. The existence of viable microorganisms in ancient sediments would have great implications with respect to our notions on evolution, the search for life in extraterrestrial environments and the long- term survival of functional biological structures. Of crucial importance is thus the question if these microorganisms existed in the salt since the time of deposition or invaded at some later date. Some suggestions to address these issues experimentally are discussed.

  13. Characterization of microorganisms isolated from lignite excavated from the Záhorie coal mine (southwestern Slovakia).

    PubMed

    Pokorný, Richard; Olejníková, Petra; Balog, Miroslav; Zifcák, Peter; Hölker, Udo; Janssen, Martina; Bend, Jutta; Höfer, Milan; Holiencin, Rudolf; Hudecová, Daniela; Varecka, L'udovít

    2005-11-01

    Microorganisms were isolated from lignite freshly excavated in the Záhorie coal mine (southwestern Slovakia) under conditions excluding contamination with either soil or air-borne microorganisms. The isolates represented both Prokarya and Eukarya (fungi). All were able to grow on standard media, although some microorganisms were unstable and became extinct during storage of coal samples. Bacteria belonged to the genera Bacillus, Staphylococcus, and Rhodococcus, according to both morphological criteria and ITS sequences. Several bacterial isolates were resistant to antibiotics. The presence of anaerobic bacteria was also documented, although they have not yet been identified. Fungal isolates were typified by using their ITS sequences. They belonged to the genera Trichoderma (Hypocrea), Penicillium, Epicoccum, Metarhizium (Cordyceps), and Cladosporium. Several fungi produced compounds with antibiotic action against standard bacterial strains. The evidence for the presence of microorganisms in native lignite was obtained by means of fluorescence microscopy, scanning electron microscopy, and electron microprobe analysis. Results demonstrated that microorganisms were able to survive in the low-rank coal over a long time period.

  14. Volatiles produced by microorganisms isolated from refrigerated chicken at spoilage.

    PubMed Central

    Freeman, L R; Silverman, G J; Angelini, P; Merritt, C; Esselen, W B

    1976-01-01

    Volatile components present at spoilage of refrigerated chicken breasts were identified using high-vacuum-low-temperature distillation techniques followed by analysis with combined temperature-programmed gas chromatography and mass spectrometry. A comparison was made of the compounds detected from both irradiated and non-irradiated muscle stored at 2 and 10 degrees C under both aerobic and anaerobic conditions. Isolates were randomly selected from the spoiled poultry, identified, and evaluated for their ability to produce volatile spoilage noted when grown on radiation-sterilized chicken. Several isolates that produced off-odors on sterile chicken breasts were examined. Twenty-two compounds were associated with spoilage. Some of the compounds found on both irradiated and unirradiated samples were considered to play only a minor role in the spoilage aroma or were present in low concentrations, since the aroma of spoiled irradiated chicken lacked the harsh odor notes typical of spoiled unirradiated chicken. Fifteen of the 22 compounds were considered to be unique to unirradiated, aerobically spoiled samples. Nine of these compounds, hydrogen sulfide, methyl mercaptan, dimethyl sulfide, dimethyl disulfide, methyl acetate, ethyl acetate, heptadiene, methanol, and ethanol, were found on chicken spoiled at both 2 and 10 degrees C. xylene, benzaldehyde, and 2,3-dithiahexane were detected only in samples stored at 2 degrees C and methyl thiolacetate, 2-butanone, and ethyl propionate were associated with 10 degrees C spoilage. Fifty-eight isolates randomly selected from fresh, radiation-pasteurized, and unirradiated spoiled poultry were classified taxonomically, and 10 of them, which produced spoilage odors on sterilized chicken breasts, were selected for subsequent analysis of their volatiles. Isolates identified as Pseudomonas putrefaciens and Pseudomonas species that were members of groups I and II of Shewan's classification, as well as Flavobacterium and oxidative

  15. Enhancement of Biodegradation of Palm Oil Mill Effluents by Local Isolated Microorganisms

    PubMed Central

    Soleimaninanadegani, Mohammadreza

    2014-01-01

    This study was designed to investigate the microorganisms associated with palm oil mill effluent (POME) in Johor Bahru state, Malaysia. Biodegradation of palm oil mill effluents (POME) was conducted to measure the discarded POME based on physicochemical quality. The bacteria that were isolated are Micrococcus species, Bacillus species, Pseudomonas species, and Staphylococcus aureus, while the fungi that were isolated are Aspergillus niger, Aspergillus fumigatus, Candida species, Fusarium species, Mucor species, and Penicillium species. The autoclaved and unautoclaved raw POME samples were incubated for 7 days and the activities of the microorganisms were observed each 12 hours. The supernatants of the digested POME were investigated for the removal of chemical oxygen demand (COD), color (ADMI), and biochemical oxygen demand (BOD) at the end of each digestion cycle. The results showed that the unautoclaved raw POME sample degraded better than the inoculated POME sample and this suggests that the microorganisms that are indigenous in the POME are more effective than the introduced microorganisms. This result, however, indicates the prospect of isolating indigenous microorganisms in the POME for effective biodegradation of POME. Moreover, the effective treatment of POME yields useful products such as reduction of BOD, COD, and color. PMID:27433516

  16. Isolation and Antibiotic Susceptibility of the Microorganisms Isolated from Diabetic Foot Infections in Nemazee Hospital, Southern Iran

    PubMed Central

    Anvarinejad, Mojtaba; Pouladfar, Gholamreza; Japoni, Aziz; Bolandparvaz, Shahram; Satiary, Zeinab; Abbasi, Pejman; Mardaneh, Jalal

    2015-01-01

    Background. Diabetic foot infections (DFIs) are a major public health issue and identification of the microorganisms causing such polymicrobial infections is useful to find out appropriate antibiotic therapy. Meanwhile, many reports have shown antibiotic resistance rising dramatically. In the present study, we sought to determine the prevalence of microorganisms detected on culture in complicated DFIs in hospitalized patients and their antibiotic sensitivity profiles. Methods. A cross-sectional study was conducted for a period of 24 months from 2012 to 2014 in Nemazee Hospital, Shiraz, Iran. The demographic and clinical features of the patients were obtained. Antimicrobial susceptibility testing to different agents was carried out using the disc diffusion method. Results. During this period, 122 aerobic microorganisms were isolated from DFIs. Among Gram-positive and Gram-negative bacteria, Staphylococcus spp. and E. coli were the most frequent organisms isolated, respectively. Of the isolates, 91% were multidrug while 78% of S. aureus isolates were methicillin resistant. 53% of Gram-negative bacteria were positive for extended-spectrum β-lactamase. Conclusion. Given the involvement of different microorganisms and emergence of multidrug resistant strains, clinicians are advised to consider culture before initiation of empirical therapy. PMID:26843987

  17. A new carotenoid glycosyl ester isolated from a marine microorganism, Fusarium strain T-1.

    PubMed

    Sakaki, Hideyuki; Kaneno, Hirokazu; Sumiya, Yasuji; Tsushima, Miyuki; Miki, Wataru; Kishimoto, Noriaki; Fujita, Tokio; Matsumoto, Sadayoshi; Komemushi, Sadao; Sawabe, Akiyoshi

    2002-11-01

    A new carotenoid glycosyl ester, neurosporaxanthin beta-D-glucopyranoside (2), together with neurosporaxanthin (1), beta-carotene, gamma-carotene, and torulene were isolated from cultured cells of a marine microorganism, strain T-1, which was identified as Fusarium sp. Their structures were determined by chemical and spectral data.

  18. Isolation and screening of microorganisms for R-(+)-limonene and (-)-beta-pinene biotransformation.

    PubMed

    Rottava, Ieda; Cortina, Priscila F; Grando, Camila E; Colla, André R S; Martello, Eduarda; Cansian, Rogério L; Toniazzo, Geciane; Treichel, Helen; Antunes, Octávio A C; Oestreicher, Enrique G; de Oliveira, Débora

    2010-10-01

    This work is focused on the biotransformation of R-(+)-limonene and (-)-beta-pinene to bioflavor production. To carry out the present study, 405 microorganisms were tested for their ability to bioconvert the substrates. From the isolated microorganisms, 193 were selected in the prescreening using mineral medium for limonene degradation. At the screening step, eight strains were able to convert R-(+)-limonene and 15 to transform (-)-beta-pinene, both in alpha-terpineol. The highest concentration in alpha-terpineol from R-(+)-limonene was about 3,450 mg/L for Penicillium sp. isolated from eucalyptus steam. From (-)-beta-pinene, the highest product concentration of 675.5 mg/L was achieved using an Aspergillus sp. strain isolated from orange tree stem.

  19. Diversity of microorganisms isolated from the soil sample surround Chroogomphus rutilus in the Beijing region.

    PubMed

    Wang, Peng; Liu, Yu; Yin, Yonggang; Jin, Haojie; Wang, Shouxian; Xu, Feng; Zhao, Shuang; Geng, Xiaoli

    2011-03-02

    Artificially cultivating Chroogomphus rutilus is too inefficient to be commercially feasible. Furthermore, isolating C. rutilus mycelia in the wild is difficult. Thus, it is important to determine the natural habitat of its fruiting body. This study focused on the ecology of the C. rutilus habitat to isolate and classify beneficial microorganisms that could affect its growth, which could be used in future research on artificial cultivation. In total, 342 isolates were isolated from soil samples collected around a C. rutilus colony in the Beijing region. Of these, 22 bacterial and 14 fungal isolates were selected for sequencing and phylogenetic analysis, based on their growth characteristics and colony morphology. Using 16S rRNA gene sequence analysis, the bacterial isolates were divided into two monophyletic clusters which had significant hits to the genera Bacillus and Pseudomonas, respectively. Using internal transcribed spacer (ITS) sequence analysis, fungal isolates were divided into four monophyletic clusters: Penicillium, Trichoderma, Mortierella, and Bionectria. Moreover, the phylogenetic diversity of these isolates was analysed. The results indicated that numerous microorganisms were present in C. rutilus habitat. This was the first reported examination of the microbiological ecology of C. rutilus.

  20. Isolation and characterization of microorganisms from instruments used by pedicurists operating within Lagos metropolis, Nigeria.

    PubMed

    Adeleye, I A; Osidipe, O O

    2004-12-01

    Eight bacterial and five fungal species were isolated from swab samples taken from instruments used by pedicurists operating at three different sites in Lagos, Nigeria. The bacterial isolates included Micrococcus luteus, Micrococcus roseus, Staphylococcus epidermidis, Staphylococcus aureus, Hafnia spp, Shigella spp, Bacillus subtilis and Bacillus spp. The five fungal isolates were identified as Aspergillus niger, Aspergillus flavus, Mucor spp, Trichophyton spp and Candida albicans. The presence of these microorganisms, some of which are pathogenic, is an indication that pedicurists could be contributing towards the spread of skin and nail infections within the Lagos metropolis.

  1. Enumeration and Characterization of Acidophilic Microorganisms Isolated from a Pilot Plant Stirred-Tank Bioleaching Operation

    PubMed Central

    Okibe, Naoko; Gericke, Mariekie; Hallberg, Kevin B.; Johnson, D. Barrie

    2003-01-01

    Microorganisms were enumerated and isolated on selective solid media from a pilot-scale stirred-tank bioleaching operation in which a polymetallic sulfide concentrate was subjected to biologically accelerated oxidation at 45°C. Four distinct prokaryotes were isolated: three bacteria (an Acidithiobacillus caldus-like organism, a thermophilic Leptospirillum sp., and a Sulfobacillus sp.) and one archaeon (a Ferroplasma-like isolate). The relative numbers of these prokaryotes changed in the three reactors sampled, and the Ferroplasma isolate became increasingly dominant as mineral oxidation progressed, eventually accounting for >99% of plate isolates in the third of three in-line reactors. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits (e.g., oxidation of iron and/or sulfur and autotrophy or heterotrophy) were examined. More detailed studies were carried out with the Leptospirillum and Ferroplasma isolates. The data presented here represent the first quantitative study of the microorganisms in a metal leaching situation and confirm that mixed cultures of iron- and sulfur-oxidizing prokaryotic acidophiles catalyze the accelerated dissolution of sulfidic minerals in industrial tank bioleaching operations. The results show that indigenous acidophilic microbial populations change as mineral dissolution becomes more extensive. PMID:12676667

  2. Micro-organisms isolated from cadaveric samples of allograft musculoskeletal tissue.

    PubMed

    Varettas, Kerry

    2013-12-01

    Allograft musculoskeletal tissue is commonly used in orthopaedic surgical procedures. Cadaveric donors of musculoskeletal tissue supply multiple allografts such as tendons, ligaments and bone. The microbiology laboratory of the South Eastern Area Laboratory Services (SEALS, Australia) has cultured cadaveric allograft musculoskeletal tissue samples for bacterial and fungal isolates since 2006. This study will retrospectively review the micro-organisms isolated over a 6-year period, 2006-2011. Swab and tissue samples were received for bioburden testing and were inoculated onto agar and/or broth culture media. Growth was obtained from 25.1 % of cadaveric allograft musculoskeletal tissue samples received. The predominant organisms isolated were coagulase-negative staphylococci and coliforms, with the heaviest bioburden recovered from the hemipelvis. The rate of bacterial and fungal isolates from cadaveric allograft musculoskeletal tissue samples is higher than that from living donors. The type of organism isolated may influence the suitability of the allograft for transplant.

  3. Toxicity of heavy metals for microorganisms isolated from slow sand filter schmutzdecke.

    PubMed

    Muhammad, N; Hooke, A M

    2003-09-01

    This paper describes the susceptibility of three species of environmental bacteria isolated from the schmutzdecke of biologically active slow sand filters to cadmium, chromium and lead. The microorganisms, all identified as members of the genus Pseudomonas, were grown in tryptic soy broth with various concentrations of the selected heavy metals. The mean generation times (MGT) of the bacteria were compared to evaluate the toxic effects of the heavy metals. All three species tolerated high doses of heavy metals and the MGTs increased exponentially as the heavy metal concentrations increased; 12 mg l(-1) was the highest dose tested and the bacteria continued to grow albeit at very slow rates. In dilute media, the toxic effects of heavy metals were enhanced, illustrating the protection effect of organic matter and heavy metal complex formation. Growth studies of the isolated microorganisms on half-strength tryptic soy agar containing 6 mg l(-1) heavy metals also proved useful in determining toxicity.

  4. Phenotypic characterization and genomic DNA polymorphisms of Escherichia coli strains isolated as the sole micro-organism from vaginal infections.

    PubMed

    Lobos, Olga; Padilla, Carlos

    2009-03-01

    Vaginal infections such as vulvovaginal candiadiasis, trichomoniasis and bacterial vaginosis are common worldwide. Accurate diagnosis and prescription of appropriate treatments are important since these infections are linked to adverse outcomes for women during pregnancy and for newborns. Several aetiological agents are responsible for these infectious diseases; however, the presence of Escherichia coli in these infections is controversial. Thus, it is important to identify some phenotypic and genotypic properties of E. coli strains isolated from vaginal infections. Forty-six E. coli strains isolated from vaginal fluid as the sole micro-organism, and 20 other E. coli strains isolated from other samples (urinary tract infections, otitis and septicaemia) were analysed by several phenotypic tests. In addition, genotypic features were studied by RAPD-PCR techniques. Biochemical tests showed that the E. coli strains isolated from vaginal fluid could be grouped into a single cluster which is subdivided into two phenogroups. Analysis of the dendrogram based on fragment length polymorphisms of genomic DNA indicated that E. coli isolates from vaginal infections form a single cluster with two subdivisions. Further studies are needed to analyse the molecular structure and virulence characteristics of these E. coli strains in order to determine their potential role in vaginal infections.

  5. Isolation of Phyllosilicate–Iron Redox Cycling Microorganisms from an Illite–Smectite Rich Hydromorphic Soil

    PubMed Central

    Shelobolina, Evgenya; Konishi, Hiromi; Xu, Huifang; Benzine, Jason; Xiong, Mai Yia; Wu, Tao; Blöthe, Marco; Roden, Eric

    2012-01-01

    The biogeochemistry of phyllosilicate–Fe redox cycling was studied in a Phalaris arundinacea (reed canary grass) dominated redoximorphic soil from Shovelers Sink, a small glacial depression near Madison, WI. The clay size fraction of Shovelers Sink soil accounts for 16% of the dry weight of the soil, yet contributes 74% of total Fe. The dominant mineral in the clay size fraction is mixed layer illite–smectite, and in contrast to many other soils and sediments, Fe(III) oxides are present in low abundance. We examined the Fe biogeochemistry of Shovelers Sink soils, estimated the abundance of Fe redox cycling microorganisms, and isolated in pure culture representative phyllosilicate–Fe oxidizing and reducing organisms. The abundance of phyllosilicate–Fe reducing and oxidizing organisms was low compared to culturable aerobic heterotrophs. Both direct isolation and dilution-to-extinction approaches using structural Fe(II) in Bancroft biotite as a Fe(II) source, and O2 as the electron acceptor, resulted in recovery of common rhizosphere organisms including Bradyrhizobium spp. and strains of Cupriavidus necator and Ralstonia solanacearum. In addition to oxidizing biotite and soluble Fe(II) with O2, each of these isolates was able to oxidize Fe(II) in reduced NAu-2 smectite with NO3- as the electron acceptor. Oxidized NAu-2 smectite or amorphous Fe(III) oxide served as electron acceptors for enrichment and isolation of Fe(III)-reducing microorganisms, resulting in recovery of a strain related to Geobacter toluenoxydans. The ability of the recovered microorganisms to cycle phyllosilicate–Fe was verified in an experiment with native Shovelers Sink clay. This study confirms that Fe in the native Shovelers Sink clay is readily available for microbial redox transformation and can be cycled by the Fe(III)-reducing and Fe(II)-oxidizing microorganisms recovered from the soil. PMID:22493596

  6. Isolation of phyllosilicate-iron redox cycling microorganisms from an illite-smectite rich hydromorphic soil.

    PubMed

    Shelobolina, Evgenya; Konishi, Hiromi; Xu, Huifang; Benzine, Jason; Xiong, Mai Yia; Wu, Tao; Blöthe, Marco; Roden, Eric

    2012-01-01

    The biogeochemistry of phyllosilicate-Fe redox cycling was studied in a Phalaris arundinacea (reed canary grass) dominated redoximorphic soil from Shovelers Sink, a small glacial depression near Madison, WI. The clay size fraction of Shovelers Sink soil accounts for 16% of the dry weight of the soil, yet contributes 74% of total Fe. The dominant mineral in the clay size fraction is mixed layer illite-smectite, and in contrast to many other soils and sediments, Fe(III) oxides are present in low abundance. We examined the Fe biogeochemistry of Shovelers Sink soils, estimated the abundance of Fe redox cycling microorganisms, and isolated in pure culture representative phyllosilicate-Fe oxidizing and reducing organisms. The abundance of phyllosilicate-Fe reducing and oxidizing organisms was low compared to culturable aerobic heterotrophs. Both direct isolation and dilution-to-extinction approaches using structural Fe(II) in Bancroft biotite as a Fe(II) source, and O(2) as the electron acceptor, resulted in recovery of common rhizosphere organisms including Bradyrhizobium spp. and strains of Cupriavidus necator and Ralstonia solanacearum. In addition to oxidizing biotite and soluble Fe(II) with O(2), each of these isolates was able to oxidize Fe(II) in reduced NAu-2 smectite with [Formula: see text] as the electron acceptor. Oxidized NAu-2 smectite or amorphous Fe(III) oxide served as electron acceptors for enrichment and isolation of Fe(III)-reducing microorganisms, resulting in recovery of a strain related to Geobacter toluenoxydans. The ability of the recovered microorganisms to cycle phyllosilicate-Fe was verified in an experiment with native Shovelers Sink clay. This study confirms that Fe in the native Shovelers Sink clay is readily available for microbial redox transformation and can be cycled by the Fe(III)-reducing and Fe(II)-oxidizing microorganisms recovered from the soil.

  7. Salt-tolerant phenol-degrading microorganisms isolated from Amazonian soil samples.

    PubMed

    Bastos, A E; Moon, D H; Rossi, A; Trevors, J T; Tsai, S M

    2000-11-01

    Two phenol-degrading microorganisms were isolated from Amazonian rain forest soil samples after enrichment in the presence of phenol and a high salt concentration. The yeast Candida tropicalis and the bacterium Alcaligenes faecoalis were identified using several techniques, including staining, morphological observation and biochemical tests, fatty acid profiles and 16S/18S rRNA sequencing. Both isolates, A. faecalis and C. tropicalis, were used in phenol degradation assays, with Rhodococcus erythropolis as a reference phenol-degrading bacterium, and compared to microbial populations from wastewater samples collected from phenol-contaminated environments. C. tropicalis tolerated higher concentrations of phenol and salt (16 mM and 15%, respectively) than A. faecalis (12 mM and 5.6%). The yeast also tolerated a wider pH range (3-9) during phenol degradation than A. faecalis (pH 7-9). Phenol degradation was repressed in C. tropicalis by acetate and glucose, but not by lactate. Glucose and acetate had little effect, while lactate stimulated phenol degradation in A. faecalis. To our knowledge, these soils had never been contaminated with man-made phenolic compounds and this is the first report of phenol-degrading microorganisms from Amazonian forest soil samples. The results support the idea that natural uncontaminated environments contain sufficient genetic diversity to make them valid choices for the isolation of microorganisms useful in bioremediation.

  8. Iron corrosion activity of anaerobic hydrogen-consuming microorganisms isolated from oil facilities.

    PubMed

    Mori, Koji; Tsurumaru, Hirohito; Harayama, Shigeaki

    2010-10-01

    The purpose of the present study was to test the hypothesis that anaerobic hydrogen-consuming microorganisms generally promote iron corrosion. We isolated 26 hydrogen-consuming microorganisms (acetogens, sulfate-reducing bacteria, and methanogens) from oil facilities in Japan using hydrogen as an electron donor. The iron corrosion activities of these microorganisms were examined using iron (Fe0) granules as the sole electron donor. Almost all the isolates consumed hydrogen that was chemically generated from iron granules but did not induce significant iron corrosion. The amount of corroded iron in the cultures of these organisms was less than 2-fold that in an abiotic chemical corrosion reaction. These results indicated that hydrogen consumption did not strongly stimulate iron corrosion. On the other hand, one isolate, namely, Methanococcus maripaludis Mic1c10, considerably corroded iron: this phenomenon was not accompanied by hydrogen consumption, methane formation, or cell growth. This finding also provided strong evidence that M. maripaludis Mic1c10 produced some material that caused iron to corrode.

  9. [Compliance with contact isolation precautions of multidrug-resistant microorganisms in a tertiary hospital].

    PubMed

    González-Estrada, A; Fernández-Prada, M; Martínez Ortega, C; Lana Pérez, A; López González, M L

    2016-01-01

    Preventive isolation of patients with multidrug-resistant microorganisms is considered an effective measure to prevent outbreaks in hospitals. The objective of this study is to assess compliance by healthcare workers and family of contact isolation precautions in colonised/infected patients with multidrug-resistant microorganisms. An observational study was conducted from October 2014 to March 2015. A checklist with a structure was designed (equipment trolley), including knowledge of the situation and compliance by the patients, families, and healthcare workers. Univariate and bivariate analyses were performed. Non-parametric tests Mann-Whitney and Kruskal-Wallis were used. Out of the 467 observations made, the equipment trolley was correctly situated in 97% (453) of cases, the sphygmomanometer in 90% (421), the stethoscope 43.9% (205), and thermometer 16.5% (77). A dustbin and the alcoholic solution were observed in over 98.7% (461) of cases. The antiseptic soap for patient hygiene was observed to be correctly placed in 348 (74%) of occasions. The situation was known by 84.9% (305) of patients and 91.4% (234) of families. As regards compliance by professionals with the placement/removal of disposable gowns and gloves was about 50% for entering the room (49.5%, 56 gown and 53.09%, 60 gloves), and 40% (28) for leaving the room by professionals. Hand hygiene compliance was 26.5% (30) for entering and 35.2% (25) when leaving. There is significant room for improvement in the compliance with isolation precautions. Non-compliance to isolation procedures is not due to a deficit of materials, but to individual behaviours. It is important to implement and evaluate programs based on psychosocial intervention models that can change attitudes and behaviours related to contact isolation precautions for multidrug-resistant microorganisms. Copyright © 2016 SECA. Publicado por Elsevier España, S.L.U. All rights reserved.

  10. Adsorption of aflatoxin B1, zearalenone and ochratoxin A by microorganisms isolated from Kefir grains.

    PubMed

    Taheur, Fadia Ben; Fedhila, Kais; Chaieb, Kamel; Kouidhi, Bochra; Bakhrouf, Amina; Abrunhosa, Luís

    2017-06-19

    A strategy to reduce the deleterious effects of mycotoxins is to use dietary supplements that contain microorganisms that bind mycotoxins and decrease their gastrointestinal absorption. Novel strains were isolated from a Kefir culture and assessed for their mycotoxin adsorption and biotransformation ability. The most active strains were identified using DNA sequencing, and the stability of microorganism/mycotoxin complexes was evaluated using buffer solutions to simulate the pH conditions in the gastrointestinal tract. Our results showed that the microorganism consortium of Kefir grains adsorbed 82 to 100% of aflatoxin B1 (AFB1), zearalenone (ZEA) and ochratoxin A (OTA) when cultivated in milk. The main strains that were capable of mycotoxin adsorption were identified as Lactobacillus kefiri, Kazachstania servazzii and Acetobacter syzygii. The strain L. kefiri KFLM3 was the most active, adsorbing 80 to 100% of the studied mycotoxins when cultivated in milk. Nonetheless, the strain K. servazzii KFGY7 retained more mycotoxin after the desorption experiments (65, 69 and 67% for AFB1, OTA and ZEA, respectively). These findings suggest that Kefir consumption may help to reduce gastrointestinal absorption of these mycotoxins and consequently reduce their toxic effects. The isolated strains may be of interest for the development of fermented dairy products for human consumption that have a new probiotic characteristic, the adsorption of mycotoxins. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Methods for Observing Microbial Biofilms Directly on Leaf Surfaces and Recovering Them for Isolation of Culturable Microorganisms

    PubMed Central

    Morris, C. E.; Monier, J.; Jacques, M.

    1997-01-01

    Epifluorescence microscopy, scanning electron microscopy, and confocal laser scanning microscopy were used to observe microbial biofilms directly on leaf surfaces. Biofilms were observed on leaves of all species sampled (spinach, lettuce, Chinese cabbage, celery, leeks, basil, parsley, and broad-leaved endive), although the epifluorescent images were clearest when pale green tissue or cuticle pieces were used. With these techniques, biofilms were observed that were about 20 (mu)m in depth and up to 1 mm in length and that contained copious exopolymeric matrices, diverse morphotypes of microorganisms, and debris. The epifluorescence techniques described here can be used to rapidly determine the abundance and localization of biofilms on leaves. An additional technique was developed to recover individual biofilms or portions of single biofilms from leaves and to disintegrate them for isolation of the culturable microorganisms they contained. Nineteen biofilms from broad-leaved endive, spinach, parsley, and olive leaves were thus isolated and characterized to illustrate the applications of this technique. PMID:16535579

  12. Nontraditional method of evaluating disinfectants: with isolated microorganisms from the food factory.

    PubMed

    Herrera, Anavella Gaitan

    2004-01-01

    Cleaning and disinfection in the food industry are critical in the production process, and the efficacy of the disinfectants used is frequently debated. Several factors are involved in the effectiveness of a disinfectant agent. It is important to consider the number and type of microorganism present as well as the physical and chemical characteristics of the water; these factors vary from industry to industry and they determine efficacious disinfection. In the laboratory it is possible to evaluate disinfectants to be used in a particular factory, even though these are different from those reported by international organizations. Some useful practices are: 1. To use cultures of microorganisms isolated in one's own lab instead of reference cultures. 2. To use as a diluter the water that is used daily in the factory under question. 3. To compare different disinfectant products under identical conditions of time and temperature.

  13. Isolation and characterization of the ethynylestradiol-biodegrading microorganism Fusarium proliferatum strain HNS-1.

    PubMed

    Shi, J H; Suzuki, Y; Lee, B D; Nakai, S; Hosomi, M

    2002-01-01

    We cultivated hundreds of sediment, soil, and manure samples taken from rivers and farms in a medium containing ethynylestradiol (EE2) as the sole source of carbon, so that microorganisms in the samples would acclimatize to the presence of EE2. Finally, we isolated an EE2-degrading microorganism, designated as strain HNS-1, from a cowshed sample. Based on its partial nucleotide sequence (563 bp) of the 28S rRNA gene, strain HNS-1 was identified as Fusarium proliferatum. Over 15 days, F. proliferatum strain HNS-1 removed 97% of EE2 at an initial concentration of 25 mg.L-1, with a first-order rate constant of 0.6 d-1. Unknown products of EE2 degradation, which may be more polar compounds that have a phenolic group, remained in the culture medium.

  14. Isolation and characterization of a novel thraustochytrid-like microorganism that efficiently produces docosahexaenoic acid.

    PubMed

    Perveen, Zakia; Ando, Hitomi; Ueno, Akio; Ito, Yukiya; Yamamoto, Yusuke; Yamada, Yohko; Takagi, Tomoko; Kaneko, Takako; Kogame, Kazuhiro; Okuyama, Hidetoshi

    2006-02-01

    A thraustochytrid-like microorganism (strain 12B) was isolated from the mangrove area of Okinawa, Japan. On the basis of its ectoplasmic net structure and biflagellate zoospores we determined strain 12B to be a novel member of the phylum Labyrinthulomycota in the kingdom Protoctista. When grown on glucose/seawater at 28 degrees C, it had a lipid content of 58% with docosahexaenoic acid (DHA; 22:6 n-3) at 43% of the total fatty acids. It had a growth rate of 0.38 h(-1). The DHA production rate of 2.8 +/- 0.7 g l(-1) day(-1) is the highest value reported for any microorganism.

  15. [Qualitative composition of dominating forms of microorganisms isolated from radionuclide contaminated soil and their ability to accumulate 137Cs].

    PubMed

    Pareniuk, O Iu; Moshynets', O V; Tytova, L V; Levchuk, S Ie

    2013-01-01

    Qualitative composition of the dominating forms of microorganisms isolated from radionuclide contaminated soils has been studied. The ability to accumulate 137Cs by freshly isolated species and collection cultures that were not adapted to the presence of the radionuclide has been analyzed. It is shown that among the analyzed microorganisms the greatest ability to accumulate the radionuclide is inherent in the collection culture Bacillus megaterium UKMV-5724.

  16. Isolation and Identification of Microorganisms in JSC Mars-1 Simulant Soil

    NASA Technical Reports Server (NTRS)

    Mendez, Claudia; Garza, Elizabeth; Gulati, Poonam; Morris, Penny A.; Allen, Carlton C.

    2005-01-01

    Microorganisms were isolated and identified in samples of JSC Mars-1, a Mars simulant soil. JSC Mars-1 is an altered volcanic ash from a cinder cone south of Mauna Kea, Hawaii. This material was chosen because of its similarity to the Martian soil in physical and chemical composition. The soil was obtained by excavating 40 cm deep in a vegetated area to prevent contamination. In previous studies, bacteria from this soil has been isolated by culturing on different types of media, including minimal media, and using biochemical techniques for identification. Isolation by culturing is successful only for a small percentage of the population. As a result, molecular techniques are being employed to identify microorganisms directly from the soil without culturing. In this study, bacteria were identified by purifying and sequencing the DNA encoding the 16s ribosomal RNA (16s rDNA). This gene is well conserved in species and demonstrates species specificity. In addition, biofilm formation, an indicator of microbial life, was studied with this soil. Biofilms are microbial communities consisting of microbes and exopolysaccharides secreted by them. This is a protective way of life for the microbes as they are more resistant to environmental pressures.

  17. Arsenic methylation by micro-organisms isolated from sheepskin bedding materials.

    PubMed

    Lehr, Corinne R; Polishchuk, Elena; Delisle, Marie-Chantal; Franz, Catherine; Cullen, William R

    2003-06-01

    Sudden infant death syndrome (SIDS) has been associated with the volatilization of arsenic, antimony or phosphorus compounds from infants' bedding material by micro-organisms, the so-called 'toxic gas hypothesis'. The volatilization of arsenic by aerobic micro-organisms isolated from new sheepskin bedding material, as well as on material used by a healthy infant and by an infant who perished of SIDS, was examined. Three fungi were isolated from a piece of sheepskin bedding material on which an infant perished of SIDS, which methylated arsenic to form trimethylarsenic(V) species, precursors to volatile trimethylarsine. These three fungi were identified as Scopulariopsis koningii, Fomitopsis pinicola and Penicillium gladioli by their 26S-ribosomal RNA polymerase chain reaction products. These fungi were not previously known to methylate arsenic. The volatilization of arsenic by these three fungi was then examined. Only P. gladioli volatilized arsenic and only under conditions such that the production of sufficient trimethylarsine to be acutely toxic to an infant is unlikely. S. brevicaulis grew on the sheepskin bedding material and evolved a trace amount of trimethylarsine. Known human pathogens such as Mycobacterium neoaurum and Acinetobacter junii were isolated from used bedding.

  18. Diversity and cold adaptation of microorganisms isolated from the Schirmacher Oasis, Antarctica

    NASA Astrophysics Data System (ADS)

    Mojib, Nazia; Bej, Asim K.; Hoover, Richard

    2008-08-01

    We have investigated the feasibility of the PCR amplification of the 16S rRNA genes from eubacteria and Archea on samples collected on Whatman FTA filters from Schirmacher Oasis for the study of culture-independent analysis of the microbial diversity. Both conventional PCR and real-time TaqmaTM PCR successfully amplified the targeted genes. A number of diverse groups of psychrotolerant microorganisms with various pigments have been isolated when cultured on agar medium. 16S rRNA gene analysis of these isolates helped us to identify closest taxonomic genus Pseudomonas, Frigoribacterium, Arthrobacter, Flavobacterium, and Janthinobacterium. It is possible that the pigments play protective role from solar UV radiation, which is prevalent in Antarctic continent especially during Austral summer months. Study of the expression of cold adaptive protein CapB and ice-binding protein IBP using western blots showed positive detection of both or either of these proteins in 6 out of 8 isolates. Since the CapB and IBP protein structure greatly varies in microorganisms, it is possible that the 2 isolates with negative results could have a different class of these proteins. The expression of the CapB and the IBP in these isolates suggest that these proteins are essential for the survival in the Antarctic cold and subzero temperatures and protect themselves from freeze-damage. The current study provided sufficient data to further investigate the rich and diverse biota of psychrotolerant extremophiles in the Antarctic Schirmacher Oasis using both culture-independent and culture-based approaches; and understand the mechanisms of cold tolerance.

  19. Isolation and Characterization of Four Gram-Positive Nickel-Tolerant Microorganisms from Contaminated Sediments

    SciTech Connect

    Van Nostrand, J. D.; Khijniak, T. V.; Gentry, T. J.; Novak, M. T.; Sowder, A. G.; Zhou, J. Z.; Bertsch, P. M.; Morris, P. J.

    2007-01-01

    Microbial communities from riparian sediments contaminated with high levels of Ni and U were examined for metal-tolerant microorganisms. Isolation of four aerobic Ni-tolerant, Gram-positive heterotrophic bacteria indicated selection pressure from Ni. These isolates were identified as Arthrobacter oxydans NR-1, Streptomyces galbus NR-2, Streptomyces aureofaciens NR-3, and Kitasatospora cystarginea NR-4 based on partial 16S rDNA sequences. A functional gene microarray containing gene probes for functions associated with biogeochemical cycling, metal homeostasis, and organic contaminant degradation showed little overlap among the four isolates. Fifteen of the genes were detected in all four isolates with only two of these related to metal resistance, specifically to tellurium. Each of the four isolates also displayed resistance to at least one of six antibiotics tested, with resistance to kanamycin, gentamycin, and ciprofloxacin observed in at least two of the isolates. Further characterization of S. aureofaciens NR-3 and K. cystarginea NR-4 demonstrated that both isolates expressed Ni tolerance constitutively. In addition, both were able to grow in higher concentrations of Ni at pH 6 as compared with pH 7 (42.6 and 8.5 mM Ni at pH 6 and 7, respectively). Tolerance to Cd, Co, and Zn was also examined in these two isolates; a similar pH-dependent metal tolerance was observed when grown with Co and Zn. Neither isolate was tolerant to Cd. These findings suggest that Ni is exerting a selection pressure at this site for metal-resistant actinomycetes.

  20. [Antibacterial activity of copper salts against microorganisms isolated from chronic infected wounds].

    PubMed

    Febré, Naldy; Silva, Viviana; Báez, Andrea; Palza, Humberto; Delgado, Katherine; Aburto, Isabel; Silva, Victor

    2016-12-01

    The antimicrobial activity of copper (Cu+2) is recognized and used as an antimicrobial agent. To evaluate the antimicrobial activity of copper against microorganisms obtained from chronic cutaneous wound infections. Five chemical products that contained copper particles in their composition were tested (zeolite, silica, acetate, nitrate and nanoparticle of copper). The antimicrobial activity against antibiotic resistant strains usually isolated from chronic cutaneous wound infections was determined for two of the products with better performance in copper release. The minimal inhibitory and minimal bactericidal concentrations of copper acetate and nitrate were similar, fluctuating between 400-2,000 µg/ml. The studied copper salts show great potential to be used to control both gram positive and gram negative, antibiotic resistant bacteria isolated from wound infections.

  1. Corrosion of aluminum alloy 2024 by microorganisms isolated from aircraft fuel tanks.

    PubMed

    McNamara, Christopher J; Perry, Thomas D; Leard, Ryan; Bearce, Ktisten; Dante, James; Mitchell, Ralph

    2005-01-01

    Microorganisms frequently contaminate jet fuel and cause corrosion of fuel tank metals. In the past, jet fuel contaminants included a diverse group of bacteria and fungi. The most common contaminant was the fungus Hormoconis resinae. However, the jet fuel community has been altered by changes in the composition of the fuel and is now dominated by bacterial contaminants. The purpose of this research was to determine the composition of the microbial community found in fuel tanks containing jet propellant-8 (JP-8) and to determine the potential of this community to cause corrosion of aluminum alloy 2024 (AA2024). Isolates cultured from fuel tanks containing JP-8 were closely related to the genus Bacillus and the fungi Aureobasidium and Penicillium. Biocidal activity of the fuel system icing inhibitor diethylene glycol monomethyl ether is the most likely cause of the prevalence of endospore forming bacteria. Electrochemical impedance spectroscopy and metallographic analysis of AA2024 exposed to the fuel tank environment indicated that the isolates caused corrosion of AA2024. Despite the limited taxonomic diversity of microorganisms recovered from jet fuel, the community has the potential to corrode fuel tanks.

  2. Amylolytic Microorganism from São Paulo Zoo Composting: Isolation, Identification, and Amylase Production

    PubMed Central

    Pascon, Renata C.; Bergamo, Rogério Faria; Spinelli, Rafael Xavier; de Souza, Elisangela Dutra; Assis, Diego Magno; Juliano, Luiz; Vallim, Marcelo Afonso

    2011-01-01

    Composting is a way of transforming the organic waste into fertilizer, minimizing the use of inorganic compounds that may contaminate the environment. This transformation is the result of the microorganism action, converting complex carbon sources into energy. Enzymes that are exported by the microorganisms to the surrounding environment mediate this process. The aiming of the present work is to prospect the compost produced by the organic composting unit (OCU) of the Fundação Parque Zoológico de São Paulo (FPZSP) to find novel starch hydrolyzing organisms (SHO) that secrete large amounts of amylases under harsh conditions, such as high temperature. We found five bacterial isolates that have amylolytic activity induced by soluble starch and 39°C temperature of growth. These bacterial strains were identified by MALDI-TOF (Matrix-assisted laser desorption/ionization-Time of Flight) analysis, a rapid and efficient methodology for microbe identification in large scale. Our results present amylolytic strains that belong to diverse taxonomic groups (Solibacillus silvestris, Arthrobacter arilaitensis, Isoptericola variabilis, and Acinetobacter calcoaceticus); some of them have never been associated with this kind of hydrolytic activity before. The information regarding enzyme induction will be important to optimize the production by the bacterial isolates, which may be a great value for biotechnological applications. PMID:21845217

  3. Growth study and hydrocarbonoclastic potential of microorganisms isolated from aviation fuel spill site in Ibeno, Nigeria.

    PubMed

    Etuk, C U; John, R C; Ekong, U E; Akpan, M M

    2012-10-01

    The growth study and hydrocarbonoclastic potential of microorganisms isolated from aviation fuel spill sites at Inua-eyet Ikot in Ibeno, Nigeria were examined using standard microbiological methods. The results of the analysis revealed that the viable plate count of microorganisms in the polluted soil ranged from 2.2 ± 0.04 × 10(3) to 3.4 ± 0.14 × 10(6) cfu/g for bacteria and 1.4 ± 0.5 × 10(2) to 2.3 ± 0.4 × 10(4) cfu/g for fungi while count of biodegraders ranged from 1.2 ± 0.4 × 10(3) to 2.1 ± 0.8 × 10(5) cfu/g. A total of 11 microbial isolates comprising of Micrococcus, Klebsiella, Flavobacterium, Bacillus, Pseudomonas, Candida, Aspergillus, Cladosporium, Penicillium, Saccharomyces and Fusarium were characterized. The ability of the selected isolates to utilize the pollutant (aviation fuel) as their sole source of carbon and energy was examined and noticed to vary in growth profiles between the isolates. The results of their degradability after 28 days of incubation shows that species of Cladosporium, Pseudomonas, Candida, Bacillus, Micrococcus and Penicillium were the most efficient Aviation fuel degraders with percentage weight loss of 86.2, 78.4, 78, 56, 53 and 50.6 respectively. Flavobacterium, Saccharomyces and Aspergillus exhibited moderate growth with percentage weight loss of 48, 45.8 and 43.4 respectively while Klebsiella and Fusarium species showed minimal growth with percentage weight loss of 20 and 18.5 respectively. The results imply that the most efficient biodegraders like Cladosporium, Pseudomonas, Candida, Bacillus and Microoccus could tolerate and remove aviation fuel from the environment.

  4. Phylogenetic and physiological diversity of microorganisms isolated from a deep greenland glacier ice core

    NASA Technical Reports Server (NTRS)

    Miteva, V. I.; Sheridan, P. P.; Brenchley, J. E.

    2004-01-01

    We studied a sample from the GISP 2 (Greenland Ice Sheet Project) ice core to determine the diversity and survival of microorganisms trapped in the ice at least 120,000 years ago. Previously, we examined the phylogenetic relationships among 16S ribosomal DNA (rDNA) sequences in a clone library obtained by PCR amplification from genomic DNA extracted from anaerobic enrichments. Here we report the isolation of nearly 800 aerobic organisms that were grouped by morphology and amplified rDNA restriction analysis patterns to select isolates for further study. The phylogenetic analyses of 56 representative rDNA sequences showed that the isolates belonged to four major phylogenetic groups: the high-G+C gram-positives, low-G+C gram-positives, Proteobacteria, and the Cytophaga-Flavobacterium-Bacteroides group. The most abundant and diverse isolates were within the high-G+C gram-positive cluster that had not been represented in the clone library. The Jukes-Cantor evolutionary distance matrix results suggested that at least 7 isolates represent new species within characterized genera and that 49 are different strains of known species. The isolates were further categorized based on the isolation conditions, temperature range for growth, enzyme activity, antibiotic resistance, presence of plasmids, and strain-specific genomic variations. A significant observation with implications for the development of novel and more effective cultivation methods was that preliminary incubation in anaerobic and aerobic liquid prior to plating on agar media greatly increased the recovery of CFU from the ice core sample.

  5. Phylogenetic and physiological diversity of microorganisms isolated from a deep greenland glacier ice core

    NASA Technical Reports Server (NTRS)

    Miteva, V. I.; Sheridan, P. P.; Brenchley, J. E.

    2004-01-01

    We studied a sample from the GISP 2 (Greenland Ice Sheet Project) ice core to determine the diversity and survival of microorganisms trapped in the ice at least 120,000 years ago. Previously, we examined the phylogenetic relationships among 16S ribosomal DNA (rDNA) sequences in a clone library obtained by PCR amplification from genomic DNA extracted from anaerobic enrichments. Here we report the isolation of nearly 800 aerobic organisms that were grouped by morphology and amplified rDNA restriction analysis patterns to select isolates for further study. The phylogenetic analyses of 56 representative rDNA sequences showed that the isolates belonged to four major phylogenetic groups: the high-G+C gram-positives, low-G+C gram-positives, Proteobacteria, and the Cytophaga-Flavobacterium-Bacteroides group. The most abundant and diverse isolates were within the high-G+C gram-positive cluster that had not been represented in the clone library. The Jukes-Cantor evolutionary distance matrix results suggested that at least 7 isolates represent new species within characterized genera and that 49 are different strains of known species. The isolates were further categorized based on the isolation conditions, temperature range for growth, enzyme activity, antibiotic resistance, presence of plasmids, and strain-specific genomic variations. A significant observation with implications for the development of novel and more effective cultivation methods was that preliminary incubation in anaerobic and aerobic liquid prior to plating on agar media greatly increased the recovery of CFU from the ice core sample.

  6. Phylogenetic and physiological diversity of microorganisms isolated from a deep greenland glacier ice core.

    PubMed

    Miteva, V I; Sheridan, P P; Brenchley, J E

    2004-01-01

    We studied a sample from the GISP 2 (Greenland Ice Sheet Project) ice core to determine the diversity and survival of microorganisms trapped in the ice at least 120,000 years ago. Previously, we examined the phylogenetic relationships among 16S ribosomal DNA (rDNA) sequences in a clone library obtained by PCR amplification from genomic DNA extracted from anaerobic enrichments. Here we report the isolation of nearly 800 aerobic organisms that were grouped by morphology and amplified rDNA restriction analysis patterns to select isolates for further study. The phylogenetic analyses of 56 representative rDNA sequences showed that the isolates belonged to four major phylogenetic groups: the high-G+C gram-positives, low-G+C gram-positives, Proteobacteria, and the Cytophaga-Flavobacterium-Bacteroides group. The most abundant and diverse isolates were within the high-G+C gram-positive cluster that had not been represented in the clone library. The Jukes-Cantor evolutionary distance matrix results suggested that at least 7 isolates represent new species within characterized genera and that 49 are different strains of known species. The isolates were further categorized based on the isolation conditions, temperature range for growth, enzyme activity, antibiotic resistance, presence of plasmids, and strain-specific genomic variations. A significant observation with implications for the development of novel and more effective cultivation methods was that preliminary incubation in anaerobic and aerobic liquid prior to plating on agar media greatly increased the recovery of CFU from the ice core sample.

  7. Susceptibility Pattern of Microorganisms Isolated by Percutaneous Needle Biopsy in Nonbacteremic Pyogenic Vertebral Osteomyelitis

    PubMed Central

    Desoutter, Sophie; Cottier, Jean-Philippe; Ghout, Idir; Issartel, Bertrand; Dinh, Aurélien; Martin, Arnaud; Carlier, Robert

    2015-01-01

    Pyogenic vertebral osteomyelitis (VO) is diagnosed according to several lines of evidence: clinical, biological, radiological, and histological. Definitive diagnosis requires the isolation of a causative pathogen or histological confirmation. The aim of our study was to describe the microorganisms isolated by percutaneous needle biopsy (PNB) and to analyze their susceptibility patterns, in order to assess the possibility of empirical combination therapy for the treatment of nonbacteremic patients without resorting to PNB. Based on a French prospective multicenter study of 351 patients with VO, we compiled clinical, biological, and radiological findings for 101 patients with microbiologically confirmed VO. Based on antibiotic susceptibility testing of PNB isolated pathogens, the suitabilities of four antibiotic combinations were analyzed: ofloxacin plus rifampin, levofloxacin plus rifampin, ciprofloxacin plus clindamycin, and ciprofloxacin plus amoxicillin-clavulanate. The main causative pathogens identified were coagulase-negative Staphylococcus spp. (26% of isolates), followed by Staphylococcus aureus (21%), Streptoccocus spp. (13%), and enterobacteria (21%). Empirical antibiotic combination therapy was effective in nearly 75% of cases, and the different combinations gave similar results, except for ofloxacin-rifampin, which was effective in only 58% of cases. A “perfect” empirical antibiotic therapy does not exist. If PNB is not possible, a combination of a fluoroquinolone with clindamycin or rifampin can be used, but the high risk of microbiological failure does not allow the exclusion of PNB. (This study has been registered with EudraCT, number 2006-000951-18, and ClinicalTrials.gov, number NCT00764114.) PMID:26438497

  8. Polymer Film-Based Screening and Isolation of Polylactic Acid (PLA)-Degrading Microorganisms.

    PubMed

    Kim, Mi Yeon; Kim, Changman; Moon, Jungheun; Heo, Jinhee; Jung, Sokhee P; Kim, Jung Rae

    2017-02-28

    Polylactic acid (PLA) has been highlighted as an alternative renewable polymer for the replacement of petroleum-based plastic materials, and is considered to be biodegradable. On the other hand, the biodegradation of PLA by terminal degraders, such as microorganisms, requires a lengthy period in the natural environment, and its mechanism is not completely understood. PLA biodegradation studies have been conducted using mainly undefined mixed cultures, but only a few bacterial strains have been isolated and examined. For further characterization of PLA biodegradation, in this study, the PLA-degrading bacteria from digester sludge were isolated and identified using a polymer film-based screening method. The enrichment of sludge on PLA granules was conducted with the serial transference of a subculture into fresh media for 40 days, and the attached biofilm was inoculated on a PLA film on an agar plate. 3D optical microscopy showed that the isolates physically degraded the PLA film due to bacterial degradation. 16S rRNA gene sequencing identified the microbial colonies to be Pseudomonas sp. MYK1 and Bacillus sp. MYK2. The two isolates exhibited significantly higher specific gas production rates from PLA biodegradation compared with that of the initial sludge inoculum.

  9. Comparison of surface swab and quantitative biopsy cultures dependent on isolated microorganisms from burn wounds.

    PubMed

    Vural, Mete Koray; Altoparlak, Ulku; Celebi, Demet; Akcay, Mufide Nuran

    2013-02-01

    Infections are one of the most important and potentially serious complications of burn wounds. Quantitative biopsy cultures are useful for showing that a burn wound infection is not present, but these cultures are not useful for showing that an infection is present. Swab cultures are used to diagnosis surface colonization and may not accurately reflect the organism(s) causing the wound infection. To perform a comparison of surface swab and biopsy cultures for isolated microorganisms from burn wounds, 160 biopsy/surface swab pairs were collected from 160 patients. Seventy-seven patients (48.1%) showed positive micro-organism cultures from the burn wounds; 19.3 percent had both positive surface cultures and significant bacterial counts from the biopsy cultures, whereas 28.8 percent of the patients had only positive surface cultures. There was moderate agreement (41%) between the results of surface swab and quantitative biopsy cultures used to diagnose burn wound infections. Pseudomonas aeruginosa (45.5%) was the most predominantly isolated bacteria from the wounds, and there was good concordance between the results of the surface swab and quantitative biopsy culture samples (78%). Coagulase-negative Staphylococci (CNS) and Escherichia coli strains were only isolated from the surface swab samples because they are colonizing bacteria. The univariate analysis revealed that there were significant associations between the results of the positive biopsy cultures and the total surface body area, open flame burns, prolonged hospitalization and female gender (p<0.05). Surface swab and quantitative biopsy cultures have a high rate of concordance in predicting P. aeruginosa invasion and the colonization of E. coli and CNS strains in burn wounds.

  10. Isolation, Culture, and Staining of Single Myofibers

    PubMed Central

    Gallot, Yann Simon; Hindi, Sajedah M.; Mann, Aman K.; Kumar, Ashok

    2016-01-01

    Adult skeletal muscle regeneration is orchestrated by a specialized population of adult stem cells called satellite cells, which are localized between the basal lamina and the plasma membrane of myofibers. The process of satellite cell-activation, proliferation, and subsequent differentiation that occurs during muscle regeneration can be recapitulated ex vivo by isolation of single myofibers from skeletal muscles and culturing them under suspension conditions. Here, we describe an improved protocol to evaluate ex vivo satellite cells activation through isolation of single myofibers from extensor digitorum longus (EDL) muscle of mice and culturing and staining of myofiber-associated satellite cells with the markers of self-renewal, proliferation, and differentiation. PMID:27819014

  11. Microorganisms isolated from root canals presenting necrotic pulp and their drug susceptibility in vitro.

    PubMed

    Lana, M A; Ribeiro-Sobrinho, A P; Stehling, R; Garcia, G D; Silva, B K; Hamdan, J S; Nicoli, J R; Carvalho, M A; Farias, L de M

    2001-04-01

    The knowledge about causative agents involved in endodontic infections is increasing, especially due to the improvement of culture techniques for anaerobic bacteria, showing that these microorganisms are predominant in this pathology. In this study, 31 canals with pulp necrosis were microbiologically analyzed before and after manipulation. Obligate and facultative anaerobes, microaerophilic bacteria and yeasts were recovered from 24, 14, 5 and 2 clinical specimens, respectively. The most frequent genera were Prevotella, Fusobacterium, Lactobacillus, Streptococcus, Clostridium and Peptostreptococcus for bacteria and Candida and Saccharomyces for yeasts. Strong positive associations, using an odds ratio system, were found between Clostridium and Prevotella and between Peptostreptococcus and Fusobacterium. Even after the instrumentation and the use of Ca(OH)2, facultative anaerobes were detected in two root canals and yeasts in three. Microorganisms were isolated from seven canals at the end of the endodontic treatment: facultative anaerobes from five and yeasts from one. The microbiological evaluation of root canals with pulp necrosis suggests the presence of polymicrobial infections, mainly involving obligate anaerobes, and shows that the infection may persist after treatment.

  12. [Resistance to UV radiation of microorganisms isolated from the rock biotopes of the Antarctic region].

    PubMed

    Romanovskaia, V A; Tashirev, A B; Shilin, S O; Chernaia, N A

    2010-01-01

    Microbiological analysis of terrestrial biotopes of the Antarctic Region has shown, that vertical rocks of the Antarctic islands open for the Sun were characterized by special microcenoses. The wide distribution of pigmented microorganisms in the rock Antarctic samples, a higher frequency of their occurrence, the total number and biologic diversity, than in other Antarctic biotopes, has been demonstrated. For the first time the presence of bacteria and yeast, resistant to high doses of UV radiation on the vertical rocks in the Antarctic Region was shown. The lethal doze of UV radiation for the Antarctic pink pigmented Methylobacterium strains exceeded 200-300 J/m2, for coal-black yeast--500-800 J/m2, for red yeast--1200-1500 J/m2. The distinctions in lethal UV effect against strains of Methylobacterium isolated from the regions with different climate have not been found. Probably, adaptation of the rock microcenosis to extreme factors of the environment proceeds by natural selection of microorganisms, which resistance to this factor is genetically determined.

  13. Degradation of vanillic acid and production of guaiacol by microorganisms isolated from cork samples.

    PubMed

    Alvarez-Rodríguez, María Luisa; Belloch, Carmela; Villa, Mercedes; Uruburu, Federico; Larriba, Germán; Coque, Juan José R

    2003-03-14

    The presence of guaiacol in cork stoppers is responsible for some cases of cork taint causing unpleasant alterations to wine. We have performed a characterization of the cork-associated microbiota by isolating 55 different microorganisms: eight yeast, 14 filamentous fungi or molds, 13 actinomycetes and 20 non-filamentous bacteria. A screening for degradation of vanillic acid and guaiacol production showed that none of the filamentous fungi could achieve any of these processes. By contrast, five of the eight yeast strains isolated were able to degrade vanillic acid, although it was not converted to guaiacol. Guaiacol production was only detected in four bacterial strains: one isolate of Bacillus subtilis and three actinomycetes, Streptomyces sp. A3, Streptomyces sp. A5 and Streptomyces sp. A13, were able to accumulate this compound in both liquid media and cultures over cork. These results suggest that guaiacol-mediated cork taint should be attributed to the degradative action of vanillic acid by bacterial strains growing on cork.

  14. Factors affecting the attachment of micro-organisms isolated from ultrafiltration and reverse osmosis membranes in dairy processing plants.

    PubMed

    Tang, X; Flint, S H; Brooks, J D; Bennett, R J

    2009-08-01

    To identify the types of micro-organisms involved in the formation of biofilms on dairy ultrafiltration and reverse osmosis membranes and investigate factors affecting the attachment of those isolates. Micro-organisms isolated from industrial membranes following standard cleaning were identified using the API culture identification system. Thirteen different isolates representing eight genera were isolated and their ability to attach to surfaces was compared using a microtitre plate assay. Three Klebsiella strains attached best, while mixed strains of Pseudomonas and Klebsiella attached better than individual strains. Whey enhanced the attachment of the isolates. The micro-organisms were characterized according to cell surface hydrophobicity using the microbial adhesion to hydrocarbon (MATH) test, and cell surface charge by measuring the zeta potential. These cell surface characteristics did not show a clear relationship with the attachment of our strains. A variety of different micro-organisms is associated with dairy ultrafiltration and reverse osmosis membranes after cleaning, suggesting several possible sources of contamination. The cleaning of these membranes may be inadequate. The attachment of the different isolates is highly variable and enhanced in the presence of whey. Knowledge of persistent microflora colonizing dairy membrane systems will help develop strategies to mitigate biofilm development in this environment, improving hygiene in membrane processing plants.

  15. Lipid extraction from isolated single nerve cells

    NASA Technical Reports Server (NTRS)

    Krasnov, I. V.

    1977-01-01

    A method of extracting lipids from single neurons isolated from lyophilized tissue is described. The method permits the simultaneous extraction of lipids from 30-40 nerve cells and for each cell provides equal conditions of solvent removal at the conclusion of extraction.

  16. The isolated pancreatic islet as a micro-organ and its transplantation to cure diabetes

    PubMed Central

    2010-01-01

    Over the past three decades the pancreatic islet of Langerhans has taken center stage as an endocrine microorgan whose glucoregulatory function is highly explicable on the basis of the increasingly well understood activities of three highly interactive secretory cells. Islet dysfunction underlies both type 1 and type 2 diabetes mellitus (DM); its protection from immune attack and gluco-and lipo-toxicity may prevent the development of DM; and its replacement by non-surgical transplantation may be curative of DM. During a career marked by vision, focus and tenacity, Paul Lacy contributed substantially to the development of each of these concepts. In this review we focus on Lacy's contribution to the development of the concept of the islet as a micro-organ, how this foreshadowed our current detailed understanding of single cell function and cell-cell interactions and how this led to a reduced model of islet function encouraging islet transplantation. Next, we examine how clinical allotransplantation, first undertaken by Lacy, has contributed to a more complex view of the interaction of islet endocrine cells with its circulation and neighboring tissues, both “in situ” and after transplantation. Lastly, we consider recent developments in some alternative approaches to treatment of DM that Lacy could glimpse on the horizon but did not have the chance to participate in. PMID:21099316

  17. Isolation of Soil Microorganisms Having Antibacterial Activity and Antimigratory Effects on Sphingosylphosphorylcholine-induced Migration of PANC-1 Cells.

    PubMed

    Kang, Jun Hee; Park, Mi Kyung; Kim, Hyun Ji; Kim, Yuri; Lee, Chang Hoon

    2011-12-01

    To obtain soil microorganisms producing antimigratory activity which is important in controlling the metastasis of cancer cells, more than three hundreds of soil microbes were isolated from sixteen soil sources including Namsan mountain and designated as DGU1001-10338. At first, their antibiotic activities were examined by paper-disc method. More than 40 soil microbes produced compounds with antibiotic activity. Then, antimigratory activities of selected soil microorganisms were examined in a sphingosylphosphorylcholine-induced migration assay in PANC-1 cells. Six of 42 soil microorganisms having antibacterial activity also had more than 45% inhibitory activity on migration of PANC-1 cells. These results suggested that selected soil microorganisms were a useful starting point to find compounds for controlling metastasis of cancer cells.

  18. Technologies for Single-Cell Isolation

    PubMed Central

    Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

    2015-01-01

    The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field. PMID:26213926

  19. Technologies for Single-Cell Isolation.

    PubMed

    Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

    2015-07-24

    The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.

  20. Quorum-sensing inhibitory compounds from extremophilic microorganisms isolated from a hypersaline cyanobacterial mat.

    PubMed

    Abed, Raeid M M; Dobretsov, Sergey; Al-Fori, Marwan; Gunasekera, Sarath P; Sudesh, Kumar; Paul, Valerie J

    2013-07-01

    In this study, extremely halophilic and moderately thermophilic microorganisms from a hypersaline microbial mat were screened for their ability to produce antibacterial, antidiatom, antialgal, and quorum-sensing (QS) inhibitory compounds. Five bacterial strains belonging to the genera Marinobacter and Halomonas and one archaeal strain belonging to the genus Haloterrigena were isolated from a microbial mat. The strains were able to grow at a maximum salinity of 22-25 % and a maximum temperature of 45-60 °C. Hexanes, dichloromethane, and butanol extracts from the strains inhibited the growth of at least one out of nine human pathogens. Only butanol extracts of supernatants of Halomonas sp. SK-1 inhibited growth of the microalga Dunaliella salina. Most extracts from isolates inhibited QS of the acyl homoserine lactone producer and reporter Chromobacterium violaceum CV017. Purification of QS inhibitory dichloromethane extracts of Marinobacter sp. SK-3 resulted in isolation of four related diketopiperazines (DKPs): cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Leu), cyclo(L-Pro-L-isoLeu), and cyclo(L-Pro-D-Phe). QS inhibitory properties of these DKPs were tested using C. violaceum CV017 and Escherichia coli-based QS reporters (pSB401 and pSB1075) deficient in AHL production. Cyclo(L-Pro-L-Phe) and cyclo(L-Pro-L-isoLeu) inhibited QS-dependent production of violacein by C. violaceum CV017. Cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Leu), and cyclo(L-Pro-L-isoLeu) reduced QS-dependent luminescence of the reporter E. coli pSB401 induced by 3-oxo-C6-HSL. Our study demonstrated the ability of halophilic and moderately thermophilic strains from a hypersaline microbial mat to produce biotechnologically relevant compounds that could be used as antifouling agents.

  1. Dissimilatory Sb(V) reduction by microorganisms isolated from Sb-contaminated sediment

    NASA Astrophysics Data System (ADS)

    Dovick, M. A.; Kulp, T. R.

    2013-12-01

    this isolate exhibited Sb(V)-dependent heterotrophic growth. These results suggest that the endogenous microbial community from this Sb-contaminated site includes anaerobic microorganisms capable of obtaining energy for growth by oxidizing heterotrophic electron donors using Sb(V) as the terminal electron acceptor. Ongoing work includes identification of the isolated organism using 16S rDNA phylogenetic markers as well as an inventory of known functional genes (e.g., arrA) within this isolate that may more typically encode for As(V)-reduction. These results elucidate the potentially significant role of microbiological transformations in controlling the speciation of Sb in the environment, and may help to identify potential bioremediation strategies for Sb contaminated waters.

  2. Conversion of sucrose into isomaltulose by Enterobacter sp. FMB1, an isomaltulose-producing microorganism isolated from traditional Korean food.

    PubMed

    Cho, Mee-Hyun; Park, Sang-Eun; Lim, Jin Kyu; Kim, Jong-Sang; Kim, Jeong Hwan; Kwon, Dae Young; Park, Cheon-Seok

    2007-03-01

    Over 500 microorganisms isolated from Korean traditional foods, Maeju (source of soybean paste) and Nuruk (Korean koji), were screened to obtain an isomaltulose-producing microorganism. It was identified as Enterobacter sp. FMB-1 by 16S rRNA sequencing and the API 20E system. It had a greater than 90% conversion of sucrose (as 4 g/l) to isomaltulose in 2 days. Small amounts of trehalulose, glucose, and fructose were produced as byproducts, implying that this strain could be possibly employed in the production of isomaltulose in industry.

  3. Comparative antimicrobial efficacy of selected root canal irrigants on commonly isolated microorganisms in endodontic infection.

    PubMed

    Dubey, Sandeep; Saha, Suparna Ganguly; Rajkumar, Balakrishnan; Dhole, Tapan Kumar

    2017-01-01

    This study aims to evaluate and compare the antimicrobial efficacy of three selected root canal irrigants (BioPure MTAD, metronidazole, aztreonam) against microbes commonly isolated from polymicrobial microbiota of root canal infection. This study was designed with four experimental groups (Group I - Bacteroides fragilis, Group II - Propionibacterium acnes, Group III - Enterococcus faecalis, Group IV - Candida albicans) based on the microbes selected for the study. Group I and Group II bacteria were used to compare and evaluate antimicrobial effect of BioPure MTAD, metronidazole, aztreonam, and normal saline. Group III and Group IV bacteria were used to compare and evaluate antimicrobial efficacy of BioPure MTAD, aztreonam, and normal saline. Normal saline was used as a control irrigant in this study. Agar disc diffusion method was applied to assess and compare the antimicrobial action of selected irrigants. Metronidazole was found to be the most effective root canal irrigant against B. fragilis and P. acnes among the tested irrigants. Mean zone of inhibition against E. faecalis has been shown to be maximum by BioPure MTAD, followed by aztreonam. Antifungal effect against C. albicans was only shown by BioPure MTAD. Overall, BioPure MTAD is the most effective root canal irrigant as it has shown an antibacterial effect against all the tested microorganisms. However, metronidazole showed maximum antibacterial effect against obligate anaerobes. Aztreonam also showed an antibacterial effect in the present study, raising its possibility to be used as a root canal irrigant in the future.

  4. [Isolation and identification of electrochemically active microorganism from micro-aerobic environment].

    PubMed

    Wu, Song; Xiao, Yong; Zheng, Zhi-Yong; Zheng, Yue; Yang, Zhao-Hui; Zhao, Feng

    2014-10-01

    Extracellular electron transfer of electrochemically active microorganism plays vital role in biogeochemical cycling of metals and carbon and in biosynthesis of bioenergy. Compared to anaerobic anode, micro-aerobic anode captures more energy from microbial fuel cell. However, most of previous researches focused on functioning bacteria in anaerobic anode, functioning bacteria in micro-aerobic anode was rarely studied. Herein, we used the traditional aerobic screening technology to isolate functioning bacteria from a micro-aerobic anode. Three pure cultures Aeromonas sp. WS-XY2, Citrobacter sp. WS-XY3 and Bacterium strain WS-XY4 were obtained. WS-XY2 and WS-XY3 were belonged to Proteobacteria, whereas WS-XY4 was possibly a new species. Cyclic voltammetry and chronoamperometry analysis demonstrated all of them showed the electrochemical activity by direct extracellular electron transfer, and micro-aerobic anode could select bacteria that have similar electrochemical activity to proliferate on the anode. We further conclude that functioning bacteria in micro-aerobic anode are more efficient than that of anaerobic anode may be the reason that micro-aerobic anode has better performance than anaerobic anode. Therefore, a thorough study of functioning bacteria in micro-aerobic anode will significantly promote the energy recovery from microbial fuel cell.

  5. Essential Oils of Plants as Biocides against Microorganisms Isolated from Cuban and Argentine Documentary Heritage.

    PubMed

    Borrego, Sofía; Valdés, Oderlaise; Vivar, Isbel; Lavin, Paola; Guiamet, Patricia; Battistoni, Patricia; Gómez de Saravia, Sandra; Borges, Pedro

    2012-01-01

    Natural products obtained from plants with biocidal activity represent an alternative and useful source in the control of biodeterioration of documentary heritage, without negative environmental and human impacts. In this work, we studied the antimicrobial activity of seven essential oils against microorganisms associated with the biodeterioration of documentary heritage. The essential oils were obtained by steam distillation. The antimicrobial activity was analyzed using the agar diffusion method against 4 strains of fungi and 6 bacterial strains isolated from repositories air and documents of the National Archive of the Republic of Cuba and the Historical Archive of the Museum of La Plata, Argentina. Anise and garlic oils showed the best antifungal activity at all concentrations studied, while oregano oil not only was effective against fungi tested but also prevented sporulation of them all. Orange sweet and laurel oils were ineffective against fungi. Clove, garlic, and oregano oils showed the highest antibacterial activity at 25% against Enterobacter agglomerans and Streptomyces sp., while only clove and oregano oils were effective against Bacillus sp. at all concentrations studied. This study has an important implication for the possible use of the natural products from plants in the control of biodeterioration of documentary heritage.

  6. Essential Oils of Plants as Biocides against Microorganisms Isolated from Cuban and Argentine Documentary Heritage

    PubMed Central

    Borrego, Sofía; Valdés, Oderlaise; Vivar, Isbel; Lavin, Paola; Guiamet, Patricia; Battistoni, Patricia; Gómez de Saravia, Sandra; Borges, Pedro

    2012-01-01

    Natural products obtained from plants with biocidal activity represent an alternative and useful source in the control of biodeterioration of documentary heritage, without negative environmental and human impacts. In this work, we studied the antimicrobial activity of seven essential oils against microorganisms associated with the biodeterioration of documentary heritage. The essential oils were obtained by steam distillation. The antimicrobial activity was analyzed using the agar diffusion method against 4 strains of fungi and 6 bacterial strains isolated from repositories air and documents of the National Archive of the Republic of Cuba and the Historical Archive of the Museum of La Plata, Argentina. Anise and garlic oils showed the best antifungal activity at all concentrations studied, while oregano oil not only was effective against fungi tested but also prevented sporulation of them all. Orange sweet and laurel oils were ineffective against fungi. Clove, garlic, and oregano oils showed the highest antibacterial activity at 25% against Enterobacter agglomerans and Streptomyces sp., while only clove and oregano oils were effective against Bacillus sp. at all concentrations studied. This study has an important implication for the possible use of the natural products from plants in the control of biodeterioration of documentary heritage. PMID:23762760

  7. Analysis of mitochondria isolated from single cells.

    PubMed

    Johnson, Ryan D; Navratil, Marian; Poe, Bobby G; Xiong, Guohua; Olson, Karen J; Ahmadzadeh, Hossein; Andreyev, Dmitry; Duffy, Ciarán F; Arriaga, Edgar A

    2007-01-01

    Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations. Similar measurements for organelles released from single cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either the fluorescent mitochondrion-specific 10-N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment, an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram, the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach.

  8. Community of thermoacidophilic and arsenic resistant microorganisms isolated from a deep profile of mine heaps.

    PubMed

    Casas-Flores, S; Gómez-Rodríguez, E Y; García-Meza, J V

    2015-12-01

    Soluble arsenic (As) in acidic feed solution may inhibit the copper (Cu) bioleaching process within mine heaps. To clarify the effect of soluble arsenic on the live biomass and bioxidative activity in heaps, toxicological assays were performed using a synthetic feed solution given by a mine company. The microorganisms had previously been isolated from two heap samples at up to 66 m depth, and cultured using specific media for chemolithotrophic acidophiles (pH 1-2) and moderate thermophiles (48°C), for arsenic tolerance assay. The four media with the highest biomass were selected to assay As-resistance; one culture (Q63h) was chosen to assay biooxidative activity, using a heap sample that contained chalcopyrite and covellite. We found that 0.5 g/L of As does not affect living biomass or biooxidative activity on Cu sulfides, but it dissolves Cu, while As precipitates as arsenic acid (H3AsO4·½H2O). The arsenic tolerant community, as identified by 16S rDNA gene sequence analysis, was composed of three main metabolic groups: chemolithotrophs (Leptospirillum, Sulfobacillus); chemolithoheterotrophs and organoheterotrophs as Acidovorax temperans, Pseudomonas alcaligenes, P. mendocina and Sphingomonas spp. Leptospirillum spp. and S. thermosulfidooxidans were the dominant taxa in the Q63-66 cultures from the deepest sample of the oldest, highest-temperature heap. The results indicated arsenic resistance in the microbial community, therefore specific primers were used to amplify ars (arsenic resistance system), aio (arsenite oxidase), or arr (arsenate respiratory reduction) genes from total sample DNA. Presence of arsB genes in S. thermosulfidooxidans in the Q63-66 cultures permits H3AsO4-As(V) detoxification and strengthens the community's response to As.

  9. Isolation of a low-temperature adapted lipolytic enzyme from uncultivated micro-organism.

    PubMed

    Roh, C; Villatte, F

    2008-07-01

    The aim of the study was to isolate a novel lipolytic enzyme from the activated sludge of uncultured micro-organisms. The metagenomic DNA was directly extracted from the activated sludge, and a metagenomic library was constructed by using the pUC vector. The library was screened for lipolytic enzyme activity on 1% tributyrin agar plate. A clone among c. 100 000 recombinant libraries showed the lipolytic activity. The putative lipolytic gene encoding lipo1 from the metagenomic library was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The expressed recombinant enzyme was purified by Ni-nitrilotriacetic acid affinity chromatography and characterized using general substrates of lipolytic property. The gene consisted of 972 bp encoding a polypeptide of 324 amino acids with a molecular mass of 35.6 kDa. Typical residues essential for lipolytic activity such as penta-peptide (GXSXG) and catalytic triad sequences (Ser166, Asp221 and His258) were detected. The deduced amino acid sequence of lipo1 showed low identity with amino acid sequences of esterase/lipase (32%, ZP_01528487) from Pseudomonas mendocina ymp and esterase (31%, AAY45707) from uncultured bacterium. This lipolytic enzyme exhibited the highest activity at pH 7.5 and 10 degrees C. At thermal stability analysis, lipo1 was more unstable at 40 degrees C than 10 degrees C. An activity based strategy has been an effective method for fishing out a low-temperature adapted lipolytic enzyme from the metagenomic library. This lipo1 enzyme can be considered to belong to the hormone-sensitive lipase family due to the enzyme's oxyanion hole by the sequence HGGG. Lipo1 is a novel psychrophilic esterase obtained directly from the metagenomic library. Owing its support of significant activity at low temperature, this enzyme is expected to be useful for potential application as a biocatalyst in organic chemistry.

  10. Comparative antimicrobial efficacy of selected root canal irrigants on commonly isolated microorganisms in endodontic infection

    PubMed Central

    Dubey, Sandeep; Saha, Suparna Ganguly; Rajkumar, Balakrishnan; Dhole, Tapan Kumar

    2017-01-01

    Objective: This study aims to evaluate and compare the antimicrobial efficacy of three selected root canal irrigants (BioPure MTAD, metronidazole, aztreonam) against microbes commonly isolated from polymicrobial microbiota of root canal infection. Materials and Methods: This study was designed with four experimental groups (Group I – Bacteroides fragilis, Group II – Propionibacterium acnes, Group III – Enterococcus faecalis, Group IV – Candida albicans) based on the microbes selected for the study. Group I and Group II bacteria were used to compare and evaluate antimicrobial effect of BioPure MTAD, metronidazole, aztreonam, and normal saline. Group III and Group IV bacteria were used to compare and evaluate antimicrobial efficacy of BioPure MTAD, aztreonam, and normal saline. Normal saline was used as a control irrigant in this study. Agar disc diffusion method was applied to assess and compare the antimicrobial action of selected irrigants. Results: Metronidazole was found to be the most effective root canal irrigant against B. fragilis and P. acnes among the tested irrigants. Mean zone of inhibition against E. faecalis has been shown to be maximum by BioPure MTAD, followed by aztreonam. Antifungal effect against C. albicans was only shown by BioPure MTAD. Conclusions: Overall, BioPure MTAD is the most effective root canal irrigant as it has shown an antibacterial effect against all the tested microorganisms. However, metronidazole showed maximum antibacterial effect against obligate anaerobes. Aztreonam also showed an antibacterial effect in the present study, raising its possibility to be used as a root canal irrigant in the future. PMID:28435359

  11. [Antimicrobial resistance of the most frequently isolated microorganisms in the Hospital General La Mancha Centro between June 2009 and May 2010].

    PubMed

    Asencio, M A; Carranza, R; Huertas, M

    2012-09-01

    During the last two decades an increased incidence of infections caused by multiresistant bacteria has been observed. The spread of these microorganisms in the hospital is a major therapeutic and epidemiological problem. The aim of this study was to determine local resistance patterns of microorganisms causative of multirresistant infections in patients admitted to our hospital. A retrospective study was designed, including Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii strains isolated from clinical and surveillance samples of patients admitted to the Hospital General La Mancha Centro, between June 2009 and May 2010. The rate of S. aureus isolates resistant to oxacillin was 50%, with 0% resistance to vancomycin. The percentage of resistance to 3rd generation cephalosporins in E. coli and K. pneumoniae was 17 and 19%, respectively; the ESBL-production in enterobacterial strains was 15 and 19%, respectively, and the quinolone resistance was 41 and 28%, respectively. The resistance of P. aeruginosa to ceftazidime and imipenem was 30 and 40%, respectively. Most strains of A. baumannii studied came from a single multidrug-resistant clone, endemic in the ICU of our hospital. Of particular concern is the high rate of MRSA, E. coli and K. pneumoniae ESBL producers and resistant to fluoroquinolones as well as P. aeruginosa multiresistant. A. baumannii isolates belong mainly to endemic multidrug-resistant clone from the ICU.

  12. Frequency of microorganisms isolated in patients with bacteremia in intensive care units in Colombia and their resistance profiles.

    PubMed

    Cortes, Jorge Alberto; Leal, Aura Lucía; Montañez, Anita María; Buitrago, Giancarlo; Castillo, Juan Sebastián; Guzman, Lucy

    2013-01-01

    The aim of this study was to describe the most frequently found bacterial microorganisms in bloodstream isolates taken from patients in intensive care units in Colombia and their resistance profiles. This was a multicentre descriptive observational study that was carried out between January 2001 and June 2008 with laboratory data from 33 participating hospitals in a surveillance network. The most frequently found microorganisms were coagulase-negative Staphylococci 39.6%, Staphylococcus aureus 12.3%, Klebsiella pneumoniae 8.2%, Escherichia coli 5.7%, Acinetobacter baumannii, 4.0% and Pseudomonas aeruginosa 3.8%. Coagulase-negative Staphylococci registered greater than 70% oxacillin resistance rate. S. aureus presented a change in its multiresistance profile during the years of follow-up. There was a trend towards a lower resistance rate among E. coli and K. pneumoniae isolates during the study period while A. baumannii carbapenem resistance rate exceeded 50%. There has been a change in the frequency of species being isolated with a higher frequency of enterobacteriaceae compared to Gram-positive microorganisms, in general with a high resistance rate. Copyright © 2013 Elsevier Editora Ltda. All rights reserved.

  13. Study of psychrophilic and psychrotolerant micro-organisms isolated in cold rooms used for pharmaceutical processing.

    PubMed

    Sandle, T; Skinner, K

    2013-04-01

    To examine for psychrophilic or psychrotolerant micro-organisms in pharmaceutical cold rooms (in relation to numbers, incidents and species) and to determine, where such micro-organisms are present, whether standard microbiological environmental monitoring regimes require modification. This is presented as a case study. Comparative environmental monitoring within different pharmaceutical facility cold rooms (using standard mesophilic and low temperature incubation). Data were collected over two periods, 5 years apart. The results indicated that psychrophilic micro-organisms were not present and that those micro-organisms deemed psychrotolerant, primarily pseudomonads, could be grown on standard media under mesophilic conditions. Psychrophilic micro-organisms were not detected and those considered to be psychrotolerant were only found in low numbers. Pyschrotolerant organisms were recovered under both low temperature incubation conditions and under standard conditions (between 20 and 35°C). Further evaluation may be required, using alternative agar, and microbiologists should regularly review the species recovered to note differences between different environments. The study came about from requests made by US and UK regulators concerning the risk of any extremophiles present in pharmaceutical manufacturing facilities upon product safety. Regulators expressed concerns about whether standard, and accepted, environmental monitoring regimes were capable of detecting such micro-organisms. The data provide a benchmark to support pharmaceutical manufacturers in relation to their existing monitoring programmes or as a case study with which to undertake a similar study. © 2012 The Society for Applied Microbiology.

  14. Alert microorganisms isolated from patients hospitalized in Małopolskie province in 2010-2012.

    PubMed

    Seweryn, Michał; Bandoła, Katarzyna; Bała, Małgorzata; Sroka, Sylwia; Koperny, Magdalena; Wszołek, Magdalena

    2014-01-01

    Healthcare centers undertake supervisory activities to control health care-associated infections (HCAIs) by elaborating procedures, identifying alert microorganisms and analyzing data collected. The aim of the study was to analyze the prevalence of alert microorganisms in hospital wards in 2010-2012. Legislation which is in force since several years introduced the principles of health care-associated infections control and reporting system. Analysis was based on annual reports on alert microorganisms provided by 19 District Sanitary and Epidemiological Stations from Małopolskie province. The data discuss positive tests results for alert microorganisms in patients who stayed in hospitals supervised by the Sanitary and Epidemiological Stations. Compared to 2010-2011, the number of tests per hospital bed in 2012 was lower, amounting to 24 (2010 - 44, 2011 - 34). Of these tests, the majority was performed in the following wards: transplantology (2010 - 339, 2011 - 354, 2012 - 330), burn care (2010 - 354, 2011 - 148, 2012 - 113) and ICUs for adults (2010 - 155, 2011 - 157, 2012 - 140). In 2010-2012, an increase in the number of positive test results for extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL+) and Clostridium difficile as well as slight decrease in the number of positive test results for other alert microorganisms were noted. The highest number of microorganisms was identified in neonatal and neonatal pathology (Enterobacteriaceae ESBL+); pediatric and infectious diseases (Rotavirus); infectious diseases (Rotavirus, C.difficile); burn care (Acinetobacter baumanii, Pseudomonas sp.) and ophthalmic and hemodialysis wards (MRSA). Irrespective of a decrease in the number of tests per hospital bed in 2012, a high number of positive test results for alert microorganisms was observed. It suggests the necessity for wider application of collected data as to improve monitoring of infections and reduce resulting threats.

  15. Biodegradation of international jet A-1 aviation fuel by microorganisms isolated from aircraft tank and joint hydrant storage systems.

    PubMed

    Itah, A Y; Brooks, A A; Ogar, B O; Okure, A B

    2009-09-01

    Microorganisms contaminating international Jet A-1 aircraft fuel and fuel preserved in Joint Hydrant Storage Tank (JHST) were isolated, characterized and identified. The isolates were Bacillus subtillis, Bacillus megaterium, Flavobacterium oderatum, Sarcina flava, Micrococcus varians, Pseudomonas aeruginosa, Bacillus licheniformis, Bacillus cereus and Bacillus brevis. Others included Candida tropicalis, Candida albicans, Saccharomyces estuari, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus, Cladosporium resinae, Penicillium citrinum and Penicillium frequentans. The viable plate count of microorganisms in the Aircraft Tank ranged from 1.3 (+/-0.01) x 104 cfu/mL to 2.2 (+/-1.6) x 104 cfu/mL for bacteria and 102 cfu/mL to 1.68 (+/-0.32) x 103 cfu/mL for fungi. Total bacterial counts of 1.79 (+/-0.2) x 104 cfu/mL to 2.58 (+/-0.04) x 104 cfu/mL and total fungal count of 2.1 (+/-0.1) x 103 cfu/mL to 2.28 (+/-0.5) x 103 cfu/mL were obtained for JHST. Selected isolates were re-inoculated into filter sterilized aircraft fuels and biodegradation studies carried out. After 14 days incubation, Cladosporium resinae exhibited the highest degradation rate with a percentage weight loss of 66 followed by Candida albicans (60.6) while Penicillium citrinum was the least degrader with a weight loss of 41.6%. The ability of the isolates to utilize the fuel as their sole source of carbon and energy was examined and found to vary in growth profile between the isolates. The results imply that aviation fuel could be biodegraded by hydrocarbonoclastic microorganisms. To avert a possible deterioration of fuel quality during storage, fuel pipe clogging and failure, engine component damage, wing tank corrosion and aircraft disaster, efficient routine monitoring of aircraft fuel systems is advocated.

  16. Flavor evaluation of yak butter in Tsinghai-Tibet Plateau and isolation of microorganisms contributing flavor.

    PubMed

    Hu, SongQing; Wei, HaiLiu; Guo, ShaSha; Li, Lin; Hou, Yi

    2011-02-01

    Yak butter in Tsinghai-Tibet Plateau possesses the characters of high energy, abundant alimentation and a special flavor with certain medical and health care functions. In this paper the organoleptic flavor of yak butter was estimated, and 28 kinds of substance with different flavors were identified with the technique of coupling gas chromatography to mass spectroscopy (GC-MS). The results showed that there are many microorganisms in yak butter with natural inoculation, which contribute to the formation of its special flavors. It was found that three of these 15 microorganisms, identified as Saccharomycetaceae, Penicillium and Asperillus separately, contributed the most to flavors. The microorganisms are expected to be applied in the food industry, especially to produce dairy food with the unique flavor of yak butter.

  17. Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to -35 degrees C.

    PubMed

    Panikov, Nicolai S; Sizova, Maria V

    2007-02-01

    We developed a procedure to culture microorganisms below freezing point on solid media (cellulose powder or plastic film) with ethanol as the sole carbon source without using artificial antifreezes. Enrichment from soil and permafrost obtained on such frozen solid media contained mainly fungi, and further purification resulted in isolation of basidiomycetous yeasts of the genera Mrakia and Leucosporidium as well as ascomycetous fungi of the genus Geomyces. Contrary to solid frozen media, the enrichment of liquid nutrient solutions at 0 degrees C or supercooled solutions stabilized by glycerol at -1 to -5 degrees C led to the isolation of bacteria representing the genera Polaromonas, Pseudomonas and Arthrobacter. The growth of fungi on ethanol-microcrystalline cellulose media at -8 degrees C was exponential with generation times of 4.6-34 days, while bacteria displayed a linear or progressively declining curvilinear dynamic. At -17 to -0 degrees C the growth of isolates and entire soil community on 14C-ethanol was continuous and characterized by yields of 0.27-0.52 g cell C (g of C-substrate)(-1), similar to growth above the freezing point. The 'state of maintenance,' implying measurable catabolic activity of non-growing cells, was not confirmed. Below -18 to -35 degrees C, the isolated organisms were able to grow only transiently for 3 weeks after cooling with measurable respiratory and biosynthetic (14CO2 uptake) activity. Then metabolic activity declined to zero, and microorganisms entered a state of reversible dormancy.

  18. Isolation of novel benzo[a]anthracene-degrading microorganisms and continuous bioremediation in an expanded-bed bioreactor.

    PubMed

    Rosales, E; Pérez-Paz, A; Vázquez, X; Pazos, M; Sanromán, M A

    2012-06-01

    In the present work, several samples from lab waste containers polluted with polycyclic aromatic hydrocarbons (PAHs) and heavy metals were investigated as potential sources of PAH-degrading microorganisms. After isolating, two fungal strains were selected as the best degrading microorganisms. Genetic identification by sequencing was carried out and they were identified as Trichoderma longibrachiatum and Byssochlamys spectabilis. Their degradation ability was determined in liquid cultures with 100 μM of benzo[a]anthracene. T. longibrachiatum cultures showed highest degradation values (around 97%) after 9 days, furthermore in a second batch the time was reduced to 6 days. To analyse the viability of industrial application, a continuous treatment in an expanded-bed bioreactor was developed operating at different residence times with T. longibrachiatum immobilised on cubes of nylon sponge. It is noticeable that the bioreactor working in continuous mode was able to operate without operational problems and attaining high degradation levels depending on the residence time.

  19. Modification of the mycotoxin deoxynivalenol using microorganisms isolated from environmental samples

    USDA-ARS?s Scientific Manuscript database

    The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of wheat, barley, and maize. New strategies are needed to reduce or eliminate DON in feed and food products. Microorganisms from plant and soil samples collected in Blacksburg, VA, USA, were screened by incubation in a mineral ...

  20. Using the second law of thermodynamics for enrichment and isolation of microorganisms to produce fuel alcohols or hydrocarbons.

    PubMed

    Kohn, Richard A; Kim, Seon-Woo

    2015-10-07

    Fermentation of crops, waste biomass, or gases has been proposed as a means to produce desired chemicals and renewable fuels. The second law of thermodynamics has been shown to determine the net direction of metabolite flow in fermentation processes. In this article, we describe a process to isolate and direct the evolution of microorganisms that convert cellulosic biomass or gaseous CO2 and H2 to biofuels such as ethanol, 1-butanol, butane, or hexane (among others). Mathematical models of fermentation elucidated sets of conditions that thermodynamically favor synthesis of desired products. When these conditions were applied to mixed cultures from the rumen of a cow, bacteria that produced alcohols or alkanes were isolated. The examples demonstrate the first use of thermodynamic analysis to isolate bacteria and control fermentation processes for biofuel production among other uses.

  1. Heterotrophic nitrogen removal by a newly-isolated alkalitolerant microorganism, Serratia marcescens W5.

    PubMed

    Wang, Teng; Dang, Qifeng; Liu, Chengsheng; Yan, Jingquan; Fan, Bing; Cha, Dongsu; Yin, Yanyan; Zhang, Yubei

    2016-07-01

    A new microbe, Serratia marcescens W5 was successfully isolated. Its feasibility in purification of excessively nitrogen-containing wastewater was evaluated using inorganic nitrogen media. Single factor tests showed that W5 exhibited high ammonium removal rates (above 80%) under different culture conditions (pH 7-10, C/N ratios of 6-20, 15-35°C, 0-2.5% of salinity, respectively). Besides various organic carbon sources, W5 was able to utilize calcium carbonate with 28.05% of ammonium removed. Further experiments indicated that W5 was capable of resisting high-strength ammonium (1200mg/L) with the maximum removal rate of 514.13mgL(-1)d(-1). The nitrogen removal pathway of W5 was also tested, showing that both nitrite and nitrate were efficiently removed only in the presence of ammonium, with hydroxylamine as intermediate, which was different from the conventional nitrogen removal pathway. All the results verified that W5 was a good candidate for the purification of excessively nitrogenous wastewater. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Isolation and characterization of chitin-degrading micro-organisms from the faeces of Goeldi's monkey, Callimico goeldii.

    PubMed

    Macdonald, C; Barden, S; Foley, S

    2014-01-01

    The objective of this study was the isolation and characterization of chitin-degrading micro-organisms from the faeces of the insectivorous Goeldi's monkey, Callimico goeldii. Faeces samples were screened for chitin-degrading bacteria using basal medium in which chitin was included as the carbon and energy source. Of fifteen bacterial isolates with chitin-degrading activity, fourteen were also capable of degrading cellulose. All isolates were either aerobes or facultative anaerobes. Phylogenetic analyses of those isolates exhibiting strongest activity, as determined by the most distinctive zones of clearing in chitin-supplemented medium, were identified as Cellulosimicrobium spp., Arthrobacter spp., Staphylococcus spp. and Enterobacteriaceae. This study reports on the isolation of chitin-degrading microflora from nonhuman primates. Considering that chitin and cellulose are the most abundant naturally occurring polymers, it is of interest to note that the majority of isolates are capable of digesting both substrates. This may be of significance given that omnivorous primates live in seasonal environments, where the availability of food items varies with the seasons. Furthermore, given the presence of a chitin-degrading microflora, this may have implications, in terms of the inclusion of fungi and/or insects in the diets of these animals in captivity, whether as part of medical research or conservation programmes. © 2013 The Society for Applied Microbiology.

  3. The isolation and characterization of simultaneous saccharification and fermentation microorganisms for Laminaria japonica utilization.

    PubMed

    Lee, Sung-Mok; Lee, Jae-Hwa

    2011-05-01

    Brown seaweed contains various carbohydrates, such as alginate, laminaran, and mannitol, therefore ethanol fermentation was attempted with Nuruk and a mixed culture that included Laminaria japonica. Nuruk is used to make Korean traditional alcohol. In the research, four microorganisms that produced ethanol and had the ability to achieve alginate degradation were obtained on the L. japonica medium. Nuruk 4 was found to produce a better result than the other tested microorganisms, and the optimal substrate for ethanol production was found to be mannitol (2.59 g/L at 96 h). Nuruk 4 was more than three times better compared with Candida tropicalis in regards to ethanol production. When alginate lyase activity occurred, it appeared as a clear zone around Nuruk 3. The maximal ethanol production yield conditions were comprised of Nuruk 3 and 4 on the anaerobic culture. In this case, 2.0 g/L of ethanol were efficiently produced under the same conditions.

  4. Screening of cloud microorganisms isolated at the Puy de Dôme (France) station for the production of biosurfactants

    NASA Astrophysics Data System (ADS)

    Renard, Pascal; Canet, Isabelle; Sancelme, Martine; Wirgot, Nolwenn; Deguillaume, Laurent; Delort, Anne-Marie

    2016-09-01

    A total of 480 microorganisms collected from 39 clouds sampled at the Puy de Dôme station (alt. 1465 m; 45°46'19'' N, 2°57'52'' E; Massif Central, France) were isolated and identified. This unique collection was screened for biosurfactant (surfactants of microbial origin) production by measuring the surface tension (σ) of the crude extracts, comprising the supernatants of the pure cultures, using the pendant drop technique. The results showed that 41 % of the tested strains were active producers (σ < 55 mN m-1), with 7 % being extremely active (σ < 30 mN m-1). The most efficient biosurfactant producers (σ < 45 mN m-1) belong to a few bacterial genera (Pseudomonas and Xanthomonas) from the Υ-Proteobacteria class (78 %) and a yeast genus (Udeniomyces) from the Basidiomycota phylum (11 %). Some Bacillus strains from the Firmicutes phylum were also active but represented a small fraction of the collected population. Strains from the Actinobacteria phylum in the collection examined in the present study showed moderate biosurfactant production (45<σ < 55 mN m-1). Pseudomonas (Υ-Proteobacteria), the most frequently detected genus in clouds, with some species issued from the phyllosphere, was the dominant group for the production of biosurfactants. We observed some correlations between the chemical composition of cloud water and the presence of biosurfactant-producing microorganisms, suggesting the "biogeography" of this production. Moreover, the potential impact of the production of biosurfactants by cloud microorganisms on atmospheric processes is discussed.

  5. Raman sorting and identification of single living micro-organisms with optical tweezers

    NASA Astrophysics Data System (ADS)

    Xie, Changan; Chen, De; Li, Yong-Qing

    2005-07-01

    We report on a novel technique for sorting and identification of single biological cells and food-borne bacteria based on laser tweezers and Raman spectroscopy (LTRS). With this technique, biological cells of different physiological states in a sample chamber were identified by their Raman spectral signatures and then they were selectively manipulated into a clean collection chamber with optical tweezers through a microchannel. As an example, we sorted the live and dead yeast cells into the collection chamber and validated this with a standard staining technique. We also demonstrated that bacteria existing in spoiled foods could be discriminated from a variety of food particles based on their characteristic Raman spectra and then isolated with laser manipulation. This label-free LTRS sorting technique may find broad applications in microbiology and rapid examination of food-borne diseases.

  6. Simulation of single microorganism motion in fluid based on granular model

    NASA Astrophysics Data System (ADS)

    Viridi, S.; Nuraini, N.

    2016-04-01

    Microorganism model for simulating its motion is proposed in this work. It consists of granular particles which can interact to each other through linear spring mimicking microorganism muscles, which is simpler than other model. As a part of the organism organ is moving, while the other remains at its position, it will push the surrounding fluid through Stoke's force and as reaction the fluid pushes back the microorganism. Contracting force is used to change the distance between two points in the organ. Gravity influence is simply neglected in this work. All the considered forces are used to get motion parameters of organism through molecular dynamics method. It is observed that the use of contracting (push-pull) organ constructs slightly more effective model than shrink- and swell-organs as previously investigated, if weighted effectiveness formula is used as function of number of considered forces and involved particles.

  7. Isolation and 16S DNA characterization of soil microorganisms from tropical soils capable of utilizing the herbicides hexazinone and tebuthiuron.

    PubMed

    Mostafa, Fadwa I Y; Helling, Charles S

    2003-11-01

    Six non-fermentative bacteria were isolated from Colombian (South America) and Hawaiian (USA) soils after enrichment with minimal medium supplemented with two herbicides, hexazinone (Hex) and tebuthiuron (Teb). Microscopic examination and physiological tests were followed by partial 16S DNA sequence analysis, using the first 527 bp of the 16S rRNA gene for bacterial identification. The isolated microorganisms (and in brackets, the herbicide that each degraded) were identified as: from Colombia. Methylobacterium organophilum [Teb], Paenibacillus pabuli [Teb], and Micrmbacterium foliorum [Hex]; and from Hawaii, Methylobacterium radiotolerans [Teb], Paenibacillus illinoisensis [Hex], and Rhodococcus equi [Hex]. The findings further explain how these herbicides, which have potential for illicit coca (Erythroxylum sp.) control, dissipate following their application to tropical soils.

  8. Metabolic response of environmentally isolated microorganisms to industrial effluents: Use of a newly described cell culture assay

    NASA Technical Reports Server (NTRS)

    Ferebee, Robert N.

    1992-01-01

    An environmental application using a microtiter culture assay to measure the metabolic sensitivity of microorganisms to petrochemical effluents will be tested. The Biomedical Operations and Research Branch at NASA JSC has recently developed a rapid and nondestructive method to measure cell growth and metabolism. Using a colorimetric procedure the uniquely modified assay allows the metabolic kinetics of prokaryotic and eukaryotic cells to be measured. Use of such an assay if adapted for the routine monitoring of waste products, process effluents, and environmentally hazardous substances may prove to be invaluable to the industrial community. The microtiter method as described will be tested using microorganisms isolated from the Galveston Bay aquatic habitat. The microbial isolates will be identified prior to testing using the automated systems available at JSC. Sodium dodecyl sulfate (SDS), cadmium, and lead will provide control toxic chemicals. The toxicity of industrial effluent from two industrial sites will be tested. An effort will be made to test the efficacy of this assay for measuring toxicity in a mixed culture community.

  9. Optical Isolator For Use With Single-Mode Fiber

    NASA Technical Reports Server (NTRS)

    Lutes, George F.

    1988-01-01

    Assembly of commercially available components acts as single-mode fiber-optic isolator with lower forward-transmission loss and higher attenuation of reverse transmission than previously achieved in single unit. New design reduces cost and improves performance of optical gyroscopes, precise time- and frequency-signal-distribution systems, and other systems that include fiber optics and isolators.

  10. [Antibacterial activity of essential oils on microorganisms isolated from urinary tract infection].

    PubMed

    Pereira, Rogério Santos; Sumita, Tânia Cristina; Furlan, Marcos Roberto; Jorge, Antonio Olavo Cardoso; Ueno, Mariko

    2004-04-01

    The antibacterial activity of essential oils extracted from medicinal plants (Ocimum gratissimum, L., Cybopogum citratus (DC) Stapf., and Salvia officinalis, L.) was assessed on bacterial strains derived from 100 urine samples. Samples were taken from subjects diagnosed with urinary tract infection living in the community. Microorganisms were plated on Müller Hinton agar. Plant extracts were applied using a Steers replicator and petri dishes were incubated at 37 degrees C for 24 hours. Salvia officinalis, L. showed enhanced inhibitory activity compared to the other two herbs, with 100% efficiency against Klebsiella and Enterobacter species, 96% against Escherichia coli, 83% against Proteus mirabilis, and 75% against Morganella morganii.

  11. Modification of the Mycotoxin Deoxynivalenol Using Microorganisms Isolated from Environmental Samples.

    PubMed

    Wilson, Nina M; McMaster, Nicole; Gantulga, Dash; Soyars, Cara; McCormick, Susan P; Knott, Ken; Senger, Ryan S; Schmale, David G

    2017-04-15

    The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of wheat, barley, and maize. New strategies are needed to reduce or eliminate DON in feed and food products. Microorganisms from plant and soil samples collected in Blacksburg, VA, USA, were screened by incubation in a mineral salt media containing 100 μg/mL DON and analysis by gas chromatography mass spectrometry (GC/MS). Two mixed cultures derived from soil samples consistently decreased DON levels in assays using DON as the sole carbon source. Nuclear magnetic resonance (NMR) analysis indicated that 3-keto-4-deoxynivalenol was the major by-product of DON. Via 16S rRNA sequencing, these mixed cultures, including mostly members of the genera Acinetobacter, Leadbetterella, and Gemmata, were revealed. Incubation of one of these mixed cultures with wheat samples naturally contaminated with 7.1 μg/mL DON indicated nearly complete conversion of DON to the less toxic 3-epimer-DON (3-epi-DON). Our work extends previous studies that have demonstrated the potential for bioprospecting for microorganisms from the environment to remediate or modify mycotoxins for commercial applications, such as the reduction of mycotoxins in fuel ethanol co-products.

  12. Modification of the Mycotoxin Deoxynivalenol Using Microorganisms Isolated from Environmental Samples

    PubMed Central

    Wilson, Nina M.; McMaster, Nicole; Gantulga, Dash; Soyars, Cara; McCormick, Susan P.; Knott, Ken; Senger, Ryan S.; Schmale, David G.

    2017-01-01

    The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of wheat, barley, and maize. New strategies are needed to reduce or eliminate DON in feed and food products. Microorganisms from plant and soil samples collected in Blacksburg, VA, USA, were screened by incubation in a mineral salt media containing 100 μg/mL DON and analysis by gas chromatography mass spectrometry (GC/MS). Two mixed cultures derived from soil samples consistently decreased DON levels in assays using DON as the sole carbon source. Nuclear magnetic resonance (NMR) analysis indicated that 3-keto-4-deoxynivalenol was the major by-product of DON. Via 16S rRNA sequencing, these mixed cultures, including mostly members of the genera Acinetobacter, Leadbetterella, and Gemmata, were revealed. Incubation of one of these mixed cultures with wheat samples naturally contaminated with 7.1 μg/mL DON indicated nearly complete conversion of DON to the less toxic 3-epimer-DON (3-epi-DON). Our work extends previous studies that have demonstrated the potential for bioprospecting for microorganisms from the environment to remediate or modify mycotoxins for commercial applications, such as the reduction of mycotoxins in fuel ethanol co-products. PMID:28420137

  13. Biosynthetic origin of natural products isolated from marine microorganism-invertebrate assemblages.

    PubMed

    Simmons, T Luke; Coates, R Cameron; Clark, Benjamin R; Engene, Niclas; Gonzalez, David; Esquenazi, Eduardo; Dorrestein, Pieter C; Gerwick, William H

    2008-03-25

    In all probability, natural selection began as ancient marine microorganisms were required to compete for limited resources. These pressures resulted in the evolution of diverse genetically encoded small molecules with a variety of ecological and metabolic roles. Remarkably, many of these same biologically active molecules have potential utility in modern medicine and biomedical research. The most promising of these natural products often derive from organisms richly populated by associated microorganisms (e.g., marine sponges and ascidians), and often there is great uncertainty about which organism in these assemblages is making these intriguing metabolites. To use the molecular machinery responsible for the biosynthesis of potential drug-lead natural products, new tools must be applied to delineate their genetic and enzymatic origins. The aim of this perspective is to highlight both traditional and emerging techniques for the localization of metabolic pathways within complex marine environments. Examples are given from the literature as well as recent proof-of-concept experiments from the authors' laboratories.

  14. Antimicrobial efficacy of Achillea ligustica All. (Asteraceae) essential oils against reference and isolated oral microorganisms.

    PubMed

    Cecchini, Cinzia; Silvi, Stefania; Cresci, Alberto; Piciotti, Andrea; Caprioli, Giovanni; Papa, Fabrizio; Sagratini, Gianni; Vittori, Sauro; Maggi, Filippo

    2012-01-01

    The aim of this study was to verify the effectiveness of Achillea ligustica essential oils against several oral microorganisms in comparison with a commercial essential oil-containing mouthrinse (Listerine(®)) and clove oil (containing 89% eugenol). The inhibition efficacy of A. ligustica essential oils alone and in combination with Listerine(®) was evaluated by the micro-dilution method. The most susceptible microorganisms were Bacillus cereus, Streptococcus pyogenes, and Candida albicans. The efficacy was similar to that of the clove oil. The antiseptic mouthwash Listerine(®) did not exert a strong inhibition on microbial strains tested, whereas its effectiveness increased significantly when essential oil was added. The study provides additional evidence for the in vitro inhibitory activity of A. ligustica essential oils on several pathogens, suggesting their usefulness in mouthrinse formulations as an adjunct to mechanical oral hygiene regimens. Essential oil-containing mouthrinses can be beneficial, safe components of daily oral health routines, representing an efficient and without side effect alternative to prevent and control oral infections. Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.

  15. Isolation and Characterization of Four Gram-PositiveNickel-Tolerant Microorganisms from Contaminated Riparian Sediments

    SciTech Connect

    Van Nostrand, Joy D.; Khijniak, Tatiana V.; Gentry, Terry J.; Novak, Michelle T.; Sowder, Andrew G.; Zhou, Jizhong Z.; Bertsch, PaulM.; Morris, Pamela J.

    2006-08-30

    Microbial communities from riparian sediments contaminatedwith high levels of Ni and U were examined for metal-tolerantmicroorganisms. Isolation of four aerobic Ni-tolerant, Gram-positiveheterotrophic bacteria indicated selection pressure from Ni. Theseisolates were identified as Arthrobacter oxydans NR-1, Streptomycesgalbus NR-2, Streptomyces aureofaciens NR-3, and Kitasatosporacystarginea NR-4 based on partial 16S rDNA sequences. A functional genemicroarray containing gene probes for functions associated withbiogeochemical cycling, metal homeostasis, and organic contaminantdegradation showed little overlap among the four isolates. Fifteen of thegenes were detected in all four isolates with only two of these relatedto metal resistance, specifically to tellurium. Each of the four isolatesalso displayed resistance to at least one of six antibiotics tested, withresistance to kanamycin, gentamycin, and ciprofloxacin observed in atleast two of the isolates. Further characterization of S. aureofaciensNR-3 and K. cystarginea NR-4 demonstrated that both isolates expressed Nitolerance constitutively. In addition, both were able to grow in higherconcentrations of Ni at pH 6 as compared to pH 7 (42.6 and 8.5 mM Ni atpH 6 and 7, respectively). Tolerance to Cd, Co, and Zn was also examinedin these two isolates; a similar pH-dependent metal tolerance wasobserved when grown with Co and Zn. Neither isolate was tolerant to Cd.These findings suggest that Ni is exerting a selection pressure at thissite for metal-resistant actinomycetes.

  16. Silicon dioxide thin film mediated single cell nucleic acid isolation.

    PubMed

    Bogdanov, Evgeny; Dominova, Irina; Shusharina, Natalia; Botman, Stepan; Kasymov, Vitaliy; Patrushev, Maksim

    2013-01-01

    A limited amount of DNA extracted from single cells, and the development of single cell diagnostics make it necessary to create a new highly effective method for the single cells nucleic acids isolation. In this paper, we propose the DNA isolation method from biomaterials with limited DNA quantity in sample, and from samples with degradable DNA based on the use of solid-phase adsorbent silicon dioxide nanofilm deposited on the inner surface of PCR tube.

  17. Silicon Dioxide Thin Film Mediated Single Cell Nucleic Acid Isolation

    PubMed Central

    Bogdanov, Evgeny; Dominova, Irina; Shusharina, Natalia; Botman, Stepan; Kasymov, Vitaliy; Patrushev, Maksim

    2013-01-01

    A limited amount of DNA extracted from single cells, and the development of single cell diagnostics make it necessary to create a new highly effective method for the single cells nucleic acids isolation. In this paper, we propose the DNA isolation method from biomaterials with limited DNA quantity in sample, and from samples with degradable DNA based on the use of solid-phase adsorbent silicon dioxide nanofilm deposited on the inner surface of PCR tube. PMID:23874571

  18. Microorganisms Isolated from Blood Cultures of Febrile Neutropenic Patients in ‹bn-i Sina Hospital.

    PubMed

    Arıkan Akan, Özay

    2003-12-05

    Patients with profound neutropenia have increased risk of septicemia associated with significant morbidity. To provide the appropriate broad-spectrum antimicrobial cover, documentation of causative agents and their antimicrobial susceptibilities should be established in each hospital. During 2001 in Ibn-i Sina Hospital Hematology unit, among 125 isolates from blood cultures of febrile neutropenic patients, gram-negative bacteria was prevalent (56.8%). Among the gram-positives (34.4% of isolates) coagulase-negative staphylococci (CNS) were the predominant bacteria (15/43) followed by Staphylococcus aureus (12/43). Escherichia coli (23/71) and Klebsiella spp. (15/71) were the most common species among 71 gram-negative bacteria. Nonfermentative gram-negative bacilli were 21.6% of the isolates. Increase in the isolation rate of Acinetobacter baumannii (7 strains) and Stenotrophomonas maltophilia (6 strains) was noticed.

  19. Anti-microorganism, anti-tumor, and immune activities of a novel polysaccharide isolated from Tricholoma matsutake

    PubMed Central

    Hou, Yiling; Ding, Xiang; Hou, Wanru; Zhong, Jie; Zhu, Hongqing; Ma, Binxiang; Xu, Ting; Li, Junhua

    2013-01-01

    Background: Many more fungal polysaccharides have been reported to exhibit a variety of biological activities, including anti-tumor, immunostimulation, anti-oxidation, and so on. The non-starch polysaccharides have emerged as an important class of bioactive natural products. Objective: To investigate the anti-microorganism, anti-tumor, and immune activities of a novel polysaccharide (TMP-A) isolated from Tricholoma matsutake. Materials and Methods: The anti-microorganism activity of purified polysaccharides (TMP-A) was evaluated by the inhibition zone diameter, the anti-tumor activity was evaluated by the S180 tumor cells that were implanted subcutaneously into the Kunming strain male mice in vivo, and the immune activity was evaluated by lymphocyte proliferation and macrophage stimulation, respectively. Results: In this study, the most susceptible bacteria of TMP-A at a concentration of 20 mg/ml was Micrococcus lysodeikticus (inhibition zone diameter 24.38 ± 1.19 mm) and the TMP-A did not show any antifungal activity for the tested stains of the fungi. In addition, the inhibitory rate in mice treated with 80 mg/kg TMP-A could reach 68.422%, being the highest in the three doses, which might be comparable to mannatide. The anti-tumor activity of the TMP-A was usually believed to be a consequence of the stimulation of the cell-mediated immune response, because it could significantly promote the lymphocyte and macrophage cells in the dose range of 50–200 μg/mL and in the dose range of 100 – 400 μg/mL in vitro, respectively. Discussion and Conclusion: The results obtained in the present study indicate that the purification polysaccharide of Tricholoma matsutake is a potential source of natural broad-spectrum, anti-microorganism, anti-tumor, and immunomodulation. PMID:23930009

  20. Anti-microorganism, anti-tumor, and immune activities of a novel polysaccharide isolated from Tricholoma matsutake.

    PubMed

    Hou, Yiling; Ding, Xiang; Hou, Wanru; Zhong, Jie; Zhu, Hongqing; Ma, Binxiang; Xu, Ting; Li, Junhua

    2013-07-01

    Many more fungal polysaccharides have been reported to exhibit a variety of biological activities, including anti-tumor, immunostimulation, anti-oxidation, and so on. The non-starch polysaccharides have emerged as an important class of bioactive natural products. To investigate the anti-microorganism, anti-tumor, and immune activities of a novel polysaccharide (TMP-A) isolated from Tricholoma matsutake. The anti-microorganism activity of purified polysaccharides (TMP-A) was evaluated by the inhibition zone diameter, the anti-tumor activity was evaluated by the S180 tumor cells that were implanted subcutaneously into the Kunming strain male mice in vivo, and the immune activity was evaluated by lymphocyte proliferation and macrophage stimulation, respectively. In this study, the most susceptible bacteria of TMP-A at a concentration of 20 mg/ml was Micrococcus lysodeikticus (inhibition zone diameter 24.38 ± 1.19 mm) and the TMP-A did not show any antifungal activity for the tested stains of the fungi. In addition, the inhibitory rate in mice treated with 80 mg/kg TMP-A could reach 68.422%, being the highest in the three doses, which might be comparable to mannatide. The anti-tumor activity of the TMP-A was usually believed to be a consequence of the stimulation of the cell-mediated immune response, because it could significantly promote the lymphocyte and macrophage cells in the dose range of 50-200 μg/mL and in the dose range of 100 - 400 μg/mL in vitro, respectively. The results obtained in the present study indicate that the purification polysaccharide of Tricholoma matsutake is a potential source of natural broad-spectrum, anti-microorganism, anti-tumor, and immunomodulation.

  1. Potential for biodegradation of hydrocarbons by microorganisms isolated from Antarctic soils.

    PubMed

    Vasileva-Tonkova, Evgenia; Gesheva, Victoria

    2004-01-01

    Seventeen pure aerobic microbial isolates were obtained from soil samples of three regions of Antarctica: Casey Station, Dewart Island and Terra Nova Bay. Most of them were gram positive coryneform bacteria. Isolates were tested for their ability to grow on mineral salt agar plates supplemented with one of the following model n-alkanes or aromatic hydrocarbons: hexane, heptane, paraffin, benzene, toluene, naphthalene and kerosene. Cell hydrophobicity, the ability to produce anionic glycolipids and extracellular emulsifying activity were also determined and assessed on the basis of growth of soil isolates on hydrocarbons. This study revealed degraders with broader abilities to grow on both types of hydrocarbons, good production of glycolipids and emulsifying activity. On this basis, a mixed culture of strains is proposed, which may find application for bioremediation at temperate temperature of soil environments polluted with different hydrocarbons.

  2. DON modification in naturally-contaminated wheat samples using microorganisms isolated from the environment

    USDA-ARS?s Scientific Manuscript database

    The fungus Fusarium graminearum produces the toxic compound deoxynivalenol (DON) that contaminates wheat, barley, and maize. New strategies are needed to mitigate DON in the United States. Microbes were isolated from different soil types, and cultured in a mineral salt media using 100 ppm DON as the...

  3. [Emission of microorganisms from sewage treatment plants depending upon construction differences of single structural parts].

    PubMed

    Eikmann, T; Schröder, S; Pieler, J; Bahr, H; Einbrodt, H J

    1986-04-01

    In order to examine the influence exerted by the differing design of individual water treatment plant units on the emission rate of micro-organisms and the associated degree of exposure to which plant personnel is subjected, measurements were taken at three different types of treatment plants. Measurements were made using "Biotest" RCS Air Samplers. The total count of colonies was determined by means of Agar Strips GK-A (tryptic soy agar). Enterobacteriaceae were quantitatively ascertained using Agar Strips C (MacConkey agar), particular attention being paid to the determination of the coliform bacteria as faeces indicators. Agar Strips S (mannitol salt agar) were used to measure the count of staphylococci using Agar Strips HS (rosa Bengal streptomycin agar). Before taking measurements, the prevailing climatic conditions were recorded. It could be ascertained that the enclosure of the inflow area (screw conveyor pump station and aerated grit removal tank) lead to a considerable increase in the concentration of microorganisms in the air within the housing. The values dropped however, when adequate ventilation was provided. Differing oxygen in the activated sludge tanks - finebubble aeration at the tank bottom or the blowing in of air via centrifugal blowers - lead to large variations in the emission rates. However, the less the waste water is agitated, the lower the emission rates. In the case of fine-bubble aeration, rates which are also normally to be found in the "non-burdened" outside air were even recorded close to the aeration tank. In cases of centrifugal blower, the aeration tank should be covered with a shield. With this type of aeration the waste water is emitted radially towards the walls of the tank. The use of a sprinkler unit on an aeration tank equipped with centrifugal blower - to avoid foam formation on the surface of the water - does not lead to an increase in the already high emission rate. An increase in air pollution through mould fungi from

  4. Study of photodynamic therapy in the control of isolated microorganisms from infected wounds--an in vitro study.

    PubMed

    Pereira de Lima Carvalho, Denise; Guerra Pinto, Juliana; Di Paula Costa Sorge, Camila; Rodrigues Benedito, Fabiana Regis; Khouri, Sonia; Ferreira Strixino, Juliana

    2014-01-01

    The effective treatment of infected wounds continues to be a serious challenge, mainly due to the rise of antibiotic-resistant bacteria. Photodynamic therapy (PDT) refers to the topical or systemic administration of a non-toxic, photosensitizing agent (PS), followed by irradiation with visible light of a suitable wavelength. The possibility of applying the PDT locally is what makes it so favorable to the treatment of infected wounds. The goal of this study was to evaluate the action of the PDT in the inactivation in vitro of microorganisms coming from infected wounds, using methylene blue (MB) and photodithazine (PDZ) as the PS and comparing the efficacy of these two compounds for PDT on bacteria. For the application of PDT, isolated microorganisms identified from material collected from wounds were suspended in a saline solution containing 10(6) viable cells/ml. Each isolated microorganism was submitted to PDT with MB and with PDZ in accordance with the following treatment groups: N/T--no treatment; T1--PDT with PDZ; T2--PDT with MB; T3--irradiation without PS; T4--treatment with PDZ without light; and T5--treatment with MB without light. As a light source, an LED-based device was used (Biopdi/Irrad-Lead 660), composed of 54 LEDs, each with 70 mW of power in the 660 nm region of the electromagnetic spectrum. Each tray of 96 wells was irradiated with an intensity of 25 mW/cm(2) and a dose of light of 50 J/cm(3) for 33 min. All the tests were made in duplicate. It was then concluded that the PDT with PDZ was capable of inhibiting the growth of gram-positive bacteria samples, however it did not have the same effect on gram-negative bacteria, which showed growth greater than 100,000 CFU; the PDT with MB showed an effectiveness on gram-positive as well as gram-negative bacteria, for it was able to inhibit bacterial growth in both cases.

  5. Enhancing pesticide degradation using indigenous microorganisms isolated under high pesticide load in bioremediation systems with vermicomposts.

    PubMed

    Castillo Diaz, Jean Manuel; Delgado-Moreno, Laura; Núñez, Rafael; Nogales, Rogelio; Romero, Esperanza

    2016-08-01

    In biobed bioremediation systems (BBSs) with vermicomposts exposed to a high load of pesticides, 6 bacteria and 4 fungus strains were isolated, identified, and investigated to enhance the removal of pesticides. Three different mixtures of BBSs composed of vermicomposts made from greenhouse (GM), olive-mill (OM) and winery (WM) wastes were contaminated, inoculated, and incubated for one month (GMI, OMI and WMI). The inoculums maintenance was evaluated by DGGE and Q-PCR. Pesticides were monitored by HPLC-DAD. The highest bacterial and fungal abundance was observed in WMI and OMI respectively. In WMI, the consortia improved the removal of tebuconazole, metalaxyl, and oxyfluorfen by 1.6-, 3.8-, and 7.7-fold, respectively. The dissipation of oxyfluorfen was also accelerated in OMI, with less than 30% remaining after 30d. One metabolite for metalaxyl and 4 for oxyfluorfen were identified by GC-MS. The isolates could be suitable to improve the efficiency of bioremediation systems.

  6. Metabolic products of microorganisms. 239. Bacimethrin isolated from Streptomyces albus identification, derivatives, synthesis and biological properties.

    PubMed

    Drautz, H; Messerer, W; Zähner, H; Breiding-Mack, S; Zeeck, A

    1987-10-01

    Bacimethrin (1), known as a thiamine antagonist produced by Bacillus megatherium, was isolated from Streptomyces albus and has been further characterized by NMR spectra and acetylation. A new easy three step synthesis for 1 is described. The biological activity of 1, and its mode of action were discussed. There are indications that bacimethrin inhibits the phosphorylation of 4-amino-5-hydroxymethyl-2-methylpyrimidine (Pyr-OH) during thiamine biosynthesis.

  7. Isolation and characterization of triacylglycerol-accumulating microorganisms which can utilize wood polysaccharide

    NASA Astrophysics Data System (ADS)

    Susanto, S. A.

    2017-05-01

    Triacylglycerol is an ester which is made of glycerol and three fatty acids. This compound is an important feedstock for biodiesel production. In this study, several strains of oleaginous bacteria were isolated from environmental sample based on their ability to grow in mineral salts medium supplemented with wood-derived sugars such as cellulose, arabinose, xylose, mannose, and galactose. The lipid accumulating bacteria were selected based on fluorescent signal from hydrophobic inclusion in the cytoplasm after incubation in selective medium containing lipophilic dye 0.5 % (w/v) nile red. The lipid content was analyzed using thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS). In this study, three bacterial isolates 2HPCS1R4, 1LPCS2R2, and 1LPCS2R14 were selected among several candidates. TLC analysis of hydrophobic substance from 1LPCS2R2 and 1LPCS2R14 showed two overlapped discrete bands corresponded to triacylglycerol reference band. While 2HPCS1R4 displayed a faint band located above the reference band. GC-MS analysis confirmed that the bands consisted of fatty acid methyl esters with alkyl length varied from C12 to C17. Kinetic study showed that the fastest growing strain was 1LPCS2R2 had the highest growth rates and when grown in glucose (µ = 0.29 h-1) and xylose (µ = 0.16 h-1). In conclusion, this study has identified of prospective bacterial isolates for commercial biodiesel production.

  8. Isolation of microorganisms from CO2 sequestration sites through enrichments under high pCO2

    NASA Astrophysics Data System (ADS)

    Peet, K. C.; Freedman, A. J.; Boreham, C.; Thompson, J. R.

    2012-12-01

    Carbon Capture and Storage (CCS) in geologic formations has the potential to reduce greenhouse gas emissions from fossil fuel processing and combustion. However, little is known about the effects that CO2 may have on biological activity in deep earth environments. To understand microorganisms associated with these environments, we have developed a simple high-pressure enrichment methodology to cultivate organisms capable of growth under supercritical CO2 (scCO2). Growth media targeting different subsurface functional metabolic groups is added to sterilized 316 stainless steel tubing sealed with quarter turn plug valves values and pressurized to 120-136 atm using a helium-padded CO2 tank, followed by incubation at 37 °C to achieve the scCO2 state. Repeated passages of crushed subsurface rock samples and growth media under supercritical CO2 headspaces are assessed for growth via microscopic enumeration. We have utilized this method to survey sandstone cores for microbes capable of growth under scCO2 from two different geologic sites targeted for carbon sequestration activities. Reproducible growth of microbial biomass under high pCO2 has been sustained from each site. Cell morphologies consist of primarily 1-2 μm rods and oval spores, with densities from 1E5-1E7 cells per ml of culture. We have purified and characterized a bacterial strain most closely related to Bacillus subterraneus (99% 16S rRNA identity) capable of growth under scCO2. Preliminary physiological characterization of this strain indicates it is a spore-forming facultative anaerobe able to grow in 0.5 to 50 ppt salinity. Genome sequencing and analysis currently in progress will help reveal genetic mechanisms of acclimation to high pCO2 conditions associated with geologic carbon sequestration.

  9. Isolation of a single rice chromosome by optical micromanipulation

    NASA Astrophysics Data System (ADS)

    Wang, Haowei; Liu, Xiaohui; Li, Yinmei; Han, Bin; Lou, Liren; Wang, Kangjun

    2004-01-01

    A new method based on optical tweezers technology is reported for the isolation of a single chromosome. A rice cell suspended in liquid was first fragmented by laser pulses (optical scalpel). Then a single released chromosome from the cell was manipulated and pulled away from other cells and oddments by optical tweezers without any direct mechanical contact. Finally the isolated single chromosome was extracted individually into a glass capillary nearby. After molecular cloning of the isolated chromosome, we obtained some specific DNA segments from the single chromosome. All these segments can be used for rice genomic sequencing. Different methods of extracting a single chromosome are compared. The advantages of optical micromanipulation method are summarized.

  10. Isolation and evaluation of native cellulose degrading microorganisms for efficient bioconversion of weed biomass and rice straw.

    PubMed

    Mahanta, K; Jha, D K; Rajkhowa, D J; Kumar, Manoj

    2014-07-01

    Cellulose decomposing microorganisms (CDMs) are important for efficient bioconversion of plant biomasses. To this end, we isolated seven fungal isolates (Aspergillus wentii, Fusarium solani, Mucor sp., Penicillum sp., Trichoderma harzaianum, Trichoderma sp.1 and Trichoderma sp.2) and three bacterial isolates (bacterial isolate I, II and III) from partially decomposed farm yard manure, rice straw and vermicompost, and evaluated them for decomposition of rice straw (Oryza sativa), Ipomoea camea and Eichhornia crassipes biomass. CDMs inoculation, in general, reduced the composting period by 14-28 days in rice straw, 14-34 days in Eichhornia and 10-28 days in Ipomoea biomass over control. Of the 10 CDMs tested, Mucor sp. was found to be the most effective as Mucor-inoculated biomass required minimum time, i.e. 84, 68 and 80 days respectively for composting of rice straw, Eichhornia and Ipomoea biomass as against 112, 102 and 108 days required under their respective control. CDMs inoculation also narrowed down the C:N ratio of the composts which ranged from 19.1-22.7, 12.9-14.7 and 10.5-13.1 in rice straw, Eichhornia and Ipomoea biomass respectively as against 24.1, 17.1 and 16.2 in the corresponding control treatments. Aspergillus wentii, Fusarium solani, Mucor sp., and Penicillum sp. were found most effective (statistically at par) in reducing C:N ratio and causing maximum loss of carbon and dry matter in composted materials. These benefits of CDMs inoculation were also accompanied by significant increase in NPK contents in the composted materials.

  11. Preliminary characterization of biosurfactants produced by microorganisms isolated from refinery wastewaters.

    PubMed

    Yalçin, Emine; Ergene, Aysun

    2010-02-01

    Some bacterial strains isolated from refinery wastewaters were identified as Pseudomonas aeruginosa RWI, Pseudomonas putida RWII, Pseudomonas fluorescens RWIII and Burkholderia cepacia RWIV, and the biosurfactants produced by these strains were coded as BS-I, BS-II, BS-III and BS-IV, respectively. The bacterial strains were characterized by the following biochemical methods: Gram stain, oxidase activity, indol, lactose and growth at 42 degrees C. Biosurfactant production was evaluated by: emulsification activity, surface tension measurement and critical micelle concentration. Chemical characterization of the biosurfactants was done by: FTIR and analysis of carbohydrate, protein and lipid content. The biosurfactants showed good emulsification activity against different hydrocarbon sources. The initial surface tension of culture broth was determined as 67.3 mN/m, and production of BS-I, BS-II, BS-III and BS-IV lowered this value to 35.9, 49.2, 51.6 and 45.7 mN/m, respectively. The critical micelle concentration of the biosurfactants was found to be in the range 10-50 mg/L. From the results of this study it was observed that the refinery wastewaters are a suitable source for isolation of biosurfactant-producing bacteria, but are not a substrate for biosurfactant production.

  12. Manganese-oxidizing and -reducing microorganisms isolated from biofilms in chlorinated drinking water systems.

    PubMed

    Cerrato, José M; Falkinham, Joseph O; Dietrich, Andrea M; Knocke, William R; McKinney, Chad W; Pruden, Amy

    2010-07-01

    The interaction of chemical, physical and biological factors that affect the fate, transport and redox cycling of manganese in engineered drinking water systems is not clearly understood. This research investigated the presence of Mn-oxidizing and -reducing bacteria in conventional water treatment plants exposed to different levels of chlorine. Mn(II)-oxidizing and Mn(IV)-reducing bacteria, principally Bacillus spp., were isolated from biofilm samples recovered from four separate drinking water systems. Rates of Mn-oxidation and -reduction for selected individual isolates were represented by pseudo-first-order kinetics. Pseudo-first-order rate constants were obtained for Mn-oxidation (range: 0.106-0.659 days(-1)), aerobic Mn-reduction (range: 0.036-0.152 days(-1)), and anaerobic Mn-reduction (range: 0.024-0.052 days(-1)). The results indicate that microbial-catalyzed Mn-oxidation and -reduction (aerobic and anaerobic) can take place simultaneously in aqueous environments exposed to considerable oxygen and chlorine levels and thus affect Mn-release and -deposition in drinking water systems. This has important implications for Mn-management strategies, which typically assume Mn-reduction is not possible in the presence of chlorine and oxidizing conditions. Copyright 2010 Elsevier Ltd. All rights reserved.

  13. Isolation and initial characterization of a novel type of Baeyer-Villiger monooxygenase activity from a marine microorganism.

    PubMed

    Willetts, Andrew; Joint, Ian; Gilbert, Jack A; Trimble, William; Mühling, Martin

    2012-07-01

    A novel type of Baeyer-Villiger monooxygenase (BVMO) has been found in a marine strain of Stenotrophomonas maltophila strain PML168 that was isolated from a temperate intertidal zone. The enzyme is able to use NADH as the source of reducing power necessary to accept the atom of diatomic oxygen not incorporated into the oxyfunctionalized substrate. Growth studies have establish that the enzyme is inducible, appears to serve a catabolic role, and is specifically induced by one or more unidentified components of seawater as well as various anthropogenic xenobiotic compounds. A blast search of the primary sequence of the enzyme, recovered from the genomic sequence of the isolate, has placed this atypical BVMO in the context of the several hundred known members of the flavoprotein monooxygenase superfamily. A particular feature of this BVMO lies in its truncated C-terminal domain, which results in a relatively small protein (357 amino acids; 38.4 kDa). In addition, metagenomic screening has been conducted on DNA recovered from an extensive range of marine environmental samples to gauge the relative abundance and distribution of similar enzymes within the global marine microbial community. Although low, abundance was detected in samples from many marine provinces, confirming the potential for biodiscovery in marine microorganisms.

  14. Isolation and identification of pathogenic microorganisms at wastewater-irrigated fields: ratios in air and wastewater

    SciTech Connect

    Teltsch, B.; Kedmi, S.; Bonnet, L.; Borenzstajn-Rotem, Y.; Katzenelson, E.

    1980-06-01

    Samples of air and corresponding wastewater samples were taken at wastewater spray-irrigated fields. The concentrations of salmonellae and enteroviruses present in these samples were determined and compared with those of coliforms, and the ratios between them were calculated. The most common Salmonella serotype in the air was Salmonella ohio, whereas in the wastewater, Salmonella anatum was the most common. Enteroviruses isolated and identified were poliovirus, echovirus, and coxsackievirus type B. From the ratios of salmonellas to coliforms and enteroviruses to coliforms in the air, as compared to these ratios in the wastewater, it was concluded that the suitability of coliforms as an indication of airborne contamination caused by spray irrigation is questionable.

  15. Isolated microorganisms in plastic biliary stents placed for benign and malignant diseases.

    PubMed

    Basioukas, Paris; Vezakis, Antonios; Zarkotou, Olympia; Fragulidis, Georgios; Themeli-Digalaki, Katerina; Rizos, Spyros; Polydorou, Andreas

    2014-01-01

    Biliary stenting is a well-established method to treat patients with malignant and benign biliary diseases. However, occlusion of plastic biliary stents is considered as a drawback and bacterial colonization seems to be the key factor in this process. During a 3-year period, 51 plastic biliary stents were extracted from 42 patients. Twenty three stents were inserted for treating malignant and 28 for benign diseases. Stent samples were taken under a strict protocol, and were immediately sent to microbiological laboratory for culturing. A polymicrobial growth was present in nearly all stents. The most frequently isolated organisms were Enterococcus spp (74%), Escherichia coli (E. coli) (62%), and Klebsiella spp (58%). E. coli was more frequently encountered in benign vs. malignant disease (78% vs. 43%, P<0.05). Klebsiella spp, Pseudomonas spp, and Candida spp were more frequently isolated in occluded vs. non-occluded stents, 68% vs. 37%, 22% vs. 0 and 40% vs. 6% respectively (P<0.05). E. coli and Pseudomonas spp had 34% and 50% resistance rate to quinolones respectively. Enterobacter spp expressed Amp-C derepression in 35%. Enterococcus spp, Klebsiella spp and Pseudomonas spp had a low resistance rate. Enterococcus spp, E. coli and Klebsiella spp are the most frequently associated organisms in plastic biliary stents. In occluded stents Pseudomonas spp and Candida spp should be taken into account. Quinolones may not be adequate for the treatment of cholangitis associated with stent occlusion. In patients under chemotherapy for malignancy and stent occlusion-related biliary sepsis, antifungal and enterococcal covering should be considered.

  16. Streptomyces mexicanus sp. nov., a xylanolytic micro-organism isolated from soil.

    PubMed

    Petrosyan, Pavel; García-Varela, Martin; Luz-Madrigal, Agustín; Huitrón, Carlos; Flores, María Elena

    2003-01-01

    The taxonomic position of a thermophilic actinomycete strain isolated from soil was examined using a polyphasic approach. The strain, designated CH-M-1035T, was assigned to the genus Streptomyces on the basis of chemical and morphological criteria. It formed Rectiflexibiles aerial hyphae that carried long chains of rounded, smooth spores. The almost complete nucleotide sequence of the 16S rRNA gene of strain CH-M-1035T was determined and its comparison with the 16S rDNA sequences of previously studied streptomycetes confirmed the assignment of the novel strain to the genus Streptomyces. Strain CH-M-1035T clustered with species belonging to the Streptomyces thermodiastaticus clade in the 1 6S-rDNA-based phylogenetic tree. However, the phenotypic properties of strain CH-M-1035T differed from those of the recognized species within this clade. Therefore, it is proposed that strain CH-M-1035T be classified as a novel species within the genus Streptomyces, as Streptomyces mexicanus (type strain CH-M-1035T =DSM 41796T =BM-B-384T =NRRL B-24196T).

  17. Microbiological and chemical properties of kefir manufactured by entrapped microorganisms isolated from kefir grains.

    PubMed

    Chen, T-H; Wang, S-Y; Chen, K-N; Liu, J-R; Chen, M-J

    2009-07-01

    In this study, various yeasts (Kluyveromyces marxianus, Saccharomyces turicensis, Pichia fermentans) and lactic acid bacteria (Lactobacillus kefiranofaciens, Lactobacillus kefiri, Leuconostoc mesenteroides) were entrapped in 2 different microspheres using an entrapment ratio for the strains that was based on the distribution ratio of these organisms in kefir grains. The purpose of this study was to develop a new technique to produce kefir using immobilized starter cultures isolated from kefir grains. An increase in cell counts with fermentation cycles was observed for both the lactic acid bacteria (LAB) and yeasts, whereas the cell counts of kefir grains were very stable during cultivation. Scanning electron microscopy showed that the short-chain lactobacilli and lactococci occupied the surface of the LAB microspheres, whereas the long-chain lactobacilli were inside the microspheres. When the yeasts were analyzed, cells at a high density were entrapped in cracks on the surface and within the microspheres, where they were surrounded by the short-chain lactobacilli. The distribution of the LAB and yeast species in kefir produced from grains and microspheres showed that there was no significant difference between the kefirs produced by the 2 methods; moreover, Leu. mesenteroides and K. marxianus were the predominating microflora in both types of kefir. There was no significant difference in the ethanol and exopolysaccharide contents between the 2 kefirs, although the acidity was different.

  18. Bioaugmentation treatment of mature landfill leachate by new isolated ammonia nitrogen and humic acid resistant microorganism.

    PubMed

    Yu, Dahai; Yang, Jiyu; Teng, Fei; Feng, Lili; Fang, Xuexun; Ren, Hejun

    2014-07-01

    The mature landfill leachate, which is characterized by a high concentration of ammonia nitrogen (NH3-N) and humic acid (HA), poses a challenge to biotreatment methods, due to the constituent toxicity and low biodegradable fraction of the organics. In this study, we applied bioaugmentation technology in landfill leachate degradation by introducing a domesticated NH3-N and HA resistant bacteria strain, which was identified as Bacillus cereus (abbreviated as B. cereus Jlu) and Enterococcus casseliflavus (abbreviated as E. casseliflavus Jlu), respectively. The isolated strains exhibited excellent tolerant ability for NH3-N and HA and they could also greatly improved the COD (chemical oxygen demand), NH3-N and HA removal rate, and efficiency of bioaugmentation degradation of landfill leachate. Only 3 days was required for the domesticated bacteria to remove about 70.0% COD, compared with 9 days' degradation for the undomesticated (autochthonous) bacteria to obtain a similar removal rate. An orthogonal array was then used to further improve the COD and NH3-N removal rate. Under the optimum condition, the COD removal rate in leachate by using E. casseliflavus Jlu and B. cereus Jlu increased to 86.0% and 90.0%, respectively after, 2 days of degradation. The simultaneous removal of NH3-N and HA with more than 50% and 40% removal rate in leachate by employing the sole screened strain was first observed.

  19. Production of exopolysaccharides from a thermophilic microorganism isolated from a marine hot spring in flegrean areas.

    PubMed

    Schiano Moriello, V; Lama, L; Poli, A; Gugliandolo, C; Maugeri, T L; Gambacorta, A; Nicolaus, B

    2003-02-01

    A thermophilic strain isolated from sea sand at Maronti, near Sant' Angelo (Ischia), is described. The organism grows well at an optimal temperature of 60 degrees C at pH 7.0. The thermophilic bacterium, named strain 4004, produces an exocellular polysaccharide (EPS) in yields of 90 mg/l. The EPS fraction was produced with all substrates tested, although a higher yield was obtained with sucrose or trehalose as sole carbon source. During growth, the EPS content was proportional to the biomass. Three fractions (EPS1, EPS2, EPS3) were obtained after purification. Quantitative monosaccharide analysis of the EPSs revealed the presence of mannose:glucose:galactose in a relative ratio of 0.5:1.0:0.3 in EPS1, mannose:glucose:galactose in a relative ratio of 1.0:0.3:trace in EPS2, and galactose:mannose:glucosamine:arabinose in a relative ratio of 1.0:0.8:0.4:0.2 in EPS3. The average molecular mass of EPS3 was determined to be 1x10(6) Da. From comparison of the chemical shift values in (1)H and (13)C spectra, we conclude that EPS3 presents a pentasaccharide repeating unit.

  20. Antibacterial and antioxidant capacity of polar microorganisms isolated from Arctic lichen Ochrolechia sp..

    PubMed

    Kim, Mi-Kyeong; Park, Hyun; Oh, Tae-Jin

    2014-01-01

    Although many scientists have been studying lichens, the bacterial communities among lichens have not been fully explored. In this study, the biological activities of bacterial isolates from Arctic lichen Ochrolechia sp. were screened. Cell cultures were extracted using ethyl acetate, and these cultures were tested for antibacterial and antioxidant activities. To evaluate the antibacterial activities, we carried out a disk diffusion test and minimum inhibitory concentration assay. The inhibition zone diameter (mm) was 8 to 12, and its MIC value ranged from 222.0 to > 1000 μg/ml (control antibiotic ampicillin ranged from 0.31 to 0.58 μg/ml). PAMC26625 (Sphingomonas sp., sequence similarity: 99.108%) showed a high amount of total phenolic and total flavonoid contents within 5 extracts and also exhibited 77.23% and 80.02% in the 1,1-diphenyl-2-picryl-hydrazyl assay and the 2,2'-azino-bis(3-ethyl benzothiazoline-6-sulphonic acid) assay, respectively. In the ferric reducing antioxidant power assay, it was determined that PAMC26625 contained a higher amount of ferrous ion [8.72 mM Fe(II)/mg extract] than control ascorbic acid [6.21 mM Fe(II)/mg extract]. Our data indicated that they had potential to be novel sources of antibacterials and antioxidants.

  1. Accumulation of High-Value Lipids in Single-Cell Microorganisms: A Mechanistic Approach and Future Perspectives

    PubMed Central

    2015-01-01

    In recent years attention has been focused on the utilization of microorganisms as alternatives for industrial and nutritional applications. Considerable research has been devoted to techniques for growth, extraction, and purification of high-value lipids for their use as biofuels and biosurfactants as well as high-value metabolites for nutrition and health. These successes argue that the elucidation of the mechanisms underlying the microbial biosynthesis of such molecules, which are far from being completely understood, now will yield spectacular opportunities for industrial scale biomolecular production. There are important additional questions to be solved to optimize the processing strategies to take advantage of the assets of microbial lipids. The present review describes the current state of knowledge regarding lipid biosynthesis, accumulation, and transport mechanisms present in single-cell organisms, specifically yeasts, microalgae, bacteria, and archaea. Similarities and differences in biochemical pathways and strategies of different microorganisms provide a diverse toolset to the expansion of biotechnologies for lipid production. This paper is intended to inspire a generation of lipid scientists to insights that will drive the biotechnologies of microbial production as uniquely enabling players of lipid biotherapeutics, biofuels, biomaterials, and other opportunity areas into the 21st century. PMID:24628496

  2. Antibiofilm activity of sandblasted and laser-modified titanium against microorganisms isolated from peri-implantitis lesions.

    PubMed

    Drago, Lorenzo; Bortolin, Monica; De Vecchi, Elena; Agrappi, Serse; Weinstein, Roberto L; Mattina, Roberto; Francetti, Luca

    2016-10-01

    Infections due to biofilm-producing microorganisms are one of the main causes for the failure of dental implants. Increasing efforts have been made in order to develop new strategies to prevent biofilm formation. In this study, the biofilm development on a newly designed laser-modified titanium implant surface was evaluated and compared to that on conventional sandblasted titanium used in implant dentistry. The amount of biofilm produced by Staphylococcus aureus, Pseudomonas aeruginosa and Porphyromonas gingivalis isolated from peri-implantitis was assessed by a semi-quantitative spectrophotometric method and by confocal laser scanning microscopy. Results showed a lower biofilm production on laser-modified surface compared to the sandblasted one. In particular, a significantly lower total volume of the biomass was observed on laser-modified surface, while no significant changes in live/dead bacteria percentages were noticed between materials. Modifying the topography of the conventional implant surface with laser ablation could represent a promising approach for inhibiting biofilm formation.

  3. Diagnostic accuracy of semi-quantitative and quantitative culture techniques for the diagnosis of catheter-related infections in newborns and molecular typing of isolated microorganisms

    PubMed Central

    2014-01-01

    Background Catheter-related bloodstream infections (CR-BSIs) have become the most common cause of healthcare-associated bloodstream infections in neonatal intensive care units (ICUs). Microbiological evidence implicating catheters as the source of bloodstream infection is necessary to establish the diagnosis of CR-BSIs. Semi-quantitative culture is used to determine the presence of microorganisms on the external catheter surface, whereas quantitative culture also isolates microorganisms present inside the catheter. The main objective of this study was to determine the sensitivity and specificity of these two techniques for the diagnosis of CR-BSIs in newborns from a neonatal ICU. In addition, PFGE was used for similarity analysis of the microorganisms isolated from catheters and blood cultures. Methods Semi-quantitative and quantitative methods were used for the culture of catheter tips obtained from newborns. Strains isolated from catheter tips and blood cultures which exhibited the same antimicrobial susceptibility profile were included in the study as positive cases of CR-BSI. PFGE of the microorganisms isolated from catheters and blood cultures was performed for similarity analysis and detection of clones in the ICU. Results A total of 584 catheter tips from 399 patients seen between November 2005 and June 2012 were analyzed. Twenty-nine cases of CR-BSI were confirmed. Coagulase-negative staphylococci (CoNS) were the most frequently isolated microorganisms, including S. epidermidis as the most prevalent species (65.5%), followed by S. haemolyticus (10.3%), yeasts (10.3%), K. pneumoniae (6.9%), S. aureus (3.4%), and E. coli (3.4%). The sensitivity of the semi-quantitative and quantitative techniques was 72.7% and 59.3%, respectively, and specificity was 95.7% and 94.4%. The diagnosis of CR-BSIs based on PFGE analysis of similarity between strains isolated from catheter tips and blood cultures showed 82.6% sensitivity and 100% specificity. Conclusion The semi

  4. Diagnostic accuracy of semi-quantitative and quantitative culture techniques for the diagnosis of catheter-related infections in newborns and molecular typing of isolated microorganisms.

    PubMed

    Riboli, Danilo Flávio Moraes; Lyra, João César; Silva, Eliane Pessoa; Valadão, Luisa Leite; Bentlin, Maria Regina; Corrente, José Eduardo; Rugolo, Ligia Maria Suppo de Souza; da Cunha, Maria de Lourdes Ribeiro de Souza

    2014-05-22

    Catheter-related bloodstream infections (CR-BSIs) have become the most common cause of healthcare-associated bloodstream infections in neonatal intensive care units (ICUs). Microbiological evidence implicating catheters as the source of bloodstream infection is necessary to establish the diagnosis of CR-BSIs. Semi-quantitative culture is used to determine the presence of microorganisms on the external catheter surface, whereas quantitative culture also isolates microorganisms present inside the catheter. The main objective of this study was to determine the sensitivity and specificity of these two techniques for the diagnosis of CR-BSIs in newborns from a neonatal ICU. In addition, PFGE was used for similarity analysis of the microorganisms isolated from catheters and blood cultures. Semi-quantitative and quantitative methods were used for the culture of catheter tips obtained from newborns. Strains isolated from catheter tips and blood cultures which exhibited the same antimicrobial susceptibility profile were included in the study as positive cases of CR-BSI. PFGE of the microorganisms isolated from catheters and blood cultures was performed for similarity analysis and detection of clones in the ICU. A total of 584 catheter tips from 399 patients seen between November 2005 and June 2012 were analyzed. Twenty-nine cases of CR-BSI were confirmed. Coagulase-negative staphylococci (CoNS) were the most frequently isolated microorganisms, including S. epidermidis as the most prevalent species (65.5%), followed by S. haemolyticus (10.3%), yeasts (10.3%), K. pneumoniae (6.9%), S. aureus (3.4%), and E. coli (3.4%). The sensitivity of the semi-quantitative and quantitative techniques was 72.7% and 59.3%, respectively, and specificity was 95.7% and 94.4%. The diagnosis of CR-BSIs based on PFGE analysis of similarity between strains isolated from catheter tips and blood cultures showed 82.6% sensitivity and 100% specificity. The semi-quantitative culture method showed higher

  5. Single-molecule detection of protein efflux from microorganisms using fluorescent single-walled carbon nanotube sensor arrays

    NASA Astrophysics Data System (ADS)

    Landry, Markita Patricia; Ando, Hiroki; Chen, Allen Y.; Cao, Jicong; Kottadiel, Vishal Isaac; Chio, Linda; Yang, Darwin; Dong, Juyao; Lu, Timothy K.; Strano, Michael S.

    2017-05-01

    A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response. Unlabelled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show that the process of E. coli induction, protein synthesis and protein export is highly stochastic, yielding variability in protein secretion, with E. coli cells undergoing division under starved conditions producing 66% fewer secreted protein products than their non-dividing counterparts. We further demonstrate the detection of a unique protein product resulting from T7 bacteriophage infection of E. coli, illustrating that nanosensor arrays can enable real-time, single-cell analysis of a broad range of protein products from various cell types.

  6. Note: Galvanic isolated voltage source using a single photodiode.

    PubMed

    Stoican, O S

    2010-04-01

    A galvanic isolated voltage source able to provide several volts by using a single photodiode is described. A pulse-modulated laser beam is sent to a photodiode. By using a step-up transformer the amplitude of the variable voltage generated by the photodiode is increased. Adding a rectifier cell the variable voltage is converted back into a dc voltage.

  7. Single-cell isolation using a DVD optical pickup

    PubMed Central

    Kasukurti, A.; Potcoava, M.; Desai, S.A.; Eggleton, C.; Marr, D. W. M.

    2011-01-01

    A low-cost single-cell isolation system incorporating a digital versatile disc burner (DVD RW) optical pickup has been developed. We show that these readily available modules have the required laser power and focusing optics to provide a steady Gaussian beam capable of optically trapping micron-sized colloids and red blood cells. Utility of the pickup is demonstrated through the non-destructive isolation of such particles in a laminar-flow based microfluidic device that captures and translates single microscale objects across streamlines into designated channel exits. In this, the integrated objective lens focusing coils are used to steer the optical trap across the channel, resulting in the isolation of colloids and red blood cells using a very inexpensive off-the-shelf optical component. PMID:21643294

  8. Microfluidic Device for Capture and Isolation of Single Cells

    PubMed Central

    Hsiao, Alexander P.; Barbee, Kristopher D.; Huang, Xiaohua

    2011-01-01

    We describe a microfluidic device capable of trapping, isolating, and lysing individual cells in parallel using dielectrophoretic forces and a system of PDMS channels and valves. The device consists of a glass substrate patterned with electrodes and two PDMS layers comprising of the microfluidic channels and valve control channels. Individual cells are captured by positive dielectrophoresis using the microfabricated electrode pairs. The cells are then isolated into nanoliter compartments using pneumatically actuated PDMS valves. Following isolation, the cells are lysed open by applying an electric field using the same electrode pairs. With the ability to capture and compartmentalize single cells our device may be combined with analytical methods for in situ molecular analysis of cellular components from single cells in a highly parallel manner. PMID:21614137

  9. Microfluidic device for capture and isolation of single cells

    NASA Astrophysics Data System (ADS)

    Hsiao, Alexander P.; Barbee, Kristopher D.; Huang, Xiaohua

    2010-08-01

    We describe a microfluidic device capable of trapping, isolating, and lysing individual cells in parallel using dielectrophoretic forces and a system of PDMS channels and valves. The device consists of a glass substrate patterned with electrodes and two PDMS layers comprising of the microfluidic channels and valve control channels. Individual cells are captured by positive dielectrophoresis using the microfabricated electrode pairs. The cells are then isolated into nanoliter compartments using pneumatically actuated PDMS valves. Following isolation, the cells are lysed open by applying an electric field using the same electrode pairs. With the ability to capture and compartmentalize single cells our device may be combined with analytical methods for in situ molecular analysis of cellular components from single cells in a highly parallel manner.

  10. [Influence of magnetic field on the growth of pathogen microorganisms isolated from the indoor environment at the Archivo Nacional de la República de Cuba].

    PubMed

    Anaya, Matilde; Barbará, Eduardo; Padrón, Jesús; Borrego, Sofía F; Valdés, Oderlaise; Molina, Alian

    2015-01-01

    Electromagnetic pollution has been detected at the Archivo Nacional de la República de Cuba and the influence of extremely low frequency magnetic fields could be quantified with pathogenic microorganisms isolated from the indoor environment. To quantify the influence of an extremely low frequency magnetic field on the growth of pathogenic microorganisms isolated from the environment at the Archivo Nacional. We used five microorganisms isolated at the Archivo Nacional: Streptococcus sp. (1), Listeria sp. (2) and Candida guillermondii (3), and Escherichia coli ATCC 25922 (4) and Saccharomyces cerevisiae (5) as references. We applied this magnetic field of extremely low frequency, 60 Hz/220 V (3 mT), for two hours to these microorganisms on three culture tubes with distilled water and nutrient broth. Then we inoculated 0.1 mL in the following solid culture mediums on Petri dishes: CromoCen SC Agar (1 and 2), Potato Dextrose Agar (3), CromoCen DC 4227 (4) and Malt Extract Agar (5). The colonies were counted (log CFU/mL) by digital processing of the images of Petri dishes using the MatLab ® tool. We observed a statistically significant stimulation (p=0.05) in the quantity of treated colonies as compared to controls, which was higher in nutrient broth than in distilled water, and in bacteria (nutrient broth and treated colonies: 9.43 to 10.62 CFU/mL) as compared with yeasts (nutrient broth-treated colonies: 8.31 to 8.79 CFU/mL). In decreasing order, stimulation was as follows: Listeria sp., E. coli ATCC 25922, Streptococcus sp., C. guillermondii and S. cerevisiae . We concluded that the magnetic field applied had a stimulating effect on the microorganisms under study, which increases the risk to the health of staff and visitors at the Archivo Nacional .

  11. Diversity of cultivable β-glycosidase-producing micro-organisms isolated from the soil of a ginseng field and their ginsenosides-hydrolysing activity.

    PubMed

    Fu, Y; Yin, Z; Wu, L; Yin, C

    2014-02-01

    This research aimed to explore the diversity of cultivable β-glycosidase-producing micro-organisms in ginseng field soil. Fifty-three strains showing β-glucosidase activity were isolated from a ginseng field, using a newly designed Esculin-R2A agar. All the isolated strains belonged to the genus Agrobacterium, Arthrobacter, Burkholderia, Dyella, Edaphobacter, Luteibacter, Mucilaginibacter, Paenibacillus, Phenylobacterium, Pseudomonas, Sphingomonas and Streptomyces. The main β-glucosidase-producing micro-organisms in the ginseng field soil were Sphingomonas, Burkholderia, Luteibacter and Streptomyces, while concentrations of Agrobacterium, Arthrobacter, Paenibacillus and Pseudomonas were relatively low. Of these micro-organisms, the strain GS 09 could hydrolyse major ginsenosides Rb1, Rb2 and Rc to the active metabolite compound K. The strain GS 09 belonged to the genus Sphingomonas, and its 16S rRNA gene sequence showed 100% similarities with that of Sphingomonas asaccharolytica. This is the first study to provide information of cultivable β-glycosidase-producing micro-organisms in ginseng field soil. The strain GS 09 has potential to be applied on the preparation for minor ginsenoside C-K in pharmaceutical industry. © 2013 The Society for Applied Microbiology.

  12. Isolation of single Chlamydia-infected cells using laser microdissection.

    PubMed

    Podgorny, Oleg V; Polina, Nadezhda F; Babenko, Vladislav V; Karpova, Irina Y; Kostryukova, Elena S; Govorun, Vadim M; Lazarev, Vassili N

    2015-02-01

    Chlamydia are obligate intracellular parasites of humans and animals that cause a wide range of acute and chronic infections. To elucidate the genetic basis of chlamydial parasitism, several approaches for making genetic modifications to Chlamydia have recently been reported. However, the lack of the available methods for the fast and effective selection of genetically modified bacteria restricts the application of genetic tools. We suggest the use of laser microdissection to isolate of single live Chlamydia-infected cells for the re-cultivation and whole-genome sequencing of single inclusion-derived Chlamydia. To visualise individual infected cells, we made use of the vital labelling of inclusions with the fluorescent Golgi-specific dye BODIPY® FL C5-ceramide. We demonstrated that single Chlamydia-infected cells isolated by laser microdissection and placed onto a host cell monolayer resulted in new cycles of infection. We also demonstrated the successful use of whole-genome sequencing to study the genomic variability of Chlamydia derived from a single inclusion. Our work provides the first evidence of the successful use of laser microdissection for the isolation of single live Chlamydia-infected cells, thus demonstrating that this method can help overcome the barriers to the fast and effective selection of Chlamydia. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Biological treatment of produced water in a sequencing batch reactor by a consortium of isolated halophilic microorganisms.

    PubMed

    Pendashteh, A R; Fakhru'l-Razi, A; Chuah, T G; Radiah, A B Dayang; Madaeni, S S; Zurina, Z A

    2010-10-01

    Produced water or oilfield wastewater is the largest volume ofa waste stream associated with oil and gas production. The aim of this study was to investigate the biological pretreatment of synthetic and real produced water in a sequencing batch reactor (SBR) to remove hydrocarbon compounds. The SBR was inoculated with isolated tropical halophilic microorganisms capable of degrading crude oil. A total sequence of 24 h (60 min filling phase; 21 h aeration; 60 min settling and 60 min decant phase) was employed and studied. Synthetic produced water was treated with various organic loading rates (OLR) (0.9 kg COD m(-3) d(-1), 1.8 kg COD m(-3) d(-1) and 3.6 kg COD m(-3) d(-1)) and different total dissolved solids (TDS) concentration (35,000 mg L(-1), 100,000 mg L(-1), 150,000 mg L(-1), 200,000 mg L(-1) and 250,000 mg L(-1)). It was found that with an OLR of 0.9 kg COD m(-3) d(-1) and 1.8 kg COD m(-3) d(-1), average oil and grease (O&G) concentrations in the effluent were 7 mg L(-1) and 12 mg L(-1), respectively. At TDS concentration of 35,000 mg L(-1) and at an OLR of 1.8 kg COD m(-3)d(-1), COD and O&G removal efficiencies were more than 90%. However, with increase in salt content to 250,000 mg L(-1), COD and O&G removal efficiencies decreased to 74% and 63%, respectively. The results of biological treatment of real produced water showed that the removal rates of the main pollutants of wastewater, such as COD, TOC and O&G, were above 81%, 83%, and 85%, respectively.

  14. Microorganism immobilization

    DOEpatents

    Compere, Alicia L.; Griffith, William L.

    1981-01-01

    Live metabolically active microorganisms are immobilized on a solid support by contacting particles of aggregate material with a water dispersible polyelectrolyte such as gelatin, crosslinking the polyelectrolyte by reacting it with a crosslinking agent such as glutaraldehyde to provide a crosslinked coating on the particles of aggregate material, contacting the coated particles with live microorganisms and incubating the microorganisms in contact with the crosslinked coating to provide a coating of metabolically active microorganisms. The immobilized microorganisms have continued growth and reproduction functions.

  15. Response of soil microorganisms to As-produced and functionalized single-wall carbon nanotubes (SWNTs).

    PubMed

    Tong, Zhonghua; Bischoff, Marianne; Nies, Loring F; Myer, Phillip; Applegate, Bruce; Turco, Ronald F

    2012-12-18

    The use of single-wall carbon nanotubes (SWNTs) in manufacturing and biomedical applications is increasing at a rapid rate; however data on the effects of a potential environmental release of the materials remain sparse. In this study, soils with either low or high organic matter contents as well as pure cultures of E. coli are challenged with either raw as-produced SWNTs (AP-SWNTs) or SWNTs functionalized with either polyethyleneglycol (PEG-SWNTs) or m-polyaminobenzene sulfonic acid (PABS-SWNTs). To mimic chronic exposure, the soil systems were challenged weekly for six weeks; microbial activities and community structures for both the prokaryote and eukaryote community were evaluated. Results show that repeated applications of AP-SWNTs can affect microbial community structures and induce minor changes in soil metabolic activity in the low organic matter systems. Toxicity of the three types of SWNTs was also assessed in liquid cultures using a bioluminescent E. coli-O157:H7 strain. Although decreases in light were detected in all treated samples, low light recovery following glucose addition in AP-SWNTs treatment and light absorption property of SWNTs particles suggest that AP-SWNTs suppressed metabolic activity of the E. coli, whereas the two functionalized SWNTs are less toxic. The metals released from the raw forms of SWNTs would not play a role in the effects seen in soil or the pure culture. We suggest that sorption to soil organic matter plays a controlling role in the soil microbiological responses to these nanomaterials.

  16. Isolation and Characterization of Single Cells from Zebrafish Embryos

    PubMed Central

    Samsa, Leigh Ann; Fleming, Nicole; Magness, Scott; Qian, Li; Liu, Jiandong

    2017-01-01

    The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies. PMID:27022828

  17. Single spore isolation and morphological characterization of local Malaysian isolates of rice blast fungus Magnoporthe grisea

    NASA Astrophysics Data System (ADS)

    Mishra, Ankitta; Ratnam, Wickneswari; Bhuiyan, Md Atiqur Rahman; Ponaya, Ariane; Jena, Khisord K.

    2015-09-01

    Rice blast is a destructive disease, caused by the fungal pathogen Magnaporthe grisea. It causes considerable damage to rice and leads to crop loss in rice growing regions worldwide. Although fungicides can be used to control rice blast, they generate additional cost in rice production and contamination of environment and food. Therefore, the use of resistant varieties is thought to be one of the most economically and environmentally efficient ways of crop protection from the disease. Six new local Malaysian isolates of M. grisea were isolated using single spore isolation method. Five isolates were from infected leaf samples collected from Kompleks Latihan MADA, Kedah and one was from Kelantan. These isolates were identified using morphological characteristics and microscopic studies and later confirmed by ITSequences. These isolates were induced to sporulate and used for greenhouse screening on two differential rice varieties: Mahsuri (susceptible) and Pongsu Seribu 2 (resistant). Among the 6 isolates, isolate number 3 was found to be the most virulent showing high sporulation while isolate number 4 was very slow growing, and the least virulent.

  18. Isolating and moving single atoms using silicon nanocrystals

    DOEpatents

    Carroll, Malcolm S.

    2010-09-07

    A method is disclosed for isolating single atoms of an atomic species of interest by locating the atoms within silicon nanocrystals. This can be done by implanting, on the average, a single atom of the atomic species of interest into each nanocrystal, and then measuring an electrical charge distribution on the nanocrystals with scanning capacitance microscopy (SCM) or electrostatic force microscopy (EFM) to identify and select those nanocrystals having exactly one atom of the atomic species of interest therein. The nanocrystals with the single atom of the atomic species of interest therein can be sorted and moved using an atomic force microscope (AFM) tip. The method is useful for forming nanoscale electronic and optical devices including quantum computers and single-photon light sources.

  19. Detection of swelling of single isolated mitochondrion with optical microscopy

    PubMed Central

    Morikawa, Daisuke; Kanematsu, Keita; Shibata, Takahiro; Haseda, Keisuke; Umeda, Norihiro; Ohta, Yoshihiro

    2014-01-01

    Volume regulation under osmotic loading is one of the most fundamental functions in cells and organelles. However, the effective method to detect volume changes of a single organelle has not been developed. Here, we present a novel technique for detecting volume changes of a single isolated mitochondrion in aqueous solution based on the transmittance of the light through the mitochondrion. We found that 70% and 21% of mitochondria swelled upon addition of a hypotonic solution and Ca2+, respectively. These results show the potential of the present technique to detect the physiological volume changes of individual small organelles such as mitochondria. PMID:24688818

  20. Single-Cell Isolation and Gene Analysis: Pitfalls and Possibilities

    PubMed Central

    Hodne, Kjetil; Weltzien, Finn-Arne

    2015-01-01

    During the last two decades single-cell analysis (SCA) has revealed extensive phenotypic differences within homogenous cell populations. These phenotypic differences are reflected in the stochastic nature of gene regulation, which is often masked by qualitatively and quantitatively averaging in whole tissue analyses. The ability to isolate transcripts and investigate how genes are regulated at the single cell level requires highly sensitive and refined methods. This paper reviews different strategies currently used for SCA, including harvesting, reverse transcription, and amplification of the RNA, followed by methods for transcript quantification. The review provides the historical background to SCA, discusses limitations, and current and future possibilities in this exciting field of research. PMID:26569222

  1. Single and Combined Effects of Pesticide Seed Dressings and Herbicides on Earthworms, Soil Microorganisms, and Litter Decomposition

    PubMed Central

    Van Hoesel, Willem; Tiefenbacher, Alexandra; König, Nina; Dorn, Verena M.; Hagenguth, Julia F.; Prah, Urša; Widhalm, Theresia; Wiklicky, Viktoria; Koller, Robert; Bonkowski, Michael; Lagerlöf, Jan; Ratzenböck, Andreas; Zaller, Johann G.

    2017-01-01

    Seed dressing, i.e., the treatment of crop seeds with insecticides and/or fungicides, aiming to protect seeds from pests and diseases, is widely used in conventional agriculture. During the growing season, those crop fields often receive additional broadband herbicide applications. However, despite this broad utilization, very little is known on potential side effects or interactions between these different pesticide classes on soil organisms. In a greenhouse pot experiment, we studied single and interactive effects of seed dressing of winter wheat (Triticum aestivum L. var. Capo) with neonicotinoid insecticides and/or strobilurin and triazolinthione fungicides and an additional one-time application of a glyphosate-based herbicide on the activity of earthworms, soil microorganisms, litter decomposition, and crop growth. To further address food-web interactions, earthworms were introduced to half of the experimental units as an additional experimental factor. Seed dressings significantly reduced the surface activity of earthworms with no difference whether insecticides or fungicides were used. Moreover, seed dressing effects on earthworm activity were intensified by herbicides (significant herbicide × seed dressing interaction). Neither seed dressings nor herbicide application affected litter decomposition, soil basal respiration, microbial biomass, or specific respiration. Seed dressing did also not affect wheat growth. We conclude that interactive effects on soil biota and processes of different pesticide classes should receive more attention in ecotoxicological research. PMID:28270821

  2. Single and Combined Effects of Pesticide Seed Dressings and Herbicides on Earthworms, Soil Microorganisms, and Litter Decomposition.

    PubMed

    Van Hoesel, Willem; Tiefenbacher, Alexandra; König, Nina; Dorn, Verena M; Hagenguth, Julia F; Prah, Urša; Widhalm, Theresia; Wiklicky, Viktoria; Koller, Robert; Bonkowski, Michael; Lagerlöf, Jan; Ratzenböck, Andreas; Zaller, Johann G

    2017-01-01

    Seed dressing, i.e., the treatment of crop seeds with insecticides and/or fungicides, aiming to protect seeds from pests and diseases, is widely used in conventional agriculture. During the growing season, those crop fields often receive additional broadband herbicide applications. However, despite this broad utilization, very little is known on potential side effects or interactions between these different pesticide classes on soil organisms. In a greenhouse pot experiment, we studied single and interactive effects of seed dressing of winter wheat (Triticum aestivum L. var. Capo) with neonicotinoid insecticides and/or strobilurin and triazolinthione fungicides and an additional one-time application of a glyphosate-based herbicide on the activity of earthworms, soil microorganisms, litter decomposition, and crop growth. To further address food-web interactions, earthworms were introduced to half of the experimental units as an additional experimental factor. Seed dressings significantly reduced the surface activity of earthworms with no difference whether insecticides or fungicides were used. Moreover, seed dressing effects on earthworm activity were intensified by herbicides (significant herbicide × seed dressing interaction). Neither seed dressings nor herbicide application affected litter decomposition, soil basal respiration, microbial biomass, or specific respiration. Seed dressing did also not affect wheat growth. We conclude that interactive effects on soil biota and processes of different pesticide classes should receive more attention in ecotoxicological research.

  3. Responses of soil ammonia-oxidizing microorganisms to repeated exposure of single-walled and multi-walled carbon nanotubes.

    PubMed

    Chen, Qinglin; Wang, Hui; Yang, Baoshan; He, Fei; Han, Xuemei; Song, Ziheng

    2015-02-01

    The impacts of carbon nanotubes (CNTs) including single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) on soil microbial biomass and microbial community composition (especially on ammonium oxidizing microorganisms) have been evaluated. The first exposure of CNTs lowered the microbial biomass immediately, but the values recovered to the level of the control at the end of the experiment despite the repeated addition of CNTs. The abundance and diversity of ammonium-oxidizing archaea (AOA) were higher than that of ammonium-oxidizing bacteria (AOB) under the exposure of CNTs. The addition of CNTs decreased Shannon-Wiener diversity index of AOB and AOA. Two-way ANOVA analysis showed that CNTs had significant effects on the abundance and diversity of AOB and AOA. Dominant terminal restriction fragments (TRFs) of AOB exhibited a positive relationship with NH4(+), while AOA was on the contrary. It implied that AOB prefer for high-NH4(+) soils whereas AOA is favored in low NH4(+) soils in the CNT-contaminated soil. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. IGF1R Variants Associated with Isolated Single Suture Craniosynostosis

    PubMed Central

    Cunningham, Michael L.; Horst, Jeremy A.; Rieder, Mark J.; Hing, Anne V.; Stanaway, Ian B.; Park, Sarah S.; Samudrala, Ram; Speltz, Matthew L.

    2010-01-01

    The genetic contribution to the pathogenesis of isolated single suture craniosynostosis is poorly understood. The role of mutations in genes known to be associated with syndromic synostosis appears to be limited. We present our findings of a candidate gene resequencing approach to identify rare variants associated with the most common forms of isolated craniosynostosis. Resequencing of the coding regions, splice junction sites, and 5′ and 3′ untranslated regions of 27 candidate genes in 186 cases of isolated nonsyndromic single suture synostosis revealed three novel and two rare sequence variants (R406H, R595H, N857S, P190S, M446V) in insulin-like growth factor I receptor (IGF1R) that are enriched relative to control samples. Mapping the resultant amino acid changes to the modeled homodimer protein structure suggests a structural basis for segregation between these and other disease-associated mutations found in IGF1R. These data suggest that IGF1R mutations may contribute to the risk and in some cases cause single suture craniosynostosis. PMID:21204214

  5. Importance of amoebae as a tool to isolate amoeba-resisting microorganisms and for their ecology and evolution: the Chlamydia paradigm.

    PubMed

    Kebbi-Beghdadi, Carole; Greub, Gilbert

    2014-08-01

    Free-living amoebae are distributed worldwide and are frequently in contact with humans and animals. As cysts, they can survive in very harsh conditions and resist biocides and most disinfection procedures. Several microorganisms, called amoeba-resisting microorganisms (ARMs), have evolved to survive and multiply within these protozoa. Among them are many important pathogens, such as Legionella and Mycobacteria, and also several newly discovered Chlamydia-related bacteria, such as Parachlamydia acanthamoebae, Estrella lausannensis, Simkania negevensis or Waddlia chondrophila whose pathogenic role towards human or animal is strongly suspected. Amoebae represent an evolutionary crib for their resistant microorganisms since they can exchange genetic material with other ARMs and develop virulence traits that will be further used to infect other professional phagocytes. Moreover, amoebae constitute an ideal tool to isolate strict intracellular microorganisms from complex microbiota, since they will feed on other fast-growing bacteria, such as coliforms potentially present in the investigated samples. The paradigm that ARMs are likely resistant to macrophages, another phagocytic cell, and that they are likely virulent towards humans and animals is only partially true. Indeed, we provide examples of the Chlamydiales order that challenge this assumption and suggest that the ability to multiply in protozoa does not strictly correlate with pathogenicity and that we should rather use the ability to replicate in multiple and diverse eukaryotic cells as an indirect marker of virulence towards mammals. Thus, cell-culture-based microbial culturomics should be used in the future to try to discover new pathogenic bacterial species.

  6. Prospect, isolation, and characterization of microorganisms for potential use in cases of oil bioremediation along the coast of Trindade Island, Brazil.

    PubMed

    Rodrigues, Edmo M; Kalks, Karlos H M; Tótola, Marcos R

    2015-06-01

    In the present study, acrylic coupons with a thin layer of oil on the surface were incubated in the coastal water of Trindade Island, Brazil, for 60 days. The microorganisms adhered to the coupons were isolated using enrichment medium with hexadecane and naphthalene as the sole carbon and energy source. A total of 15 bacterial isolates were obtained, and the ability of these isolates to use different hydrocarbons as the source of carbon and energy was investigated. None of the isolates produced biosurfactants under our experimental conditions. Subsequently, identification methods such as partial sequencing of the 16S rRNA gene and analysis of fatty acids (MIDI) profile were employed. Among the 15 isolates, representatives of Actinobacteria, Firmicutes, and Alphaproteobacteria were detected. The isolates Rhodococcus rhodochrous TRN7 and Nocardia farcinica TRH1 were able to use all the hydrocarbons added to the culture medium (toluene, octane, xylene, naphthalene, phenanthrene, pyrene, hexadecane, anthracene, eicosane, tetracosane, triacontane, and pentacontane). Polymerase chain reaction amplification of the DNA isolated by employing primers for catechol 2,3-dioxygenase, alkane dehydrogenase and the alpha subunit of hydroxylating dioxygenases polycyclic aromatic hydrocarbon rings genes demonstrated that various isolates capable of utilizing hydrocarbons do not exhibit genes of known routes of catabolism, suggesting the existence of unknown catabolic pathways in these microorganisms. Our findings suggest that the microbiota associated to the coast of tropical oceanic islands has the ability to assist in environmental regeneration in cases of accidents involving oil spills in its shore. Thus, it motivates studies to map bioremediation strategies using the autochthonous microbiota from these environments.

  7. Selective single cell isolation for genomics using microraft arrays

    PubMed Central

    Welch, Joshua D.; Williams, Lindsay A.; DiSalvo, Matthew; Brandt, Alicia T.; Marayati, Raoud; Sims, Christopher E.; Allbritton, Nancy L.; Prins, Jan F.; Yeh, Jen Jen; Jones, Corbin D.

    2016-01-01

    Genomic methods are used increasingly to interrogate the individual cells that compose specific tissues. However, current methods for single cell isolation struggle to phenotypically differentiate specific cells in a heterogeneous population and rely primarily on the use of fluorescent markers. Many cellular phenotypes of interest are too complex to be measured by this approach, making it difficult to connect genotype and phenotype at the level of individual cells. Here we demonstrate that microraft arrays, which are arrays containing thousands of individual cell culture sites, can be used to select single cells based on a variety of phenotypes, such as cell surface markers, cell proliferation and drug response. We then show that a common genomic procedure, RNA-seq, can be readily adapted to the single cells isolated from these rafts. We show that data generated using microrafts and our modified RNA-seq protocol compared favorably with the Fluidigm C1. We then used microraft arrays to select pancreatic cancer cells that proliferate in spite of cytotoxic drug treatment. Our single cell RNA-seq data identified several expected and novel gene expression changes associated with early drug resistance. PMID:27530426

  8. Selection of prophylactic antibiotics according to the microorganisms isolated from surgical site infections (SSIs) in a previous series of surgeries reduces SSI incidence after pancreaticoduodenectomy.

    PubMed

    Kondo, Kazuhiro; Chijiiwa, Kazuo; Ohuchida, Jiro; Kai, Masahiro; Fujii, Yoshiro; Otani, Kazuhiro; Hiyoshi, Masahide; Nagano, Motoaki; Imamura, Naoya

    2013-03-01

    The incidence of surgical site infections (SSIs) is high after pancreaticoduodenectomy (PD). We divided 116 consecutive patients who underwent PD into an early group (n = 58) and a later group (n = 58) according to time of surgery. In both groups, endoscopic retrograde biliary drainage was mainly employed for the patients with obstructive jaundice. In the later group, prophylactic antibiotics were selected according to the susceptibility of microorganisms isolated from SSIs in the early group. The incidence of SSIs was compared between the groups. The background characteristics (including methods of preoperative biliary drainage and microorganisms in the bile obtained before or during operation) of the patients were not significantly different between the groups, except for the serum albumin level, which was lower in the later group than in the early group (P = 0.0026). The incidence of SSIs was significantly lower in the later group (24.1 %) than in the early group (46.6 %) (P = 0.0116). Belonging to the later group was one independent negative risk factor for SSI. Selection of prophylactic antibiotics on the basis of microorganisms isolated from SSIs in the early group contributed to the reduced incidence of SSIs in the later group after PD.

  9. Analytical model of an isolated single-atom electron source.

    PubMed

    Engelen, W J; Vredenbregt, E J D; Luiten, O J

    2014-12-01

    An analytical model of a single-atom electron source is presented, where electrons are created by near-threshold photoionization of an isolated atom. The model considers the classical dynamics of the electron just after the photon absorption, i.e. its motion in the potential of a singly charged ion and a uniform electric field used for acceleration. From closed expressions for the asymptotic transverse electron velocities and trajectories, the effective source temperature and the virtual source size can be calculated. The influence of the acceleration field strength and the ionization laser energy on these properties has been studied. With this model, a single-atom electron source with the optimum electron beam properties can be designed. Furthermore, we show that the model is also applicable to ionization of rubidium atoms, and thus also describes the ultracold electron source, which is based on photoionization of laser-cooled alkali atoms.

  10. Quantifying Glosair™ 400 efficacy for surface disinfection of American Type Culture Collection strains and micro-organisms recently isolated from intensive care unit patients.

    PubMed

    Herruzo, R; Vizcaíno, M J; Herruzo, I

    2014-07-01

    Microbial contamination of hospital surfaces may be a source of infection for hospitalized patients. We evaluated the efficacy of Glosair™ 400 against two American Type Culture Collection strains and 18 clinical isolates, placed on glass germ-carriers. Carriers were left to air-dry for 60 min and then exposed to a cycle before detection of any surviving micro-organisms. Antibiotic-susceptible Gram-negative bacilli were less susceptible (although not significantly) to this technique than resistant Gram-negative bacilli or Gram-positive cocci and yeasts (3, 3.4 and 4.6 log10 reduction, respectively). In conclusion, in areas that had not been cleaned, aerosolized hydrogen peroxide obtained >3 log10 mean destruction of patients' micro-organisms. Copyright © 2014 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

  11. Surface Disinfectants for Burn Units Evaluated by a New Double Method, Using Microorganisms Recently Isolated From Patients, on a Surface Germ-Carrier Model.

    PubMed

    Herruzo, Rafael; Vizcaino, Maria Jose; Herruzo, Irene; Sanchez, Manuel

    Assessment methods of surface disinfection based on international standards (Environmental Protection Agency, European Norms, etc) do not correspond to hospital reality. New evaluation methods of surfaces disinfection are proposed to choose the most suitable disinfectant to act against clinically relevant microorganisms detected on the surfaces of burn units. 1) "Immediate effect": 6 products were compared using a glass germ-carrier and 20 recently isolated microorganisms from different patients in the intensive care units. Disinfectants were applied with microfiber cloths. Log10 reductions were calculated for colony forming units produced after 15 minutes of disinfectant application. 2) "Residual effect": the glass germ-carriers were previously impregnated with one of the studied disinfectants. After a 30-minute wait period, they were then contaminated with 1 microorganism (from the 20 above-mentioned). After 15 minutes, the disinfectant was inhibited and the log10 reduction of colony forming units was assessed. The immediate effect (disinfection and microorganism dragging and transferring from the surface to the cloth) produced complete elimination of the inoculums for all products used except one (a diluted quaternary ammonium). The average residual effect found on the 20 microorganisms was moderate: 2 to 3 log10 colony forming unit reduction with chlorine dioxide or 0.5% chlorhexidine (and lower with the other products), obtaining surfaces refractory to recontamination, at least, during 30 minutes. Two tests should be performed before advising surface disinfectant: 1) direct effect and 2) residual efficacy. These characteristics should be considered when a new surface disinfectant is chosen. Chlorine dioxide has a similar or better direct effect than sodium hypochlorite and a similar residual effect than chlorhexidine.

  12. Computational/experimental studies of isolated, single component droplet combustion

    NASA Technical Reports Server (NTRS)

    Dryer, Frederick L.

    1993-01-01

    Isolated droplet combustion processes have been the subject of extensive experimental and theoretical investigations for nearly 40 years. The gross features of droplet burning are qualitatively embodied by simple theories and are relatively well understood. However, there remain significant aspects of droplet burning, particularly its dynamics, for which additional basic knowledge is needed for thorough interpretations and quantitative explanations of transient phenomena. Spherically-symmetric droplet combustion, which can only be approximated under conditions of both low Reynolds and Grashof numbers, represents the simplest geometrical configuration in which to study the coupled chemical/transport processes inherent within non-premixed flames. The research summarized here, concerns recent results on isolated, single component, droplet combustion under microgravity conditions, a program pursued jointly with F.A. Williams of the University of California, San Diego. The overall program involves developing and applying experimental methods to study the burning of isolated, single component droplets, in various atmospheres, primarily at atmospheric pressure and below, in both drop towers and aboard space-based platforms such as the Space Shuttle or Space Station. Both computational methods and asymptotic methods, the latter pursued mainly at UCSD, are used in developing the experimental test matrix, in analyzing results, and for extending theoretical understanding. Methanol, and the normal alkanes, n-heptane, and n-decane, have been selected as test fuels to study time-dependent droplet burning phenomena. The following sections summarizes the Princeton efforts on this program, describe work in progress, and briefly delineate future research directions.

  13. A technique to dissect the alimentary canal of the coffee berry borer (Hypothenemus hampei), with isolation of internal microorganisms

    USDA-ARS?s Scientific Manuscript database

    A novel technique has been developed to dissect the alimentary canal of the coffee berry borer, Hypothenemus hampei. The technique allows recovering bacteria and fungi present inside the alimentary canal. These microorganisms will be the subjects of studies aimed at elucidating how the insect brea...

  14. Antibiotic susceptibility and imaging findings of the causative microorganisms responsible for acute urinary tract infection in children: a five-year single center study

    PubMed Central

    Yoon, Ji Eun; Kim, Wun Kon; Lee, Jin Seok; Shin, Kyeong-Seob

    2011-01-01

    Purpose We studied the differences in the antibiotic susceptibilities of the microorganisms that causeing urinary tract infections (UTI) in children to obtain useful information on appropriate drug selection for childhood UTI. Methods We retrospectively analyzed the antibiotic susceptibilities of 429 microorganisms isolated from 900 patients diagnosed with UTI in the Department of Pediatrics, Chungbuk National University Hospital, from 2003 to 2008. Results The most common causative microorganisms for UTI were Escherichia coli (81.4%), Klebsiella pneumoniae (8.4%), Enterobacter spp. (1.7%), and Proteus spp. (0.4%). E. coli showed relatively high susceptibility as compared to imipenem (100%), amikacin (97.7%), aztreonam (97.9%), cefepime (97.7%), and ceftriaxone (97.1%), while it showed relatively low susceptibility to gentamicin (GM) (79.0%), trimethoprim/sulfamethoxazole (TMP/SMX) (68.7%), ampicillin/sulbactam (33.0%), and ampicillin (AMP) (28.6%). There were no significant differences in the image findings for causative microorganisms. Conclusion Gram-negative organisms showed high susceptibility to amikacin and third-generation cephalosporins, and low susceptibility to AMP, GM, and TMP/SMX. Therefore, the use of AMP or TMP/SMX as the first choice in empirical and prophylactic treatment of childhood UTI in Korea should be reconsidered and investigated further. PMID:21503201

  15. Single radial immunodiffusion method for screening Staphylococcal isolates for enterotoxin.

    PubMed Central

    Meyer, R F; Palmieri, M J

    1980-01-01

    A direct system for screening large numbers of staphylococcal isolates for enterotoxin production has been developed. The system employs polyvalent (serotypes A, B, C, D, and E) immunodiffusion assay slides in conjunction with a multiple-culturing system for toxin production. With the combined system, as many as 50 cultures can be screened simultaneously on a single assay slide having a sensitivity of about 0.3 microgram/ml. The system should be useful for detecting potential enterotoxin in foods containing a predominance of non-enterotoxigenic strains. Images PMID:6779703

  16. Prevalence of extended-spectrum beta-lactamases-producing microorganisms in nosocomial patients and molecular characterization of the shv type isolates

    PubMed Central

    de Oliveira, Caio Fernando; Salla, Adenilde; Lara, Valéria Maria; Rieger, Alexandre; Horta, Jorge André; Alves, Sydney Hartz

    2010-01-01

    The emergence of Extended-Spectrum Beta-Lactamase (ESBL)-producing microorganisms in Brazilian hospitals is a challenge that concerns scientists, clinicians and healthcare institutions due to the serious risk they pose to confined patients. The goal of this study was the detection of ESBL production by clinical strains of Escherichia coli and Klebsiella sp. isolated from pus, urine and blood of patients at Hospital Universitário Santa Maria, Rio Grande Sul, RS, Brazil and the genotyping of the isolates based on bla SHV genes. The ESBL study was carried out using the Combined Disc Method, while Polymerase Chain Reaction (PCR) was used to study the bla SHV genes. Of the 90 tested isolates, 55 (61.1%) were identified as ESBL-producing by the combined disk method. The bla SHV genes were found in 67.8% of these microorganisms. K. pneumoniae predominated in the samples, presenting the highest frequency of positive results from the combined disk and PCR. PMID:24031491

  17. Sequencing viral genomes from a single isolated plaque.

    PubMed

    Depew, Jessica; Zhou, Bin; McCorrison, Jamison M; Wentworth, David E; Purushe, Janaki; Koroleva, Galina; Fouts, Derrick E

    2013-06-06

    Whole genome sequencing of viruses and bacteriophages is often hindered because of the need for large quantities of genomic material. A method is described that combines single plaque sequencing with an optimization of Sequence Independent Single Primer Amplification (SISPA). This method can be used for de novo whole genome next-generation sequencing of any cultivable virus without the need for large-scale production of viral stocks or viral purification using centrifugal techniques. A single viral plaque of a variant of the 2009 pandemic H1N1 human Influenza A virus was isolated and amplified using the optimized SISPA protocol. The sensitivity of the SISPA protocol presented here was tested with bacteriophage F_HA0480sp/Pa1651 DNA. The amplified products were sequenced with 454 and Illumina HiSeq platforms. Mapping and de novo assemblies were performed to analyze the quality of data produced from this optimized method. Analysis of the sequence data demonstrated that from a single viral plaque of Influenza A, a mapping assembly with 3590-fold average coverage representing 100% of the genome could be produced. The de novo assembled data produced contigs with 30-fold average sequence coverage, representing 96.5% of the genome. Using only 10 pg of starting DNA from bacteriophage F_HA0480sp/Pa1651 in the SISPA protocol resulted in sequencing data that gave a mapping assembly with 3488-fold average sequence coverage, representing 99.9% of the reference and a de novo assembly with 45-fold average sequence coverage, representing 98.1% of the genome. The optimized SISPA protocol presented here produces amplified product that when sequenced will give high quality data that can be used for de novo assembly. The protocol requires only a single viral plaque or as little as 10 pg of DNA template, which will facilitate rapid identification of viruses during an outbreak and viruses that are difficult to propagate.

  18. Resistance to tetracycline and β-lactams and distribution of resistance markers in enteric microorganisms and pseudomonads isolated from the oral cavity.

    PubMed

    Ramos, Marcelle Marie Buso; Gaetti-Jardim, Ellen Cristina; Gaetti-Jardim Junior, Elerson

    2009-01-01

    This study evaluated the occurrence of enteric bacteria and pseudomonads resistant to tetracycline and β-lactams in the oral cavity of patients exhibiting gingivitis (n=89), periodontitis (n=79), periodontally healthy (n=50) and wearing complete dentures (n=41). Microbial identification and presence of resistance markers associated with the production of β-lactamases and tetracycline resistance were performed by using biochemical tests and PCR. Susceptibility tests were carried out in 201 isolates of enteric cocci and rods. Resistance to ampicillin, amoxicillin/clavulanic acid, imipenem, meropenem and tetracycline was detected in 57.4%, 34.6%, 2.4%, 1.9% and 36.5% of the isolates, respectively. β-lactamase production was observed in 41.2% of tested microorganisms, while the most commonly found β-lactamase genetic determinant was gene blaTEM. Tetracycline resistance was disseminated and a wide scope of tet genes were detected in all studied microbial genus.

  19. Ionic currents in single isolated bullfrog atrial cells

    PubMed Central

    1983-01-01

    Enzymatic dispersion has been used to yield single cells from segments of bullfrog atrium. Previous data (Hume and Giles, 1981) have shown that these individual cells are quiescent and have normal resting potentials and action potentials. The minimum DC space constant is approximately 920 microns. The major goals of the present study were: (a) to develop and refine techniques for making quantitative measurements of the transmembrane ionic currents, and (b) to identify the individual components of ionic current which generate different phases of the action potential. Initial voltage-clamp experiments made using a conventional two-microelectrode technique revealed a small tetrodotoxin (TTX)-insensitive inward current. The small size of this current (2.5-3.0 X 10(-10)A) and the technical difficulty of the two- microelectrode experiments prompted the development of a one- microelectrode voltage-clamp technique which requires impalements using a low-resistance (0.5-2 M omega) micropipette. Voltage-clamp experiments using this new technique in isolated single atrial cells reveal five distinct ionic currents: (a) a conventional transient Na+ current, (b) a TTX-resistant transient inward current, carried mainly by Ca++, (c) a component of persistent inward current, (d) a slowly developing outward K+ current, and (e) an inwardly rectifying time- independent background current. The single suction micropipette technique appears well-suited for use in the quantitative study of ionic currents in these cardiac cells, and in other small cells having similar electrophysiological properties. PMID:6302197

  20. Practical, Microfabrication-Free Device for Single-Cell Isolation

    PubMed Central

    Lin, Liang-I; Chao, Shih-hui; Meldrum, Deirdre R.

    2009-01-01

    Microfabricated devices have great potential in cell-level studies, but are not easily accessible for the broad biology community. This paper introduces the Microscale Oil-Covered Cell Array (MOCCA) as a low-cost device for high throughput single-cell analysis that can be easily produced by researchers without microengineering knowledge. Instead of using microfabricated structures to capture cells, MOCCA isolates cells in discrete aqueous droplets that are separated by oil on patterned hydrophilic areas across a relatively more hydrophobic substrate. The number of randomly seeded Escherichia coli bacteria in each discrete droplet approaches single-cell levels. The cell distribution on MOCCA is well-fit with Poisson distribution. In this pioneer study, we created an array of 900-picoliter droplets. The total time needed to seed cells in ∼3000 droplets was less than 10 minutes. Compared to traditional microfabrication techniques, MOCCA dramatically lowers the cost of microscale cell arrays, yet enhances the fabrication and operational efficiency for single-cell analysis. PMID:19696926

  1. Pseudometallophytes colonising Pb/Zn mine tailings: a description of the plant-microorganism-rhizosphere soil system and isolation of metal-tolerant bacteria.

    PubMed

    Becerra-Castro, C; Monterroso, C; Prieto-Fernández, A; Rodríguez-Lamas, L; Loureiro-Viñas, M; Acea, M J; Kidd, P S

    2012-05-30

    The plant-microorganism-soil system of three pseudometallophytes (Betula celtiberica, Cytisus scoparius and Festuca rubra) growing in a Pb/Zn mine was characterised. Plant metal accumulation, soil metal fractions (rhizosphere and non-vegetated) and bacterial densities were determined. Total Cd, Pb and Zn in non-vegetated soils was up to 50, 3000 and 20,000 mg kg(-1) dry weight, respectively. The residual fraction dominated non-vegetated soils, whereas plant-available fractions became important in rhizosphere soils. All plant species effectively excluded metals from the shoot. F. rubra presented a shoot:root transport factor of ≤0.2 and this population could be useful in future phytostabilisation trials. Culturable bacterial densities and diversity were low (predominantly Actinobacteria). Rhizosphere soils hosted higher total and metal-tolerant bacterial densities. Seventy-four metal-tolerant rhizobacteria were isolated, and characterised genotypically (BOX-PCR, 16S rDNA) and phenotypically [Cd/Zn tolerance, biosurfactant production and plant growth promoting (PGP) traits]. Several isolates resisted high concentrations of Cd and Zn, and only a few presented PGP traits. Fourteen isolates were evaluated for promoting plant growth of two species (Salix viminalis and Festuca pratensis). Thirteen inoculants enhanced growth of F. pratensis, while only three enhanced growth of S. viminalis. Growth enhancement could not always be related to isolate PGP traits. In conclusion, some isolates show potential application in phytostabilisation or phytoextraction techniques.

  2. Method for isolating two aquifers in a single borehole

    DOEpatents

    Burklund, P.W.

    1984-01-20

    A method for isolating and individually instrumenting separate aquifers within a single borehole is disclosed. A borehole is first drilled from the ground surface, through an upper aquifer, and into a separating confining bed. A casing, having upper and lower sections separated by a coupling collar, is lowered into the borehole. The borehole is grouted in the vicinity of the lower section of the casing. A borehole is then drilled through the grout plug and into a lower aquifer. After the lower aquifer is instrumented, the borehole is grouted back into the lower portion of the casing. Then the upper section of the casing is unscrewed via the coupling collar and removed from the borehole. Finally, instrumentation is added to the upper aquifer and the borehole is appropriately grouted. The coupling collar is designed to have upper right-hand screw threads and lower left-hand screw thread, whereby the sections of the casing can be readily separated.

  3. Method for isolating two aquifers in a single borehole

    DOEpatents

    Burklund, Patrick W.

    1985-10-22

    A method for isolating and individually instrumenting separate aquifers within a single borehole. A borehole is first drilled from the ground surface, through an upper aquifer, and into a separating confining bed. A casing, having upper and lower sections separated by a coupling collar, is lowered into the borehole. The borehole is grouted in the vicinity of the lower section of the casing. A borehole is then drilled through the grout plug and into a lower aquifer. After the lower aquifer is instrumented, the borehole is grouted back into the lower portion of the casing. Then the upper section of the casing is unscrewed via the coupling collar and removed from the borehole. Finally, instrumentation is added to the upper aquifer and the borehole is appropriately grouted. The coupling collar is designed to have upper right-hand screw threads and lower left-hand screw thread, whereby the sections of the casing can be readily separated.

  4. Biotransformation of organic-rich copper-bearing black shale by indigenous microorganisms isolated from lubin copper mine (Poland).

    PubMed

    Matlakowska, Renata; Narkiewicz, Wanda; Sklodowska, Aleksandra

    2010-04-01

    The role of indigenous microorganisms in the biotransformation of refractory organic-rich copper-bearing black shale ore (Kupferschiefer) was confirmed in laboratory experiments. The persistent shale's organic matter was utilized by a mixture of bacterial strains as the sole carbon and energy source, and bacterial growth was accompanied by chemical and structural changes of black shale. The release of metallic elements and organic compounds into the aqueous phase was shown. Chemical analysis revealed the presence of long-chain aliphatic hydrocarbons and further biodegradation of these compounds by bacterial action. In this study, the release of metals from metalloorganic compounds present in organic-rich copper-bearing black shale was shown for the first time. The results have also confirmed the biotransformation of metalloporphyrins naturally occurring in black shale by indigenous microorganisms. Moreover, changes in the surface area and quantitative mineral composition of black shale were detected following bacterial treatment. This biotransformation activity is of potential use in biotechnological procedures for the recovery of copper and other valuable metals from tailings that contain up to 16% black shale. On the other hand, the release of organic carbon and heavy metals from black shale by biodegradation may significantly add to anthropogenic pollution.

  5. Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

    PubMed Central

    Bailes, Graham; Lind, Margaret; Ely, Andrew; Powell, Marianne; Moore-Kucera, Jennifer; Miles, Carol; Inglis, Debra; Brodhagen, Marion

    2013-01-01

    Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g. fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation. PMID:23712218

  6. Isolation of native soil microorganisms with potential for breaking down biodegradable plastic mulch films used in agriculture.

    PubMed

    Bailes, Graham; Lind, Margaret; Ely, Andrew; Powell, Marianne; Moore-Kucera, Jennifer; Miles, Carol; Inglis, Debra; Brodhagen, Marion

    2013-05-10

    Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g. fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.

  7. Insulin independence following isolated islet transplantation and single islet infusions.

    PubMed

    Markmann, James F; Deng, Shaoping; Huang, Xiaolun; Desai, Niraj M; Velidedeoglu, Ergun H; Lui, Chengyang; Frank, Adam; Markmann, Eileen; Palanjian, Maral; Brayman, Kenneth; Wolf, Bryan; Bell, Ewan; Vitamaniuk, Marko; Doliba, Nicolai; Matschinsky, Franz; Barker, Clyde F; Naji, Ali

    2003-06-01

    To restore islet function in patients whose labile diabetes subjected them to frequent dangerous episodes of hypoglycemic unawareness, and to determine whether multiple transplants are always required to achieve insulin independence. The recent report by the Edmonton group documenting restoration of insulin independence by islet transplantation in seven consecutive patients with type 1 diabetes differed from previous worldwide experience of only sporadic success. In the Edmonton patients, the transplanted islet mass critical for success was approximately more than 9,000 IEq/kg of recipient body weight and required two or three separate transplants of islets isolated from two to four cadaveric donors. Whether the success of the Edmonton group can be recapitulated by others, and whether repeated transplants using multiple donors will be a universal requirement for success have not been reported. The authors report their treatment with islet transplantation of nine patients whose labile type 1 diabetes was characterized by frequent episodes of dangerous hypoglycemia. In each of the seven patients who have completed the treatment protocol (i.e., one or if necessary a second islet transplant), insulin independence has been achieved. In five of the seven patients only a single infusion of islets was required. To date, only one recipient has subsequently lost graft function, after an initially successful transplant. This patient suffered recurrent hyperglycemia 9 months after the transplant. This report confirms the efficacy of the Edmonton immunosuppressive regimen and indicates that insulin independence can often be achieved by a single transplant of sufficient islet mass.

  8. Isolation and characterization of an ether-type polyurethane-degrading micro-organism and analysis of degradation mechanism by Alternaria sp.

    PubMed

    Matsumiya, Y; Murata, N; Tanabe, E; Kubota, K; Kubo, M

    2010-06-01

    To degrade ether-type polyurethane (ether-PUR), ether-PUR-degrading micro-organism was isolated. Moreover, ether-PUR-degrading mechanisms were analysed using model compounds of ether-PUR. A fungus designated as strain PURDK2, capable of changing the configuration of ether-PUR, has been isolated. This isolated fungus was identified as Alternaria sp. Using a scanning electron microscope, the grid structure of ether-PUR was shown to be melted and disrupted by the fungus. The degradation of ether-PUR by the fungus was analysed, and the ether-PUR was degraded by the fungus by about 27.5%. To analyse the urethane-bond degradation by the fungus, a degraded product of ethylphenylcarbamate was analysed using GC/MS. Aniline and ethanol were detected by degradation with the supernatant, indicating that the fungus secreted urethane-bond-degrading enzyme(s). PURDK2 also degraded urea bonds when diphenylmethane-4,4'-dibutylurea was used as a substrate. The enzyme(s) from PURDK2 degraded urethane and urea bonds to convert the high molecular weight structure of ether-PUR to small molecules; and then the fungus seems to use the small molecules as an energy source. Ether-PUR-degrading fungus, strain PURDK2, was isolated, and the urethane- and urea-bonds-degrading enzymes from strain PURDK2 could contribute to the material recycling of ether-PUR.

  9. A novel lineage of sulfate-reducing microorganisms: Thermodesulfobiaceae fam. nov., Thermodesulfobium narugense, gen. nov., sp. nov., a new thermophilic isolate from a hot spring.

    PubMed

    Mori, Koji; Kim, Hongik; Kakegawa, Takeshi; Hanada, Satoshi

    2003-08-01

    A novel type of a sulfate-reducing microorganism, represented by strain Na82T, was isolated from a hot spring in Narugo, Japan. The isolate was a moderate thermophilic autotroph that was able to grow on H2/CO2 by sulfate respiration. The isolate could grow with nitrate in place of sulfate, and possessed menaquinone-7 and menaquinone-7(H2) as respiratory quinones. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain Na82T was a member of the domain Bacteria and distant from any known bacteria, as well as from other sulfate-reducing bacteria (sequence similarities less than 80%). The phylogenetic analysis of the dsrAB gene (alpha and beta subunits of dissimilatory sulfite reductase) sequence also suggested that strain Na82T was not closely related to other sulfate reducers. On the basis of the phenotypic and phylogenetic data, a new taxon is established for the isolate. We proposed the name Thermodesulfobium narugense gen. nov., sp. nov. with strain Na82T (=DSM 14796T=JCM 11510T) as the type strain. Furthermore, a new family, Thermodesulfobiaceae fam. nov., is proposed for the genus.

  10. A high-performance single-mode fiber-optic isolator assembly

    NASA Technical Reports Server (NTRS)

    Lutes, G.

    1987-01-01

    A high-isolation single-mode fiber optic isolator assembly was designed and fabricated. The measured forward loss is 2.6 dB and the reverse loss (isolation) is greater than 70 dB. This is a 30-dB higher isolation than the isolation of the best fiber optic isolator previously reported. This isolator provides isolation between the semiconductor laser diode and the optical fiber in a precise reference frequency transmission system. The isolation of the laser greatly reduces the system's sensitivity to microphonics.

  11. Isolates identification and characteristics of microorganisms in biotrickling filter and biofilter system treating H2S and NH3.

    PubMed

    Guang-Hui, Yu; Xiao-Jun, Xu; Pin-Jing, He

    2007-01-01

    A combination system of biotrickling filter (BTF) and biofilter (BF), adopting surfactant-modified clinoptilolite and surfactant-modified wood chip as the media respectively, was applied to treat H2S and NH3 simultaneously. The identification and sole carbon sources utilization patterns of isolates in the combination system were studied by Biolog system. The isolates were identified as Bacillus sphaericus, Geobacillus themoglucosidasius (55 degrees C) and Micrococcus luteus (ATCC 9341) in BTF, and Aspergillus sydowii (Bainier & Sartory) Thorm & Church in BF. Among 95 substrate classes supplied by Biolog system, the carboxylic acids and methyl esters had the highest utilization extent for the four species, followed by the amino acids and peptides. The descending sequence of carbon sources utilization capability of isolates was A. sydowii (52.6%), M. luteus (39.5%), B. sphaericus (21.6%), and G. thermoglucosidasius (17.7%).

  12. Isolation and identification of spoilage microorganisms using food-based media combined with rDNA sequencing: ranch dressing as a model food.

    PubMed

    Waite, Joy G; Jones, Joseph M; Yousef, Ahmed E

    2009-05-01

    Investigating microbial spoilage of food is hampered by the lack of suitable growth media and protocols to characterize the causative agents. Microbial spoilage of salad dressing is sporadic and relatively unpredictable, thus processors struggle to develop strategies to minimize or prevent spoilage of this product. The objectives of this study were to (i) induce and characterize spoilage events in ranch-style dressing as a model food, and (ii) isolate and identify the causative microorganisms using traditional and food-based media, coupled with rDNA sequence analysis. Ranch dressing (pH 4.4) was prepared and stored at 25 degrees C for 14 d and microbial populations were recovered on MRS agar and ranch dressing agar (RDA), a newly formulated food-based medium. When isolates suspected as the spoilage agents were inoculated into ranch dressing and held at 25 degrees C for 9-10 d, three unique spoilage events were characterized. Using rDNA sequence comparisons, spoilage organisms were identified as Lactobacillus brevis, Pediococcus acidilactici, and Torulaspora delbrueckii. P. acidilactici produced flat-sour spoilage, whereas Lb. brevis resulted in product acidification and moderate gas production. The RDA medium allowed for optimum recovery of the excessive gas-producing spoilage yeast, T. delbrueckii. The isolation and identification strategy utilized in this work should assist in the characterization of spoilage organisms in other food systems.

  13. Incidence, etiology, and antibiotic resistance patterns of gram-negative microorganisms isolated from patients with ventilator-associated pneumonia in a medical-surgical intensive care unit of a teaching hospital in istanbul, Turkey (2004-2006).

    PubMed

    Erdem, Ilknur; Ozgultekin, Asu; Inan, Asuman Sengoz; Dincer, Emine; Turan, Guldem; Ceran, Nurgul; Ozturk Engin, Derya; Senbayrak Akcay, Seniha; Akgun, Nur; Goktas, Pasa

    2008-09-01

    The identification of microorganisms causing ventilator-associated pneumonia (VAP) is important for formulating appropriate therapies. In this study, we report the incidence, etiology, and antibiotic resistance patterns of Gram-negative microorganisms isolated from patients diagnosed with VAP in our medical-surgical intensive care unit (ICU) during the years 2004-2006. VAP was diagnosed by using the clinical criteria of the Centers for Disease Control and Prevention. Antibiotic resistance patterns of isolated microorganisms were defined by standard methods. The VAP incidence rate was 22.6/1,000 ventilator days. The most frequently isolated pathogens were Acinetobacter spp., methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa. Ninety percent of Acinetobacter spp. isolates were resistant to ceftazidime, 64% to imipenem, and 80% to ciprofloxacin. Fifty-nine percent of P. aeruginosa isolates were resistant to ceftazidime, 32% to imipenem, and 62% to ciprofloxacin. Cefoperazone-sulbactam was the most active agent against Acinetobacter spp. In conclusion, the incidence of VAP and the prevalence of multidrug-resistant microorganisms are quite high in our ICU. Comparison of the resistance rates of isolates demonstrates that certain antibiotic agents are more effective than others.

  14. How do hatcheries influence embryonic development of sea turtle eggs? Experimental analysis and isolation of microorganisms in leatherback turtle eggs.

    PubMed

    Patino-Martinez, Juan; Marco, Adolfo; Quiñones, Liliana; Abella, Elena; Abad, Roberto Muriel; Diéguez-Uribeondo, Javier

    2012-01-01

    Many conservation programs consider translocation of turtle nests to hatcheries as a useful technique. The repeated use of the same incubation substrate over several seasons in these hatcheries could, however, be harmful to embryos if pathogens were able to accumulate or if the physical and chemical characteristics of the incubation environment were altered. However, this hypothesis has yet to be tested. We conducted two field experiments to evaluate the effects of hatchery sand and eggshell decay on the embryonic development of leatherback sea turtle eggs in Colombia. We identified the presence of both fungi and bacteria species on leatherback turtle eggs. Sea turtle eggs exposed to previously used hatchery substrates or to decaying eggshells during the first and middle third of the embryonic development produced hatchlings that were smaller and/or weighed less than control eggs. However, this did not negatively influence hatching success. The final third of embryonic development seems to be less susceptible to infection by microorganisms associated with decaying shells. We discuss the mechanisms that could be affecting sea turtle egg development when in contact with fungi. Further studies should seek to understand the infection process and the stages of development in which the fungi are more virulent to the eggs of this critically endangered species.

  15. Cellulolytic Microorganisms from Thermal Environments

    SciTech Connect

    Vishnivetskaya, Tatiana A; Raman, Babu; Phelps, Tommy Joe; Podar, Mircea; Elkins, James G

    2012-01-01

    Thermal, anaerobic environments rich in decaying plant material are a potential source of novel cellulolytic bacteria. Samples collected from geothermal aquifers in the Yellowstone National Park (YNP) were used to select for cellulolytic thermophiles. Laboratory enrichments on dilute-acid pretreated plant biomass (switchgrass, Populus), and crystalline cellulose (Avicel) resulted in the isolation of 247 environmental clones. The majority of individual clones were affiliated with the cellulolytic bacteria of phylum Firmicutes, followed by xylanolytic and saccharolytic members of the phylum Dictyoglomi. Among the Firmicutes, the clones were affiliated with the genera Caldicellulosiruptor (54.4%), Caloramator (11.5%), Thermoanaerobacter (8.8%), Thermovenabulum (4.1%), and Clostridium (2.0%). From established anaerobic thermophilic enrichments a total of 81 single strains of the genera Caldicellulosiruptor (57%) and Thermoanaerobacter (43%) were isolated. With continuous flow enrichment on Avicel, increases in the relative abundance of Caloramator sp. was observed over clones detected from the Caldicellulosiruptor. Complex communities of interacting microorganisms bring about cellulose decomposition in nature, therefore using up-to-date approaches may yield novel cellulolytic microorganisms with high activity and a rapid rate of biomass conversion to biofuels.

  16. [Metagenomics as a Tool for the Investigation of Uncultured Microorganisms].

    PubMed

    Ravin, N V; Mardanova, A V; Skryabin, K G

    2015-05-01

    Uncultured microorganisms represent a significant part of the Earth's biodiversity. Natural ecosystems contain less than 0.1-1% of the microorganisms that can be cultured in the laboratory. Therefore, new methodological approaches are required for the identification and description of uncultured microorganisms, for studies of their genetic diversity and the structure of microbial associations, and for an understanding of their ecological importance in the biosphere. Metagenomics, a method of analyzing the collective genome.of a microbial community without cultivation, makes it possible to unravel fundamental matters of the microbiology and ecology of microorganisms. Another efficient method of analysis of uncultured forms of microorganisms is "single cell genomics," which involves the isolation of single cells from microbial communities and the sequencing of their genomes. Developed in the last decade, the high throughput technologies of next-generation sequencing provide important input into the investigation of genome reconstruction for all of the microorganisms residing and interacting within ecosystems. This review describes the major methodological approaches used in metagenomic analysis of microbial communities, as well as accomplishments in the search for new uncultured microorganism, the unraveling of their genomes, and an elucidation of their role in ecosystems.

  17. PFLOTRAN Simulation of Waste Isolation Pilot Plant Single Waste Panel

    NASA Astrophysics Data System (ADS)

    Park, H.; Hammond, G. E.

    2015-12-01

    The Waste Isolation Pilot Plant (WIPP), located in southeastern New Mexico, has been developed by the U.S. Department of Energy (DOE) for the deep geologic disposal of transuranic (TRU) waste. WIPP performance assessment (PA) calculations estimate the probability and consequence of potential radionuclide releases from the repository to the accessible environment arising from events and processes that could occur over the 10,000 year regulatory period. The conceptual model estimates three possible cases and the combinations of these cases: 1) undisturbed condition of the repository, 2) human borehole intrusion condition that penetrates the repository, and 3) human borehole intrusion that penetrates pressurized brine underlying the repository. To date, WIPP PA calculations have employed multiple two-dimensional (2D) numerical models requiring simplification of the mesh and processes including homogenization of materials and regions while maintaining volume aspect ratio. Introducing three-dimensional (3D) numerical models within WIPP PA enables increasingly realistic representations of the WIPP subsurface domain and improved flexibility for incorporating relevant features. PFLOTRAN is a state-of-art massively parallel subsurface flow and reactive transport code that will be implemented to enhance PA with more physically realistic 3D flow and transport models; eliminating the need for multiple related, but decoupled 2D models. This paper demonstrates PFLOTRAN simulation of a single waste panel of the WIPP undisturbed condition in 3D. The simulation also employs newly implemented WIPP specific functionalities to PFLOTRAN: 1) gas generation from the wastes, 2) creep closure of bedded salt formation, 3) fractures of marker beds near the excavation, 4) Klinkenberg effect on gas permeability in low-permeable materials, and 5) Redlich-Kwong-Soave equation of state for gas density.

  18. Characterization of microorganisms isolated from the black dirt of toilet bowls and componential analysis of the black dirt.

    PubMed

    Mori, Miho; Nagata, Yusuke; Niizeki, Kazuma; Gomi, Mitsuhiro; Sakagami, Yoshikazu

    2014-01-01

    We have previously conducted a microflora analysis and examined the biofilm-forming activity of bacteria isolated from toilet bowl biofilms. In the present investigation, to reveal the strain involved in the formation of black dirt in toilet bowls, we performed a microflora analysis of the bacteria and fungi isolated from the black dirt of toilet bowls at ten homes. Among samples from different isolation sites and sampling seasons, although a similar tendency was not seen in bacterial microflora, Exophiala sp. was detected in the fungal microflora from all samples of black dirt except for one, and constituted the major presence. By scanning electron microscope (SEM) analysis of the formed black dirt, SEM image at × 1,000 and × 5,000 magnification showed objects like hyphae and many bacteria adhering to them, respectively. Micro fourier transform infrared spectroscopy (micro FT-IR) and SEM with X-ray microanalysis (SEM-XMA) were used to investigate the components of black dirt. IR spectra of micro-FT-IR showed typical absorptions associated with amide compounds and protein, and the elements such as C, N, O, Na, Mg, Al, Si, P, S, K, and Ba were detected with SEM-XMA. These results showed that black dirt had living body ingredients. Furthermore, Exophiala sp. and Cladosporium sp. strains, which were observed at a high frequency, accumulated 2-hydroxyjuglone (2-HJ) and flaviolin as one of the intermediates in the melanin biosynthetic pathway by the addition of a melanin synthesis inhibitor (tricyclazole) at the time of cultivation. These results suggested strongly that the pigment of black dirt in toilet bowls was melanin produced by Exophiala sp. and Cladosporium sp. strains.

  19. Isolation and identification of microorganisms including lactic acid bacteria and their use in microbial deacidification of wines from domestic vineyards.

    PubMed

    Drozdz, Iwona; Makarewicz, Malgorzata; Tuszyński, Tadeusz

    2013-01-01

    The aim of this study was to identify various bacteria isolated from grapes and their wines. Additionally we investigated the capacity of lactic acid bacteria for microbiological deacidification of wines produced in Poland. We have identified Oenococcus oeni, Lactobacillus acidophilus and Lactobacillus delbrueckii. During the microbial deacidification process, we observed decreases of total acidity and increases of volatile acidity, with statistically significant changes noted for O. oeni in Marechal Foch and Seyval Blanc, and for Lb. acidophilus in Frontenac. On the other hand, a statistically significant increase in pH was observed in Marechal Foch and Seyval Blanc following deacidification by O. oeni.

  20. Safety assessment of dairy microorganisms: aerobic coryneform bacteria isolated from the surface of smear-ripened cheeses.

    PubMed

    Denis, Catherine; Irlinger, Françoise

    2008-09-01

    The group of "coryneform bacteria" belongs to the class of Actinobacteria including a diverse and heterogeneous collection of bacteria of various genera. Most of them are known as environmental residents and/or commensal flora of humans and they are isolated frequently in clinical studies. Actinobacteria include also several aerobic species, present at the surface of smear-ripened cheeses for decades and used as ripening culture in the dairy industry. Their clinical significance is controversial because an easy combination of phenotypic and molecular methods to characterize Actinobacteria at the species level is still lacking. A bibliographical survey was conducted to assess the safety status of Actinobacteria species used as starter culture in fermented dairy foods, according to their technological interest. Aerobic coryneform bacteria isolated from smear-ripened cheeses are most commonly recovered from soil, the environment or food. To date, no clinical infection or food toxi-infection related to smear cheese coryneform bacteria ingestion has been reported. From a taxonomic viewpoint, dairy species are distant from the reference species associated with known pathologies. From a physiological viewpoint, cheese smear coryneform bacteria appear to be related to particular ecological niches: they are all oxidative species, and most are psychrotrophic and unable to grow at 37 degrees C whereas medically relevant coryneform bacteria are facultative anaerobes and grow at 35-37 degrees C. Consequently, technological strains must be selected according to taxonomic criteria (nonpathogenic species) and ecological criteria.

  1. Classifying Microorganisms.

    ERIC Educational Resources Information Center

    Baker, William P.; Leyva, Kathryn J.; Lang, Michael; Goodmanis, Ben

    2002-01-01

    Focuses on an activity in which students sample air at school and generate ideas about how to classify the microorganisms they observe. The results are used to compare air quality among schools via the Internet. Supports the development of scientific inquiry and technology skills. (DDR)

  2. Classifying Microorganisms.

    ERIC Educational Resources Information Center

    Baker, William P.; Leyva, Kathryn J.; Lang, Michael; Goodmanis, Ben

    2002-01-01

    Focuses on an activity in which students sample air at school and generate ideas about how to classify the microorganisms they observe. The results are used to compare air quality among schools via the Internet. Supports the development of scientific inquiry and technology skills. (DDR)

  3. Antimicrobial Activity of neo-Clerodane Diterpenoids isolated from Lamiaceae Species against Pathogenic and Food Spoilage Microorganisms.

    PubMed

    Bozov, Petko; Girova, Tania; Prisadova, Natalia; Hristova, Yana; Gochev, Velizar

    2015-11-01

    Antimicrobial activity of nineteen neo-clerodane diterpenoids, isolated from the acetone extracts of the aerial parts of Scutellaria and Salvia species (Lamiaceae) were tested against thirteen strains belonging to nine different species of pathogenic and food spoilage bacteria Aeromonas hydrophila, Bacillus cereus, Escherichia coli, Listeria monocytogenes, Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonas fluorescens, Salmonella abony and Staphylococcus aureus as well as against two yeast strains belonging to species Candida albicans. Seven of the evaluated compounds scutalpin A, scutalpin E, scutalpin F, salviarin, splenolide A, splenolide B and splendidin demonstrated antimicrobial activity against used test microbial strains, the rest of the compounds were inactive within the studied concentration range. Among all of the tested compounds the highest antimicrobial activity was detected for scutalpin A against Staphylococcus aureus (MIC 25 µg/mL).

  4. Degradation of Di- Through Hepta-Chlorobiphenyls in Clophen Oil Using Microorganisms Isolated from Long Term PCBs Contaminated Soil.

    PubMed

    Sharma, Jitendra K; Gautam, Ravindra K; Misra, Rashmi R; Kashyap, Sanjay M; Singh, Sanjeev K; Juwarkar, Asha A

    2014-09-01

    Present work describes microbial degradation of selected polychlorinated biphenyls (PCBs) congeners in Clophen oil which is used as transformer oil and contains high concentration of PCBs. Indigenous PCBs degrading bacteria were isolated from Clophen oil contaminated soil using enrichment culture technique. A 15 days study was carried out to assess the biodegradation potential of two bacterial cultures and their consortium for Clophen oil with a final PCBs concentration of 100 mg kg(-1). The degradation capability of the individual bacterium and the consortium towards the varying range of PCBs congeners (di- through hepta-chlorobiphenyls) was determined using GCMS. Also, dehydrogenase enzyme was estimated to assess the microbial activity. Maximum degradation was observed in treatment containing consortium that resulted in up to 97 % degradation of PCB-44 which is a tetra chlorinated biphenyl whereas, hexa chlorinated biphenyl congener (PCB-153) was degraded up to 90 % by the consortium. This indicates that the degradation capability of microbial consortium was significantly higher than that of individual cultures. Furthermore, the results suggest that for degradation of lower as well as higher chlorinated PCB congeners; a microbial consortium is required rather than individual cultures.

  5. Environmentally relevant microorganisms.

    PubMed

    Watanabe, K; Baker, P W

    2000-01-01

    The development of molecular microbial ecology in the 1990s has allowed scientists to realize that microbial populations in the natural environment are much more diverse than microorganisms so far isolated in the laboratory. This finding has exerted a significant impact on environmental biotechnology, since knowledge in this field has been largely dependent on studies with pollutant-degrading bacteria isolated by conventional culture methods. Researchers have thus started to use molecular ecological methods to analyze microbial populations relevant to pollutant degradation in the environment (called environmentally relevant microorganisms, ERMs), although further effort is needed to gain practical benefits from these studies. This review highlights the utility and limitations of molecular ecological methods for understanding and advancing environmental biotechnology processes. The importance of the combined use of molecular ecological and physiological methods for identifying ERMs is stressed.

  6. Precipitation of Phosphate Minerals by Microorganisms Isolated from a Fixed-Biofilm Reactor Used for the Treatment of Domestic Wastewater

    PubMed Central

    Rivadeneyra, Almudena; Gonzalez-Martinez, Alejandro; Gonzalez-Lopez, Jesus; Martin-Ramos, Daniel; Martinez-Toledo, Maria Victoria; Rivadeneyra, Maria Angustias

    2014-01-01

    The ability of bacteria isolated from a fixed-film bioreactor to precipitate phosphate crystals for the treatment of domestic wastewater in both artificial and natural media was studied. When this was demonstrated in artificial solid media for crystal formation, precipitation took place rapidly, and crystal formation began 3 days after inoculation. The percentage of phosphate-forming bacteria was slightly higher than 75%. Twelve major colonies with phosphate precipitation capacity were the dominant heterotrophic platable bacteria growing aerobically in artificial media. According to their taxonomic affiliations (based on partial sequencing of the 16S rRNA), the 12 strains belonged to the following genera of Gram-negative bacteria: Rhodobacter, Pseudoxanthobacter, Escherichia, Alcaligenes, Roseobacter, Ochrobactrum, Agromyce, Sphingomonas and Paracoccus. The phylogenetic tree shows that most of the identified populations were evolutionarily related to the Alphaproteobacteria (91.66% of sequences). The minerals formed were studied by X-ray diffraction, scanning electron microscopy (SEM), and energy dispersive X-ray microanalysis (EDX). All of these strains formed phosphate crystals and precipitated struvite (MgNH4PO4·6H2O), bobierrite [Mg3(PO4)2·8H2O] and baricite [(MgFe)3(PO4)2·8H2O]. The results obtained in this study show that struvite and spherulite crystals did not show any cell marks. Moreover, phosphate precipitation was observed in the bacterial mass but also near the colonies. Our results suggest that the microbial population contributed to phosphate precipitation by changing the media as a consequence of their metabolic activity. Moreover, the results of this research suggest that bacteria play an active role in the mineral precipitation of soluble phosphate from urban wastewater in submerged fixed-film bioreactors. PMID:24699031

  7. Drop-on-Demand Single Cell Isolation and Total RNA Analysis

    PubMed Central

    Moon, Sangjun; Kim, Yun-Gon; Dong, Lingsheng; Lombardi, Michael; Haeggstrom, Edward; Jensen, Roderick V.; Hsiao, Li-Li; Demirci, Utkan

    2011-01-01

    Technologies that rapidly isolate viable single cells from heterogeneous solutions have significantly contributed to the field of medical genomics. Challenges remain both to enable efficient extraction, isolation and patterning of single cells from heterogeneous solutions as well as to keep them alive during the process due to a limited degree of control over single cell manipulation. Here, we present a microdroplet based method to isolate and pattern single cells from heterogeneous cell suspensions (10% target cell mixture), preserve viability of the extracted cells (97.0±0.8%), and obtain genomic information from isolated cells compared to the non-patterned controls. The cell encapsulation process is both experimentally and theoretically analyzed. Using the isolated cells, we identified 11 stem cell markers among 1000 genes and compare to the controls. This automated platform enabling high-throughput cell manipulation for subsequent genomic analysis employs fewer handling steps compared to existing methods. PMID:21412416

  8. Evaluation of epidemiological cut-off values indicates that biocide resistant subpopulations are uncommon in natural isolates of clinically-relevant microorganisms.

    PubMed

    Morrissey, Ian; Oggioni, Marco Rinaldo; Knight, Daniel; Curiao, Tania; Coque, Teresa; Kalkanci, Ayse; Martinez, Jose Luis

    2014-01-01

    To date there are no clear criteria to determine whether a microbe is susceptible to biocides or not. As a starting point for distinguishing between wild-type and resistant organisms, we set out to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) distributions for four common biocides; triclosan, benzalkonium chloride, chlorhexidine and sodium hypochlorite for 3319 clinical isolates, with a particular focus on Staphylococcus aureus (N = 1635) and Salmonella spp. (N = 901) but also including Escherichia coli (N = 368), Candida albicans (N = 200), Klebsiella pneumoniae (N = 60), Enterobacter spp. (N = 54), Enterococcus faecium (N = 53), and Enterococcus faecalis (N = 56). From these data epidemiological cut-off values (ECOFFs) are proposed. As would be expected, MBCs were higher than MICs for all biocides. In most cases both values followed a normal distribution. Bimodal distributions, indicating the existence of biocide resistant subpopulations were observed for Enterobacter chlorhexidine susceptibility (both MICs and MBCs) and the susceptibility to triclosan of Enterobacter (MBC), E. coli (MBC and MIC) and S. aureus (MBC and MIC). There is a concern on the potential selection of antibiotic resistance by biocides. Our results indicate however that resistance to biocides and, hence any potential association with antibiotic resistance, is uncommon in natural populations of clinically relevant microorganisms.

  9. Halophilic microorganisms

    NASA Astrophysics Data System (ADS)

    Kunte, Hans Jörg; Trüper, Hans G.; Stan-Lotter, Helga

    We consider the different mechanisms of osmoadaptation, the environment of halophiles, especially of subterranean halophilic isolates, and the relevance of microbial survival in high saline environments to astrobiology.

  10. Study of dynamical process of heat denaturation in optically trapped single microorganisms by near-infrared Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Xie, Changan; Li, Yong-qing; Tang, Wei; Newton, Ronald J.

    2003-11-01

    The development of laser traps has made it possible to investigate single cells and record real-time Raman spectra during a heat-denaturation process when the temperature of the surrounding medium is increased. Large changes in the phenylalanine band (1004 cm-1) of near-infrared spectra between living and heat-treated cells were observed in yeast and Escerichia coli and Enterobacter aerogenes bacteria. This change appears to reflect the change in environment of phenylalanine as proteins within the cells unfold as a result of increasing temperatures. As a comparison, we measured Raman spectra of native and heat-denatured solutions of bovine serum albumin proteins, and a similar change in the phenylalanine band of spectra was observed. In addition, we measured Raman spectra of native and heat-treated solutions of pure phenylalanine molecules; no observable difference in vibrational spectra was observed. These findings may make it possible to study conformational changes in proteins within single cells.

  11. Single-cell analysis and isolation for microbiology and biotechnology: methods and applications.

    PubMed

    Ishii, Satoshi; Tago, Kanako; Senoo, Keishi

    2010-05-01

    Various single-cell isolation techniques, including dilution, micromanipulation, flow cytometry, microfluidics, and compartmentalization, have been developed. These techniques can be used to cultivate previously uncultured microbes, to assess and monitor cell physiology and function, and to screen for novel microbiological products. Various other techniques, such as viable staining, in situ hybridization, and those using autofluorescence proteins, are frequently combined with these single-cell isolation techniques depending on the purpose of the study. In this review article, we summarize currently available single-cell isolation techniques and their applications, when used in combination with other techniques, in microbiological and biotechnological studies.

  12. Microorganism billiards

    NASA Astrophysics Data System (ADS)

    Spagnolie, Saverio E.; Wahl, Colin; Lukasik, Joseph; Thiffeault, Jean-Luc

    2017-02-01

    Recent experiments and numerical simulations have shown that certain types of microorganisms "reflect" off of a flat surface at a critical angle of departure, independent of the angle of incidence. The nature of the reflection may be active (cell and flagellar contact with the surface) or passive (hydrodynamic) interactions. We explore the billiard-like motion of a body with this empirical reflection law inside a regular polygon and show that the dynamics can settle on a stable periodic orbit or can be chaotic, depending on the swimmer's departure angle and the domain geometry. The dynamics are often found to be robust to the introduction of weak random fluctuations. The Lyapunov exponent of swimmer trajectories can be positive or negative, can have extremal values, and can have discontinuities depending on the degree of the polygon. A passive sorting device is proposed that traps swimmers of different departure angles into separate bins. We also study the external problem of a microorganism swimming in a patterned environment of square obstacles, where the departure angle dictates the possibility of trapping or diffusive trajectories.

  13. Isolated paroxysmal dysarthria caused by a single demyelinating midbrain lesion.

    PubMed

    Codeluppi, Luca; Bigliardi, Guido; Chiari, Annalisa; Meletti, Stefano

    2013-10-16

    Paroxysmal dysarthria is an unusual condition characterised by brief episodes of dysarthria with the sudden onset and frequent recurrence. It has been mainly reported in multiple sclerosis and an association with midbrain lesions has been claimed; however, most of the reported patients had multiple brain alterations so it was difficult to associate this symptom with a specific lesion site. We illustrate the cases of two patients with an isolated demyelinating midbrain lesion presenting paroxysmal dysarthria as the only symptom; both participants had oligoclonal bands in the cerebrospinal fluid and an unremarkable follow-up. Both patients had benefit from carbamazepine treatment, similarly to previously reported cases. Our report confirms that a demyelinating midbrain lesion is sufficient to provoke paroxysmal dysarthria. It is noteworthy that an erroneous diagnosis of psychogenic disorders was initially made in both cases, highlighting the importance not to underestimate isolated paroxysmal symptoms in clinical practice.

  14. Isolated single digit Porokeratosis of Mibelli: an unusual case.

    PubMed

    Odeyinde, Samuel; Belcher, Harry

    2012-10-15

    Porokeratosis of Mibelli is an uncommon dermatosis, which may be associated with immunosuppression and which may undergo malignant transformation. We report a patient with a chronic history of a skin lesion of his right 5th finger that was histologically confirmed to be Porokeratosis of Mibelli. Although it commonly affects the extremities, the isolated involvement of an individual digit of the hand has not been previously reported.

  15. Experimental measurement of the flow field around a freely swimming microorganism

    NASA Astrophysics Data System (ADS)

    Polin, Marco; Drescher, Knut; Goldstein, Raymond; Michel, Nicolas; Tuval, Idan

    2010-03-01

    Despite their small size, the fluid flows produced by billions of microscopic swimmers in nature can have dramatic macroscopic effects (e.g. biogenic mixing in the ocean). Understanding the flow structure of a single swimming microorganism is essential to explain and model these macroscopic phenomena. Here we report the first detailed measurement of the flow field around an isolated, freely swimming microorganism, the spherical alga Volvox, and discuss the implications of this measurement for other species.

  16. High-isolation optical isolator using a BiCalnVIG single crystal.

    PubMed

    Ma, X; Tao, S

    1992-07-20

    To the best of our knowledge this is the first time a new optical isolator has been developed that uses BiCaInVIG, a non-rare-earth iron garnet. The device has high isolation > 40 dB with an insertion loss of 1.0 dB at 1.3-microm wavelength and > 43 dB with 1.1 dB at 1.52-microm wavelength, including Fresnel reflection losses of ~0.8 dB for two polarizers. In addition, the device is inexpensive. In order to adjust maximum isolation when the isolator is assembled, a theoretical basis is presented, and the calculated values are in good agreement with the experimental results.

  17. Natural Isolates of Salmonella enterica Serovar Dublin Carry a Single nadA Missense Mutation

    PubMed Central

    Bergthorsson, Ulfar; Roth, John R.

    2005-01-01

    Nicotinic acid is required by most isolates of Salmonella enterica (serovar Dublin), a pathogen of cattle. A single nadA missense mutation causes the nutritional requirement of all serovar Dublin isolates tested. Models for persistence of this allele are tested and discussed. PMID:15601727

  18. Natural isolates of Salmonella enterica serovar Dublin carry a single nadA missense mutation.

    PubMed

    Bergthorsson, Ulfar; Roth, John R

    2005-01-01

    Nicotinic acid is required by most isolates of Salmonella enterica (serovar Dublin), a pathogen of cattle. A single nadA missense mutation causes the nutritional requirement of all serovar Dublin isolates tested. Models for persistence of this allele are tested and discussed.

  19. Genotyping of Toxoplasma gondii isolates with 15 microsatellite markers in a single multiplex PCR assay.

    PubMed

    Ajzenberg, Daniel; Collinet, Frédéric; Mercier, Aurélien; Vignoles, Philippe; Dardé, Marie-Laure

    2010-12-01

    We developed an easy-to-use method for genotyping Toxoplasma gondii isolates in a single multiplex PCR assay with 15 microsatellite markers. This method was validated by testing 26 reference isolates that had been characterized with other sets of markers.

  20. Relationships among North American and Japanese Laetiporus isolates inferred from molecular phylogenetics and single-spore incompatibility reactions

    Treesearch

    Mark T. Banik; Daniel L. Lindner; Yuko Ota; Tsutomu. Hattori

    2010-01-01

    Relationships were investigated among North American and Japanese isolates of Laetiporus using phylogenetic analysis of ITS sequences and single-spore isolate incompatibility. Single-spore isolate pairings revealed no significant compatibility between North American and Japanese isolates. ITS analysis revealed 12 clades within the core ...

  1. Characterization of single spore isolates of Agaricus bisporus (Lange) Imbach using conventional and molecular methods.

    PubMed

    Sharma, Manju; Suman, B C; Gupta, Dharmesh

    2014-10-01

    Strains A-15, S11, S-140, and U3 of Agaricus bisporus (Lange) Imbach, were used as parent strains for raising single spore homokaryotic isolates. Out of total 1,642 single spore isolates, only 36 single spore isolates were homokaryons and exhibited slow mycelial growth rate (≤2.0 mm/day) and appressed colony morphology. All these SSIs failed to produce pinheads in Petri plates even after 65 days of incubation, whereas the strandy slow growing SSIs along with parent strains were able to form the fructification in petriplates after 30 days. Out of 24, six ISSR primers, exhibited scorable bands. In the ISSR fingerprints, single spore isolates, homokaryons, lacked amplification products at multiple loci; they grow slowly and all of them had appressed types of colony morphology. The study revealed losses of ISSR polymorphic patterns in non-fertile homokaryotic single spore isolates compared to the parental control or fertile heterokaryotic single spore isolates.

  2. Isolating single cells in a neurosphere assay using inertial microfluidics

    PubMed Central

    Nathamgari, S. Shiva P.; Dong, Biqin; Zhou, Fan; Kang, Wonmo; Giraldo-Vela, Juan P.; McGuire, Tammy; McNaughton, Rebecca L.; Sun, Cheng; Kessler, John A.; Espinosa, Horacio D.

    2015-01-01

    Sphere forming assays are routinely used for in vitro propagation and differentiation of stem cells. Because the stem cell clusters can become heterogeneous and polyclonal, they must first be dissociated into a single cell suspension for further clonal analysis or differentiation studies. The dissociated population is marred by the presence of doublets, triplets and semi-cleaved/intact clusters which makes identification and further analysis of differentiation pathways difficult. In this work, we use inertial microfluidics to separate the single cells and clusters in a population of chemically dissociated neurospheres. In contrast to previous microfluidic sorting technologies which operated at high flow rates, we implement the spiral microfluidic channel in a novel focusing regime that occurs at lower flow rates. In this regime, the curvature-induced Dean’s force focuses the smaller, single cells towards the inner wall and the larger clusters towards the center. We further demonstrate that sorting in this low flow rate (and hence low shear stress) regime yields a high percentage (> 90%) of viable cells and preserves multipotency by differentiating the sorted neural stem cell population into neurons and astrocytes. The modularity of the device allows easy integration with other lab-on-a-chip devices for upstream mechanical dissociation and downstream high-throughput clonal analysis, localized electroporation and sampling. Although demonstrated in the case of the neurosphere assay, the method is equally applicable to other sphere forming assays. PMID:26511875

  3. Mechanism of single-event transient pulse quenching between dummy gate isolated logic nodes

    NASA Astrophysics Data System (ADS)

    Chen, Jian-Jun; Chi, Ya-Qing; Liang, Bin

    2015-01-01

    As integrated circuits scale down in size, a single high-energy ion strike often affects multiple adjacent logic nodes. The so-called single-event transient (SET) pulse quenching induced by single-event charge sharing collection has been widely studied. In this paper, SET pulse quenching enhancement is found in dummy gate isolated adjacent logic nodes compared with that isolated by the common shallow trench isolation (STI). The physical mechanism is studied in depth and this isolation technique is explored for SET mitigation in combinational standard cells. Three-dimensional (3D) technology computer-aided design simulation (TCAD) results show that this technique can achieve efficient SET mitigation. Project supported by the National Natural Science Foundation of China (Grant No. 61376109) and the Opening Project of National Key Laboratory of Science and Technology on Reliability Physics and Application Technology of Electrical Component, China (Grant No. ZHD201202).

  4. Photoluminescence Brightening of Isolated Single-Walled Carbon Nanotubes

    DOE PAGES

    Hou, Zhentao; Krauss, Todd D.

    2017-09-22

    Addition of dithiothreitol (DTT) to a suspension consisting of either DNA or sodium dodecyl sulfate (SDS) wrapped single-walled carbon nanotubes (SWCNTs) caused significant photoluminescence (PL) brightening from the SWCNTs, while PL quenching to different extents was observed for other surfactant-SWCNT suspensions. PL lifetime studies with high temporal resolution show that addition of DTT mitigates non-radiative decay processes, but also surprisingly increases the radiative decay rate for DNA- and SDS-SWCNTs. There are completely opposite effects on the decay rates found for the other surfactant-SWCNTs and show PL quenching. Here, we propose that the PL brightening results from a surfactant reorganization uponmore » DTT addition. TOC« less

  5. Is screening for renal anomalies warranted in neonates with isolated single umbilical artery?

    PubMed

    de Boom, M L; Kist-van Holthe, J E; Sramek, A; Lardenoye, S W J; Walther, F J; Lopriore, E

    2010-01-01

    To determine the prevalence of renal anomalies in patients with an isolated single umbilical artery (SUA). We performed a retrospective study of all renal ultrasound examinations assessed at our centre between January 1998 and December 2008 in neonates with SUA with or without associated anomalies. Renal ultrasound examination was performed in 65 neonates with SUA (57 neonates with isolated SUA and 8 neonates with nonisolated SUA). The prevalence of renal anomalies in the group with and without isolated SUA was 2% (1/57) and 38% (3/8), respectively. Only one patient with isolated SUA had a mild renal abnormality without clinical consequences. The prevalence of renal anomalies in neonates with isolated SUA is low. We suggest that routine ultrasound screening for renal anomalies is not warranted in neonates with isolated SUA. Copyright 2009 S. Karger AG, Basel.

  6. Single nucleotide polymorphism genotyping of Erysipelothrix rhusiopathiae isolates from pigs affected with chronic erysipelas in Japan.

    PubMed

    Shiraiwa, Kazumasa; Ogawa, Yohsuke; Nishikawa, Sayaka; Kusumoto, Masahiro; Eguchi, Masahiro; Shimoji, Yoshihiro

    2017-04-05

    Over the past decades, Erysipelothrix rhusiopathiae strains displaying similar phenotypic and genetic profiles of the attenuated, acriflavine-resistant E. rhusiopathiae Koganei 65-0.15 strain (serovar 1a) have been frequently isolated from pigs affected with chronic erysipelas in Japan. In this study, using the conventional PCR assay that was designed to detect strain-specific single nucleotide polymorphism (SNP) sites found in the genome of the vaccine strain, we analyzed E. rhusiopathiae isolates from pigs with chronic disease in farms where the Koganei vaccine was used. Out of a total of 155 isolates, 101 isolates (65.2%) were determined to be the vaccine strain by SNP-based PCR. Among the 101 PCR-positive isolates, four isolates were found to be sensitive to acriflavine.

  7. Single-cell PCR of genomic DNA enabled by automated single-cell printing for cell isolation.

    PubMed

    Stumpf, F; Schoendube, J; Gross, A; Rath, C; Niekrawietz, S; Koltay, P; Roth, G

    2015-07-15

    Single-cell analysis has developed into a key topic in cell biology with future applications in personalized medicine, tumor identification as well as tumor discovery (Editorial, 2013). Here we employ inkjet-like printing to isolate individual living single human B cells (Raji cell line) and load them directly into standard PCR tubes. Single cells are optically detected in the nozzle of the microfluidic piezoelectric dispenser chip to ensure printing of droplets with single cells only. The printing process has been characterized by using microbeads (10µm diameter) resulting in a single bead delivery in 27 out of 28 cases and relative positional precision of ±350µm at a printing distance of 6mm between nozzle and tube lid. Process-integrated optical imaging enabled to identify the printing failure as void droplet and to exclude it from downstream processing. PCR of truly single-cell DNA was performed without pre-amplification directly from single Raji cells with 33% success rate (N=197) and Cq values of 36.3±2.5. Additionally single cell whole genome amplification (WGA) was employed to pre-amplify the single-cell DNA by a factor of >1000. This facilitated subsequent PCR for the same gene yielding a success rate of 64% (N=33) which will allow more sophisticated downstream analysis like sequencing, electrophoresis or multiplexing.

  8. Assessment of the Geographic Origins of Pinewood Nematode Isolates via Single Nucleotide Polymorphism in Effector Genes

    PubMed Central

    Figueiredo, Joana; Simões, Maria José; Gomes, Paula; Barroso, Cristina; Pinho, Diogo; Conceição, Luci; Fonseca, Luís; Abrantes, Isabel; Pinheiro, Miguel; Egas, Conceição

    2013-01-01

    The pinewood nematode, Bursaphelenchus xylophilus, is native to North America but it only causes damaging pine wilt disease in those regions of the world where it has been introduced. The accurate detection of the species and its dispersal routes are thus essential to define effective control measures. The main goals of this study were to analyse the genetic diversity among B. xylophilus isolates from different geographic locations and identify single nucleotide polymorphism (SNPs) markers for geographic origin, through a comparative transcriptomic approach. The transcriptomes of seven B. xylophilus isolates, from Continental Portugal (4), China (1), Japan (1) and USA (1), were sequenced in the next generation platform Roche 454. Analysis of effector gene transcripts revealed inter-isolate nucleotide diversity that was validated by Sanger sequencing in the genomic DNA of the seven isolates and eight additional isolates from different geographic locations: Madeira Island (2), China (1), USA (1), Japan (2) and South Korea (2). The analysis identified 136 polymorphic positions in 10 effector transcripts. Pairwise comparison of the 136 SNPs through Neighbor-Joining and the Maximum Likelihood methods and 5-mer frequency analysis with the alignment-independent bilinear multivariate modelling approach correlated the SNPs with the isolates geographic origin. Furthermore, the SNP analysis indicated a closer proximity of the Portuguese isolates to the Korean and Chinese isolates than to the Japanese or American isolates. Each geographic cluster carried exclusive alleles that can be used as SNP markers for B. xylophilus isolate identification. PMID:24391785

  9. Has the introduction of antibiotic-impregnated external ventricular drain catheters changed the nature of the microorganisms cultured in patients with drain-related infection? A single neurosurgical centre's experience.

    PubMed

    Talibi, S; Tarnaris, A; Shaw, S A

    2016-10-01

    The introduction of antibiotic-impregnated catheters (AICs) has significantly reduced external ventricular drain (EVD) infection rates, inhibiting in particular Gram-positive microbial infection. There is a theoretical increased risk of selection of resistant and Gram-negative microorganisms. The aim of this study is to look at the impact of the introduction of AICs on microorganism susceptibility of infected EVDs and to determine whether the use of such devices results in shift from Gram-positive to Gram-negative microorganisms. A secondary aim is to determine if a change in routine empiric antimicrobial therapy is required because of a change in the microorganism(s) causing infection. Retrospective analysis of EVDs inserted as a primary procedure or part of a concomitant neurosurgical procedure in 2006 (pre-AIC) and 2012 (post-AIC Codman Bactiseal(©) impregnated with clindamycin 0.15% and rifampicin 0.054%). EVD-related infection was defined as a cerebral spinal fluid sample with a positive culture with some patients having one or more microorganisms infecting the same EVD at the same time. Sixty-five EVDs over 843 days and 66 EVDs over 619 days were inserted respectively in each cohort. In 2006, 9 of 35 patients developed EVD-related infection and 13 microorganisms were cultured; 8 of which were Gram-positive and 4 Gram-negative. Four of 49 patients developed EVD-related infection in 2012 and six microorganisms were cultured; four Gram-positive and two Gram-negative. The cumulative incidence of EVDs becoming infected is 18.5% (12/65) and 6.1% (4/66; p = 0.03) in 2012. The EVD-associated infection rate was 14.2 per 1000 EVD days and 6.5 per 1000 EVD days in 2012. There was no change in mortality. The study demonstrates that within a single neurosurgical centre there have been neither changes in susceptibility of microorganisms nor a change from predominately Gram-positive to Gram-negative microorganisms infecting EVDs following the switch to AIC. The

  10. Current recordings at the single channel level in adult mammalian isolated cardiomyocytes.

    PubMed

    Guinamard, Romain; Hof, Thomas; Sallé, Laurent

    2014-01-01

    This chapter describes appropriate methods to investigate mammalian cardiac channels properties at the single channel level. Cell isolation is performed from new born or adult heart by enzymatic digestion on minced tissue or using the Langendorff apparatus. Isolation proceeding is suitable for rabbit, rat, and mouse hearts. In addition, isolation of human atrial cardiomyocytes is described. Such freshly isolated cells or cells maintained in primary culture are suitable for patch-clamp studies. Here we describe the single channel variants of the patch-clamp technique (cell-attached, inside-out, outside-out) used to investigate channel properties. Proceedings for the evaluation of biophysical properties such as conductance, ionic selectivity, regulations by extracellular and intracellular mechanisms are described. To illustrate the study, we provide an example by the characterization of a calcium-activated non-selective cation channel (TRPM4).

  11. Single-mode waveguide optical isolator based on direction-dependent cutoff frequency.

    PubMed

    Tang, Lingling; Drezdzon, Samuel M; Yoshie, Tomoyuki

    2008-09-29

    A single-mode-waveguide optical isolator based on propagation direction dependent cut-off frequency is proposed. The isolation bandwidth is the difference between the cut-off frequencies of the lowest forward and backward propagating modes. Perturbation theory is used for analyzing the correlation between the material distribution and the bandwidth. The mode profile determines an appropriate distribution of non-reciprocal materials.

  12. Simultaneous isolation of intact mitochondria and chloroplasts from a single pulping of plant tissue.

    PubMed

    Rödiger, Anja; Baudisch, Bianca; Klösgen, Ralf Bernd

    2010-05-15

    Isolated organelles are suitable tools for the investigation of organelle function. However, if the properties of different organelles are to be compared, analysis is generally impeded by the fact that the organelles are isolated independently from each other from different specimens, different tissues or even different plants, i.e. the organelles have been exposed to different conditions during growth and development. Here we describe a method to isolate intact chloroplasts and mitochondria simultaneously from a single pulping of pea leaves, which results in organelles with an essentially identical physiological background. The functionality of the isolated chloroplasts and mitochondria is demonstrated by protein transport experiments, which yield results identical to those obtained with independently isolated organelles. With slight modifications, the method is also successfully applied to organelles from potato and spinach, which implies that it may be generally applicable to organelles from many different species. (c) 2009 Elsevier GmbH. All rights reserved.

  13. In Situ Detection, Isolation, and Physiological Properties of a Thin Filamentous Microorganism Abundant in Methanogenic Granular Sludges: a Novel Isolate Affiliated with a Clone Cluster, the Green Non-Sulfur Bacteria, Subdivision I

    PubMed Central

    Sekiguchi, Yuji; Takahashi, Hiroki; Kamagata, Yoichi; Ohashi, Akiyoshi; Harada, Hideki

    2001-01-01

    We previously showed that very thin filamentous bacteria affiliated with the division green non-sulfur bacteria were abundant in the outermost layer of thermophilic methanogenic sludge granules fed with sucrose and several low-molecular-weight fatty acids (Y. Sekiguchi, Y. Kamagata, K. Nakamura, A. Ohashi, H. Harada, Appl. Environ. Microbiol. 65:1280–1288, 1999). Further 16S ribosomal DNA (rDNA) cloning-based analysis revealed that the microbes were classified within a unique clade, green non-sulfur bacteria (GNSB) subdivision I, which contains a number of 16S rDNA clone sequences from various environmental samples but no cultured representatives. To investigate their function in the community and physiological traits, we attempted to isolate the yet-to-be-cultured microbes from the original granular sludge. The first attempt at isolation from the granules was, however, not successful. In the other thermophilic reactor that had been treating fried soybean curd-manufacturing wastewater, we found filamentous microorganisms to outgrow, resulting in the formation of projection-like structures on the surface of granules, making the granules look like sea urchins. 16S rDNA-cloning analysis combined with fluorescent in situ hybridization revealed that the projections were comprised of the uncultured filamentous cells affiliated with the GNSB subdivision I and Methanothermobacter-like cells and the very ends of the projections were comprised solely of the filamentous cells. By using the tip of the projection as the inoculum for primary enrichment, a thermophilic, strictly anaerobic, filamentous bacterium, designated strain UNI-1, was successfully isolated with a medium supplemented with sucrose and yeast extract. The strain was a very slow growing bacterium which is capable of utilizing only a limited range of carbohydrates in the presence of yeast extract and produced hydrogen from these substrates. The growth was found to be significantly stimulated when the strain was

  14. Single CD271 marker isolates mesenchymal stem cells from human dental pulp.

    PubMed

    Alvarez, Ruth; Lee, Hye-Lim; Hong, Christine; Wang, Cun-Yu

    2015-12-18

    Mesenchymal stem cells (MSCs) are a promising tool in regenerative medicine due to their capacity to differentiate into multiple lineages. In addition to MSCs isolated from bone marrow (BMSCs), adult MSCs are isolated from craniofacial tissues including dental pulp tissues (DPs) using various stem cell surface markers. However, there has been a lack of consensus on a set of surface makers that are reproducibly effective at isolating putative multipotent dental mesenchymal stem cells (DMSCs). In this study, we used different combinations of surface markers (CD51/CD140α, CD271, and STRO-1/CD146) to isolate homogeneous populations of DMSCs from heterogeneous dental pulp cells (DPCs) obtained from DP and compared their capacity to undergo multilineage differentiation. Fluorescence-activated cell sorting revealed that 27.3% of DPCs were CD51(+)/CD140α(+), 10.6% were CD271(+), and 0.3% were STRO-1(+)/CD146(+). Under odontogenic conditions, all three subsets of isolated DMSCs exhibited differentiation capacity into odontogenic lineages. Among these isolated subsets of DMSCs, CD271(+) DMSCs demonstrated the greatest odontogenic potential. While all three combinations of surface markers in this study successfully isolated DMSCs from DPCs, the single CD271 marker presents the most effective stem cell surface marker for identification of DMSCs with high odontogenic potential. Isolated CD271(+) DMSCs could potentially be utilized for future clinical applications in dentistry and regenerative medicine.

  15. Beating irregularity of single pacemaker cells isolated from the rabbit sinoatrial node.

    PubMed Central

    Wilders, R; Jongsma, H J

    1993-01-01

    Single pacemaker heart cells discharge irregularly. Data on fluctuations in interbeat interval of single pacemaker cells isolated from the rabbit sinoatrial node are presented. The coefficient of variation of the interbeat interval is quite small, approximately 2%, even though the coefficient of variation of diastolic depolarization rate is approximately 15%. It has been hypothesized that random fluctuations in interbeat interval arise from the stochastic behavior of the membrane ionic channels. To test this hypothesis, we constructed a single channel model of a single pacemaker cell isolated from the rabbit sinoatrial node, i.e., a model into which the stochastic open-close kinetics of the individual membrane ionic channels are incorporated. Single channel conductances as well as single channel open and closed lifetimes are based on experimental data from whole cell and single channel experiments that have been published in the past decade. Fluctuations in action potential parameters of the model cell are compared with those observed experimentally. It is concluded that fluctuations in interbeat interval of single sinoatrial node pacemaker cells indeed are due to the stochastic open-close kinetics of the membrane ionic channels. PMID:8312495

  16. Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing

    NASA Astrophysics Data System (ADS)

    Riba, J.; Gleichmann, T.; Zimmermann, S.; Zengerle, R.; Koltay, P.

    2016-09-01

    The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry.

  17. Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing

    PubMed Central

    Riba, J.; Gleichmann, T.; Zimmermann, S.; Zengerle, R.; Koltay, P.

    2016-01-01

    The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry. PMID:27596612

  18. Detection and isolation of single tumor cells containing mutated DNA sequences

    NASA Astrophysics Data System (ADS)

    Leary, James F.; He, Feng; Reece, Lisa M.

    1999-04-01

    One of the problems in treating breast cancer patients is discovering the gene rearrangements that are occurring while the patient is in apparent remission. Spontaneous mutations in DNA sequences, particularly in tumor suppressor genes, can lead to the evolution of new clones of tumor cells that may be able to evade both clinical treatments and the patient's immune surveillance system. Isolation of these tumor clones is extremely difficult. Rare-event analysis and single-cell sorting techniques must be used to successfully detect and isolate these tumor clones. PCR amplification of selected gene sequences followed by TA cloning techniques can then be used to perform single-cell DNA sequencing in those gene regions. In this paper we present preliminary data showing successful detection and single-cell sorting of rare tumor clones from defined cell mixtures. Using TA cloning techniques and PCR we have been able to detect a single base-pair mutation in the PTEN tumor suppressor gene in single cells from a breast cancer cell line. Thus, while extremely difficult, it should in the future be possible to isolate tumor clones form a patient for subsequent molecular analyses of DNA mutations in critical gene regions.

  19. Single-dot optical emission from ultralow density well-isolated InP quantum dots

    SciTech Connect

    Ugur, A.; Hatami, F.; Masselink, W. T.; Vamivakas, A. N.; Lombez, L.; Atatuere, M.

    2008-10-06

    We demonstrate a straightforward way to obtain single well-isolated quantum dots emitting in the visible part of the spectrum and characterize the optical emission from single quantum dots using this method. Self-assembled InP quantum dots are grown using gas-source molecular-beam epitaxy over a wide range of InP deposition rates, using an ultralow growth rate of about 0.01 atomic monolayers/s, a quantum-dot density of 1 dot/{mu}m{sup 2} is realized. The resulting isolated InP quantum dots embedded in an InGaP matrix are individually characterized without the need for lithographical patterning and masks on the substrate. Such low-density quantum dots show excitonic emission at around 670 nm with a linewidth limited by instrument resolution. This system is applicable as a single-photon source for applications such as quantum cryptography.

  20. Single Nisoldipine-Sensitive Calcium Channels in Smooth Muscle Cells Isolated from Rabbit Mesenteric Artery

    NASA Astrophysics Data System (ADS)

    Worley, Jennings F.; Deitmer, Joachim W.; Nelson, Mark T.

    1986-08-01

    Single smooth muscle cells were enzymatically isolated from the rabbit mesenteric artery. At physiological levels of external Ca, these cells were relaxed and contracted on exposure to norepinephrine, caffeine, or high levels of potassium. The patch-clamp technique was used to measure unitary currents through single channels in the isolated cells. Single channels were selective for divalent cations and exhibited two conductance levels, 8 pS and 15 pS. Both types of channels were voltage-dependent, and channel activity occurred at potentials positive to -40 mV. The activity of both channel types was almost completely inhibited by 50 nM nisoldipine. These channels appear to be the pathways for voltage-dependent Ca influx in vascular smooth muscle and may be the targets of the clinically used dihydropyridines.

  1. [Sherry wine microorganisms].

    PubMed

    García Maiquez, E

    1995-03-01

    Sherry wine presents, during all its wine-making and aging process, a great diversity of yeast and bacteria, as well as in the wine itself; its particular wine-making system, with traditional and legal additions to correct the acidity and to get a final alcoholic content of 15%, originates a selection of accompanying microorganisms. Species of the genera Kloeckera, Candida, Saccharomyces, Pichia, Hansenula and Saccharomycodes, have been isolated during the fermentation process in different proportions. This fact confirms that, besides S. cerevisiae, strains of S. chevalieri and S. fermentati have an important role in the fermentative process, and that the film-forming Saccharomyces have great activity in the fermentation. The biological aging of the Sherry wine, carried out by S. cheresiensis, S. beticus, S. feduchii and S. rouxii, has been studied in "finos" and "manzanillas". Different species and percentages in both wines have been described.

  2. Congenital heart defects in newborns with apparently isolated single gastrointestinal malformation: A retrospective study.

    PubMed

    Schierz, Ingrid Anne Mandy; Pinello, Giuseppa; Giuffrè, Mario; La Placa, Simona; Piro, Ettore; Corsello, Giovanni

    2016-12-01

    Congenital gastrointestinal system malformations/abdominal wall defects (GISM) may appear as isolated defects (single or complex), or in association with multiple malformations. The high incidence of association of GISM and congenital heart defects (CHD) in patients with syndromes and malformative sequences is known, but less expected is the association of apparently isolated single GISM and CHD. The aim of this study was to investigate the frequency of CHD in newborns with isolated GISM, and the possibility to modify the diagnostic-therapeutic approach just before the onset of cardiac symptoms or complications. Anamnestic, clinical, and imaging data of newborns requiring abdominal surgery for GISM, between 2009 and 2014, were compared with a control group of healthy newborns. Distribution of GISM and cardiovascular abnormalities were analyzed, and risk factors for adverse outcomes were identified. Seventy-one newborns with isolated GISM were included in this study. More frequent GISM were intestinal rotation and fixation disorders. CHD were observed in 15.5% of patients, augmenting their risk for morbidity. Risk factors for morbidity related to sepsis were identified in central venous catheter, intestinal stoma, and H2-inhibitor-drugs. Moreover, 28.2% of newborns presented only functional cardiac disorders but an unexpectedly higher mortality. The high incidence of congenital heart disease in infants with apparently isolated GISM confirms the need to perform an echocardiographic study before surgery to improve perioperative management and prevent complications such as sepsis and endocarditis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Studies on formation, control and application of biofilm formed by food related microorganisms.

    PubMed

    Furukawa, Soichi

    2015-01-01

    Biofilms are sessile microbial aggregates on the interfaces, and they were usually considered as microbial contamination sources in medical care and various industries. We studied the control and application of biofilms formed by food-related microorganisms, and mechanism of the biofilm formation was also investigated. We studied the biofilm formation in mixed cultures using various combinations of two strains of food-related microorganisms. There were various microorganisms that showed decreased or increased biofilm formation in the mixed culture in comparison with that in a single culture. Biofilm formed by lactic acid bacteria and yeast isolated from traditional fermented food, Fukuyama pot vinegar, exhibited unique feature in that structure and formation mechanism, and expected to be used as an immobilized microorganism in fermentation production. Here our studies on the control and application of biofilms and the mechanisms of its formation were described.

  4. Single nucleotide polymorphisms in the bovine Histophilus somni genome; a comparison of new and old isolates.

    PubMed

    Madampage, Claudia Avis; Rawlyk, Neil; Crockford, Gordon; Van Donkersgoed, Joyce; Dorin, Craig; Potter, Andrew

    2015-07-01

    Histophilus somni, a causative agent of the bovine respiratory disease complex, can also cause a variety of systemic disorders, including bronchopneumonia, myocarditis, pericarditis, arthritis, pleuritis, and infectious thrombotic meningoencephalitis. The purpose of this study was to determine if currently circulating strains differ from those of the 1980s by identifying genomic changes. Single nucleotide polymorphisms (SNPs) and insertion and deletion (INDEL) sites were examined by whole-genome sequencing in 12 samples, 6 old and 6 new. The 31 028 SNP/INDELs recorded were compared against the reference genome sequence of the pathogenic H. somni strain 2336. The distribution of about 75% of these SNPs within a specified gene differed between old and new isolates and did not follow any particular pattern. The other 25% clustered into 2 groups containing the same SNPs in various genes: group I included 5 old isolates and 1 new isolate; group II included 5 new isolates and 1 old isolate. For putative virulence genes there were more SNPs in group I compared with strain 2336, itself an older isolate, than in group II. Although only 25% of all the SNPs formed 2 clusters, the results suggest some genetic difference in various genes between old and new strains.

  5. Single nucleotide polymorphisms in the bovine Histophilus somni genome; a comparison of new and old isolates

    PubMed Central

    Madampage, Claudia Avis; Rawlyk, Neil; Crockford, Gordon; Van Donkersgoed, Joyce; Dorin, Craig; Potter, Andrew

    2015-01-01

    Histophilus somni, a causative agent of the bovine respiratory disease complex, can also cause a variety of systemic disorders, including bronchopneumonia, myocarditis, pericarditis, arthritis, pleuritis, and infectious thrombotic meningoencephalitis. The purpose of this study was to determine if currently circulating strains differ from those of the 1980s by identifying genomic changes. Single nucleotide polymorphisms (SNPs) and insertion and deletion (INDEL) sites were examined by whole-genome sequencing in 12 samples, 6 old and 6 new. The 31 028 SNP/INDELs recorded were compared against the reference genome sequence of the pathogenic H. somni strain 2336. The distribution of about 75% of these SNPs within a specified gene differed between old and new isolates and did not follow any particular pattern. The other 25% clustered into 2 groups containing the same SNPs in various genes: group I included 5 old isolates and 1 new isolate; group II included 5 new isolates and 1 old isolate. For putative virulence genes there were more SNPs in group I compared with strain 2336, itself an older isolate, than in group II. Although only 25% of all the SNPs formed 2 clusters, the results suggest some genetic difference in various genes between old and new strains. PMID:26130851

  6. Evaluation of the Bruker Biotyper and VITEK MS MALDI-TOF MS systems for the identification of unusual and/or difficult-to-identify microorganisms isolated from clinical specimens.

    PubMed

    McElvania TeKippe, E; Burnham, C-A D

    2014-12-01

    The purpose of this investigation was to evaluate the analytical performance characteristics of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of unusual organisms. We evaluated the accuracy of two MALDI-TOF MS systems, bioMérieux VITEK MS (database v2.0) and Bruker Biotyper (software version 3.0), for the identification of the most difficult and/or unusual microorganisms isolated from clinical specimens. Our study included 174 bacterial isolates recovered from clinical cultures at Barnes-Jewish Hospital, St. Louis, MO, from 2009 to 2013, representing 50 genera and 52 species. MS identifications were compared to the identification reported by the reference laboratory. Discrepancies were resolved using molecular methods, including 16S rRNA gene sequencing and additional molecular methods. When performed, molecular methods were considered the gold standard. Of the 168 isolates resolved to the genus level, VITEK MS identified 145 (86.3 %), and of the 114 isolates resolved to the species level, 97 (85.1 %) were correctly identified. Bruker Biotyper identified 155 (92.3 %) of 168 isolates to the genus level and 97 (85.1 %) of 114 isolates to the species level. VITEK MS and Bruker Biotyper provided no identification for 17 (10.1 %) and 12 (7.1 %) organisms, respectively, and misidentified six (3.6 %) and one (0.6 %) isolate, respectively. Six isolates (3.6 %) were not resolvable to the genus level and were excluded from data analysis due to the lack of a gold standard for comparison. There was no significant difference in the number of organisms identified to the genus level, species level, unidentified, or misidentified by the two MALDI-TOF MS systems (p = 0.11, 1.0, 0.44, and 0.12, respectively).

  7. Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.

    PubMed

    Monsanto, Megan M; White, Kevin S; Kim, Taeyong; Wang, Bingyan J; Fisher, Kristina; Ilves, Kelli; Khalafalla, Farid G; Casillas, Alexandria; Broughton, Kathleen; Mohsin, Sadia; Dembitsky, Walter P; Sussman, Mark A

    2017-07-07

    The relative actions and synergism between distinct myocardial-derived stem cell populations remain obscure. Ongoing debates on optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration. Establish a robust cardiac stem cell isolation and culture protocol to consistently generate 3 distinct stem cell populations from a single human heart biopsy. Isolation of 3 endogenous cardiac stem cell populations was performed from human heart samples routinely discarded during implantation of a left ventricular assist device. Tissue explants were mechanically minced into 1 mm(3) pieces to minimize time exposure to collagenase digestion and preserve cell viability. Centrifugation removes large cardiomyocytes and tissue debris producing a single cell suspension that is sorted using magnetic-activated cell sorting technology. Initial sorting is based on tyrosine-protein kinase Kit (c-Kit) expression that enriches for 2 c-Kit(+) cell populations yielding a mixture of cardiac progenitor cells and endothelial progenitor cells. Flowthrough c-Kit(-) mesenchymal stem cells are positively selected by surface expression of markers CD90 and CD105. After 1 week of culture, the c-Kit(+) population is further enriched by selection for a CD133(+) endothelial progenitor cell population. Persistence of respective cell surface markers in vitro is confirmed both by flow cytometry and immunocytochemistry. Three distinct cardiac cell populations with individualized phenotypic properties consistent with cardiac progenitor cells, endothelial progenitor cells, and mesenchymal stem cells can be successfully concurrently isolated and expanded from a single tissue sample derived from human heart failure patients. © 2017 American Heart Association, Inc.

  8. Magnetic cooling at a single molecule level: a spectroscopic investigation of isolated molecules on a surface.

    PubMed

    Corradini, Valdis; Ghirri, Alberto; Candini, Andrea; Biagi, Roberto; del Pennino, Umberto; Dotti, Gianluca; Otero, Edwige; Choueikani, Fadi; Blagg, Robin J; McInnes, Eric J L; Affronte, Marco

    2013-05-28

    A sub-monolayer distribution of isolated molecular Fe14 (bta)6 nanomagnets is deposited intact on a Au(111) surface and investigated by X-ray magnetic circular dichroism spectroscopy. The entropy variation with respect to the applied magnetic field is extracted from the magnetization curves and evidences high magnetocaloric values at the single molecule level. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Fatty acid profiles of marine benthic microorganisms isolated from the continental slope of bay of bengal: a possible implications in the benthic Food web

    NASA Astrophysics Data System (ADS)

    Das, Surajit; Lyla, P. S.; Khan, S. Ajmal

    2007-12-01

    Marine bacteria, actinomycetes and fungal strains were isolated from continental slope sediment of the Bay of Bengal and studied for fatty acid profile to investigate their involvement in the benthic food-web. Fifteen different saturated and unsaturated fatty acids from bacterial isolates, 14 from actinomycetes and fungal isolates were detected. The total unsaturated fatty acids in bacterial isolates ranged from 11.85 to 37.26%, while the saturated fatty acid ranged between 42.34 and 80.74%. In actinomycetes isolates, total unsaturated fatty acids varied from 27.86 to 38.85% and saturated fatty acids ranged from 35.29 to 51.25%. In fungal isolates unsaturated fatty acids ranged between 44.62 and 65.52% while saturated FA ranged from 20.80 to 46.30%. The higher percentages of unsaturated fatty acids from the microbial isolates are helpful in anticipating the active participation in the benthic food-web of Bay of Bengal.

  10. On-chip waveguide isolator based on bismuth iron garnet operating via nonreciprocal single-mode cutoff.

    PubMed

    Drezdzon, Samuel M; Yoshie, Tomoyuki

    2009-05-25

    We analyze an on-chip optical isolator based on direction dependent single-mode cutoff, which is described in 1D and 2D momentum space. Isolation is shown using 3D finite difference time domain (FDTD) where the magnetization is represented by imaginary off-diagonal permittivity tensor elements. The isolator designs are optimized using perturbation theory, which successfully predicts increased isolation for rib waveguides and structures with non-magnetic dielectric layers. Our isolators are based on bismuth iron garnet and its compatible substrates; an isolation ratio of 10.7 dB/mm is achieved for TM modes.

  11. Beneficial microorganisms [Chapter 14

    Treesearch

    Kim M. Wilkinson

    2009-01-01

    The web of life depends on microorganisms, a vast network of small and unseen allies that permeate the soil, water, and air of our planet. For people who work with plants, the greatest interest in microorganisms is in the complex communities that are part of the soil. Beneficial microorganisms are naturally occurring bacteria, fungi, and other microbes that play a...

  12. Single prokaryotic cell isolation and total transcript amplification protocol for transcriptomic analysis.

    PubMed

    Kang, Yun; McMillan, Ian; Norris, Michael H; Hoang, Tung T

    2015-07-01

    Until recently, transcriptome analyses of single cells have been confined to eukaryotes. The information obtained from single-cell transcripts can provide detailed insight into spatiotemporal gene expression, and it could be even more valuable if expanded to prokaryotic cells. Transcriptome analysis of single prokaryotic cells is a recently developed and powerful tool. Here we describe a procedure that allows amplification of the total transcript of a single prokaryotic cell for in-depth analysis. This is performed by using a laser-capture microdissection instrument for single-cell isolation, followed by reverse transcription via Moloney murine leukemia virus, degradation of chromosomal DNA with McrBC and DpnI restriction enzymes, single-stranded cDNA (ss-cDNA) ligation using T4 polynucleotide kinase and CircLigase, and polymerization of ss-cDNA to double-stranded cDNA (ds-cDNA) by Φ29 polymerase. This procedure takes ∼5 d, and sufficient amounts of ds-cDNA can be obtained from single-cell RNA template for further microarray analysis.

  13. Thermophilic microorganisms in biomining.

    PubMed

    Donati, Edgardo Rubén; Castro, Camila; Urbieta, María Sofía

    2016-11-01

    Biomining is an applied biotechnology for mineral processing and metal extraction from ores and concentrates. This alternative technology for recovering metals involves the hydrometallurgical processes known as bioleaching and biooxidation where the metal is directly solubilized or released from the matrix for further solubilization, respectively. Several commercial applications of biomining can be found around the world to recover mainly copper and gold but also other metals; most of them are operating at temperatures below 40-50 °C using mesophilic and moderate thermophilic microorganisms. Although biomining offers an economically viable and cleaner option, its share of the world´s production of metals has not grown as much as it was expected, mainly considering that due to environmental restrictions in many countries smelting and roasting technologies are being eliminated. The slow rate of biomining processes is for sure the main reason of their poor implementation. In this scenario the use of thermophiles could be advantageous because higher operational temperature would increase the rate of the process and in addition it would eliminate the energy input for cooling the system (bioleaching reactions are exothermic causing a serious temperature increase in bioreactors and inside heaps that adversely affects most of the mesophilic microorganisms) and it would decrease the passivation of mineral surfaces. In the last few years many thermophilic bacteria and archaea have been isolated, characterized, and even used for extracting metals. This paper reviews the current status of biomining using thermophiles, describes the main characteristics of thermophilic biominers and discusses the future for this biotechnology.

  14. Electrode Cultivation and Interfacial Electron Transport in Subsurface Microorganisms

    NASA Astrophysics Data System (ADS)

    Karbelkar, A. A.; Jangir, Y.; Reese, B. K.; Wanger, G.; Anderson, C.; El-Naggar, M.; Amend, J.

    2016-12-01

    Continental subsurface environments can present significant energetic challenges to the resident microorganisms. While these environments are geologically diverse, potentially allowing energy harvesting by microorganisms that catalyze redox reactions, many of the abundant electron donors and acceptors are insoluble and therefore not directly bioavailable. Microbes can use extracellular electron transfer (EET) as a metabolic strategy to interact with redox active surfaces. This process can be mimicked on electrode surfaces and hence can lead to enrichment and quantification of subsurface microorganisms A primary bioelectrochemical enrichment with different oxidizing and reducing potentials set up in a single bioreactor was applied in situ to subsurface microorganisms residing in iron oxide rich deposits in the Sanford Underground Research Facility. Secondary enrichment revealed a plethora of classified and unclassified subsurface microbiota on both oxidizing and reducing potentials. From this enrichment, we have isolated a Gram-positive Bacillus along with Gram-negative Cupriavidus and Anaerospora strains (as electrode reducers) and Comamonas (as an electrode oxidizer). The Bacillus and Comamonas isolates were subjected to a detailed electrochemical characterization in half-reactors at anodic and cathodic potentials, respectively. An increase in cathodic current upon inoculation and cyclic voltammetry measurements confirm the hypothesis that Comamonas is capable of electron uptake from electrodes. In addition, measurements of Bacillus on anodes hint towards novel mechanisms that allow EET from Gram-positive bacteria. This study suggests that electrochemical approaches are well positioned to dissect such extracellular interactions that may be prevalent in the subsurface, while using physical electrodes to emulate the microhabitats, redox and geochemical gradients, and the spatially dependent interspecies interactions encountered in the subsurface. Electrochemical

  15. Single prokaryotic cell isolation and total transcript amplification protocol for transcriptomic analysis

    PubMed Central

    Kang, Yun; McMillan, Ian; Norris, Michael H; Hoang, Tung T.

    2015-01-01

    Until recently, transcriptome analyses of single cells have been confined to eukaryotes. The information obtained from single cell transcripts can provide detailed insight into spatiotemporal gene-expression, and could be even more valuable if expanded to prokaryotic cells. Transcriptome analysis of single prokaryotic cells is a recently developed and powerful tool. Here, we describe a procedure that allows amplification of the total transcript of a single prokaryotic cell for in-depth analysis. This is performed by utilizing a laser capture microdissection instrument for single cell isolation, followed by reverse transcription via Moloney Murine Leukemia virus, degradation of chromosomal DNA with McrBC and DpnI restriction enzymes, ss-cDNA ligation using T4 polynucleotide kinase and CircLigase, and polymerization of ss-cDNA to ds-cDNA by ϕ 29 polymerase. This procedure takes ~5 days, and sufficient amounts of ds-cDNA can be obtained from single cell RNA template for further microarray analysis. PMID:26042386

  16. Amplification of multiple genomic loci from single cells isolated by laser micro-dissection of tissues

    PubMed Central

    Frumkin, Dan; Wasserstrom, Adam; Itzkovitz, Shalev; Harmelin, Alon; Rechavi, Gideon; Shapiro, Ehud

    2008-01-01

    Background Whole genome amplification (WGA) and laser assisted micro-dissection represent two recently developed technologies that can greatly advance biological and medical research. WGA allows the analysis of multiple genomic loci from a single genome and has been performed on single cells from cell suspensions and from enzymatically-digested tissues. Laser micro-dissection makes it possible to isolate specific single cells from heterogeneous tissues. Results Here we applied for the first time WGA on laser micro-dissected single cells from stained tissue sections, and developed a protocol for sequentially performing the two procedures. The combined procedure allows correlating the cell's genome with its natural morphology and precise anatomical position. From each cell we amplified 122 genomic and mitochondrial loci. In cells obtained from fresh tissue sections, 64.5% of alleles successfully amplified to ~700000 copies each, and mitochondrial DNA was amplified successfully in all cells. Multiplex PCR amplification and analysis of cells from pre-stored sections yielded significantly poorer results. Sequencing and capillary electrophoresis of WGA products allowed detection of slippage mutations in microsatellites (MS), and point mutations in P53. Conclusion Comprehensive genomic analysis of single cells from stained tissue sections opens new research opportunities for cell lineage and depth analyses, genome-wide mutation surveys, and other single cell assays. PMID:18284708

  17. Synergistic effects of honey and propolis toward drug multi-resistant Staphylococcus aureus, Escherichia coli and Candida albicans isolates in single and polymicrobial cultures.

    PubMed

    Al-Waili, Noori; Al-Ghamdi, Ahmad; Ansari, Mohammad Javed; Al-Attal, Y; Salom, Khelod

    2012-01-01

    Propolis and honey are natural bee products with wide range of biological and medicinal properties. The study investigated antimicrobial activity of ethyl alcohol extraction of propolis collected from Saudi Arabia (EEPS) and from Egypt (EEPE), and their synergistic effect when used with honey. Single and polymicrobial cultures of antibiotic resistant human pathogens were tested. Staphylococcus aureus (S. aureus),), Escherichia coli (E. coli) and Candida albicans (C.albicans) were cultured in 10-100% (v/v) honey diluted in broth, or 0.08-1.0% (weight/volume) EEPS and EEPE diluted in broth. Four types of polymicrobial cultures were prepared by culturing the isolates with each other in broth (control) and broth containing various concentrations of honey or propolis. Microbial growth was assessed on solid plate media after 24 h incubation. EEPS and EEPE inhibited antibiotic resistant E.coli, and S.aureus, and C.albicans in single and polymicrobial cultures. S.aureus became more susceptible when it was cultured with E.coli or C.albicans or when all cultured together. C.albicans became more susceptible when it was cultured with S.aureus or with E.coli and S. aureus together. The presence of ethyl alcohol or honey potentiated antimicrobial effect of propolis toward entire microbes tested in single or polymicrobial cultures. EEPS had lower MIC toward E.coli and C.albicans than EEPE. When propolis was mixed with honey, EEPS showed lower MIC than EEPE. In addition, honey showed lower MIC toward entire microbes when mixed with EEPS than when it was mixed with EEPE. 1) propolis prevents the growth of the microorganisms in single and mixed microbial cultures, and has synergistic effect when used with honey or ethyl alcohol, 2) the antimicrobial property of propolis varies with geographical origin, and 3) this study will pave the way to isolate active ingredients from honey and propolis to be further tested individually or in combination against human resistant infections.

  18. Deformation of isolated single-wall carbon nanotubes in electrospun polymer nanofibres

    NASA Astrophysics Data System (ADS)

    Kannan, Prabhakaran; Eichhorn, Stephen J.; Young, Robert J.

    2007-06-01

    Electrospinning has been used to prepare poly(vinyl alcohol) (PVA) nanofibres, with diameters ranging from 1 µm down to 20 nm, that contain dispersions of isolated, well-aligned, single-wall carbon nanotubes (SWNTs). The nanofibres were characterized by electron microscopy and Raman spectroscopy. Single Raman radial breathing modes (RBMs) were found for the SWNTs in the nanofibres which allowed the identification of particular nanotubes and indicated debundling/separation of the original SWNT ropes. Moreover the results of polarized Raman spectroscopy were consistent with the presence of isolated SWNTs, well-aligned along the nanofibre axes. The nanofibres were subjected to deformation and the position of the G and G' bands was followed as a function of strain. It was found that large band shifts were obtained, indicating that there was good stress transfer from the PVA matrix to the nanotubes. A band shift of up to 40 cm-1 for 1% strain was found for the G' band which is similar to that reported for the deformation of isolated nanotubes. This indicates that the Young's modulus of SWNTs is in excess of 800 GPa.

  19. Microorganisms responsible for periprosthetic knee infections in England and Wales.

    PubMed

    Holleyman, Richard J; Baker, Paul; Charlett, Andre; Gould, Kate; Deehan, David J

    2016-10-01

    This study aimed to delineate epidemiology of infecting microorganism genus in first-time revision knee arthroplasty for indication of periprosthetic joint infection in England and Wales using linked registry data. From the National Joint Registry database for England and Wales, a consecutive series of primary knee arthroplasties performed between April 2003 and January 2014 that went on to have a revision for periprosthetic infection were identified (n = 2810). Each case was then linked to microbiology data held by Public Health England in order to identify infecting microorganism at time of revision surgery established from intra-operative cultures. Following data linkage, 403 culture results at time of revision surgery were identified in a group of 331 patients. The demographic characteristics of five microorganism groups were compared: pure staphylococcus (single genus), pure streptococcus (single genus), other gram-positive infections (single genus), gram-negative infections (single genus) and mixed genus infections. Staphylococcus species was the most common organism genus isolated after revision of a primary implant for infection and present in 72 % of cases overall (71.3 % of patients with a single-genus infection and 76.8 % of patients with mixed genus infection). A pure staphylococcal infection was present in 59 % of all cases. A single-genus infection was responsible for infection in 83.1 % of cases, and mixed genera were responsible in 16.9 % of cases. A significant difference was observed for mean age at primary procedure in the cohort of patients where there was an isolated pure streptococcal infection (73.2 years) when compared to gram-negative infections (65.0 years). No other significant differences were observed between microorganism groups in terms of BMI, gender, ASA grade, indication for primary procedure and primary implant characteristics. Staphylococci were the most commonly isolated organism species responsible for periprosthetic

  20. Pregnancy prognosis associated with an isolated single umbilical artery in twin pregnancy.

    PubMed

    Cade, Thomas J; Da Silva Costa, Fabricio; Reidy, Karen; Doyle, Lex W; Mitchell, Sarah E; Palma-Dias, Ricardo; Umstad, Mark P

    2014-12-01

    To determine the prognosis of an isolated single umbilical artery (SUA) in a twin pregnancy, we selected twin pregnancies with a second trimester ultrasound diagnosing a SUA in at least one fetus at our tertiary hospital. This was confirmed by placental histopathology or by expert review of ultrasound images. Cases were identified by searching the hospital ultrasound database over a period of 7.5 years. Higher order multiples or coexistent aneuploidy or major anomalies were excluded. Each case of an isolated SUA was assigned three consecutive twin pregnancy controls paired for chorionicity and maternal age. Primary outcomes were preterm birth <34 weeks, small for gestational age (SGA) or perinatal death. Other outcomes included antenatal growth restriction, mode of delivery, and admission to neonatal intensive care or special care nursery. Nine pregnancies (18 fetuses) were identified for analysis as cases. Isolated SUA was associated with preterm birth <34 weeks (odds ratio = 12.2; 95% CI = 2.0-75.2; p = .005) but not for SGA. There was also no difference in SGA between the affected twin and its normal co-twin. Perinatal death was increased but after controlling for gestational age and clustering this finding was no longer significant. We conclude that isolated SUA in twins adds a degree of risk to an already high-risk pregnancy but does not increase the need for surveillance for growth restriction.

  1. Wavelet methodology to improve single unit isolation in primary motor cortex cells

    PubMed Central

    Ortiz-Rosario, Alexis; Adeli, Hojjat; Buford, John A.

    2016-01-01

    The proper isolation of action potentials recorded extracellularly from neural tissue is an active area of research in the fields of neuroscience and biomedical signal processing. This paper presents an isolation methodology for neural recordings using the wavelet transform (WT), a statistical thresholding scheme, and the principal component analysis (PCA) algorithm. The effectiveness of five different mother wavelets was investigated: biorthogonal, Daubachies, discrete Meyer, symmetric, and Coifman; along with three different wavelet coefficient thresholding schemes: fixed form threshold, Stein’s unbiased estimate of risk, and minimax; and two different thresholding rules: soft and hard thresholding. The signal quality was evaluated using three different statistical measures: mean-squared error, root-mean squared, and signal to noise ratio. The clustering quality was evaluated using two different statistical measures: isolation distance, and L-ratio. This research shows that the selection of the mother wavelet has a strong influence on the clustering and isolation of single unit neural activity, with the Daubachies 4 wavelet and minimax thresholding scheme performing the best. PMID:25794461

  2. Isolation of satellite cells from single muscle fibers from young, aged, or dystrophic muscles.

    PubMed

    Di Foggia, Valentina; Robson, Lesley

    2012-01-01

    Skeletal muscle contains an identified resident stem cell population called the satellite cells. This cell is responsible for the majority of the postnatal growth and regenerative potential of skeletal muscle. Other cells do contribute to skeletal muscle regeneration and in cultures of minced whole muscle these cells are cultured along with the satellite cells and it is impossible to dissect out their contribution compared to the satellite cells. Therefore, a method to culture pure satellite cells has been developed to study the signaling pathways that control their proliferation and differentiation. In our studies into the role of the resident myogenic stem cells in regeneration, myopathic conditions, and aging, we have optimized the established techniques that already exist to isolate pure satellite cell cultures from single muscle fibers. We have successfully isolated satellite cells from young adults through to 24-month-old muscles and obtained populations of cells that we are studying for the signaling events that regulate their proliferative potential.

  3. Rapid identification of micro-organisms from urinary tract infections by beta-glucuronidase, phenylalanine deaminase, cytochrome oxidase and indole tests on isolation media.

    PubMed

    Giammanco, G; Pignato, S

    1994-12-01

    Two commercially available media recommended for the isolation and rapid identification of Escherichia coli from urinary tract infections were supplemented with L-phenylalanine and L-tryptophan. The non-selective medium proved suitable for the direct detection of lactose fermentation, beta-glucuronidase and phenylalanine deaminase activities, indole production and the oxidase test. It was highly efficient in making a presumptive identification at species level of the most common gram-negative urinary pathogens, E. coli, Proteus mirabilis and Pseudomonas aeruginosa, that account for c. 85% of all urinary isolates. Among the gram-positive isolates, most colonies were non-fluorescent and could be separated into staphylococci and enterococci on the basis of the catalase test. Fluorescent colonies were found to be Staphylococcus haemolyticus isolates, 61% of which were fluorescent. The selective medium proved suitable for the same biochemical tests, with the exception of indole, which was not visible against the red colour of the medium. Therefore, the differentiation of P. mirabilis from other Proteus-Providencia species was impossible on this medium.

  4. Differentiation of Plum pox virus isolates by single-strand conformation polymorphism and low-stringency single specific primer PCR analysis of HC-Pro genome region.

    PubMed

    Gadiou, S; Safárová, D; Navrátil, M

    2009-01-01

    Single-strand conformation polymorphism (SSCP) and low-stringency single specific primer (LSSP)-PCR were assessed for suitability and reliability in genotyping of Plum pox virus (PPV) isolates. Examined PPV isolates included 16 PPV-D, 12 PPV-M, and 14 PPV-Rec isolates collected in Czech Republic. The analysis was performed on the helper component protease (HC-Pro) region of the PPV genome. SSCP and LSSP-PCR allowed the differentiation of PPV strain, but SSCP was not able to distinguish isolates within the same strain. The individual genotyping of each PPV isolate was obtained by LSSP-PCR. Nevertheless, both SSCP and LSSP-PCR techniques are suitable for preliminary screening of genetic variability of plant RNA viruses.

  5. Microorganisms in closed periapical lesions.

    PubMed

    Abou-Rass, M; Bogen, G

    1998-01-01

    The purpose of this study was to investigate the microorganisms of strictly selected closed periapical lesions associated with both refractory endodontic therapy and pulpal calcification. Definitive criteria were established that assured complete clinical isolation of the periapical lesion from the oral and periodontal environment. A total of 13 criteria-referenced lesions were selected from 70 patients with endodontic surgical indications. A well controlled culturing method was used in all cases and samples were taken by one clinician at three separate sites during each surgery. Samples taken at the surgical window and within the body of the lesion served as controls, whilst a third sample was taken at the apex. In all 13 cases, samples taken from the apex yielded microorganisms comprising 63.6% obligate anaerobes and 36.4% facultative anaerobes. Prevalence of the isolated species was 31.8% for Actinomyces sp., 22.7% Propionibacterium sp., 18.2% Streptococcus sp., 13.6% Staphlyococcus sp., 4.6% Porphyromonas gingivalis, 4.6% Peptostreptococcus micros and 4.6% Gram-negative enterics. The results of this investigation indicate that closed periapical lesions associated with calcified teeth or those resistant to root canal treatment harbour bacteria. The inability to eradicate all root canal microorganisms during root canal treatment, along with anatomical factors, may allow further bacterial colonization of the root apex and surrounding periapical tissues, and consequently prevent healing.

  6. In Vitro Fertilization with Isolated, Single Gametes Results in Zygotic Embryogenesis and Fertile Maize Plants.

    PubMed Central

    Kranz, E; Lorz, H

    1993-01-01

    We demonstrate here the possibility of regenerating phenotypically normal, fertile maize plants via in vitro fertilization of isolated, single sperm and egg cells mediated by electrofusion. The technique leads to the highly efficient formation of polar zygotes, globular structures, proembryos, and transition-phase embryos and to the formation of plants from individually cultured fusion products. Regeneration of plants occurs via embryogenesis and occasionally by polyembryony and organogenesis. Flowering plants can be obtained within 100 days of gamete fusion. Regenerated plants were studied by karyological and morphological analyses, and the segregation of kernel color was determined. The hybrid nature of the plants was confirmed. PMID:12271084

  7. Shape of isolated domains in lithium tantalate single crystals at elevated temperatures

    SciTech Connect

    Shur, V. Ya. Akhmatkhanov, A. R.; Baturin, I. S.; Chezganov, D. S.; Lobov, A. I.; Smirnov, M. M.

    2013-12-09

    The shape of isolated domains has been investigated in congruent lithium tantalate (CLT) single crystals at elevated temperatures and analyzed in terms of kinetic approach. The obtained temperature dependence of the growing domain shape in CLT including circular shape at temperatures above 190 °C has been attributed to increase of relative input of isotropic ionic conductivity. The observed nonstop wall motion and independent domain growth after merging in CLT as opposed to stoichiometric lithium tantalate have been attributed to difference in wall orientation. The computer simulation has confirmed applicability of the kinetic approach to the domain shape explanation.

  8. Large Vessel Vasculitis with an Isolated Lesion of a Single-lobe Pulmonary Artery.

    PubMed

    Kitajima, Takamasa; Marumo, Satoshi; Shoji, Tsuyoshi; Huang, Cheng-Long; Yuba, Yoshiaki; Fukui, Motonari

    2016-01-01

    Chronic pulmonary arterial obstructions are caused mostly by chronic pulmonary artery thromboembolism and rarely by vasculitis or intimal sarcoma of the pulmonary artery. We herein report an unusual case of a 42-year-old woman with a solitary obstruction of the pulmonary artery in the right lower lobe of her lung. Because we could not exclude the possibility of intimal sarcoma, middle and lower lobectomy was performed. The resected specimens revealed large vessel vasculitis (LVV) and an isolated lesion in the right lower lobe pulmonary artery. LVV should therefore be considered in the differential diagnosis for single pulmonary arterial stenosis or obstruction.

  9. Isolation and epitope mapping of staphylococcal enterotoxin B single-domain antibodies.

    PubMed

    Turner, Kendrick B; Zabetakis, Dan; Legler, Patricia; Goldman, Ellen R; Anderson, George P

    2014-06-19

    Single-domain antibodies (sdAbs), derived from the heavy chain only antibodies found in camelids such as llamas have the potential to provide rugged detection reagents with high affinities, and the ability to refold after denaturation. We have isolated and characterized sdAbs specific to staphylococcal enterotoxin B (SEB) which bind to two distinct epitopes and are able to function in a sandwich immunoassay for toxin detection. Characterization of these sdAbs revealed that each exhibited nanomolar binding affinities or better.  Melting temperatures for the sdAbs ranged from approximately 60 °C to over 70 °C, with each demonstrating at least partial refolding after denaturation and several were able to completely refold. A first set of sdAbs was isolated by panning the library using adsorbed antigen, all of which recognized the same epitope on SEB. Epitope mapping suggested that these sdAbs bind to a particular fragment of SEB (VKSIDQFLYFDLIYSI) containing position L45 (underlined), which is involved in binding to the major histocompatibility complex (MHC). Differences in the binding affinities of the sdAbs to SEB and a less-toxic vaccine immunogen, SEBv (L45R/Y89A/Y94A) were also consistent with binding to this epitope. A sandwich panning strategy was utilized to isolate sdAbs which bind a second epitope. This epitope differed from the initial one obtained or from that recognized by previously isolated anti-SEB sdAb A3. Using SEB-toxin spiked milk we demonstrated that these newly isolated sdAbs could be utilized in sandwich-assays with each other, A3, and with various monoclonal antibodies.

  10. Deciphering Single Nucleotide Polymorphisms and Evolutionary Trends in Isolates of the Cydia pomonella granulovirus

    PubMed Central

    Wennmann, Jörg T.; Radtke, Pit; Eberle, Karolin E.; Gueli Alletti, Gianpiero

    2017-01-01

    Six complete genome sequences of Cydia pomonella granulovirus (CpGV) isolates from Mexico (CpGV-M and CpGV-M1), England (CpGV-E2), Iran (CpGV-I07 and CpGV-I12), and Canada (CpGV-S) were aligned and analyzed for genetic diversity and evolutionary processes. The selected CpGV isolates represented recently identified phylogenetic lineages of CpGV, namely, the genome groups A to E. The genomes ranged from 120,816 bp to 124,269 bp. Several common differences between CpGV-M, -E2, -I07, -I12 and -S to CpGV-M1, the first sequenced and published CpGV isolate, were highlighted. Phylogenetic analysis based on the aligned genome sequences grouped CpGV-M and CpGV-I12 as the most derived lineages, followed by CpGV-E2, CpGV-S and CpGV-I07, which represent the most basal lineages. All of the genomes shared a high degree of co-linearity, with a common setup of 137 (CpGV-I07) to 142 (CpGV-M and -I12) open reading frames with no translocations. An overall trend of increasing genome size and a decrease in GC content was observed, from the most basal lineage (CpGV-I07) to the most derived (CpGV-I12). A total number of 788 positions of single nucleotide polymorphisms (SNPs) were determined and used to create a genome-wide SNP map of CpGV. Of the total amount of SNPs, 534 positions were specific for exactly one of either isolate CpGV-M, -E2, -I07, -I12 or -S, which allowed the SNP-based detection and identification of all known CpGV isolates. PMID:28820456

  11. Deciphering Single Nucleotide Polymorphisms and Evolutionary Trends in Isolates of the Cydia pomonella granulovirus.

    PubMed

    Wennmann, Jörg T; Radtke, Pit; Eberle, Karolin E; Gueli Alletti, Gianpiero; Jehle, Johannes A

    2017-08-18

    Six complete genome sequences of Cydia pomonella granulovirus (CpGV) isolates from Mexico (CpGV-M and CpGV-M1), England (CpGV-E2), Iran (CpGV-I07 and CpGV-I12), and Canada (CpGV-S) were aligned and analyzed for genetic diversity and evolutionary processes. The selected CpGV isolates represented recently identified phylogenetic lineages of CpGV, namely, the genome groups A to E. The genomes ranged from 120,816 bp to 124,269 bp. Several common differences between CpGV-M, -E2, -I07, -I12 and -S to CpGV-M1, the first sequenced and published CpGV isolate, were highlighted. Phylogenetic analysis based on the aligned genome sequences grouped CpGV-M and CpGV-I12 as the most derived lineages, followed by CpGV-E2, CpGV-S and CpGV-I07, which represent the most basal lineages. All of the genomes shared a high degree of co-linearity, with a common setup of 137 (CpGV-I07) to 142 (CpGV-M and -I12) open reading frames with no translocations. An overall trend of increasing genome size and a decrease in GC content was observed, from the most basal lineage (CpGV-I07) to the most derived (CpGV-I12). A total number of 788 positions of single nucleotide polymorphisms (SNPs) were determined and used to create a genome-wide SNP map of CpGV. Of the total amount of SNPs, 534 positions were specific for exactly one of either isolate CpGV-M, -E2, -I07, -I12 or -S, which allowed the SNP-based detection and identification of all known CpGV isolates.

  12. Single-step isolation of extracellular vesicles by size-exclusion chromatography.

    PubMed

    Böing, Anita N; van der Pol, Edwin; Grootemaat, Anita E; Coumans, Frank A W; Sturk, Auguste; Nieuwland, Rienk

    2014-01-01

    Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively. To develop a single-step protocol to isolate vesicles from human body fluids. Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n=3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction. Fractions 9-12 contained the highest concentrations of particles larger than 70 nm and platelet-derived vesicles (46%±6 and 61%±2 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8%±1 and 0.65%±0.3, respectively). HDL was present mainly in fractions 18-20 (32%±2 of total), and protein in fractions 19-21 (36%±2 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43%±23 in fractions 9-12, with an 8-fold and 70-fold enrichment compared to HDL and protein. SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles.

  13. Antimicrobial property of (+)-lyoniresinol-3alpha-O-beta-D-glucopyranoside isolated from the root bark of Lycium chinense Miller against human pathogenic microorganisms.

    PubMed

    Lee, Dong Gun; Jung, Hyun Jun; Woo, Eun-Rhan

    2005-09-01

    (+)-Lyoniresinol-3alpha-O-beta-D-glucopyranoside (1) was isolated from an ethyl acetate extract of the root bark from Lycium chinense Miller, and its structure was determined using 1D and 2D NMR spectroscopy including DEPT, HMQC, and HMBC. (+)-Lyoniresinol-3alpha-O-beta-D-glucopyranoside exhibited potent antimicrobial activity against antibiotic-resistant bacterial strains, methicillin-resistant Staphylococcus aureus (MRSA) isolated from patients, and human pathogenic fungi without having any hemolytic effect on human erythrocytes. In particular, compound 1 induced the accumulation of intracellular trehalose on C. albicans as stress response to the drug, and disrupted the dimorphic transition that forms pseudo-hyphae caused by the pathogenesis. This indicates that (+)-lyoniresinol-3alpha-O-beta-D-glucopyranoside has excellent potential as a lead compound for the development of antibiotic agents.

  14. Microbial iron cycling in acidic geothermal springs of yellowstone national park: integrating molecular surveys, geochemical processes, and isolation of novel fe-active microorganisms.

    PubMed

    Kozubal, Mark A; Macur, Richard E; Jay, Zackary J; Beam, Jacob P; Malfatti, Stephanie A; Tringe, Susannah G; Kocar, Benjamin D; Borch, Thomas; Inskeep, William P

    2012-01-01

    Geochemical, molecular, and physiological analyses of microbial isolates were combined to study the geomicrobiology of acidic iron oxide mats in Yellowstone National Park. Nineteen sampling locations from 11 geothermal springs were studied ranging in temperature from 53 to 88°C and pH 2.4 to 3.6. All iron oxide mats exhibited high diversity of crenarchaeal sequences from the Sulfolobales, Thermoproteales, and Desulfurococcales. The predominant Sulfolobales sequences were highly similar to Metallosphaera yellowstonensis str. MK1, previously isolated from one of these sites. Other groups of archaea were consistently associated with different types of iron oxide mats, including undescribed members of the phyla Thaumarchaeota and Euryarchaeota. Bacterial sequences were dominated by relatives of Hydrogenobaculum spp. above 65-70°C, but increased in diversity below 60°C. Cultivation of relevant iron-oxidizing and iron-reducing microbial isolates included Sulfolobus str. MK3, Sulfobacillus str. MK2, Acidicaldus str. MK6, and a new candidate genus in the Sulfolobales referred to as Sulfolobales str. MK5. Strains MK3 and MK5 are capable of oxidizing ferrous iron autotrophically, while strain MK2 oxidizes iron mixotrophically. Similar rates of iron oxidation were measured for M. yellowstonensis str. MK1 and Sulfolobales str. MK5. Biomineralized phases of ferric iron varied among cultures and field sites, and included ferric oxyhydroxides, K-jarosite, goethite, hematite, and scorodite depending on geochemical conditions. Strains MK5 and MK6 are capable of reducing ferric iron under anaerobic conditions with complex carbon sources. The combination of geochemical and molecular data as well as physiological observations of isolates suggests that the community structure of acidic Fe mats is linked with Fe cycling across temperatures ranging from 53 to 88°C.

  15. Microbial Iron Cycling in Acidic Geothermal Springs of Yellowstone National Park: Integrating Molecular Surveys, Geochemical Processes, and Isolation of Novel Fe-Active Microorganisms

    PubMed Central

    Kozubal, Mark A.; Macur, Richard E.; Jay, Zackary J.; Beam, Jacob P.; Malfatti, Stephanie A.; Tringe, Susannah G.; Kocar, Benjamin D.; Borch, Thomas; Inskeep, William P.

    2012-01-01

    Geochemical, molecular, and physiological analyses of microbial isolates were combined to study the geomicrobiology of acidic iron oxide mats in Yellowstone National Park. Nineteen sampling locations from 11 geothermal springs were studied ranging in temperature from 53 to 88°C and pH 2.4 to 3.6. All iron oxide mats exhibited high diversity of crenarchaeal sequences from the Sulfolobales, Thermoproteales, and Desulfurococcales. The predominant Sulfolobales sequences were highly similar to Metallosphaera yellowstonensis str. MK1, previously isolated from one of these sites. Other groups of archaea were consistently associated with different types of iron oxide mats, including undescribed members of the phyla Thaumarchaeota and Euryarchaeota. Bacterial sequences were dominated by relatives of Hydrogenobaculum spp. above 65–70°C, but increased in diversity below 60°C. Cultivation of relevant iron-oxidizing and iron-reducing microbial isolates included Sulfolobus str. MK3, Sulfobacillus str. MK2, Acidicaldus str. MK6, and a new candidate genus in the Sulfolobales referred to as Sulfolobales str. MK5. Strains MK3 and MK5 are capable of oxidizing ferrous iron autotrophically, while strain MK2 oxidizes iron mixotrophically. Similar rates of iron oxidation were measured for M. yellowstonensis str. MK1 and Sulfolobales str. MK5. Biomineralized phases of ferric iron varied among cultures and field sites, and included ferric oxyhydroxides, K-jarosite, goethite, hematite, and scorodite depending on geochemical conditions. Strains MK5 and MK6 are capable of reducing ferric iron under anaerobic conditions with complex carbon sources. The combination of geochemical and molecular data as well as physiological observations of isolates suggests that the community structure of acidic Fe mats is linked with Fe cycling across temperatures ranging from 53 to 88°C. PMID:22470372

  16. Technological characterization and probiotic traits of yeasts isolated from Altamura sourdough to select promising microorganisms as functional starter cultures for cereal-based products.

    PubMed

    Perricone, Marianne; Bevilacqua, Antonio; Corbo, Maria Rosaria; Sinigaglia, Milena

    2014-04-01

    The main topic of this research was to select some suitable functional starter cultures for cereal-based food or beverages. This aim was achieved through a step-by step approach focused on the technological characterization, as well as on the evaluation of the probiotic traits of yeasts; the technological characterization relied on the assessment of enzymatic activities (catalase, urease, β-glucosidase), growth under various conditions (pH, temperature, addition of salt, lactic and acetic acids) and leavening ability. The results of this step were used as input data for a Principal Component Analysis; thus, the most technologically relevant 18 isolates underwent a second selection for their probiotic traits (survival at pH 2.5 and with bile salts added, antibiotic resistance, antimicrobial activity towards foodborne pathogens, hydrophobic properties and biofilm production) and were identified through genotyping. Two isolates (Saccharomyces cerevisiae strain 2 and S. cerevisiae strain 4) were selected and analyzed in the last step for the simulation of the gastric transit; these isolates showed a trend similar to S. cerevisiae var. boulardii ATCC MYA-796, a commercial probiotic yeast used as control. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Enrichment of chitinolytic microorganisms: isolation and characterization of a chitinase exhibiting antifungal activity against phytopathogenic fungi from a novel Streptomyces strain.

    PubMed

    Hoster, Frank; Schmitz, Jessica E; Daniel, Rolf

    2005-01-01

    Thirteen different chitin-degrading bacteria were isolated from soil and sediment samples. Five of these strains (SGE2, SGE4, SSL3, MG1, and MG3) exhibited antifungal activity against phytopathogenic fungi. Analyses of the 16S rRNA genes and the substrate spectra revealed that the isolates belong to the genera Bacillus or Streptomyces. The closest relatives were Bacillus chitinolyticus (SGE2, SGE4, and SSL3), B. ehimensis (MG1), and Streptomyces griseus (MG3). The chitinases present in the culture supernatants of the five isolates revealed optimal activity between 45 degrees C and 50 degrees C and at pH values of 4 (SSL3), 5 (SGE2 and MG1), 6 (SGE4), and 5-7 (MG3). The crude chitinase preparations of all five strains possessed antifungal activity. The chitinase of MG3 (ChiIS) was studied further, since the crude enzyme conferred strong growth suppression of all fungi tested and was very active over the entire pH range tested. The chiIS gene was cloned and the gene product was purified. The deduced protein consisted of 303 amino acids with a predicted molecular mass of 31,836 Da. Sequence analysis revealed that ChiIS of MG3 is similar to chitinases of Streptomyces species, which belong to family 19 of glycosyl hydrolases. Purified ChiIS showed remarkable antifungal activity and stability.

  18. Structure-Based Systematic Isolation of Conditional-Lethal Mutations in the Single Yeast Calmodulin Gene

    PubMed Central

    Ohya, Y.; Botstein, D.

    1994-01-01

    Conditional-lethal mutations of the single calmodulin gene in Saccharomyces cerevisiae have been very difficult to isolate by random and systematic methods, despite the fact that deletions cause recessive lethality. We report here the isolation of numerous conditional-lethal mutants that were recovered by systematically altering phenylalanine residues. The phenylalanine residues of calmodulin were implicated in function both by structural studies of calmodulin bound to target peptides and by their extraordinary conservation in evolution. Seven single and 26 multiple Phe -> Ala mutations were constructed. Mutant phenotypes were examined in a haploid cmd1 disrupted strain under three conditions: single copy, low copy, and overexpressed. Whereas all but one of the single mutations caused no obvious phenotype, most of the multiple mutations caused obvious growth phenotypes. Five were lethal, 6 were lethal only in synthetic medium, 13 were temperature-sensitive lethal and 2 had no discernible phenotypic consequences. Overexpression of some of the mutant genes restored the phenotype to nearly wild type. Several temperature-sensitive calmodulin mutations were suppressed by elevated concentration of CaCl(2) in the medium. Mutant calmodulin protein was detected at normal levels in extracts of most of the lethal mutant cells, suggesting that the deleterious phenotypes were due to loss of the calmodulin function and not protein instability. Analysis of diploid strains heterozygous for all combinations of cmd1-ts alleles revealed four intragenic complementation groups. The contributions of individual phe->ala changes to mutant phenotypes support the idea of internal functional redundancy in the symmetrical calmodulin protein molecule. These results suggest that the several phenylalanine residues in calmodulin are required to different extents in different combinations in order to carry out each of the several essential tasks. PMID:7896089

  19. Prevalence, antimicrobial resistance and relation to indicator and pathogenic microorganisms of Salmonella enterica isolated from surface waters within an agricultural landscape.

    PubMed

    Economou, Vangelis; Gousia, Panagiota; Kansouzidou, Athina; Sakkas, Hercules; Karanis, Panagiotis; Papadopoulou, Chrissanthy

    2013-07-01

    During a 12 month period (June 2007-May 2008), the prevalence and susceptibility of Salmonella serovars and their relation to specific pathogenic and indicator bacteria in river and coastal waters was investigated. A total of 240 water samples were collected from selected sites in Acheron and Kalamas Rivers and the Ionian Sea coast in north western Greece. The samples were analyzed for Salmonella spp., Listeria spp., Campylobacter spp., Escherichia coli O157, Staphylococci, Pseudomonas spp., Total Coliforms, Fecal Coliforms, Fecal Streptococci, Total Heterotrophic Flora at 20°C and at 37°C, fungi and protozoa (Cryptosporidium, Giardia). Susceptibility tests to nine antimicrobials (ampicillin, amikacin, amoxicillin/clavulavic acid, cefuroxime, ciprofloxacin, cefoxitin, tetracycline, ticarcillin/clavulanic acid, ampicillin/sulbactam) were performed using the disk diffusion method for Salmonella isolates. We isolated 28 serovars of Salmonella spp. identified as Salmonella enteritidis (23), Salmonella thompson (3) and Salmonella virchow (2). Multi-drug resistant Salmonella serovars were isolated from both river and marine waters, with 34.8% of S. enteritidis and 100% of S. virchow being resistant to more than 3 antibiotics. Also we isolated 42 strains of Listeria spp. identified as L. monocytogenes (20), L. innocua (9), L. seeligeri (2) and L. ivanovii (11). All the Listeria isolates were susceptible to the tested antibiotics. No Campylobacter spp., E. coli O157, Cryptosporidium and Giardia were detected. The overall ranges (and average counts) of the indicator bacteria were: Total Coliforms 0-4×10(4)cfu/100ml (3.7×10(3)cfu/100ml), Fecal Coliforms 0-9×10(3)cfu/100ml (9.2×10(2)cfu/100ml), Fecal Streptococci 0-3.5×10(4)cfu/100ml (1.4×10(3)cfu/100ml), Total Heterotrophic Flora at 20°C 0-6×10(3)cfu/ml (10(3)cfu/ml) and at 37°C 0-5×10(3)cfu/ml (4.9×10(2)cfu/ml). Weak or non significant positive Spearman correlations (p<0.05, rs range: 0.13-0.77) were obtained

  20. Is genotyping of single isolates sufficient for population structure analysis of Pseudomonas aeruginosa in cystic fibrosis airways?

    PubMed

    Sommer, Lea M; Marvig, Rasmus L; Luján, Adela; Koza, Anna; Pressler, Tacjana; Molin, Søren; Johansen, Helle K

    2016-08-09

    The primary cause of morbidity and mortality in cystic fibrosis (CF) patients is lung infection by Pseudomonas aeruginosa. Therefore much work has been done to understand the adaptation and evolution of P. aeruginosa in the CF lung. However, many of these studies have focused on longitudinally collected single isolates, and only few have included cross-sectional analyses of entire P. aeruginosa populations in sputum samples. To date only few studies have used the approach of metagenomic analysis for the purpose of investigating P. aeruginosa populations in CF airways. We analysed five metagenomes together with longitudinally collected single isolates from four recently chronically infected CF patients. With this approach we were able to link the clone type and the majority of SNP profiles of the single isolates to that of the metagenome(s) for each individual patient. Based on our analysis we find that when having access to comprehensive collections of longitudinal single isolates it is possible to rediscover the genotypes of the single isolates in the metagenomic samples. This suggests that information gained from genome sequencing of comprehensive collections of single isolates is satisfactory for many investigations of adaptation and evolution of P. aeruginosa to the CF airways.

  1. Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

    PubMed Central

    Kandasamy, Kathirvel; Parthasarathi, Kaushik

    2014-01-01

    The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement. PMID:25045895

  2. Application of a single-colony coculture technique to the isolation of hitherto unculturable gut bacteria.

    PubMed

    Tanaka, Yoshiki; Benno, Yoshimi

    2015-02-01

    Molecular studies have led to postulation of a relationship between gut microbiota and certain diseases. However, because studies of hitherto uncultured species in vivo are essential for characterizing the biology and pathogenic properties of gut bacteria, techniques for culturing and isolating such bacteria must be developed. Here, a technique is described that partially overcomes the obstacles that prevent detection of interbacterial communication in vitro and are thus responsible for the failure to culture certain bacterial species. For this purpose, a ring with a membrane filter at the bottom was designed and a relatively simple nutrient medium was used instead of conventional media. Gut bacteria were cocultivated in soft agar separated by the membrane filter to simulate interbacterial communication in vitro. Use of this soft agar coculture technique led to the successful isolation of hitherto uncultured bacteria and the demonstration of multistage interbacterial communication among gut bacteria in vitro. Cultivation and isolation of single colonies of bacteria that require other bacteria for growth will enhance efforts to better understand the physiological and pathogenic roles of gut microbiota.

  3. Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution.

    PubMed

    Hu, Wen-Yang; Hu, Dan-Ping; Xie, Lishi; Li, Ye; Majumdar, Shyama; Nonn, Larisa; Hu, Hong; Shioda, Toshi; Prins, Gail S

    2017-08-01

    Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways analysis identified ribosome biogenesis and membrane estrogen-receptor signaling enriched in stem cells with NF-ĸB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Purification of Helicobacter suis Strains From Biphasic Cultures by Single Colony Isolation: Influence on Strain Characteristics.

    PubMed

    Liang, Jungang; De Bruyne, Ellen; Ducatelle, Richard; Smet, Annemieke; Haesebrouck, Freddy; Flahou, Bram

    2015-06-01

    Helicobacter (H.) suis causes gastritis and decreased weight gain in pigs. It is also the most prevalent non-Helicobacter pylori Helicobacter species in humans with gastric disease. H. suis is extremely fastidious, and so far, biphasic culture conditions were essential for isolation and culture, making it impossible to obtain single colonies. Hence, cultures obtained from an individual animal may contain multiple H. suis strains, which is undesirable for experiments aiming for instance at investigating H. suis strain differences. Pure cultures of H. suis were established by growing bacteria as colonies on 1% brucella agar plates, followed by purification and enrichment by biphasic subculture. Characteristics of these single colony-derived strains were compared with those of their parent strains using multilocus sequence typing (MLST) and by studying bacterium-host interactions using a gastric epithelial cell line and Mongolian gerbil model. The purification/enrichment procedure required a nonstop culture of several weeks. For 4 of 17 H. suis strains, MLST revealed differences between parental and single colony-derived strains. For three of four single colony-derived strains tested, the cell death-inducing capacity was higher than for the parental strain. One single colony-derived strain lost its capacity to colonize Mongolian gerbils. For the four other strains tested, colonization capacity and histopathologic changes were similar to what has been described when using strains with only a history of limited biphasic culture. A method was developed to obtain single colony-derived H. suis strains, but this procedure may affect the bacterial genotype and phenotype. © 2015 John Wiley & Sons Ltd.

  5. Automatic Myonuclear Detection in Isolated Single Muscle Fibers Using Robust Ellipse Fitting and Sparse Representation

    PubMed Central

    Su, Hai; Xing, Fuyong; Lee, Jonah D.; Peterson, Charlotte A.; Yang, Lin

    2015-01-01

    Accurate and robust detection of myonuclei in isolated single muscle fibers is required to calculate myonuclear domain size. However, this task is challenging because: 1) shape and size variations of the nuclei, 2) overlapping nuclear clumps, and 3) multiple z-stack images with out-of-focus regions. In this paper, we have proposed a novel automatic detection algorithm to robustly quantify myonuclei in isolated single skeletal muscle fibers. The original z-stack images are first converted into one all-in-focus image using multi-focus image fusion. A sufficient number of ellipse fitting hypotheses are then generated from them yonuclei contour segments using heteroscedastic errors-invariables (HEIV) regression. A set of representative training samples and a set of discriminative features are selected by a two-stage sparse model. The selected samples with representative features are utilized to train a classifier to select the best candidates. A modified inner geodesic distance based mean-shift clustering algorithm is used to produce the final nuclei detection results. The proposed method was extensively tested using 42 sets of z-stack images containing over 1,500 myonuclei. The method demonstrates excellent results that are better than current state-of-the-art approaches. PMID:26356342

  6. Isolation, sequencing and overproduction of the single-stranded DNA binding protein from Pseudomonas aeruginosa PAO.

    PubMed

    Genschel, J; Litz, L; Thole, H; Roemling, U; Urbanke, C

    1996-12-05

    The gene (ssb) encoding the single-stranded DNA binding (SSB) protein from Pseudomonas aeruginosa PAO was detected on a 2.1 kbp PstI-fragment of chromosomal DNA. The protein (PaeSSB) encoded by this gene consists of 165 aa and has a M(r) of 18549. The genomic sequence was confirmed by amino acid sequencing of the amino terminus of SSB protein isolated from P. aeruginosa PAO. PaeSSB shows 68% homology to the respective protein of E. coli. The nucleotide sequence upstream of the P. aeruginosa ssb gene shows little homology to the regulatory region upstream of the ssb gene of E. coli. The ssb gene was located at a distance of 690-870 kbp from the origin of replication on a physical map of P. aeruginosa PAO. In vivo PaeSSB could replace the SSB protein of E. coli (EcoSSB) if its production was controlled by the lac promoter on a high-copy vector. PaeSSB was overproduced in E. coli. Both the overproduced protein and PaeSSB isolated from Pseudomonas aeruginosa PAO are post-translationally modified by cleavage of the first methionine. Analytical ultracentrifugation shows that PaeSSB is a stable homotetramer. The copy number of PaeSSB in P. aeruginosa is 1200 +/- 250 tetramers per cell. Preliminary characterization of the DNA binding properties shows PaeSSB to have a lower affinity for single-stranded DNA than EcoSSB.

  7. Thoracoscopy without lung isolation utilizing single lumen endotracheal tube intubation and carbon dioxide insufflation.

    PubMed

    Sancheti, Manu S; Dewan, Brendan P; Pickens, Allan; Fernandez, Felix G; Miller, Daniel L; Force, Seth D

    2013-08-01

    This study evaluated the feasibility of performing thoracoscopy without lung isolation employing single lumen endotracheal tube (SLET) intubation and carbon dioxide insufflation. Eighty-two patients underwent a variety of thoracoscopic procedures without lung isolation using SLET intubation and carbon dioxide (CO2) insufflation between January and December 2012. Sixty-five of these patients underwent wedge resections and were isolated for analysis. Operations were accomplished using percutaneously placed laparoscopic trocars and insufflation up to 15 mm Hg. Operative times, length of stay, and vital signs were compared with 52 patients who underwent thoracoscopic wedge resections with double lumen endotracheal tube (DLET) intubation. A retrospective analysis was performed on 65 patients (30 females, mean age 58) who underwent thoracoscopic wedge resections with SLET intubation compared with 52 patients undergoing the same procedure with DLET intubation. Operating room time (111 ± 4.74 minutes), time to incision (49 ± 1.91 minutes), and operative time (48 ± 2.89 minutes) were significantly decreased in the SLET group (p < 0.05). Intraoperative hemodynamic parameters showed no significant aberrations. Two postoperative complications (3.1%) were identified in the SLET group. Length of stay was similar (3 ± 0.49 days versus 3 ± 0.23 days). Single lumen endotracheal tube intubation is a feasible and safe airway management alternative for thoracoscopic procedures. This method resulted in shorter operative times, no aberrant hemodynamic shifts, low complication rates, and similar hospital stays as compared with traditional DLET intubation. Copyright © 2013 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

  8. Pythium kandovanense sp. nov., a fungus-like eukaryotic micro-organism (Stramenopila, Pythiales) isolated from snow-covered ryegrass leaves.

    PubMed

    Chenari Bouket, Ali; Arzanlou, Mahdi; Tojo, Motoaki; Babai-Ahari, Asadollah

    2015-08-01

    Pythiumkandovanense sp. nov. (ex-type culture CCTU 1813T = OPU 1626T = CBS 139567T) is a novel oomycete species isolated from Lolium perenne with snow rot symptoms in a natural grassland in East-Azarbaijan province, Iran. Phylogenetic analyses based on sequence data from internal transcribed spacer (ITS)-rDNA, coxI and coxII mitochondrial genes clustered our isolates in Pythium group E as a unique, well supported clade. Pythium kandovanense sp. nov. is phylogenetically and morphologically distinct from the other closely related species in this clade, namely Pythium rostratifingens and Pythium rostratum. Pythium kandovanense sp. nov. can be distinguished from these two species by its cylindrical sporangia and lower temperatures for optimum and maximum growth rate. The development of zoospores released through a shorter discharge tube is an additional morphological feature which can be used to differentiate Pythium kandovanense sp. nov. from Pythium rostratifingens. Laboratory inoculation tests demonstrated the pathogenicity of Pythium kandovanense sp. nov. to L. perenne under wet cold (0-3 °C) conditions.

  9. Siderophores in Cloud Waters and Potential Impact on Atmospheric Chemistry: Production by Microorganisms Isolated at the Puy de Dôme Station.

    PubMed

    Vinatier, Virginie; Wirgot, Nolwenn; Joly, Muriel; Sancelme, Martine; Abrantes, Magali; Deguillaume, Laurent; Delort, Anne-Marie

    2016-09-06

    A total of 450 bacteria and yeast strains isolated from cloud waters sampled at the puy de Dôme station in France (1465 m) were screened for their ability to produce siderophores. To achieve this, a high-throughput method in 96-well plates was adapted from the CAS (chrome azurol S) method. Notably, 42% of the isolates were siderophore producers. This production was examined according to the phyla of the tested strains and the type of chelating functional groups (i.e., hydroxamate, catechol, and mixed type). The most active bacteria in the clouds belong to the γ-Proteobacteria class, among which the Pseudomonas genus is the most frequently encountered. γ-Proteobacteria are produced in the majority of mixed function siderophores, such as pyoverdines, which bear a photoactive group. Finally, siderophore production was shown to vary with the origin of the air masses. The organic speciation of iron remains largely unknown in warm clouds. Our results suggest that siderophores could partly chelate Fe(III) in cloud waters and thus potentially impact the chemistry of the atmospheric aqueous phase.

  10. Physiologically anaerobic microorganisms of the deep subsurface

    SciTech Connect

    Stevens, S.E. Jr.; Chung, K.T.

    1991-06-01

    This study seeks to determine numbers, diversity, and morphology of anaerobic microorganisms in 15 samples of subsurface material from the Idaho National Engineering Laboratory, in 18 samples from the Hanford Reservation and in 1 rock sample from the Nevada Test Site; set up long term experiments on the chemical activities of anaerobic microorganisms based on these same samples; work to improve methods for the micro-scale determination of in situ anaerobic microbial activity;and to begin to isolate anaerobes from these samples into axenic culture with identification of the axenic isolates.

  11. Electrical characterization of single cells using polysilicon wire ion sensor in an isolation window.

    PubMed

    Wu, You-Lin; Hsu, Po-Yen; Hsu, Chung-Ping; Wang, Chih-Cheng; Lee, Li-Wen; Lin, Jing-Jenn

    2011-10-01

    A polysilicon wire (PSW) sensor can detect the H(+) ion density (pH value) of the medium coated on its surface, and different cells produce different extracellular acidification and hence different H(+) ion densities. Based on this, we used a PSW sensor in combination with a mold-cast polydimethylsiloxane (PDMS) isolation window to detect the adhesion, apoptosis and extracellular acidification of single normal cells and single cancer cells. Single living human normal cells WI38, MRC5, and BEAS-2B as well as non-small-cell lung cancer (NSCLC) cells A549, H1299, and CH27 were cultivated separately inside the isolation window. The current flowing through the PSW channel was measured. From the PSW channel current change as a function of time, we determined the cell adhesion time by observing the time required for the current change to saturate, since a stable extracellular ion density was established after the cells were completely adhered to the PSW surface. The apoptosis of cells can also be determined when the channel current change drops to zero. We found that all the NSCLC cells had a higher channel current change and hence a lower pH value than the normal cells anytime after they were seeded. The corresponding average pH values were 5.86 for A549, 6.00 for H1299, 6.20 for CH27, 6.90 for BEAS-2B, 6.96for MRC5, and 7.02 for WI38, respectively, after the cells were completely adhered to the PSW surface. Our results show that NSCLC cells have a stronger cell-substrate adhesion and a higher extracellular acidification rate than normal cells.

  12. Microorganisms and Chemical Pollution

    ERIC Educational Resources Information Center

    Alexander, M.

    1973-01-01

    Discusses the importance of microorganisms in chemical pollution and pollution abatement. Selected chemical pollutants are chosen to illustrate that microorganisms synthesize hazardous substances from reasonably innocuous precursors, while others act as excellent environmental decontaminating agents by removing undesirable natural and synthetic…

  13. Microorganisms and Chemical Pollution

    ERIC Educational Resources Information Center

    Alexander, M.

    1973-01-01

    Discusses the importance of microorganisms in chemical pollution and pollution abatement. Selected chemical pollutants are chosen to illustrate that microorganisms synthesize hazardous substances from reasonably innocuous precursors, while others act as excellent environmental decontaminating agents by removing undesirable natural and synthetic…

  14. Microorganisms of the San Francisco sour dough bread process. II. Isolation and characterization of undescribed bacterial species responsible for the souring activity.

    PubMed

    Kline, L; Sugihara, T F

    1971-03-01

    A medium was developed which permitted isolation, apparently for the first time, of the bacteria responsible for the acid production in the 100-year-old San Francisco sour dough French bread process. Some of the essential ingredients of this medium included a specific requirement for maltose at a high level, Tween 80, freshly prepared yeast extractives, and an initial pH of not over 6.0. The bacteria were gram-positive, nonmotile, catalase-negative, short to medium slender rods, indifferent to oxygen, and producers of lactic and acetic acids with the latter varying from 3 to 26% of the total. Carbon dioxide was also produced. Their requirement for maltose for rapid and heavy growth and a proclivity for forming involuted, filamentous, and pleomorphic forms raises a question as to whether they should be properly grouped with the heterofermentative lactobacilli.

  15. Tensile properties of single stress fibers isolated from cultured vascular smooth muscle cells.

    PubMed

    Deguchi, Shinji; Ohashi, Toshiro; Sato, Masaaki

    2006-01-01

    Stress fibers (SFs), a contractile bundle of actin filaments, play a critical role in mechanotransduction in adherent cells; yet, the mechanical properties of SFs are poorly understood. Here, we measured tensile properties of single SFs by in vitro manipulation with cantilevers. SFs were isolated from cultured vascular smooth muscle cells with a combination of low ionic-strength extraction and detergent extraction and were stretched until breaking. The breaking force and the Young's modulus (assuming that SFs were isotropic) were, on average, 377 nN and 1.45 MPa, which were approximately 600-fold greater and three orders of magnitude lower, respectively, than those of actin filaments reported previously. Strain-induced stiffening was observed in the force-strain curve. We also found that the extracted SFs shortened to approximately 80% of the original length in an ATP-independent manner after they were dislodged from the substrate, suggesting that SFs had preexisting strain in the cytoplasm. The force required for stretching the single SFs from the zero-stress length back to the original length was approximately 10 nN, which was comparable with the traction force level applied by adherent cells at single adhesion sites to maintain cell integrity. These results suggest that SFs can bear intracellular stresses that may affect overall cell mechanical properties and will impact interpretation of intracellular stress distribution and force-transmission mechanism in adherent cells.

  16. Multiple infection and microdiversity among Helicobacter pylori isolates in a single host in India.

    PubMed

    Patra, Rajashree; Chattopadhyay, Santanu; De, Ronita; Ghosh, Prachetash; Ganguly, Mou; Chowdhury, Abhijit; Ramamurthy, T; Nair, G B; Mukhopadhyay, Asish K

    2012-01-01

    Helicobacter pylori is one of the most diverse bacterial species that chronically infects more than 70% of Indian population. Interestingly, data showing microdiversity of the H. pylori strains within a particular gastric niche remained scarce. To understand the extent of genetic diversity among H. pylori strains within a given host, 30 patients with gastro-duodenal problems were subjected to endoscopy and from each patient 10 single colonies were isolated. Characterization of each of these 10 single colonies by DNA fingerprinting as well as genotyping of several important genetic markers viz. cagA, vacA, iceA, vapD, cag PAI empty site, IS605, RFLP and two other genetic segments within cag PAI revealed that all of the 30 patients were infected with more than one strain and sometimes strains with 5 to 6 types of genetic variants. Analyses of certain genetic loci showed the microdiversity among the colonies from single patient, which may be due to the recombination events during long-term carriage of the pathogen. These results suggest that most of the patients have acquired H. pylori due to repeated exposure to this pathogen with different genetic make-up, which may increase the possibility of super infections. Genetic exchanges between these unrelated H. pylori strains may support certain H. pylori variant to grow better in a given host than the parental strain and thereby increasing the possibility for the severity of the infection.

  17. Antifungal and antibacterial activity of marine microorganisms.

    PubMed

    El Amraoui, B; El Amraoui, M; Cohen, N; Fassouane, A

    2014-03-01

    In order to explore marine microorganisms with pharmaceutical potential, marine bacteria, collected from different coastal areas of the Moroccan Atlantic Ocean, were previously isolated from seawater, sediment, marine invertebrates and seaweeds. The antimicrobial activities of these microorganisms were investigated against the pathogens involved in human pathologies. Whole cultures of 34 marine microorganisms were screened for antimicrobial activities using the method of agar diffusion against three Gram-positive bacteria, two Gram-negative bacteria, and against yeast. The results showed that among the 34 isolates studied, 28 (82%) strains have antimicrobial activity against at least one pathogen studied, 11 (32%) strains have antifungal activity and 24 (76%) strains are active against Gram-positive bacteria, while 21 (62%) strains are active against Gram-negative bacteria. Among isolates having antimicrobial activity, 14 were identified and were assigned to the genera Acinetobacter, Aeromonas, Alcaligenes, Bacillus, Chromobacterium, Enterococcus, Pantoea and Pseudomonas. Due to a competitive role for space and nutrient, the marine microorganisms can produce antibiotic substance; therefore, these marine microorganisms were expected to be potential resources of natural antibiotic products.

  18. Generation and isolation of target-specific single-domain antibodies from shark immune repertoires.

    PubMed

    Müller, Mischa Roland; O'Dwyer, Ronan; Kovaleva, Marina; Rudkin, Fiona; Dooley, Helen; Barelle, Caroline Jane

    2012-01-01

    The drive to exploit novel targets and biological pathways has lead to the expansion of classical antibody research into innovative fragment adaptations and novel scaffolds. The hope being that alternative or cryptic epitopes may be targeted, tissue inaccessibility may be overcome, and easier engineering options will facilitate multivalent, multi-targeting approaches. To this end, we have been isolating shark single domains to gain a greater understanding of their potential as therapeutic agents. Their unique shape, small size, inherent stability, and simple molecular architecture make them attractive candidates from a drug discovery perspective. Here we describe protocols to capture the immune repertoire of an immunized shark species and to build and select via phage-display target-specific IgNAR variable domains (VNARs).

  19. Transfer doping of single isolated nanodiamonds, studied by scanning probe microscopy techniques.

    PubMed

    Bolker, Asaf; Saguy, Cecile; Kalish, Rafi

    2014-09-26

    The transfer doping of diamond surfaces has been applied in various novel two-dimensional electronic devices. Its extension to nanodiamonds (ND) is essential for ND-based applications in many fields. In particular, understanding the influence of the crystallite size on transfer doping is desirable. Here, we report the results of a detailed study of the electronic energetic band structure of single, isolated transfer-doped nanodiamonds with nanometric resolution using a combination of scanning tunneling spectroscopy and Kelvin force microscopy measurements. The results show how the band gap, the valence band maximum, the electron affinity and the work function all depend on the ND's size and nanoparticle surface properties. The present analysis, which combines information from both scanning tunneling spectroscopy and Kelvin force microscopy, should be applicable to any nanoparticle or surface that can be measured with scanning probe techniques.

  20. Transfer doping of single isolated nanodiamonds, studied by scanning probe microscopy techniques

    NASA Astrophysics Data System (ADS)

    Bolker, Asaf; Saguy, Cecile; Kalish, Rafi

    2014-09-01

    The transfer doping of diamond surfaces has been applied in various novel two-dimensional electronic devices. Its extension to nanodiamonds (ND) is essential for ND-based applications in many fields. In particular, understanding the influence of the crystallite size on transfer doping is desirable. Here, we report the results of a detailed study of the electronic energetic band structure of single, isolated transfer-doped nanodiamonds with nanometric resolution using a combination of scanning tunneling spectroscopy and Kelvin force microscopy measurements. The results show how the band gap, the valence band maximum, the electron affinity and the work function all depend on the ND’s size and nanoparticle surface properties. The present analysis, which combines information from both scanning tunneling spectroscopy and Kelvin force microscopy, should be applicable to any nanoparticle or surface that can be measured with scanning probe techniques.

  1. Controlling field-emission patterns of isolated single-walled carbon nanotube rope

    NASA Astrophysics Data System (ADS)

    Tong, Yu; Lim, Seong Chu; Park, Kyung Ah; Jeong, Hee Jin; Jeong, Seung Yoi; Lee, Young Hee; Liu, Chang; Cheng, Hui-Ming; Choi, Yoon

    2005-07-01

    We report a method of controlling field-emission patterns from an isolated single-walled carbon nanotube (SWCNT) rope. By positioning two soda-lime glass flakes on both sides of a SWCNT rope, we found an anomalous current jump, enlarging the field emission current above the threshold bias voltage. The electron trajectories were systematically controlled with different configurations of glass flakes. This was explained by the induced charges on the surface of the dielectric that modified the electric field distribution near the cathode and anode, and hence, the electron trajectories and the field emission patterns as well. This opens a possibility of tuning electron beam trajectories in field emission that can be applied to various electron sources such as field emission displays and cold cathode lamps.

  2. Extensive proliferative capacity of single isolated CD34 human cord blood cells in suspension culture.

    PubMed

    Xiao, M; Broxmeyer, H E; Horie, M; Grigsby, S; Lu, L

    1994-01-01

    Nonadherent, low-density T-lymphocyte-depleted (NALT-) CD34 cells from normal human cord blood were assessed in suspension culture for the effects of recombinant cytokines on their proliferation, differentiation, and generation of myeloid progenitor cells. In this cell population, 82% of cells expressed c-kit protein as assessed by in situ hybridization, and their cloning efficiency was 85% when cells were plated at low cell numbers with combinations of growth factors. CD34 cells were sorted as 1, 5, or 10 cell(s) per well and also at 5000 cells per dish to initiate stromal-free suspension cultures in the presence of steel factor (SLF), interleukin (IL)-1 alpha, and IL-3. Forty-eight percent of the wells started with a single CD34 cell were positive for growth after 14 days, and the wells contained greater than 5 x 10(3) cells by 21-28 days. Progenitors were assayed weekly with cultures initiated with 1 or 5000 cells. While the fold expansion of nucleated cells was greater in cultures initiated with one cell per well (> 5000 compared to 791-fold expansion for 5000 cells), the fold expansion of progenitors was greater than 5000 cells were used to initiate cultures. Under optimal conditions, there was, respectively, a 160-, 164-, and 57-fold output of high proliferative potential colony-forming cells, granulocyte-macrophage colony-forming units, and erythroid burst-forming units/granulocyte erythroid macrophage megakaryocyte colony-forming units within 1-3 weeks for cultures initiated with 5000 CD34 cells compared with respective fold increases of 29, 16, and 1, for single-initiated cultures. These results demonstrate the expansion capacity of single CD34 cord blood cells and demonstrate that factors in addition to SLF, IL-1 alpha, and IL-3 are necessary for optimal expansion of progenitors from single isolated CD34 cells.

  3. Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli.

    PubMed

    Ilatovskaya, Daria V; Palygin, Oleg; Levchenko, Vladislav; Staruschenko, Alexander

    2015-06-27

    Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca(2+) concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

  4. G-band resonant Raman study of 62 isolated single-wall carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Jorio, A.; Souza Filho, A. G.; Dresselhaus, G.; Dresselhaus, M. S.; Swan, A. K.; Ünlü, M. S.; Goldberg, B. B.; Pimenta, M. A.; Hafner, J. H.; Lieber, C. M.; Saito, R.

    2002-04-01

    We report G-band resonance Raman spectra of single-wall carbon nanotubes (SWNTs) at the single-nanotube level. By measuring 62 different isolated SWNTs resonant with the incident laser, and having diameters dt ranging between 0.95 nm and 2.62 nm, we have conclusively determined the dependence of the two most intense G-band features on the nanotube structure. The higher-frequency peak is not diameter dependent (ω+G=1591 cm-1), while the lower-frequency peak is given by ω-G=ω+G-C/d2t, with C being different for metallic and semiconducting SWNTs (CM>CS). The peak frequencies do not depend on nanotube chiral angle. The intensity ratio between the two most intense features is in the range 0.1isolated SWNTs (~90%). Unusually high or low Iω-G/Iω+G ratios are observed for a few spectra coming from SWNTs under special resonance conditions, i.e., SWNTs for which the incident photons are in resonance with the ES44 interband transition and scattered photons are in resonance with ES33. Since the Eii values depend sensitively on both nanotube diameter and chirality, the (n,m) SWNTs that should exhibit such a special G-band spectra can be predicted by resonance Raman theory. The agreement between theoretical predictions and experimental observations about these special G-band phenomena gives additional support for the (n,m) assignment from resonance Raman spectroscopy.

  5. Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

    PubMed Central

    Ilatovskaya, Daria V.; Palygin, Oleg; Levchenko, Vladislav; Staruschenko, Alexander

    2015-01-01

    Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels. PMID:26167808

  6. Rapid discrimination of Salmonella isolates by single-strand conformation polymorphism analysis.

    PubMed

    Al-Adhami, Batol H; Huby-Chilton, Florence; Blais, Burton W; Martinez-Perez, Amalia; Chilton, Neil B; Gajadhar, Alvin A

    2008-10-01

    A molecular typing technique was developed for the differentiation of Salmonella isolates based on single-strand conformation polymorphism (SSCP) analysis of amplicons generated by PCR. Amplicons from parts of the fimA (both the 5' and 3' ends), mdh, invA, and atpD genes were generated separately from a panel of Salmonella strains representing Salmonella bongori, and four subspecies and 17 serovars of Salmonella enterica. These amplicons were subjected to SSCP analysis for differentiation of the salmonellae on the basis of different conformational forms arising due to nucleotide sequence variations in the target genes. Several distinct SSCP banding patterns (a maximum of 14 each for atpD and fimA 3' end) were observed with this panel of Salmonella strains for amplicons generated from each target gene. The best discrimination of Salmonella subspecies and serovar was achieved from the SSCP analysis of a combination of at least three gene targets: atpD, invA, and either mdh or fimA 3' end. This demonstrates the applicability of SSCP analysis as an important additional method to classical typing approaches for the differentiation of foodborne Salmonella isolates. SSCP is simple to perform and should be readily transferable to food microbiology laboratories with basic PCR capability.

  7. New Method to Disaggregate and Analyze Single Isolated Helminthes Cells Using Flow Cytometry: Proof of Concept

    PubMed Central

    Nava-Castro, Karen; Hernández-Bello, Romel; Muñiz-Hernández, Saé; Escobedo, Galileo; Morales-Montor, Jorge

    2011-01-01

    In parasitology, particularly in helminthes studies, several methods have been used to look for the expression of specific molecules, such as RT-PCR, western blot, 2D-electrophoresis, and microscopy, among others. However, these methods require homogenization of the whole helminth parasite, preventing evaluation of individual cells or specific cell types in a given parasite tissue or organ. Also, the extremely high interaction between helminthes and host cells (particularly immune cells) is an important point to be considered. It is really hard to obtain fresh parasites without host cell contamination. Then, it becomes crucial to determine that the analyzed proteins are exclusively from parasitic origin, and not a consequence of host cell contamination. Flow cytometry is a fluorescence-based technique used to evaluate the expression of extra-and intracellular proteins in different type cells, including protozoan parasites. It also allows the isolation and recovery of single-cell populations. Here, we describe a method to isolate and obtain purified helminthes cells. PMID:22187522

  8. Perventricular device closure of isolated muscular ventricular septal defect in infants: A single centre experience☆

    PubMed Central

    Thakkar, Bhavesh; Patel, Nehal; Shah, Shaunak; Poptani, Vishal; Madan, Tarun; Shah, Chirag; Shukla, Anand; Prajapati, Vaishali

    2012-01-01

    Objectives To evaluate prospective single centre experience of mid-term safety and efficacy of perventricular device closure of isolated large muscular ventricular septal defect (mVSD) in high-risk infants. Background Surgical closures of large mVSD in infants represent a challenge with significant morbidity. Methods Between August 2008–2010, perventricular closure was attempted in 24 infants of 6.01 ± 2.37 months age and 4.27 ± 0.56 kg weight under TEE guidance. Results The device was successfully deployed in 21/24 infants. Size of mVSD was 8.42 ± 1.46 mm (6.1–12 mm). Mean procedure time was 28.8 ± 11.7 min. The closure rate was 84% immediately and 100% at 6 months. Four patients suffered major complications: 2-died, 1-esophageal perforation, 1-persistent CHB. At 26.23 ± 6.63 months follow-up two patients were symptomatic: 1-required device retrieval, 1-died of severe gastroenteritis. Conclusion Perventricular device closure of isolated mVSD appears feasible option at mid-term follow-up and may either substitute or complement the conventional surgical technique in selected cases depending on institutional paediatric cardiac surgery performance. PMID:23253407

  9. New method to disaggregate and analyze single isolated helminthes cells using flow cytometry: proof of concept.

    PubMed

    Nava-Castro, Karen; Hernández-Bello, Romel; Muñiz-Hernández, Saé; Escobedo, Galileo; Morales-Montor, Jorge

    2011-01-01

    In parasitology, particularly in helminthes studies, several methods have been used to look for the expression of specific molecules, such as RT-PCR, western blot, 2D-electrophoresis, and microscopy, among others. However, these methods require homogenization of the whole helminth parasite, preventing evaluation of individual cells or specific cell types in a given parasite tissue or organ. Also, the extremely high interaction between helminthes and host cells (particularly immune cells) is an important point to be considered. It is really hard to obtain fresh parasites without host cell contamination. Then, it becomes crucial to determine that the analyzed proteins are exclusively from parasitic origin, and not a consequence of host cell contamination. Flow cytometry is a fluorescence-based technique used to evaluate the expression of extra-and intracellular proteins in different type cells, including protozoan parasites. It also allows the isolation and recovery of single-cell populations. Here, we describe a method to isolate and obtain purified helminthes cells.

  10. The isolation of an RNA aptamer targeting to p53 protein with single amino acid mutation

    PubMed Central

    Chen, Liang; Rashid, Farooq; Shah, Abdullah; Awan, Hassaan M.; Wu, Mingming; Liu, An; Wang, Jun; Zhu, Tao; Luo, Zhaofeng; Shan, Ge

    2015-01-01

    p53, known as a tumor suppressor, is a DNA binding protein that regulates cell cycle, activates DNA repair proteins, and triggers apoptosis in multicellular animals. More than 50% of human cancers contain a mutation or deletion of the p53 gene, and p53R175 is one of the hot spots of p53 mutation. Nucleic acid aptamers are short single-stranded oligonucleotides that are able to bind various targets, and they are typically isolated from an experimental procedure called systematic evolution of ligand exponential enrichment (SELEX). Using a previously unidentified strategy of contrast screening with SELEX, we have isolated an RNA aptamer targeting p53R175H. This RNA aptamer (p53R175H-APT) has a significantly stronger affinity to p53R175H than to the wild-type p53 in both in vitro and in vivo assays. p53R175H-APT decreased the growth rate, weakened the migration capability, and triggered apoptosis in human lung cancer cells harboring p53R175H. Further analysis actually indicated that p53R175H-APT might partially rescue or correct the p53R175H to function more like the wild-type p53. In situ injections of p53R175H-APT to the tumor xenografts confirmed the effects of this RNA aptamer on p53R175H mutation in mice. PMID:26216949

  11. The isolation of an RNA aptamer targeting to p53 protein with single amino acid mutation.

    PubMed

    Chen, Liang; Rashid, Farooq; Shah, Abdullah; Awan, Hassaan M; Wu, Mingming; Liu, An; Wang, Jun; Zhu, Tao; Luo, Zhaofeng; Shan, Ge

    2015-08-11

    p53, known as a tumor suppressor, is a DNA binding protein that regulates cell cycle, activates DNA repair proteins, and triggers apoptosis in multicellular animals. More than 50% of human cancers contain a mutation or deletion of the p53 gene, and p53R175 is one of the hot spots of p53 mutation. Nucleic acid aptamers are short single-stranded oligonucleotides that are able to bind various targets, and they are typically isolated from an experimental procedure called systematic evolution of ligand exponential enrichment (SELEX). Using a previously unidentified strategy of contrast screening with SELEX, we have isolated an RNA aptamer targeting p53R175H. This RNA aptamer (p53R175H-APT) has a significantly stronger affinity to p53R175H than to the wild-type p53 in both in vitro and in vivo assays. p53R175H-APT decreased the growth rate, weakened the migration capability, and triggered apoptosis in human lung cancer cells harboring p53R175H. Further analysis actually indicated that p53R175H-APT might partially rescue or correct the p53R175H to function more like the wild-type p53. In situ injections of p53R175H-APT to the tumor xenografts confirmed the effects of this RNA aptamer on p53R175H mutation in mice.

  12. Improvement of phytoplankton culture isolation using single cell sorting by flow cytometry.

    PubMed

    Marie, Dominique; Le Gall, Florence; Edern, Roseline; Gourvil, Priscillia; Vaulot, Daniel

    2017-04-01

    Flow cytometry provides a tool to physically sort single algal cells in order to obtain clonal cultures. During sorting, cells are submitted to physical stress factors such as high fluidic pressure, exposure to the laser beam, electrostatic charges, deflection through high voltage fields, and collisions with container surfaces. All of these can damage the cells of interest and success rates for initiation of cultures from flow-sorted cells are generally very low. We found that the addition of bovine serum albumin in the culture medium into which cells were sorted drastically improved the success of initiation of pico- and nano-eukaryotic phytoplankton strains. Adding a mixture of antibiotics (Penicillin, Neomycin, Streptomycin) to the medium in order to slow down bacterial growth further improved culture development. This approach was successfully used to isolate taxonomically diverse strains, including novel taxa, from a fresh sample obtained in the English Channel and from enrichment cultures established during an Atlantic meridional transect cruise. We anticipate that these improvements will be useful to clone or purify existing cultures and to isolate novel cultures from oceanic samples. © 2016 Phycological Society of America.

  13. Genotyping of Single Nucleotide Polymorphisms in DNA Isolated from Serum Using Sequenom MassARRAY Technology.

    PubMed

    Clendenen, Tess V; Rendleman, Justin; Ge, Wenzhen; Koenig, Karen L; Wirgin, Isaac; Currie, Diane; Shore, Roy E; Kirchhoff, Tomas; Zeleniuch-Jacquotte, Anne

    2015-01-01

    Large epidemiologic studies have the potential to make valuable contributions to the assessment of gene-environment interactions because they prospectively collected detailed exposure data. Some of these studies, however, have only serum or plasma samples as a low quantity source of DNA. We examined whether DNA isolated from serum can be used to reliably and accurately genotype single nucleotide polymorphisms (SNPs) using Sequenom multiplex SNP genotyping technology. We genotyped 81 SNPs using samples from 158 participants in the NYU Women's Health Study. Each participant had DNA from serum and at least one paired DNA sample isolated from a high quality source of DNA, i.e. clots and/or cell precipitates, for comparison. We observed that 60 of the 81 SNPs (74%) had high call frequencies (≥95%) using DNA from serum, only slightly lower than the 85% of SNPs with high call frequencies in DNA from clots or cell precipitates. Of the 57 SNPs with high call frequencies for serum, clot, and cell precipitate DNA, 54 (95%) had highly concordant (>98%) genotype calls across all three sample types. High purity was not a critical factor to successful genotyping. Our results suggest that this multiplex SNP genotyping method can be used reliably on DNA from serum in large-scale epidemiologic studies.

  14. Molecular Genetic Characterization of Individual Cancer Cells Isolated via Single-Cell Printing

    PubMed Central

    Riba, Julian; Renz, Nathalie; Niemöller, Christoph; Bleul, Sabine; Pfeifer, Dietmar; Stosch, Juliane M.; Metzeler, Klaus H.; Hackanson, Björn; Lübbert, Michael; Duyster, Justus; Koltay, Peter; Zengerle, Roland; Claus, Rainer

    2016-01-01

    Intratumoral genetic heterogeneity may impact disease outcome. Gold standard for dissecting clonal heterogeneity are single-cell analyses. Here, we present an efficient workflow based on an advanced Single-Cell Printer (SCP) device for the study of gene variants in single cancer cells. To allow for precise cell deposition into microwells the SCP was equipped with an automatic dispenser offset compensation, and the 384-microwell plates were electrostatically neutralized. The ejection efficiency was 99.7% for fluorescent beads (n = 2304) and 98.7% for human cells (U-2 OS or Kasumi-1 cancer cell line, acute myeloid leukemia [AML] patient; n = 150). Per fluorescence microscopy, 98.8% of beads were correctly delivered into the wells. A subset of single cells (n = 81) was subjected to whole genome amplification (WGA), which was successful in all cells. On empty droplets, a PCR on LINE1 retrotransposons yielded no product after WGA, verifying the absence of free-floating DNA in SCP-generated droplets. Representative gene variants identified in bulk specimens were sequenced in single-cell WGA DNA. In U-2 OS, 22 of 25 cells yielded results for both an SLC34A2 and TET2 mutation site, including cells harboring the SLC34A2 but not the TET2 mutation. In one cell, the TET2 mutation analysis was inconclusive due to allelic dropout, as assessed via polymorphisms located close to the mutation. Of Kasumi-1, 23 of 33 cells with data on both the KIT and TP53 mutation site harbored both mutations. In the AML patient, 21 of 23 cells were informative for a TP53 polymorphism; the identified alleles matched the loss of chromosome arm 17p. The advanced SCP allows efficient, precise and gentle isolation of individual cells for subsequent WGA and routine PCR/sequencing-based analyses of gene variants. This makes single-cell information readily accessible to a wide range of applications and can provide insights into clonal heterogeneity that were indeterminable solely by analyses of bulk

  15. Molecular Genetic Characterization of Individual Cancer Cells Isolated via Single-Cell Printing.

    PubMed

    Riba, Julian; Renz, Nathalie; Niemöller, Christoph; Bleul, Sabine; Pfeifer, Dietmar; Stosch, Juliane M; Metzeler, Klaus H; Hackanson, Björn; Lübbert, Michael; Duyster, Justus; Koltay, Peter; Zengerle, Roland; Claus, Rainer; Zimmermann, Stefan; Becker, Heiko

    Intratumoral genetic heterogeneity may impact disease outcome. Gold standard for dissecting clonal heterogeneity are single-cell analyses. Here, we present an efficient workflow based on an advanced Single-Cell Printer (SCP) device for the study of gene variants in single cancer cells. To allow for precise cell deposition into microwells the SCP was equipped with an automatic dispenser offset compensation, and the 384-microwell plates were electrostatically neutralized. The ejection efficiency was 99.7% for fluorescent beads (n = 2304) and 98.7% for human cells (U-2 OS or Kasumi-1 cancer cell line, acute myeloid leukemia [AML] patient; n = 150). Per fluorescence microscopy, 98.8% of beads were correctly delivered into the wells. A subset of single cells (n = 81) was subjected to whole genome amplification (WGA), which was successful in all cells. On empty droplets, a PCR on LINE1 retrotransposons yielded no product after WGA, verifying the absence of free-floating DNA in SCP-generated droplets. Representative gene variants identified in bulk specimens were sequenced in single-cell WGA DNA. In U-2 OS, 22 of 25 cells yielded results for both an SLC34A2 and TET2 mutation site, including cells harboring the SLC34A2 but not the TET2 mutation. In one cell, the TET2 mutation analysis was inconclusive due to allelic dropout, as assessed via polymorphisms located close to the mutation. Of Kasumi-1, 23 of 33 cells with data on both the KIT and TP53 mutation site harbored both mutations. In the AML patient, 21 of 23 cells were informative for a TP53 polymorphism; the identified alleles matched the loss of chromosome arm 17p. The advanced SCP allows efficient, precise and gentle isolation of individual cells for subsequent WGA and routine PCR/sequencing-based analyses of gene variants. This makes single-cell information readily accessible to a wide range of applications and can provide insights into clonal heterogeneity that were indeterminable solely by analyses of bulk

  16. Enumeration of petroleum-degrading microorganisms.

    PubMed Central

    Walker, J D; Colwell, R R

    1976-01-01

    A variety of factors, including concentration of oil, antibiotics, dyes, and inoculum washes, were examined to determine their effect on the total counts of microorganisms on oil-containing media. The media found to be best for enumerating petroleum-degrading microorganisms contained 0.5% (vol/vol) oil and 0.003% phenol red, with Fungizone added for isolating bacteria and streptomycin and tetracycline added for isolating yeasts and fungi. Washing the inoculum did not improve recovery of petroleum degraders. Specifically, silica gel-oil medium and a yeast medium are recommended for enumeration of petroleum-degrading bacteria and yeasts and fungi, respectively. It is suggested that counts of petroleum degraders be expressed as percentage of the total population rather than total numbers of petroleum degraders per se. Incubation temperature and presence of oil was found to influence the numbers of petroleum-degrading microorganisms at a given sampling site. PMID:999272

  17. An adaptation of the Wiener filter suitable for analyzing images of isolated single particles

    PubMed Central

    Sindelar, Charles V.; Grigorieff, Nikolaus

    2011-01-01

    The Wiener filter is a standard means of optimizing the signal in sums of aligned, noisy images obtained by electron cryo-microscopy (cryo-EM). However, estimation of the resolution-dependent (“spectral”) signal-to-noise ratio (SSNR) from the input data has remained problematic, and error reduction due to specific application of the SSNR term within a Wiener filter has not been reported. Here we describe an adjustment to the Wiener filter for optimal summation of images of isolated particles surrounded by large regions of featureless background, as is typically the case in single-particle cryo-EM applications. We show that the density within the particle area can be optimized, in the least-squares sense, by scaling the SSNR term found in the conventional Wiener filter by a factor that reflects the fraction of the image field occupied by the particle. We also give related expressions that allow the SSNR to be computed for application in this new filter, by incorporating a masking step into a Fourier Ring Correlation (FRC), a standard resolution measure. Furthermore, we show that this masked FRC estimation scheme substantially improves on the accuracy of conventional SSNR estimation methods. We demonstrate the validity of our new approach in numeric tests with simulated data corresponding to realistic cryo-EM imaging conditions. This variation of the Wiener filter and accompanying derivation should prove useful for a variety of single-particle cryo-EM applications, including 3D reconstruction. PMID:21757012

  18. Microfluidic cell isolation technology for drug testing of single tumor cells and their clusters

    PubMed Central

    Bithi, Swastika S.; Vanapalli, Siva A.

    2017-01-01

    Drug assays with patient-derived cells such as circulating tumor cells requires manipulating small sample volumes without loss of rare disease-causing cells. Here, we report an effective technology for isolating and analyzing individual tumor cells and their clusters from minute sample volumes using an optimized microfluidic device integrated with pipettes. The method involves using hand pipetting to create an array of cell-laden nanoliter-sized droplets immobilized in a microfluidic device without loss of tumor cells during the pipetting process. Using this technology, we demonstrate single-cell analysis of tumor cell response to the chemotherapy drug doxorubicin. We find that even though individual tumor cells display diverse uptake profiles of the drug, the onset of apoptosis is determined by accumulation of a critical intracellular concentration of doxorubicin. Experiments with clusters of tumor cells compartmentalized in microfluidic drops reveal that cells within a cluster have higher viability than their single-cell counterparts when exposed to doxorubicin. This result suggests that circulating tumor cell clusters might be able to better survive chemotherapy drug treatment. Our technology is a promising tool for understanding tumor cell-drug interactions in patient-derived samples including rare cells. PMID:28150812

  19. Archival Isolates Confirm a Single Topotype of West Nile Virus in Australia

    PubMed Central

    Huang, Bixing; Prow, Natalie A; van den Hurk, Andrew F.; Allcock, Richard J. N.; Moore, Peter R.; Doggett, Stephen L.; Warrilow, David

    2016-01-01

    West Nile virus is globally wide-spread and causes significant disease in humans and animals. The evolution of West Nile virus Kunjin subtype in Australia (WNVKUN) was investigated using archival samples collected over a period of 50 years. Based on the pattern of fixed amino acid substitutions and time-stamped molecular clock analyses, a single long-term lineage (or topotype) was inferred. This implies that a bottleneck exists such that regional strains eventually die out and are replaced with strains from a single source. This was consistent with current hypotheses regarding the distribution of WNVKUN, whereby the virus is enzootic in northern Australia and is disseminated to southern states by water-birds or mosquitoes after flooding associated with above average rainfall. In addition, two previous amino acid changes associated with pathogenicity, an N-Y-S glycosylation motif in the envelope protein and a phenylalanine at amino acid 653 in the RNA polymerase, were both detected in all isolates collected since the 1980s. Changes primarily occurred due to stochastic drift. One fixed substitution each in NS3 and NS5, subtly changed the chemical environment of important functional groups, and may be involved in fine-tuning RNA synthesis. Understanding these evolutionary changes will help us to better understand events such as the emergence of the virulent strain in 2011. PMID:27906966

  20. Micro-Organ Device

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R. (Inventor); Chang, Robert C. (Inventor); Starly, Binil (Inventor); Culbertson, Christopher (Inventor); Holtorf, Heidi L. (Inventor); Sun, Wei (Inventor); Leslie, Julia (Inventor)

    2013-01-01

    A method for fabricating a micro-organ device comprises providing a microscale support having one or more microfluidic channels and one or more micro-chambers for housing a micro-organ and printing a micro-organ on the microscale support using a cell suspension in a syringe controlled by a computer-aided tissue engineering system, wherein the cell suspension comprises cells suspended in a solution containing a material that functions as a three-dimensional scaffold. The printing is performed with the computer-aided tissue engineering system according to a particular pattern. The micro-organ device comprises at least one micro-chamber each housing a micro-organ; and at least one microfluidic channel connected to the micro-chamber, wherein the micro-organ comprises cells arranged in a configuration that includes microscale spacing between portions of the cells to facilitate diffusion exchange between the cells and a medium supplied from the at least one microfluidic channel.

  1. Micro-organ device

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R. (Inventor); von Gustedt-Gonda, legal representative, Iris (Inventor); Chang, Robert C. (Inventor); Starly, Binil (Inventor); Culbertson, Christopher (Inventor); Holtorf, Heidi L. (Inventor); Sun, Wei (Inventor); Leslie, Julia (Inventor)

    2013-01-01

    A method for fabricating a micro-organ device comprises providing a microscale support having one or more microfluidic channels and one or more micro-chambers for housing a micro-organ and printing a micro-organ on the microscale support using a cell suspension in a syringe controlled by a computer-aided tissue engineering system, wherein the cell suspension comprises cells suspended in a solution containing a material that functions as a three-dimensional scaffold. The printing is performed with the computer-aided tissue engineering system according to a particular pattern. The micro-organ device comprises at least one micro-chamber each housing a micro-organ; and at least one microfluidic channel connected to the micro-chamber, wherein the micro-organ comprises cells arranged in a configuration that includes microscale spacing between portions of the cells to facilitate diffusion exchange between the cells and a medium supplied from the at least one microfluidic channel.

  2. Resistance profiles and risk factors of resistant microorganisms in bacteraemia of abdominal origin.

    PubMed

    Martín Jaramago, J; Armero Ibáñez, R; Camarena Miñana, J J; Morales Suárez-Varela, M

    2017-04-20

    The presence of resistant microorganisms is a major cause of failure in initial empirical antimicrobial therapy. The objectives of this study are to determine the resistance profile of microorganisms that cause bacteraemia of abdominal origin and to identify whether the previous use of antibiotics and the place of acquisition of bacteraemia are risk factors associated with the presence of resistant organisms. A clinical, observational, epidemiological, retrospective cohort study was conducted with all the adult patients admitted to a university hospital from 2011-2013. Antimicrobial resistance profiles were described and a 95% confidence interval chi-square test was used to determine whether the variables studied were risk factors in the isolation of resistant microorganisms. Of the 1245 patients with bacteraemia, 212 (17%) presented bacteraemia of abdominal origin. The resistance profile highlights the incidence of methicillin resistant Staphylococcus aureus (50%), coagulase-negative staphylococci resistant to linezolid (20.58%), enterococci resistant to vancomycin (3.12%), Escherichia coli resistant to third-generation cephalosporins (9.9%) and fluoroquinolones (35.64%), Klebsiella pneumoniae resistant to third-generation cephalosporins (8.33%), Pseudomonas aeruginosa resistant to fluoroquinolones and carbapenem (25% and 25% respectively) and Acinetobacter baumanii resistant to fluoroquinolones and carbapenem (100% and 100% respectively), Candida albicans resistant to fluconazole (11.11%), single Candida krusei isolate resistant to fluconazole and Candida parapsilosis resistant to echinocandins (12.5%). In our study, previous use of antibiotics had a statistically significant association with the isolation of resistant microorganisms (P=.013) but not the place of acquisition of bacteraemia (P=.239). Establishing the incidence of resistant organisms can improve empirical antimicrobial therapy in patients with bacteraemia of abdominal origin. Previous use of

  3. Linezolid minimum inhibitory concentration (MIC) creep in methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates at a single Japanese center.

    PubMed

    Miyazaki, Motoyasu; Nagata, Nobuhiko; Miyazaki, Hiroyuki; Matsuo, Koichi; Takata, Tohru; Tanihara, Shinichi; Kamimura, Hidetoshi

    2014-01-01

    The aim of this study was to evaluate whether linezolid minimum inhibitory concentration (MIC) creep occurred in Staphylococcus aureus clinical isolates, including methicillin-resistant S. aureus (MRSA), over a recent 5-year period at a single Japanese center. A total of 453 MRSA and 195 methicillin-susceptible S. aureus (MSSA) isolates recovered from inpatients from April 1, 2008 to March 31, 2013 were analyzed. The MIC of linezolid was determined by automated Vitek-2 system. The modal MIC, MIC range, MIC50 and MIC90 (MICs required to inhibit the growth of 50% and 90% of organisms, respectively), geometric mean MIC and percentages of susceptible and resistant isolates were evaluated for each fiscal year. None of the S. aureus isolates were resistant to linezolid. Isolates with an MIC of >1 µg/mL were more common in the MSSA samples than in the MRSA samples (91.3% versus 38.2%, p<0.001). The linezolid geometric mean MIC increased by 0.403 µg/mL (from 1.178 in 2008 to 1.582 in 2012) in the MRSA isolates (p=0.006, r(2)=0.945 according to a linear regression analysis) over the 5-year period; however, no increase was observed in the MSSA isolates. The frequency of MRSA isolates with an MIC of 1 µg/mL decreased (from 76.3% in 2008 to 35.4% in 2012) and the isolates with MICs of >1 µg/mL increased over time (from 23.7% in 2008 to 64.6% in 2012). This report demonstrates the occurrence of linezolid MIC creep, as determined using the geometric mean MIC, in MRSA clinical isolates at a single Japanese center.

  4. Application of thermotolerant microorganisms for biofertilizer preparation.

    PubMed

    Chen, Kuo-Shu; Lin, Yann-Shying; Yang, Shang-Shyng

    2007-12-01

    Intensive agriculture is practised in Taiwan, and compost application is very popular as a means of improving the soil physical properties and supplying plant nutrition. We tested the potential of inoculation with thermotolerant microorganisms to shorten the maturity and improve the quality of biofertilizer prepared by composting. Thermotolerant microorganisms were isolated from compost and reinoculated for the preparation of biofertilizer. The physical, chemical and biological properties of the biofertilizer were determined during composting. The effects of biofertilizer application on the growth and yield of rape were also studied. Among 3823 colonies of thermotolerant microorganisms, Streptomyces thermonitrificans NTU-88, Streptococcus sp. NTU-130 and Aspergillus fumigatus NTU-132 exhibited high growth rates and cellulolytic and proteolytic activities. When a mixture of rice straw and swine manure were inoculated with these isolates and composted for 61 days, substrate temperature increased initially and then decreased gradually during composting. Substrate pH increased from 7.3 to 8.5. Microbial inoculation enhanced the rate of maturity, and increased the content of ash and total and immobilized nitrogen, improved the germination rate of alfalfa seed, and decreased the content of total organic carbon and the carbon/nitrogen ratio. Biofertilizer application increased the growth and yield of rape. Inoculation of thermotolerant and thermophilic microorganisms to agricultural waste for biofertilizer preparation enhances the rate of maturity and improves the quality of the resulting biofertilizer. Inoculation of appropriate microorganisms in biofertilizer preparation might be usefully applied to agricultural situations.

  5. Reliable genotyping of the koala (Phascolarctos cinereus) using DNA isolated from a single faecal pellet.

    PubMed

    Wedrowicz, Faye; Karsa, Mawar; Mosse, Jennifer; Hogan, Fiona E

    2013-07-01

    The koala, an Australian icon, has been added to the threatened species list. Rationale for the listing includes proposed declines in population size, threats to populations (e.g. disease) and loss and fragmentation of habitat. There is now an urgent need to obtain accurate data to assess the status of koala populations in Australia, to ensure the long-term viability of this species. Advances in genetic techniques have enabled DNA analysis to study and inform the management of wild populations; however, sampling of individual koalas is difficult in tall, often remote, eucalypt forest. The collection of faecal pellets (scats) from the forest floor presents an opportunistic sampling strategy, where DNA can be collected without capturing or even sighting an individual. Obtaining DNA via noninvasive sampling can be used to rapidly sample a large proportion of a population; however, DNA from noninvasively collected samples is often degraded. Factors influencing DNA quality and quantity include environmental exposure, diet and methods of sample collection, storage and DNA isolation. Reduced DNA quality and quantity can introduce genotyping errors and provide inaccurate DNA profiles, reducing confidence in the ability of such data to inform management/conservation strategies. Here, we present a protocol that produces a reliable individual koala genotype from a single faecal pellet and highlight the importance of optimizing DNA isolation and analysis for the species of interest. This method could readily be adapted for genetic studies of mammals other than koalas, particularly those whose diet contains high proportions of volatile materials that are likely to induce DNA damage.

  6. Isolation and Characterization of a Single-Stranded DNA Virus Infecting Chaetoceros lorenzianus Grunow▿

    PubMed Central

    Tomaru, Yuji; Takao, Yoshitake; Suzuki, Hidekazu; Nagumo, Tamotsu; Koike, Kanae; Nagasaki, Keizo

    2011-01-01

    Diatoms are one of the most significant primary producers in the ocean, and the importance of viruses as a potential source of mortality for diatoms has recently been recognized. Thus far, eight different diatom viruses infecting the genera Rhizosolenia and Chaetoceros have been isolated and characterized to different extents. We report the isolation of a novel diatom virus (ClorDNAV), which causes the lysis of the bloom-forming species Chaetoceros lorenzianus, and show its physiological, morphological, and genomic characteristics. The free virion was estimated to be ∼34 nm in diameter. The arrangement of virus particles appearing in cross-section was basically a random aggregation in the nucleus. Occasionally, distinctive formations such as a ring-like array composed of 9 or 10 spherical virions or a centipede-like array composed of rod-shaped particles were also observed. The latent period and the burst size were estimated to be <48 h and 2.2 × 104 infectious units per host cell, respectively. ClorDNAV harbors a covalently closed circular single-stranded DNA (ssDNA) genome (5,813 nucleotides [nt]) that includes a partially double-stranded DNA region (979 nt). At least three major open reading frames were identified; one showed a high similarity to putative replicase-related proteins of the other ssDNA diatom viruses, Chaetoceros salsugineum DNA virus (previously reported as CsNIV) and Chaetoceros tenuissimus DNA virus. ClorDNAV is the third member of the closed circular ssDNA diatom virus group, the genus Bacilladnavirus. PMID:21666026

  7. Isolation and characterization of Microbulbifer species 6532A degrading seaweed thalli to single cell detritus particles.

    PubMed

    Wakabayashi, Masayuki; Sakatoku, Akihiro; Noda, Fumio; Noda, Minoru; Tanaka, Daisuke; Nakamura, Shogo

    2012-02-01

    To reduce the volume of seaweed wastes and extract polysaccharides, seaweed-degrading bacteria were isolated from drifting macroalgae harvested along the coast of Toyama Bay, Japan. Sixty-four bacterial isolates were capable of degrading "Wakame" (Undaria pinnatifida) thallus fragments into single cell detritus (SCD) particles. Amongst these, strain 6532A was the most active degrader of thallus fragments, and was capable of degrading thallus fragments to SCD particles within a day. Although the sequence similarity of the 16S rRNA gene of strain 6532A was 100% similar to that of Microbulbifer elongatus JAMB-A7, several distinct differences were observed between strains, including motility, morphology, and utilization of D: -arabinose and gelatin. Consequently, strain 6532A was classified as a new Microbulbifer strain, and was designated Microbulbifer sp. 6532A. Strain 6532A was capable of degrading both alginate and cellulose in the culture medium, zymogram analysis of which revealed the presence of multiple alginate lyases and cellulases. To the best of our knowledge, this is the first study to directly demonstrate the existence of these enzymes in Microbulbifer species. Shotgun cloning and sequencing of the alginate lyase gene in 6532A revealed a 1,074-bp open reading frame, which was designated algMsp. The reading frame encoded a PL family seven enzyme composed of 358 amino acids (38,181 Da). With a similarity of 74.2%, the deduced amino acid sequence was most similar to a Saccharophagus enzyme (alg 7C). These findings suggest that algMsp in strain 6532A is a novel alginate lyase gene.

  8. Single-Cell Metabolomics: Changes in the Metabolome of Freshly Isolated and Cultured Neurons

    PubMed Central

    2012-01-01

    Metabolites are involved in a diverse range of intracellular processes, including a cell’s response to a changing extracellular environment. Using single-cell capillary electrophoresis coupled to electrospray ionization mass spectrometry, we investigated how placing individual identified neurons in culture affects their metabolic profile. First, glycerol-based cell stabilization was evaluated using metacerebral neurons from Aplysia californica; the measurement error was reduced from ∼24% relative standard deviation to ∼6% for glycerol-stabilized cells compared to those isolated without glycerol stabilization. In order to determine the changes induced by culturing, 14 freshly isolated and 11 overnight-cultured neurons of two metabolically distinct cell types from A. californica, the B1 and B2 buccal neurons, were characterized. Of the more than 300 distinctive cell-related signals detected, 35 compounds were selected for their known biological roles and compared among each measured cell. Unsupervised multivariate and statistical analysis revealed robust metabolic differences between these two identified neuron types. We then compared the changes induced by overnight culturing; metabolite concentrations were distinct for 26 compounds in the cultured B1 cells. In contrast, culturing had less influence on the metabolic profile of the B2 neurons, with only five compounds changing significantly. As a result of these culturing-induced changes, the metabolic composition of the B1 neurons became indistinguishable from the cultured B2 cells. This observation suggests that the two cell types differentially regulate their in vivo or in vitro metabolomes in response to a changing environment. PMID:23077722

  9. Sexual isolation of male moths explained by a single pheromone response QTL containing four receptor genes.

    PubMed

    Gould, Fred; Estock, Marie; Hillier, N Kirk; Powell, Bekah; Groot, Astrid T; Ward, Catherine M; Emerson, Jennifer L; Schal, Coby; Vickers, Neil J

    2010-05-11

    Long distance sexual communication in moths has fascinated biologists because of the complex, precise female pheromone signals and the extreme sensitivity of males to specific pheromone molecules. Progress has been made in identifying some genes involved in female pheromone production and in male response. However, we have lacked information on the genetic changes involved in evolutionary diversification of these mate-finding mechanisms that is critical to understanding speciation in moths and other taxa. We used a combined quantitative trait locus (QTL) and candidate gene approach to determine the genetic architecture of sexual isolation in males of two congeneric moths, Heliothis subflexa and Heliothis virescens. We report behavioral and neurophysiological evidence that differential male responses to three female-produced chemicals (Z9-14:Ald, Z9-16:Ald, Z11-16:OAc) that maintain sexual isolation of these species are all controlled by a single QTL containing at least four odorant receptor genes. It is not surprising that pheromone receptor differences could control H. subflexa and H. virescens responses to Z9-16:Ald and Z9-14:Ald, respectively. However, central rather than peripheral level control over the positive and negative responses of H. subflexa and H. virescens to Z11-16:OAc had been expected. Tight linkage of these receptor genes indicates that mutations altering male response to complex blends could be maintained in linkage disequilibrium and could affect the speciation process. Other candidate genes such as those coding for pheromone binding proteins did not map to this QTL, but there was some genetic evidence of a QTL for response to Z11-16:OH associated with a sensory neuron membrane protein gene.

  10. Sexual isolation of male moths explained by a single pheromone response QTL containing four receptor genes

    PubMed Central

    Gould, Fred; Estock, Marie; Hillier, N. Kirk; Powell, Bekah; Groot, Astrid T.; Ward, Catherine M.; Emerson, Jennifer L.; Schal, Coby; Vickers, Neil J.

    2010-01-01

    Long distance sexual communication in moths has fascinated biologists because of the complex, precise female pheromone signals and the extreme sensitivity of males to specific pheromone molecules. Progress has been made in identifying some genes involved in female pheromone production and in male response. However, we have lacked information on the genetic changes involved in evolutionary diversification of these mate-finding mechanisms that is critical to understanding speciation in moths and other taxa. We used a combined quantitative trait locus (QTL) and candidate gene approach to determine the genetic architecture of sexual isolation in males of two congeneric moths, Heliothis subflexa and Heliothis virescens. We report behavioral and neurophysiological evidence that differential male responses to three female-produced chemicals (Z9-14:Ald, Z9-16:Ald, Z11-16:OAc) that maintain sexual isolation of these species are all controlled by a single QTL containing at least four odorant receptor genes. It is not surprising that pheromone receptor differences could control H. subflexa and H. virescens responses to Z9-16:Ald and Z9-14:Ald, respectively. However, central rather than peripheral level control over the positive and negative responses of H. subflexa and H. virescens to Z11-16:OAc had been expected. Tight linkage of these receptor genes indicates that mutations altering male response to complex blends could be maintained in linkage disequilibrium and could affect the speciation process. Other candidate genes such as those coding for pheromone binding proteins did not map to this QTL, but there was some genetic evidence of a QTL for response to Z11-16:OH associated with a sensory neuron membrane protein gene. PMID:20404144

  11. Synergistic Effects of Honey and Propolis toward Drug Multi-Resistant Staphylococcus Aureus, Escherichia Coli and Candida Albicans Isolates in Single and Polymicrobial Cultures

    PubMed Central

    AL-Waili, Noori; Al-Ghamdi, Ahmad; Ansari, Mohammad Javed; Al-Attal, Y.; Salom, Khelod

    2012-01-01

    Background: Propolis and honey are natural bee products with wide range of biological and medicinal properties. The study investigated antimicrobial activity of ethyl alcohol extraction of propolis collected from Saudi Arabia (EEPS) and from Egypt (EEPE), and their synergistic effect when used with honey. Single and polymicrobial cultures of antibiotic resistant human pathogens were tested. Material and methods; Staphylococcus aureus (S. aureus),), Escherichia coli (E. coli) and Candida albicans (C.albicans) were cultured in 10-100% (v/v) honey diluted in broth, or 0.08-1.0% (weight/volume) EEPS and EEPE diluted in broth. Four types of polymicrobial cultures were prepared by culturing the isolates with each other in broth (control) and broth containing various concentrations of honey or propolis. Microbial growth was assessed on solid plate media after 24 h incubation. Results; EEPS and EEPE inhibited antibiotic resistant E.coli, and S.aureus, and C.albicans in single and polymicrobial cultures. S.aureus became more susceptible when it was cultured with E.coli or C.albicans or when all cultured together. C.albicans became more susceptible when it was cultured with S.aureus or with E.coli and S. aureus together. The presence of ethyl alcohol or honey potentiated antimicrobial effect of propolis toward entire microbes tested in single or polymicrobial cultures. EEPS had lower MIC toward E.coli and C.albicans than EEPE. When propolis was mixed with honey, EEPS showed lower MIC than EEPE. In addition, honey showed lower MIC toward entire microbes when mixed with EEPS than when it was mixed with EEPE. Conclusion; 1) propolis prevents the growth of the microorganisms in single and mixed microbial cultures, and has synergistic effect when used with honey or ethyl alcohol, 2) the antimicrobial property of propolis varies with geographical origin, and 3) this study will pave the way to isolate active ingredients from honey and propolis to be further tested individually or

  12. Contribution of osmotic changes to disintegrative globulization of single cortical fibers isolated from rat lens.

    PubMed

    Wang, L F; Dhir, P; Bhatnagar, A; Srivastava, S K

    1997-08-01

    In this study the contribution of osmotic changes to disintegrative globulization of lens cortical fibers was examined. Single fiber cells were isolated by trypsinization of adult rat lens cortex, and morphological changes elicited by exposure to different external solutions were monitored optically. The survival of the fiber-shaped cells was analysed in accordance with the Weibull distribution. Changes in [Ca2+]i were measured using the fluorescent calcium-sensitive dye-Fluo-3. Exposure of isolated fiber cells to Ringer's solution (containing 2 mm Ca2+) led to an exponential increase in [Ca2+]i with a time constant of 10.2+/-0.8 min, and caused disintegrative globulization in 25+/-4 min (=Tg). The process of globulization as well as the rate of increase in [Ca2+]i was delayed by removing Cl- ions from the external media. Globulization was also delayed by adding 20% bovine serum albumin (Tg=107+/-3 min) or chloride channel inhibitors 5, nitro-2-(3-phenylpropylamino) benzoate (NPPB), dideoxyforskolin, niflumic acid, and tamoxifen. When the fiber cells were suspended in isotonic (280 mm sucrose) HEPES-sucrose (HS) or HEPES-EDTA-sucrose (HES) solution, no globulization was observed for an observation time of 120 min. However, exposure to hypotonic (180 mm) HES solution led to disintegration of fiber cells in 75+/-7 min. Disintegration of the fiber induced by hypotonic HES solution could be delayed by either 0. 05 mm leupeptin (Tg=97+/-6 min) or by pre-loading the fibers with BAPTA (Tg=100+/-4 min). Inhibition of membrane calcium transport by 0.5 mm La3+ had no effect on Tg in hypotonic HES. Addition of 2 mm Ca2+ to HES solution accelerated globulization, and Tg was 57+/-4, 69+/-5 and 102+/-6 min for hypo-, iso- and hyper- tonic solutions, respectively. Transient exposure to calcium also accelerated disintegrative globulization of fiber cells exposed subsequently to HES solution. These results suggest that in ionic media, part of the calcium influx in isolated fiber

  13. Fossil Microorganisms in Archaean

    NASA Technical Reports Server (NTRS)

    Astafleva, Marina; Hoover, Richard; Rozanov, Alexei; Vrevskiy, A.

    2006-01-01

    Ancient Archean and Proterozoic rocks are the model objects for investigation of rocks comprising astromaterials. The first of Archean fossil microorganisms from Baltic shield have been reported at the last SPIE Conference in 2005. Since this confeence biomorphic structures have been revealed in Archean rocks of Karelia. It was determined that there are 3 types of such bion structures: 1. structures found in situ, in other words microorganisms even-aged with rock matrix, that is real Archean fossils biomorphic structures, that is to say forms inhabited early formed rocks, and 3. younger than Archean-Protherozoic minerali microorganisms, that is later contamination. We made attempt to differentiate these 3 types of findings and tried to understand of burial of microorganisms. The structures belongs (from our point of view) to the first type, or real Archean, forms were under examination. Practical investigation of ancient microorganisms from Green-Stone-Belt of Northern Karelia turns to be very perspective. It shows that even in such ancient time as Archean ancient diverse world existed. Moreover probably such relatively highly organized cyanobacteria and perhaps eukaryotic formes existed in Archean world.

  14. Fossil Microorganisms in Archaean

    NASA Technical Reports Server (NTRS)

    Astafleva, Marina; Hoover, Richard; Rozanov, Alexei; Vrevskiy, A.

    2006-01-01

    Ancient Archean and Proterozoic rocks are the model objects for investigation of rocks comprising astromaterials. The first of Archean fossil microorganisms from Baltic shield have been reported at the last SPIE Conference in 2005. Since this confeence biomorphic structures have been revealed in Archean rocks of Karelia. It was determined that there are 3 types of such bion structures: 1. structures found in situ, in other words microorganisms even-aged with rock matrix, that is real Archean fossils biomorphic structures, that is to say forms inhabited early formed rocks, and 3. younger than Archean-Protherozoic minerali microorganisms, that is later contamination. We made attempt to differentiate these 3 types of findings and tried to understand of burial of microorganisms. The structures belongs (from our point of view) to the first type, or real Archean, forms were under examination. Practical investigation of ancient microorganisms from Green-Stone-Belt of Northern Karelia turns to be very perspective. It shows that even in such ancient time as Archean ancient diverse world existed. Moreover probably such relatively highly organized cyanobacteria and perhaps eukaryotic formes existed in Archean world.

  15. Microorganism identification technique

    SciTech Connect

    Sillman, R. E.

    1985-07-02

    An identification technique for micro-organisms in which a dilute solution of a culture medium containing an unknown micro-organism has added thereto an emissive agent such as a radioactive amino acid to produce a mix of emissive products that depends on the metabolic mechanism of the micro-organism. After a predetermined incubation period, the reaction is arrested and the solution layered onto a gel plate where it is subjected to electrophoresis. The plate is then autoradiographed by exposing the gel to a sensitive photographic film for a period sufficient to produce thereon a characteristic band pattern functioning as an identifier for the micro-organism. Identification may be effected by comparing the identifier for the unknown with a collection of identifiers for known micro-organisms to find a match with one of these known identifiers. The comparison is preferably carried out by scanning the unknown identifier to produce a signal which is compared with signals representing known identifiers stored in a computer which, when a match is found, yields identification data. Alternatively, the emissive products, after separation, may be detected by direct scanning to provide an identifier signal for computer processing.

  16. Absorption by DNA single strands of adenine isolated in vacuo: The role of multiple chromophores

    NASA Astrophysics Data System (ADS)

    Nielsen, Lisbeth Munksgaard; Pedersen, Sara Øvad; Kirketerp, Maj-Britt Suhr; Nielsen, Steen Brøndsted

    2012-02-01

    The degree of electronic coupling between DNA bases is a topic being up for much debate. Here we report on the intrinsic electronic properties of isolated DNA strands in vacuo free of solvent, which is a good starting point for high-level excited states calculations. Action spectra of DNA single strands of adenine reveal sign of exciton coupling between stacked bases from blueshifted absorption bands (˜3 nm) relative to that of the dAMP mononucleotide (one adenine base). The bands are blueshifted by about 10 nm compared to those of solvated strands, which is a shift similar to that for the adenine molecule and the dAMP mononucleotide. Desolvation has little effect on the bandwidth, which implies that inhomogenous broadening of the absorption bands in aqueous solution is of minor importance compared to, e.g., conformational disorder. Finally, at high photon energies, internal conversion competes with electron detachment since dissociation of the bare photoexcited ions on the microsecond time scale is measured.

  17. Association Between Isolated Single Umbilical Artery and Perinatal Outcomes: A Meta-Analysis.

    PubMed

    Xu, Yajuan; Ren, Lidan; Zhai, Shanshan; Luo, Xiaohua; Hong, Teng; Liu, Rui; Ran, Limin; Zhang, Yingying

    2016-04-30

    BACKGROUND To evaluate the association between the isolated single umbilical artery (iSUA) and perinatal outcomes, including pregnancy outcomes and perinatal complications. MATERIAL AND METHODS We performed a meta-analysis of 15 eligible studies regarding the relationship between the iSUA and perinatal outcomes, including gestational age at delivery, nuchal cord, placental weight, small for gestational age (SGA), oligohydramnios, polyhydramnios, pregnancy-induced hypertension (PIH), gestational diabetes mellitus (GDM), preeclampsia, and perinatal mortality. The overall odds ratios (OR) or standardized mean difference (SMD) were calculated. RESULTS The occurrence of nuchal cord was not found to be different between an iSUA and a three-vessel cord (TVC) fetus. For perinatal complications, the SGA, oligohydramnios, polyhydramnios, GDM, and perinatal mortality showed dramatic difference between women with an iSUA and women with a TVC fetus, which implied that the presence of iSUA significantly increased the risk of perinatal complications. For other perinatal complications, such as PIH and preeclampsia, no significant association was detected. CONCLUSIONS Our meta-analysis suggests that the presence of iSUA would increase the risk of perinatal complications such as SGA, oligohydramnios, polyhydramnios, GDM, and perinatal mortality. Therefore, pregnant women with an iSUA fetus have poorer perinatal outcomes and more attention should be given to the management of their pregnancy compared to women with a TVC fetus.

  18. Isolation of BNYVV coat protein-specific single chain Fv from a mouse phage library antibody.

    PubMed

    Jahromi, Zahra Moghaddassi; Salmanian, Ali Hatef; Rastgoo, Nasrin; Arbabi, Mehdi

    2009-10-01

    Beet necrotic yellow vein virus (BNYVV) infects sugar beet plants worldwide and is responsible for the rhizomania disease and severe economic losses. Disease severity and lack of naturally occurring resistant plants make it very difficult to control the virus, both from epidemiological and economic standpoints. Therefore, early detection is vital to impose hygiene restrictions and prevent further spread of the virus in the field. Immunoassays are one of the most popular methodologies for the primary identification of plant pathogens including BNYVV since they are robust, sensitive, fast, and inexpensive. In this study, the major coat protein (CP21) of BNYVV was cloned and expressed in Escherichia coli. Thereafter, mice were immunized with purified CP21 and a phage antibody library was constructed from their PCR-amplified immunoglobulin repertoire. Following filamentous phage rescue of the library and four rounds of panning against recombinant CP21 antigen, several specific single chain Fv fragments were isolated and characterized. This approach may pave the way to develop novel immunoassays for a rapid detection of viral infection. Moreover, it will likely provide essential tools to establish antibody-mediated resistant transgenic technology in sugar beet plants.

  19. Isolation of osteogenic progenitors from human amniotic fluid using a single step culture protocol

    PubMed Central

    Antonucci, Ivana; Iezzi, Irene; Morizio, Elisena; Mastrangelo, Filiberto; Pantalone, Andrea; Mattioli-Belmonte, Monica; Gigante, Antonio; Salini, Vincenzo; Calabrese, Giuseppe; Tetè, Stefano; Palka, Giandomenico; Stuppia, Liborio

    2009-01-01

    Background Stem cells isolated from amniotic fluid are known to be able to differentiate into different cells types, being thus considered as a potential tool for cellular therapy of different human diseases. In the present study, we report a novel single step protocol for the osteoblastic differentiation of human amniotic fluid cells. Results The described protocol is able to provide osteoblastic cells producing nodules of calcium mineralization within 18 days from withdrawal of amniotic fluid samples. These cells display a complete expression of osteogenic markers (COL1, ONC, OPN, OCN, OPG, BSP, Runx2) within 30 days from withdrawal. In order to test the ability of these cells to proliferate on surfaces commonly used in oral osteointegrated implantology, we carried out cultures onto different test disks, namely smooth copper, machined titanium and Sandblasted and Acid Etching titanium (SLA titanium). Electron microscopy analysis evidenced the best cell growth on this latter surface. Conclusion The described protocol provides an efficient and time-saving tool for the production of osteogenic cells from amniotic fluid that in the future could be used in oral osteointegrated implantology. PMID:19220883

  20. Isolation and characterization of anti ROR1 single chain fragment variable antibodies using phage display technique.

    PubMed

    Aghebati-Maleki, Leili; Younesi, Vahid; Jadidi-Niaragh, Farhad; Baradaran, Behzad; Majidi, Jafar; Yousefi, Mehdi

    2017-01-01

    Receptor tyrosine kinase-like orphan receptor (ROR1) belongs to one of the families of receptor tyrosine kinases (RTKs). RTKs are involved in the various physiologic cellular functions including proliferation, migration, survival, signaling and differentiation. Several RTKs are deregulated in various cancers implying the targeting potential of these molecules in cancer therapy. ROR1 has recently been shown to be expressed in various types of cancer cells but not in normal adult cells. Hence a molecular inhibitor of extracellular domain of ROR1 that inhibits ROR1-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of ROR1, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I + J, against one specific synthetic oligopeptide from extracellular domain of ROR1 and selected scFvs were characterized using various immunological techniques. Several ROR1 specific scFvs were selected following five rounds of panning procedure. The scFvs showed specific binding to ROR1 using immunological techniques. Our results demonstrate successful isolation and characterization of specific ROR1 scFvs that may have great therapeutic potential in cancer immunotherapy.

  1. Single-walled carbon nanotubes (SWCNTs) induce vasodilation in isolated rat aortic rings.

    PubMed

    Gutiérrez-Hernández, J M; Ramirez-Lee, M A; Rosas-Hernandez, H; Salazar-García, S; Maldonado-Ortega, D A; González, F J; Gonzalez, C

    2015-06-01

    Single-walled carbon nanotubes (SWCNTs) are used in biological systems with impact in biomedicine in order to improve diagnostics and treatment of diseases. However, their effects upon the vascular system, are not fully understood. Endothelium and smooth muscle cells (SMC) communicate through release of vasoactive factors as nitric oxide (NO) to maintain vascular tone. The aim of this study was to evaluate the effect of SWCNTs on vascular tone using isolated rat aortic rings, which were exposed to SWCNTs (0.1, 1 and 10 μg/mL) in presence and absence of endothelium. SWCNTs induced vasodilation in both conditions, indicating that this effect was independent on endothelium; moreover that vasodilation was NO-independent, since its blockage with L-NAME did not modify the observed effect. Together, these results indicate that SWCNTs induce vasodilation in the macrovasculature, may be through a direct interaction with SMC rather than endothelium independent of NO production. Further investigation is required to fully understand the mechanisms of action and mediators involved in the signaling pathway induced by SWCNTs on the vascular system. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Micro-Organ Devices

    NASA Technical Reports Server (NTRS)

    Gonda, Steven R.; Leslie, Julia; Chang, Robert C.; Starly, Binil; Sun, Wei; Culbertson, Christopher; Holtorf, Heidi

    2009-01-01

    Micro-organ devices (MODs) are being developed to satisfy an emerging need for small, lightweight, reproducible, biological-experimentati on apparatuses that are amenable to automated operation and that imp ose minimal demands for resources (principally, power and fluids). I n simplest terms, a MOD is a microfluidic device containing a variety of microstructures and assemblies of cells, all designed to mimic a complex in vivo microenvironment by replicating one or more in vivo micro-organ structures, the architectures and composition of the extr acellular matrices in the organs of interest, and the in vivo fluid flows. In addition to microscopic flow channels, a MOD contains one or more micro-organ wells containing cells residing in microscopic e xtracellular matrices and/or scaffolds, the shapes and compositions o f which enable replication of the corresponding in vivo cell assembl ies and flows.

  3. Elastohydrodynamics of flagellated microorganisms

    NASA Astrophysics Data System (ADS)

    Li, Gaojin; Ardekani, Arezoo

    2016-11-01

    The swimming motion of many microorganisms and cells are achieved by the waving deformation of their cilia and flagella. The typical structure of flagella and cilia contains nine doublets of parallel microtubules in a cylindrical arrangement surrounding one pair of microtubules in the center. The dynein molecular motors internally drive the sliding motion between the neighboring microtubules and cause the bending motion of the flagella and cilia and drive the microorganism swimming motion. In this work, we develop a numerical model for a microorganism swimming by an internally self-driven filament. Our numerical method captures the interaction between the elasticity of the flagellum and the surround fluid. The no-slip boundary conditions are satisfied by an iterative distributed Lagrangian multiplier method. We also investigate the effects of the non-Newtonian fluid rheology on the motion of an elastic flagellum near a wall.

  4. Genotyping single spore isolates of a Pasteuria penetrans population occurring in Florida using SNP-based markers

    USDA-ARS?s Scientific Manuscript database

    The aim of this study was to examine genotypic variation and virulence characteristics of a population of bacterial parasite of root-knot nematode (RKN), Pasteuria penetrans, isolated from Florida. Six single spore lines (ssp), 16ssp, 17ssp, 18ssp, 25ssp, 26ssp, and 30ssp were generated by infecting...

  5. Isolation of Resistance-Bearing Microorganisms

    NASA Technical Reports Server (NTRS)

    Venkateswaran, Kasthuri, J.; Probst, Alexander; Vaishampayan, Parang A.; Ghosh, Sudeshna; Osman, Shariff

    2010-01-01

    To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid dynamic shear (i.e., as required for viability of shear-sensitive cells) to the developing engineered tissue construct. This bioreactor was recently utilized to show independent and interactive effects of a growth factor (IGF-I) and slow bidirectional perfusion on the survival, differentiation, and contractile performance of 3D tissue engineering cardiac constructs. The main application of this system is within the tissue engineering industry. The ideal final application is within the automated mass production of tissue- engineered constructs. Target industries could be both life sciences companies as well as bioreactor device producing companies.

  6. Bioplastics from microorganisms.

    PubMed

    Luengo, José M; García, Belén; Sandoval, Angel; Naharro, Germán; Olivera, Elías R

    2003-06-01

    The term 'biomaterials' includes chemically unrelated products that are synthesised by microorganisms (or part of them) under different environmental conditions. One important family of biomaterials is bioplastics. These are polyesters that are widely distributed in nature and accumulate intracellularly in microorganisms in the form of storage granules, with physico-chemical properties resembling petrochemical plastics. These polymers are usually built from hydroxy-acyl-CoA derivatives via different metabolic pathways. Depending on their microbial origin, bioplastics differ in their monomer composition, macromolecular structure and physical properties. Most of them are biodegradable and biocompatible, which makes them extremely interesting from the biotechnological point of view.

  7. [SCREENING AND SELECTION OF THE SOIL MICROORGANISMS ON THE ABILITY OF "NITROGEN-FIXING ACTIVITY"].

    PubMed

    Patyka V Ph; Kyrychenko, O V; Kots, S Ya

    2015-01-01

    The isolates of microorganisms from the rhizosphere of spring barley plants and soil at the use of analytical selection method was isolated. Its isolates on the ability of "nitrogen-fixing activity" was tested. It was shown that isolates of microorganisms had different of the colonies formed and cultural growth on the Eshbi's selective medium as well as the ability to fixing of molecular nitrogen. The different levels of intensity and dynamics of isolates nitrogenase activity in vitro were identified. New isolates of the soil microorganisms complement of the gene pool diazotrophic bacteria. Its isolates are perspectivity for the study as the basis or components of the bacterial fertilizers for the crops.

  8. Determination of nanomolar levels of reactive oxygen species in microorganisms and aquatic environments using a single nanoparticle-based optical sensor.

    PubMed

    Kim, Yura; Kim, Youngho; Choi, Jinhee; Kang, Taewook; Choi, Inhee

    2017-05-15

    Reactive oxygen species (ROS) are strong oxidants, and have attracted considerable attention in both biological and environmental fields. Although various methods for ROS detection, including optical and electrochemical techniques, have been developed, they still face challenging issues in terms of poor sensitivity, reproducibility, stability, and in vivo applicability. Here, we present a sensitive and selective optical sensor for ROS detection, based on single plasmonic nanoprobes and redox-active cytochrome c (Cyt c)-mediated plasmon resonance energy transfer. By measuring the spectral changes of plasmonic nanoprobes, derived from the unique molecular absorption of Cyt c in accordance with the redox state, calibration curves for H2O2, a representative ROS, in various media were obtained over a wide concentration range from 100 mM to 1 nM. Limit of detection and limit of quantification in accordance with the used medium were determined from 8.3 to 12.8 nM and from 27.6 to 42.7 nM, respectively. Selectivity coefficients for major interfering solutes were much lower than 0.1 indicating a good selectivity for ROS. From the dynamic spectral changes, we sensitively monitored ROS generated in Caenorhabditis elegans (C. elegans) exposed to graphene oxide. Based on the calibration curves, we also determined ROS levels in various aquatic environments, such as river streams and a small pond, as a way of environmental monitoring. We believe that our approach could provide an avenue for achieving dynamic and sensitive monitoring of ROS in toxicological, biological, and environmental fields in the future.

  9. Flow cytometry in environmental microbiology: a rapid approach for the isolation of single cells for advanced molecular biology analysis.

    PubMed

    Ferrari, Belinda C; Winsley, Tristrom J; Bergquist, Peter L; Van Dorst, Josie

    2012-01-01

    The isolation and subsequent characterization of microbial cells from within environmental samples is a difficult process. Flow cytometry and cell sorting, when combined with the application of fluorescent probes, have the capability for the detection and separation of diverse microbial populations from within complex mixtures. The isolation of single cells allows for downstream investigations towards system-level characterization of unknown Bacterial Phyla to occur. We describe here the combination of fluorescent in situ hybridization and cell sorting for the detection and isolation of Candidate Division TM7 bacteria from an enriched soil sample. The result is the isolation of rare cells suitable for advanced molecular analysis including whole genome amplification and high-throughput pyrosequencing.

  10. Fruiting Body Formation of Cordyceps militaris from Multi-Ascospore Isolates and Their Single Ascospore Progeny Strains

    PubMed Central

    Shrestha, Bhushan; Han, Sang-Kuk; Sung, Jae-Mo

    2012-01-01

    Interest in commercial cultivation and product development of Cordyceps species has shown a recent increase. Due to its biochemical and pharmacological effects, Cordyceps militaris, commonly known as orange caterpillar fungus, is being investigated with great interest. Cultivation of C. militaris has been practiced on a large scale in order to fulfill a demand for scientific investigation and product development. Isolates of C. militaris can be easily established from both spores and tissue. For isolation of spores, ascospores released from mature stromata are trapped in sterile medium. Multi-ascospore isolates, as well as combinations of single ascospore strains, are used for production of fruiting bodies. Progeny ascospore strains can be isolated from artificial fruiting bodies, thus, the cycle of fruiting body production can be continued for a long period of time. In this study, we examined fruiting body production from multi-ascospore isolates and their progeny strains for three generations. F1 progeny strains generally produced a larger number of fruiting bodies, compared with their mother multi-ascospore isolates; however, F2 and F3 progeny strains produced fewer fruiting bodies. Optimum preservation conditions could help to increase the vitality of the progeny strains. In order to retain the fruiting ability of the strains, further testing of various methods of preservation and different methods for isolation should be performed. PMID:22870051

  11. Continuing fascination of exploration in natural substances from microorganisms.

    PubMed

    Takahashi, Yoko

    2017-01-01

    In the search for novel organic compounds, I think it is of paramount importance not to overlook the pursuit of microorganism diversity and the abilities those microorganisms hold as a resource. In commemoration of Professor Satoshi Ōmura's Nobel Prize in Physiology or Medicine, I will briefly describe the microorganism that produces avermectin and then discuss how innovating isolation methods and pioneering isolation sources have opened the door to numerous new microorganism resources. Furthermore, as exploratory research of substances views the world from many different angles-from biological activity to a compound's physiochemical properties-it is possible to discover a novel compound from a well-known microorganism. Based on this, I will discuss the future prospects of exploratory research.

  12. Antimicrobial Compounds from Marine Invertebrates-Derived Microorganisms.

    PubMed

    Liu, Juan; Jung, Jee H; Liu, Yonghong

    2016-01-01

    It is known that marine invertebrates, including sponges, tunicates, cnidaria or mollusks, host affluent and various communities of symbiotic microorganisms. The microorganisms associated with the invertebrates metabolized various biologically active compounds, which could be an important resource for the discovery and development of potentially novel drugs. In this review, the new compounds with antimicrobial activity isolated from marine invertebrate-derived microorganisms in the last decade (2004-2014) will be presented, with focus on the relevant antimicrobial activities, origin of isolation, and information of strain species. New compounds without antimicrobial activity were not revealed.

  13. [Biotechnology using modified microorganisms].

    PubMed

    Deshayes, A F

    1992-11-01

    Few microorganisms, as compare to their high diversity, are used for human needs. They can produce molecules of interest, process fermentation, protect crops, treat wastes or clean environment. Molecular technics and genetic engineering are new tools offer to geneticists which breed microorganisms for years. Using them, it is now possible, theoretically, to introduce any gene in any organism. Some examples are given concerning genetic modifications in yeasts and lactic acid bacteria to optimize agrofood processes and to improve nutritive and flavour characteristics of fermented products like bread, beer, wine, cheese, meat, vegetable juices... In spite of scientific and industrial interest of the new technologies, limiting factors can explain that genetically modified microorganisms are not routinely used in agrofood yet. First, risks assessment on human health and environment are still in debate, but their is a consensus, within the scientific community, to consider that new characteristics of improved microorganisms are more important than the technics used for their construction. Second, regulations turn out to impose constraints susceptible to discourage technological innovations. At least, the public perception about the new technologies appears, actually, as the major factor to limit their development.

  14. Microorganisms and Man.

    ERIC Educational Resources Information Center

    Noble, W. C.

    1983-01-01

    Provides information to update Institute of Biology's Studies in Biology No. 111, "Microorganisms and Man," by W. C. Noble and Jay Naidoo (Edward Arnold, 1979). Topics include: (1) food poisoning; (2) airborn infections in man; (3) infection in animals and plants; and (4) biodegradation and biosynthesis. (JN)

  15. Microorganisms and Man.

    ERIC Educational Resources Information Center

    Noble, W. C.

    1983-01-01

    Provides information to update Institute of Biology's Studies in Biology No. 111, "Microorganisms and Man," by W. C. Noble and Jay Naidoo (Edward Arnold, 1979). Topics include: (1) food poisoning; (2) airborn infections in man; (3) infection in animals and plants; and (4) biodegradation and biosynthesis. (JN)

  16. Microorganisms within Human Follicular Fluid: Effects on IVF

    PubMed Central

    Pelzer, Elise S.; Allan, John A.; Waterhouse, Mary A.; Ross, Tara; Beagley, Kenneth W.; Knox, Christine L.

    2013-01-01

    Our previous study reported microorganisms in human follicular fluid. The objective of this study was to test human follicular fluid for the presence of microorganisms and to correlate these findings with the in vitro fertilization (IVF) outcomes. In this study, 263 paired follicular fluids and vaginal swabs were collected from women undergoing IVF cycles, with various causes for infertility, and were cultured to detect microorganisms. The cause of infertility and the IVF outcomes for each woman were correlated with the microorganisms detected within follicular fluid collected at the time of trans-vaginal oocyte retrieval. Microorganisms isolated from follicular fluids were classified as: (1) ‘colonizers’ if microorganisms were detected within the follicular fluid, but not within the vaginal swab (at the time of oocyte retrieval); or (2) ‘contaminants’ if microorganisms detected in the vagina at the time of oocyte retrieval were also detected within the follicular fluid. The presence of Lactobacillus spp. in ovarian follicular fluids was associated with embryo maturation and transfer. This study revealed microorganisms in follicular fluid itself and that the presence of particular microorganisms has an adverse affect on IVF outcomes as seen by an overall decrease in embryo transfer rates and pregnancy rates in both fertile and infertile women, and live birth rates in women with idiopathic infertility. Follicular fluid microorganisms are a potential cause of adverse pregnancy outcomes in IVF in both infertile women and in fertile women with infertile male partners. PMID:23554970

  17. Microorganisms within human follicular fluid: effects on IVF.

    PubMed

    Pelzer, Elise S; Allan, John A; Waterhouse, Mary A; Ross, Tara; Beagley, Kenneth W; Knox, Christine L

    2013-01-01

    Our previous study reported microorganisms in human follicular fluid. The objective of this study was to test human follicular fluid for the presence of microorganisms and to correlate these findings with the in vitro fertilization (IVF) outcomes. In this study, 263 paired follicular fluids and vaginal swabs were collected from women undergoing IVF cycles, with various causes for infertility, and were cultured to detect microorganisms. The cause of infertility and the IVF outcomes for each woman were correlated with the microorganisms detected within follicular fluid collected at the time of trans-vaginal oocyte retrieval. Microorganisms isolated from follicular fluids were classified as: (1) 'colonizers' if microorganisms were detected within the follicular fluid, but not within the vaginal swab (at the time of oocyte retrieval); or (2) 'contaminants' if microorganisms detected in the vagina at the time of oocyte retrieval were also detected within the follicular fluid. The presence of Lactobacillus spp. in ovarian follicular fluids was associated with embryo maturation and transfer. This study revealed microorganisms in follicular fluid itself and that the presence of particular microorganisms has an adverse affect on IVF outcomes as seen by an overall decrease in embryo transfer rates and pregnancy rates in both fertile and infertile women, and live birth rates in women with idiopathic infertility. Follicular fluid microorganisms are a potential cause of adverse pregnancy outcomes in IVF in both infertile women and in fertile women with infertile male partners.

  18. Mycobacterium tuberculosis Isolates from Single Outpatient Clinic in Panama City Exhibit Wide Genetic Diversity

    PubMed Central

    Sambrano, Dilcia; Correa, Ricardo; Almengor, Pedro; Domínguez, Amada; Vega, Silvio; Goodridge, Amador

    2014-01-01

    Understanding Mycobacterium tuberculosis biodiversity and transmission is significant for tuberculosis control. This short report aimed to determine the genetic diversity of M. tuberculosis isolates from an outpatient clinic in Panama City. A total of 62 M. tuberculosis isolates were genotyped by 12 loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and Spoligotyping. Forty-five (72.6%) of the isolates showed unique MIRU-VNTR genotypes, and 13 (21%) of the isolates were grouped into four clusters. Four isolates showed polyclonal MIRU-VNTR genotypes. The MIRU-VNTR Hunter-Gaston discriminatory index reached 0.988. The Spoligotyping analysis revealed 16 M. tuberculosis families, including Latin American-Mediterranean, Harlem, and Beijing. These findings suggest a wide genetic diversity of M. tuberculosis isolates at one outpatient clinic. A detailed molecular epidemiology survey is now warranted, especially following second massive immigration for local Panama Canal expansion activities. PMID:24865686

  19. Interaction between propranolol and amino acids in the single-pass isolated, perfused rat liver.

    PubMed

    Semple, H A; Xia, F

    1995-08-01

    Propranolol (PL) bioavailability has been shown to increase substantially when it is administered with a protein-rich meal. A change in metabolic capacity or tissue uptake, induced by amino acids (AAs) released as a result of digestion of dietary protein, is a possible contributing mechanism to the food effect. This hypothesis was tested in isolated, perfused rat livers in the single-pass mode. Rac-PL (20 micrograms/ml) was infused to steady-state at 3 ml/min/g liver for 150 min. A balanced mixture of I-AA was coinfused from 70 to 110 min. The AA reversibly increased the steady-state concentration of PL by 18% and reduced steady-state concentrations of 4-hydroxypropranolol, N-deisopropylpranolol, PL glycol, naphthoxylactic acid, and naphthoxyacetic acid by an average of 41% and propanolol conjugates by almost 100%, indicating metabolic inhibition. In a second experiment, PL was coinfused with AAs from the beginning of the experiment, and tissue binding was compared with control livers. There was no significant effect of AAs on PL tissue binding. In a third study, the effect of four different concentrations of AAs coinfused from 70 to 110 min was assessed. The percentage change in PL and phase I metabolite levels was linearly correlated to the influent AA concentration. The large magnitude, reversibility, lack of pathway specificity, and concentration dependence of the AA interaction in the perfused liver are also features of food interaction in humans. These similarities constitute evidence that metabolic inhibition by AAs originating from dietary protein could contribute to the PL-food interaction.

  20. Isolation and characterization of anti c-met single chain fragment variable (scFv) antibodies.

    PubMed

    Qamsari, Elmira Safaie; Sharifzadeh, Zahra; Bagheri, Salman; Riazi-Rad, Farhad; Younesi, Vahid; Abolhassani, Mohsen; Ghaderi, Sepideh Safaei; Baradaran, Behzad; Somi, Mohammad Hossein; Yousefi, Mehdi

    2017-12-01

    The receptor tyrosine kinase (RTK) Met is the cell surface receptor for hepatocyte growth factor (HGF) involved in invasive growth programs during embryogenesis and tumorgenesis. There is compelling evidence suggesting important roles for c-Met in colorectal cancer proliferation, migration, invasion, angiogenesis, and survival. Hence, a molecular inhibitor of an extracellular domain of c-Met receptor that blocks c-Met-cell surface interactions could be of great thera-peutic importance. In an attempt to develop molecular inhibitors of c-Met, single chain variable fragment (scFv) phage display libraries Tomlinson I + J against a specific synthetic oligopeptide from the extracellular domain of c-Met receptor were screened; selected scFv were then characterized using various immune techniques. Three c-Met specific scFv (ES1, ES2, and ES3) were selected following five rounds of panning procedures. The scFv showed specific binding to c-Met receptor, and significantly inhibited proliferation responses of a human colorectal carcinoma cell line (HCT-116). Moreover, anti- apoptotic effects of selected scFv antibodies on the HCT-116 cell line were also evaluated using Annexin V/PI assays. The results demonstrated rates of apoptotic cell death of 46.0, 25.5, and 37.8% among these cells were induced by use of ES1, ES2, and ES3, respectively. The results demonstrated ability to successfully isolate/char-acterize specific c-Met scFv that could ultimately have a great therapeutic potential in immuno-therapies against (colorectal) cancers.

  1. Adenylate cyclase activity along the rabbit nephron as measured in single isolated segments.

    PubMed

    Imbert, M; Chabardès, D; Montégut, M; Clique, A; Morel, F

    1975-01-01

    A method is described, which allows adenylate cyclase activity measurement in single pieces of various nephron segments. Tubular samples of 0.5 to 2 mm length were isolated by microdissection from collagenase treated slices of rabbit kidney. A photograph of each piece was taken in order to measure its length. After a permeabilisation treatment involving preincubation in a hypoosmotic medium and a freezing step, each sample was incubated for 30 mm at 30 degrees C in a medium containing high specific (alpha-32-P)-ATP 3-10-4 M, final volume 2.5 mu 1. The (32P)-cAMP formed was separated from the other labelled nucleotides by filtering the incubate on a dry aluminium oxide microcolumn, 3H cAMP was added as a tracer for measuring cAMP recovery. The sensitivity of the method was found to be a few fentomoles (10-15 M) cAMP. cAMP generation increased linearly as a function of the incubation time up to more than 30 min, and as a function of the length of the segment used. Control and fluoride (5 mM) stimulated adenvlate cyclase activities were measured in the following segments of the nephron: early proximal convoluted tubule (PCT), pars recta of the proximal tubule (PR), thin descending limb of the loop (TDL), cortical portion of the thick ascending limb (CAL), distal convoluted tubule (dct), first branched portion of the collecting tubule (BCT), further cortical (CCT) and medullary (MCT) portions of the collecting tubule. Mean control adenylate cyclase activity varied from 7 (PR) to 75 (BCT) fmoles/mm/30 min. Flouride addition resulted in a 10 (BCT) to 50 (PR) fold increase in enzyme activity. Series of replicates gave a scatter equal to plus or minus 20% (S.D. as a per cent of the mean). The method described appears to be suitable to determine which nephron segments contain hormone-dependent adenylate cyclase.

  2. Single step biotransformation of corn oil phytosterols to boldenone by a newly isolated Pseudomonas aeruginosa.

    PubMed

    Eisa, Mohamed; El-Refai, Heba; Amin, Magdy

    2016-09-01

    A new potent Pseudomonas aeruginosa isolate capable for biotransformation of corn oil phytosterol (PS) to 4-androstene-3, 17-dione (AD), testosterone (T) and boldenone (BOL) was identified by phenotypic analysis and 16S rRNA gene sequencing. Sequential statistical strategy was used to optimize the biotransformation process mainly concerning BOL using Factorial design and response surface methodology (RSM). The production of BOL in single step microbial biotransformation from corn oil phytosterols by P. aeruginosa was not previously reported. Results showed that the pH concentration of the medium, (NH4)2SO4 and KH2PO4 were the most significant factors affecting BOL production. By analyzing the statistical model of three-dimensional surface plot, BOL production increased from 36.8% to 42.4% after the first step of optimization, and the overall biotransformation increased to 51.9%. After applying the second step of the sequential statistical strategy BOL production increased to 53.6%, and the overall biotransformation increased to 91.9% using the following optimized medium composition (g/l distilled water) (NH4)2SO4, 2; KH2PO4, 4; Na2HPO4. 1; MgSO4·7H2O, 0.3; NaCl, 0.1; CaCl2·2H2O, 0.1; FeSO4·7H2O, 0.001; ammonium acetate 0.001; Tween 80, 0.05%; corn oil 0.5%; 8-hydroxyquinoline 0.016; pH 8; 200 rpm agitation speed and incubation time 36 h at 30 °C. Validation experiments proved the adequacy and accuracy of model, and the results showed the predicted value agreed well with the experimental values.

  3. Rapid isolation and single-molecule analysis of ribonucleoproteins from cell lysate by SNAP-SiMPull.

    PubMed

    Rodgers, Margaret L; Paulson, Joshua; Hoskins, Aaron A

    2015-05-01

    Large macromolecular complexes such as the spliceosomal small nuclear ribonucleoproteins (snRNPs) play a variety of roles within the cell. Despite their biological importance, biochemical studies of snRNPs and other machines are often thwarted by practical difficulties in the isolation of sufficient amounts of material. Studies of the snRNPs as well as other macromolecular machines would be greatly facilitated by new approaches that enable their isolation and biochemical characterization. One such approach is single-molecule pull-down (SiMPull) that combines in situ immunopurification of complexes from cell lysates with subsequent single-molecule fluorescence microscopy experiments. We report the development of a new method, called SNAP-SiMPull, that can readily be applied to studies of splicing factors and snRNPs isolated from whole-cell lysates. SNAP-SiMPull overcomes many of the limitations imposed by conventional SiMPull strategies that rely on fluorescent proteins. We have used SNAP-SiMPull to study the yeast branchpoint bridging protein (BBP) as well as the U1 and U6 snRNPs. SNAP-SiMPull will likely find broad use for rapidly isolating complex cellular machines for single-molecule fluorescence colocalization experiments. © 2015 Rodgers et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  4. Rapid isolation and single-molecule analysis of ribonucleoproteins from cell lysate by SNAP-SiMPull

    PubMed Central

    Rodgers, Margaret L.; Paulson, Joshua; Hoskins, Aaron A.

    2015-01-01

    Large macromolecular complexes such as the spliceosomal small nuclear ribonucleoproteins (snRNPs) play a variety of roles within the cell. Despite their biological importance, biochemical studies of snRNPs and other machines are often thwarted by practical difficulties in the isolation of sufficient amounts of material. Studies of the snRNPs as well as other macromolecular machines would be greatly facilitated by new approaches that enable their isolation and biochemical characterization. One such approach is single-molecule pull-down (SiMPull) that combines in situ immunopurification of complexes from cell lysates with subsequent single-molecule fluorescence microscopy experiments. We report the development of a new method, called SNAP-SiMPull, that can readily be applied to studies of splicing factors and snRNPs isolated from whole-cell lysates. SNAP-SiMPull overcomes many of the limitations imposed by conventional SiMPull strategies that rely on fluorescent proteins. We have used SNAP-SiMPull to study the yeast branchpoint bridging protein (BBP) as well as the U1 and U6 snRNPs. SNAP-SiMPull will likely find broad use for rapidly isolating complex cellular machines for single-molecule fluorescence colocalization experiments. PMID:25805862

  5. A decade of change in susceptibility patterns of Gram-negative blood culture isolates: a single center study

    PubMed Central

    Alagesan, Murali; Gopalakrishnan, Ram; Panchatcharam, Senthur Nambi; Dorairajan, Sureshkumar; Mandayam Ananth, Thirunarayan; Venkatasubramanian, Ramasubramanian

    2015-01-01

    Background Gram-negative bacteremia is one of the leading causes of mortality and morbidity in Indian hospitals. We hereby describe changing trends in Gram-negative isolates from blood cultures from a single center over a ten-year period. Methods Antibiotic susceptibility patterns were collected for a total of 4128 non-repetitive blood culture isolates from 2003 to 2013. We analyzed clinically important Gram-negative isolates (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii) and their susceptibility pattern. A. baumannii was studied between 2009 and 2013 only. Results There was a steady increase in extended-spectrum beta-lactamase (ESBL) production in E. coli (56% to 80%) and an even steeper increase in K. pneumoniae (50% to 81%). Susceptibility to carbapenems fell marginally for E. coli (p = .242) but significantly for K. pneumoniae (p = .000) and P. aeruginosa (.0005). All these changes were seen irrespective of the source of the isolate (outpatient, inpatient and critical care unit – CCU), with a statistically significant fall among CCU isolates of K. pneumoniae and P. aeruginosa. P. aeruginosa was more susceptible to carbapenems than beta-lactam /beta-lactamase inhibitors until 2009, but thereafter the pattern reversed. A. baumannii was isolated from the CCU only: 75% were resistant to carbapenems and susceptible only to polymyxin E and tigecycline. Conclusion There was a progressive increase in antimicrobial resistance in isolates of E. coli, K. pneumoniae, P. aeruginosa and A. baumannii isolated from blood cultures. ESBL production was seen in the majority of isolates of E. coli and K. pneumoniae. Carbapenem resistance in K. pneumoniae and E. coli is increasing rapidly. Resistance to even tigecycline and polymyxin E, antibiotics of last resort, has begun to emerge. There is an urgent need for antimicrobial stewardship and other measures to limit worsening of Gram-negative resistance in India. PMID:26405674

  6. A decade of change in susceptibility patterns of Gram-negative blood culture isolates: a single center study.

    PubMed

    Alagesan, Murali; Gopalakrishnan, Ram; Panchatcharam, Senthur Nambi; Dorairajan, Sureshkumar; Mandayam Ananth, Thirunarayan; Venkatasubramanian, Ramasubramanian

    2015-09-01

    Gram-negative bacteremia is one of the leading causes of mortality and morbidity in Indian hospitals. We hereby describe changing trends in Gram-negative isolates from blood cultures from a single center over a ten-year period. Antibiotic susceptibility patterns were collected for a total of 4128 non-repetitive blood culture isolates from 2003 to 2013. We analyzed clinically important Gram-negative isolates (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii) and their susceptibility pattern. A. baumannii was studied between 2009 and 2013 only. There was a steady increase in extended-spectrum beta-lactamase (ESBL) production in E. coli (56% to 80%) and an even steeper increase in K. pneumoniae (50% to 81%). Susceptibility to carbapenems fell marginally for E. coli (p = .242) but significantly for K. pneumoniae (p = .000) and P. aeruginosa (.0005). All these changes were seen irrespective of the source of the isolate (outpatient, inpatient and critical care unit - CCU), with a statistically significant fall among CCU isolates of K. pneumoniae and P. aeruginosa. P. aeruginosa was more susceptible to carbapenems than beta-lactam /beta-lactamase inhibitors until 2009, but thereafter the pattern reversed. A. baumannii was isolated from the CCU only: 75% were resistant to carbapenems and susceptible only to polymyxin E and tigecycline. There was a progressive increase in antimicrobial resistance in isolates of E. coli, K. pneumoniae, P. aeruginosa and A. baumannii isolated from blood cultures. ESBL production was seen in the majority of isolates of E. coli and K. pneumoniae. Carbapenem resistance in K. pneumoniae and E. coli is increasing rapidly. Resistance to even tigecycline and polymyxin E, antibiotics of last resort, has begun to emerge. There is an urgent need for antimicrobial stewardship and other measures to limit worsening of Gram-negative resistance in India.

  7. Enhanced anti-Diastereo- and Enantioselectivity in Alcohol Mediated Carbonyl Crotylation Using an Isolable Single Component Iridium Catalyst

    PubMed Central

    Gao, Xin; Townsend, Ian A.; Krische, Michael J.

    2011-01-01

    The cyclometallated iridium complex (S)-I derived from [Ir(cod)Cl]2, 4-cyano-3-nitrobenzoic acid, allyl acetate and (S)-SEGPHOS is conveniently isolated by precipitation or through conventional silica gel flash chromatography. This single component precatalyst allows alcohol mediated carbonyl crotylations to be performed at significantly lower temperature, resulting in enhanced levels of anti-diastereo- and enantioselectivity. Most significantly, the chromatographically isolated precatalyst (S)-I enables carbonyl crotylations that are not possible under previously reported conditions involving in situ generation of (S)-I. PMID:21375283

  8. Determination of risk factors and perinatal outcomes of singleton pregnancies complicated by isolated single umbilical artery in Turkish population.

    PubMed

    Tülek, Fırat; Kahraman, Alper; Taşkın, Salih; Özkavukçu, Esra; Söylemez, Feride

    2015-01-01

    To address the possible risk factors, eventual pregnancy outcomes, and probable troubles in follow-ups of pregnancies complicated by an isolated single umbilical artery and to provide data on Turkish cases in such an aspect that ethnic divergences may have influence. A total of 16568 singleton pregnancies that were delivered between May 2006 and May 2013 were retrospectively screened. Ninety-three fetuses were found to have an isolated single umbilical artery. One-hundred pregnancies that did not show any structural or chromosomal abnormalities were randomly selected from the rest of the cases to establish the control group. IBM SPSS Statistics 20.0 software was utilized for statistical analysis. Non-parametric data were analyzed with Mann-Whitney U test and were presented as means±standard deviations. P values less than 0.05 were statistically significant. For the adjustment of confounding factors, odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by multiple logistic regression analysis. The incidence of small for gestational age (SGA) fetuses and hypertensive disorders in pregnancy was found to be significantly higher in cases with an isolated single umbilical artery (p<0.001 and p=0.022, respectively). Maternal smoking was found to be independently associated with the occurrence of an isolated single umbilical artery (OR: 3.556; 95% CI: 1.104-11.45). The risk of preterm birth was not higher in the study group (OR: 0.538; 95% CI: 0.576-2.873). The incidence of cases who underwent cesarean delivery because of non-reassuring fetal heart trace was similar in the study and control groups (p=0.499). Attention should be paid to the development of hypertensive disorders in cases with a diagnosis of an isolated single umbilical artery, and parents should be counseled properly, including the information on increased risk of SGA. Strict follow-up of pregnancies complicated with an isolated single umbilical artery in terms of preterm birth seems

  9. Determination of risk factors and perinatal outcomes of singleton pregnancies complicated by isolated single umbilical artery in Turkish population

    PubMed Central

    Tülek, Fırat; Kahraman, Alper; Taşkın, Salih; Özkavukçu, Esra; Söylemez, Feride

    2015-01-01

    Objective To address the possible risk factors, eventual pregnancy outcomes, and probable troubles in follow-ups of pregnancies complicated by an isolated single umbilical artery and to provide data on Turkish cases in such an aspect that ethnic divergences may have influence. Material and Methods A total of 16568 singleton pregnancies that were delivered between May 2006 and May 2013 were retrospectively screened. Ninety-three fetuses were found to have an isolated single umbilical artery. One-hundred pregnancies that did not show any structural or chromosomal abnormalities were randomly selected from the rest of the cases to establish the control group. IBM SPSS Statistics 20.0 software was utilized for statistical analysis. Non-parametric data were analyzed with Mann-Whitney U test and were presented as means±standard deviations. P values less than 0.05 were statistically significant. For the adjustment of confounding factors, odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by multiple logistic regression analysis. Results The incidence of small for gestational age (SGA) fetuses and hypertensive disorders in pregnancy was found to be significantly higher in cases with an isolated single umbilical artery (p<0.001 and p=0.022, respectively). Maternal smoking was found to be independently associated with the occurrence of an isolated single umbilical artery (OR: 3.556; 95% CI: 1.104–11.45). The risk of preterm birth was not higher in the study group (OR: 0.538; 95% CI: 0.576–2.873). The incidence of cases who underwent cesarean delivery because of non-reassuring fetal heart trace was similar in the study and control groups (p=0.499). Conclusion Attention should be paid to the development of hypertensive disorders in cases with a diagnosis of an isolated single umbilical artery, and parents should be counseled properly, including the information on increased risk of SGA. Strict follow-up of pregnancies complicated with an isolated single

  10. Genome sequences of ten Salmonella enterica serovars isolated from a single dairy farm

    USDA-ARS?s Scientific Manuscript database

    Here we report draft genomes of twenty-seven isolates of Salmonella enterica subsp. enterica representing the seven serotypes isolated from cows in a Pennsylvania dairy herd, the farm on which they were reared, and the associated off-site heifer-raising facility over an eight year sampling period. ...

  11. Genome Sequences of 30 Escherichia coli O157:H7 Isolates Recovered from a Single Dairy Farm and Its Associated Off-Site Heifer-Raising Facility.

    PubMed

    Kim, Seon-Woo; Karns, Jeffrey S; Van Kessel, Jo Ann S; Haley, Bradd J

    2017-08-31

    Cattle are the primary reservoir of Escherichia coli O157:H7, the most frequently isolated serotype of enterohemorrhagic E. coli infections among humans in North America. To evaluate the diversity of E. coli O157:H7 isolates within a single dairy herd, the genomes of 30 isolates collected over a 7-year period were sequenced.

  12. Genome sequences of thirty Escherichia coli O157:H7 isolates recovered from a single dairy farm and its associated off-site heifer raising facility

    USDA-ARS?s Scientific Manuscript database

    Cattle are the primary reservoir of Escherichia coli O157:H7, the most frequently isolated serotype of enterohemorrhagic E. coli infections among humans in North America. To evaluate the diversity of E. coli O157:H7 isolates within a single dairy herd the genomes of 30 isolates collected over a 7-ye...

  13. Complete genome assemblies for two single-chromosome Vibrio cholerae isolates, strains 1154-74 (serogroup O49) and 10432-62 (serogroup O27)

    DOE PAGES

    Johnson, Shannon Lyn; Khiani, A.; Bishop-Lilly, K. A.; ...

    2015-05-14

    We report the completed genome sequences for two non-O1/non-O139 Vibrio cholerae isolates. Each isolate has only a single chromosome, as opposed to the normal paradigm of two chromosomes found in all other V. cholerae isolates.

  14. Radiation-resistant microorganism

    DOEpatents

    Fliermans, Carl B.

    2010-06-15

    An isolated and purified bacterium is provided which was isolated from a high-level radioactive waste site of mixed waste. The isolate has the ability to degrade a wide variety of organic contaminants while demonstrating high tolerance to ionizing radiation. The organism is uniquely suited to bioremediation of a variety or organic contaminants while in the presence of ionizing radiation.

  15. Radiation-resistant microorganism

    DOEpatents

    Fliermans, Carl B.

    2007-01-09

    An isolated and purified bacterium is provided which was isolated from a high-level radioactive waste site of mixed waste. The isolate has the ability to degrade a wide variety of organic contaminants while demonstrating high tolerance to ionizing radiation. The organism is uniquely suited to bioremediation of a variety or organic contaminants while in the presence of ionizing radiation.

  16. Inactivation of Microorganisms

    NASA Astrophysics Data System (ADS)

    Alzamora, Stella Maris; Guerrero, Sandra N.; Schenk, Marcela; Raffellini, Silvia; López-Malo, Aurelio

    Minimal processing techniques for food preservation allow better retention of product flavor, texture, color, and nutrient content than comparable conventional treatments. A wide range of novel alternative physical factors have been intensely investigated in the last two decades. These physical factors can cause inactivation of microorganisms at ambient or sublethal temperatures (e.g., high hydrostatic pressure, pulsed electric fields, ultrasound, pulsed light, and ultraviolet light). These technologies have been reported to reduce microorganism population in foods while avoiding the deleterious effects of severe heating on quality. Among technologies, high-energy ultrasound (i.e., intensities higher than 1 W/cm2, frequencies between 18 and 100 kHz) has attracted considerable interest for food preservation applications (Mason et al., 1996; Povey and Mason, 1998).

  17. Microorganisms and psoriasis.

    PubMed Central

    Rosenberg, E. W.; Noah, P. W.; Skinner, R. B.

    1994-01-01

    It has been suggested previously that psoriasis is best explained as a distinctive inflammatory response to a variety of microbial stimuli, all acting primarily through activation of the alternative complement pathway. For the past several years we have conducted a "Problem Psoriasis Clinic" based on that premise. Patients are questioned, examined, and subjected to microbiologic laboratory investigations in an attempt to identify possibly relevant microorganisms, and then are treated with antibiotics. This article lists the most commonly found microorganisms in psoriasis patients and describes the usual treatment for each. Results obtained with this approach compare favorably with those achieved with more usual anti-psoriasis treatments. We recommend that a microbiologic investigation and a trial of antimicrobial treatment should precede any plan to treat psoriasis patients with anything more than the simplest topical agents. PMID:8040907

  18. Interactions between plants and microorganisms

    USDA-ARS?s Scientific Manuscript database

    Allelopathic microorganisms comprise rhizobacteria and fungi that colonize the surfaces of plant roots, and produce and release phytotoxic metabolites, similar to allelochemicals, that detrimentally affect growth of their host plants. The allelopathic microorganisms are grouped separately from typic...

  19. Saturation of calcium channels in single isolated smooth muscle cells of guinea-pig taenia caeci.

    PubMed Central

    Ganitkevich VYa; Shuba, M F; Smirnov, S V

    1988-01-01

    1. Calcium channel currents were recorded in Cs+-dialysed voltage-clamped single smooth muscle cells isolated from the guinea-pig taenia caeci to evaluate the current-carrying ability of Ca2+, Ba2+, Sr2+ and Mg2+ ions. 2. Ba2+ and Sr2+ ions, as well as Ca2+ ions, were able to carry an inward current through calcium channels. Calcium channel current was not observed when Mg2+ was the only divalent cation in the external solution. 3. Concentration dependences of calcium (ICa), barium (IBa) and strontium (ISr) currents were studied. It was found that currents through calcium channels saturated with increasing the extracellular concentration of a current carrier. Saturation of each current can be fitted with a Langmuir curve with apparent dissociation constants of 1.2 mM for Ca2+, 1.8 mM for Sr2+ and 9.6 mM for Ba2+ ions. 4. External Mg2+ ions reduced both ICa and IBa.IBa was depressed to a greater extent than ICa by Mg2+ ions. Reduction of ICa by Mg2+ ions seems to agree with competitive antagonism between Ca2+ and Mg2+ ions (Hagiwara & Takahashi, 1967). 5. When the external divalent cation concentration [( C2+]o) was changed, the current-voltage relationship of currents through calcium channels was shifted along the potential axis suggesting that activation gating of calcium channels was affected by [C2+]o. These voltage shifts can be fitted with the Gouy-Chapman theory supposing the density of surface charges near calcium channels to be 0.5 e nm-2 and including more potent binding of Ca2+ ions to surface charges than of Ba2+, Sr2+ and Mg2+ ions. 6. The changes in the Ca2+, Ba2+ and Sr2+ concentrations at the surface of the membrane were calculated. It was found that saturation of IBa can be explained by saturation of Ba2+ surface concentration while saturation of ICa and ISr cannot. 7. It was suggested that barium ions were able to carry the larger current through calcium channels in smooth muscle cells due to their much weaker binding within the calcium channel

  20. On-chip single-copy real-time reverse-transcription PCR in isolated picoliter droplets

    SciTech Connect

    Beer, N R; Wheeler, E; Lee-Houghton, L; Watkins, N; Nasarabadi, S; Hebert, N; Leung, P; Arnold, D; Bailey, C; Colston, B

    2007-12-19

    The first lab-on-chip system for picoliter droplet generation and RNA isolation, followed by reverse transcription, and PCR amplification with real-time fluorescence detection in the trapped droplets has been developed. The system utilized a shearing T-junction in a fused silica device to generate a stream of monodisperse picoliter-scale droplets that were isolated from the microfluidic channel walls and each other by the oil phase carrier. An off-chip valving system stopped the droplets on-chip, allowing thermal cycling for reverse transcription and subsequent PCR amplification without droplet motion. This combination of the established real-time reverse transcription-PCR assay with digital microfluidics is ideal for isolating single-copy RNA and virions from a complex environment, and will be useful in viral discovery and gene-profiling applications.

  1. Microchannel-free collection and single-cell isolation of yeast cells in a suspension using liquid standing wave

    NASA Astrophysics Data System (ADS)

    Matsutani, Akihiro; Takada, Ayako

    2016-11-01

    We demonstrate a microchannel-free collection method at nodes of liquid standing waves by the vertical vibration of a suspension including yeast cells. The pattern formation of the collection of cells using standing waves in a suspension was investigated by varying the frequency and waveform of vibrations. The single-cell isolation of yeast cells was achieved using a microenclosure array set at the nodes. In addition, we succeeded in the microchannel-free collection of yeast cells in a suspension, where patterns were formed by tapping vibration. The proposed technique is very simple and we believe that it will be useful for single-cell analysis and investigation.

  2. Host Defense Against Opportunist Microorganisms Following Trauma.

    DTIC Science & Technology

    1980-09-01

    candidemia or evidence of systemic candidosis were to be correlated with changes in the numbers of Candida isolated from the serial quantitative cultures...compared for their sensitivity and specificity for the detection of Candida antigenemia prior to and during candidemia and systemic candidosis. The...sera contained inhibitory activity had pneumonia, candidemia , and multiple episodes of bacteremia caused by more than one microorganism, and 2 had a

  3. Survival of vaginal microorganisms in three commercially available transport systems.

    PubMed

    DeMarco, Allison L; Rabe, Lorna K; Austin, Michele N; Stoner, Kevin A; Avolia, Hilary A; Meyn, Leslie A; Hillier, Sharon L

    2017-02-24

    Transport systems are used to collect and maintain the viability of microorganisms. Two Amies media based transport systems, BD CultureSwab™ MaxV(+) Amies Medium without Charcoal (MaxV(+)) and Fisherfinest(®) with Amies gel Transport Medium without charcoal (Fisherfinest(®)) were compared to a Cary-Blair media based transport system, Starswab(®) Anaerobic Transport System (Starswab(®)), for their capacity to maintain the viability of 17 clinical microorganisms commonly isolated from the vagina (Lactobacillus crispatus, L. jensenii, L. iners, group B streptococci, Candida albicans, Escherichia coli, Enterococcus faecalis, Atopobium vaginae, Peptoniphilus harei, Mycoplasma hominis, Gardnerella vaginalis, Dialister microaerophilus, Mobiluncus curtisii, Prevotella amnii, P. timonensis, P. bivia, and Porphyromonas uenonis). Single swabs containing mixtures of up to five different species were inoculated in triplicate and held at 4 °C and room temperature for 24, 48, 72, and 96 h (h). At each time point, swabs were eluted into a sterile salt solution, serially diluted, inoculated onto selected media, and incubated. Each colony type was quantified and identified. A change in sample stability was reported as a ≥1 log increase or decrease in microorganism density from baseline. Overall, the viability of fastidious anaerobes was maintained better at 4 °C than room temperature. At 4 °C all three transport systems maintained the viability and prevented replication of C. albicans, E. faecalis, GBS, and E. coli. Microorganisms having a ≥1 log decrease in less than 24 h at 4 °C included A. vaginae, G. vaginalis, and P. uenonis in Starswab(®), L. iners, A. vaginae, and P. amnii in MaxV(+), and A. vaginae, G. vaginalis, P. bivia, and P. amnii in Fisherfinest(®). At 48 h at 4 °C, a ≥1 log decrease in concentration density was observed for P. harei and P. amnii in Starswab(®), G. vaginalis, P. bivia and P. uenonis in MaxV(+), and L

  4. Detecting the presence of microorganisms

    NASA Technical Reports Server (NTRS)

    Wilkins, Judd R. (Inventor); Stoner, Glenn E. (Inventor)

    1977-01-01

    The presence of microorganisms in a sample is determined by culturing microorganisms in a growth medium which is in contact with a measuring electrode and a reference electrode and detecting a change in potential between the electrodes caused by the presence of the microorganisms in the medium with a high impedance potentiometer.

  5. Low vibration laboratory with a single-stage vibration isolation for microscopy applications.

    PubMed

    Voigtländer, Bert; Coenen, Peter; Cherepanov, Vasily; Borgens, Peter; Duden, Thomas; Tautz, F Stefan

    2017-02-01

    The construction and the vibrational performance of a low vibration laboratory for microscopy applications comprising a 100 ton floating foundation supported by passive pneumatic isolators (air springs), which rest themselves on a 200 ton solid base plate, are discussed. The optimization of the air spring system leads to a vibration level on the floating floor below that induced by an acceleration of 10 ng for most frequencies. Additional acoustic and electromagnetic isolation is accomplished by a room-in-room concept.

  6. Low vibration laboratory with a single-stage vibration isolation for microscopy applications

    NASA Astrophysics Data System (ADS)

    Voigtländer, Bert; Coenen, Peter; Cherepanov, Vasily; Borgens, Peter; Duden, Thomas; Tautz, F. Stefan

    2017-02-01

    The construction and the vibrational performance of a low vibration laboratory for microscopy applications comprising a 100 ton floating foundation supported by passive pneumatic isolators (air springs), which rest themselves on a 200 ton solid base plate, are discussed. The optimization of the air spring system leads to a vibration level on the floating floor below that induced by an acceleration of 10 ng for most frequencies. Additional acoustic and electromagnetic isolation is accomplished by a room-in-room concept.

  7. High-throughput de novo screening of receptor agonists with an automated single-cell analysis and isolation system

    PubMed Central

    Yoshimoto, Nobuo; Tatematsu, Kenji; Iijima, Masumi; Niimi, Tomoaki; Maturana, Andrés D.; Fujii, Ikuo; Kondo, Akihiko; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

    2014-01-01

    Reconstitution of signaling pathways involving single mammalian transmembrane receptors has not been accomplished in yeast cells. In this study, intact EGF receptor (EGFR) and a cell wall-anchored form of EGF were co-expressed on the yeast cell surface, which led to autophosphorylation of the EGFR in an EGF-dependent autocrine manner. After changing from EGF to a conformationally constrained peptide library, cells were fluorescently labeled with an anti-phospho-EGFR antibody. Each cell was subjected to an automated single-cell analysis and isolation system that analyzed the fluorescent intensity of each cell and automatically retrieved each cell with the highest fluorescence. In ~3.2 × 106 peptide library, we isolated six novel peptides with agonistic activity of the EGFR in human squamous carcinoma A431 cells. The combination of yeast cells expressing mammalian receptors, a cell wall-anchored peptide library, and an automated single-cell analysis and isolation system might facilitate a rational approach for de novo drug screening. PMID:24577528

  8. A planar microelectrode array for simultaneous detection of electrically evoked dopamine release from distinct locations of a single isolated neuron.

    PubMed

    Patel, Bhavik Anil; Luk, Collin C; Leow, Pei Ling; Lee, Arthur J; Zaidi, Wali; Syed, Naweed I

    2013-05-21

    Neurotransmission is a key process of communication between neurons. Although much is known about this process and the influence it has on the function of the body, little is understood about the dynamics of signalling from structural regions of a single neuron. In this study we have fabricated and characterised a microelectrode array (MEA) which was utilised for simultaneous multi-site recordings of dopamine release from an isolated single neuron. The MEA consisted of gold electrodes that were created in plane with the insulation layer using a chemical mechanical planarization process. The detection limit for dopamine measurements was 11 ± 3 nM and all the gold electrodes performed in a consistent fashion during amperometric recordings of 100 nM dopamine. Fouling of the gold electrode was investigated, where no significant change in the current was observed over 4 hours when monitoring 100 nM dopamine. The MEA was accessed using freshly isolated dopaminergic somas from the pond snail, Lymnaea stagnalis, where electrically evoked dopamine release was clearly observed. Measurements were conducted at four structural locations of a single isolated neuron, where electrically evoked dopamine release was observed from the cell body, axonal regions and the terminal. Over time, the release of dopamine varied over the structural regions of the neuron. Such information can provide an insight into the signalling mechanism of neurons and how they potentially form synaptic connections.

  9. Mycobacterium tuberculosis isolates from single outpatient clinic in Panama City exhibit wide genetic diversity.

    PubMed

    Sambrano, Dilcia; Correa, Ricardo; Almengor, Pedro; Domínguez, Amada; Vega, Silvio; Goodridge, Amador

    2014-08-01

    Understanding Mycobacterium tuberculosis biodiversity and transmission is significant for tuberculosis control. This short report aimed to determine the genetic diversity of M. tuberculosis isolates from an outpatient clinic in Panama City. A total of 62 M. tuberculosis isolates were genotyped by 12 loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and Spoligotyping. Forty-five (72.6%) of the isolates showed unique MIRU-VNTR genotypes, and 13 (21%) of the isolates were grouped into four clusters. Four isolates showed polyclonal MIRU-VNTR genotypes. The MIRU-VNTR Hunter-Gaston discriminatory index reached 0.988. The Spoligotyping analysis revealed 16 M. tuberculosis families, including Latin American-Mediterranean, Harlem, and Beijing. These findings suggest a wide genetic diversity of M. tuberculosis isolates at one outpatient clinic. A detailed molecular epidemiology survey is now warranted, especially following second massive immigration for local Panama Canal expansion activities. © The American Society of Tropical Medicine and Hygiene.

  10. Evaluation of a Single Procedure Allowing the Isolation of Enteropathogenic Yersinia along with Other Bacterial Enteropathogens from Human Stools

    PubMed Central

    Savin, Cyril; Leclercq, Alexandre; Carniel, Elisabeth

    2012-01-01

    Enteropathogenic Yersinia are among the most frequent agents of human diarrhea in temperate and cold countries. However, the incidence of yersiniosis is largely underestimated because of the peculiar growth characteristics of pathogenic Yersinia, which make their isolation from poly-contaminated samples difficult. The use of specific procedures for Yersinia isolation is required, but is expensive and time consuming, and therefore is not systematically performed in clinical pathology laboratories. A means to circumvent this problem would be to use a single procedure for the isolation of all bacterial enteropathogens. Since the Statens Serum Institut enteric medium (SSI) has been reported to allow the growth at 37°C of most Gram-negative bacteria, including Yersinia, our study aimed at evaluating its performances for Yersinia isolation, as compared to the commonly used Yersinia-specific semi-selective Cefsulodin-Irgasan-Novobiocin medium (CIN) incubated at 28°C. Our results show that Yersinia pseudotuberculosis growth was strongly inhibited on SSI at 37°C, and therefore that this medium is not suitable for the isolation of this species. All Yersinia enterocolitica strains tested grew on SSI, while some non-pathogenic Yersinia species were inhibited. The morphology of Y. enterocolitica colonies on SSI allowed their differentiation from various other Gram-negative bacteria commonly isolated from stool samples. However, in artificially contaminated human stools, the recovery of Y. enterocolitica colonies on SSI at 37°C was difficult and was 3 logs less sensitive than on CIN at 28°C. Therefore, despite its limitations, the use of a specific procedure (CIN incubated at 28°C) is still required for an efficient isolation of enteropathogenic Yersinia from stools. PMID:22911756

  11. Evaluation of a single procedure allowing the isolation of enteropathogenic Yersinia along with other bacterial enteropathogens from human stools.

    PubMed

    Savin, Cyril; Leclercq, Alexandre; Carniel, Elisabeth

    2012-01-01

    Enteropathogenic Yersinia are among the most frequent agents of human diarrhea in temperate and cold countries. However, the incidence of yersiniosis is largely underestimated because of the peculiar growth characteristics of pathogenic Yersinia, which make their isolation from poly-contaminated samples difficult. The use of specific procedures for Yersinia isolation is required, but is expensive and time consuming, and therefore is not systematically performed in clinical pathology laboratories. A means to circumvent this problem would be to use a single procedure for the isolation of all bacterial enteropathogens. Since the Statens Serum Institut enteric medium (SSI) has been reported to allow the growth at 37°C of most gram-negative bacteria, including Yersinia, our study aimed at evaluating its performances for Yersinia isolation, as compared to the commonly used Yersinia-specific semi-selective Cefsulodin-Irgasan-Novobiocin medium (CIN) incubated at 28°C. Our results show that Yersinia pseudotuberculosis growth was strongly inhibited on SSI at 37°C, and therefore that this medium is not suitable for the isolation of this species. All Yersinia enterocolitica strains tested grew on SSI, while some non-pathogenic Yersinia species were inhibited. The morphology of Y. enterocolitica colonies on SSI allowed their differentiation from various other gram-negative bacteria commonly isolated from stool samples. However, in artificially contaminated human stools, the recovery of Y. enterocolitica colonies on SSI at 37°C was difficult and was 3 logs less sensitive than on CIN at 28°C. Therefore, despite its limitations, the use of a specific procedure (CIN incubated at 28°C) is still required for an efficient isolation of enteropathogenic Yersinia from stools.

  12. Microorganisms for producing organic acids

    DOEpatents

    Pfleger, Brian Frederick; Begemann, Matthew Brett

    2014-09-30

    Organic acid-producing microorganisms and methods of using same. The organic acid-producing microorganisms comprise modifications that reduce or ablate AcsA activity or AcsA homolog activity. The modifications increase tolerance of the microorganisms to such organic acids as 3-hydroxypropionic acid, acrylic acid, propionic acid, lactic acid, and others. Further modifications to the microorganisms increase production of such organic acids as 3-hydroxypropionic acid, lactate, and others. Methods of producing such organic acids as 3-hydroxypropionic acid, lactate, and others with the modified microorganisms are provided. Methods of using acsA or homologs thereof as counter-selectable markers are also provided.

  13. Microorganisms for producing organic acids

    SciTech Connect

    Pfleger, Brian Frederick; Begemann, Matthew Brett

    2014-09-30

    Organic acid-producing microorganisms and methods of using same. The organic acid-producing microorganisms comprise modifications that reduce or ablate AcsA activity or AcsA homolog activity. The modifications increase tolerance of the microorganisms to such organic acids as 3-hydroxypropionic acid, acrylic acid, propionic acid, lactic acid, and others. Further modifications to the microorganisms increase production of such organic acids as 3-hydroxypropionic acid, lactate, and others. Methods of producing such organic acids as 3-hydroxypropionic acid, lactate, and others with the modified microorganisms are provided. Methods of using acsA or homologs thereof as counter-selectable markers are also provided.

  14. Streptomyces sp. 173, an insecticidal micro-organism from marine.

    PubMed

    Xiong, L; Li, J; Kong, F

    2004-01-01

    To find new insecticidal antibiotics from marine micro-organisms. Strains isolated from seawater and sea sediments from Beidiahe and Dagang of the east coast of China were screened for their insecticidal qualities. The screening was carried out using bioassay of brine shrimp and the insect pest Helicoverpa armigera. The fermentation, preliminary extraction and isolation of Streptomyces sp.173 were carried out. In total 331 isolates were examined through bioassay of brine shrimp and 40 isolates (12.08%) showed potential insecticidal activities. Of the 40 isolates, one isolate, designated Streptomyces sp.173, was found to have strong insecticidal activity against both brine shrimp and H. armigera, similar to that of avermectin B1. The isolated Streptomyces sp.173 has great insecticidal potency. This work indicated that marine micro-organisms could be an important source of insecticidal antibiotics and the improved anti-brine shrimp bioassay is suitable for primary screening.

  15. Inner structural vibration isolation method for a single control moment gyroscope

    NASA Astrophysics Data System (ADS)

    Zhang, Jingrui; Guo, Zixi; Zhang, Yao; Tang, Liang; Guan, Xin

    2016-01-01

    Assembling and manufacturing errors of control moment gyros (CMG) often generate high frequency vibrations which are detrimental to spacecrafts with high precision pointing requirement. In this paper, some design methods of vibration isolation between CMG and spacecraft is dealt with. As a first step, the dynamic model of the CMG with and without supporting isolation structures is studied and analyzed. Subsequently, the frequency domain analysis of CMG with isolation system is performed and the effectiveness of the designed system is ascertained. Based on the above studies, an adaptive design suitable with appropriate design parameters are carried out. A numerical analysis is also performed to understand the effectiveness of the system and the comparison made. The simulation results clearly indicate that when the ideal isolation structure was implemented in the spacecraft, the vibrations generated by the rotor were found to be greatly reduced, while the capacity of the output torque was not lost, which means that the isolation system will not affect the performance of attitude control.

  16. Functional expression and affinity selection of single-chain cro by phage display: isolation of novel DNA-binding proteins.

    PubMed

    Nilsson, M T; Mossing, M C; Widersten, M

    2000-07-01

    A robust selection system affording phage display of the DNA-binding helix-turn-helix protein Cro is presented. The aim of the work was to construct an experimental system allowing for the construction and isolation of Cro-derived protein with new DNA-binding properties. A derivative of the phage lambda Cro repressor, scCro8, in which the protein subunits had been covalently connected via a peptide linker was expressed in fusion with the gene 3 protein of Escherichia coli filamentous phage. The phage-displayed single-chain Cro was shown to retain the DNA binding properties of its wild-type Cro counterpart regarding DNA sequence specificity and binding affinity. A kinetic analysis revealed the rate constant of dissociation of the single-chain Cro-phage/DNA complex to be indistinguishable from that of the free single-chain Cro. Affinity selection using a biotinylated DNA with a target consensus operator sequence allowed for a 3000-fold enrichment of phages displaying single-chain Cro over control phages. The selection was based on entrapment of phage/DNA complexes formed in solution on streptavidin-coated paramagnetic beads. The expression system was subsequently used to isolate variant scCro8 proteins, mutated in their DNA-binding residues, that specifically recognized new, unnatural target DNA ligands.

  17. Effect of physiological levels of caffeine on Ca2+ handling and fatigue development in Xenopus isolated single myofibers

    PubMed Central

    Rosser, Joelle I.; Walsh, Brandon; Hogan, Michael C.

    2009-01-01

    The purpose of the present study was to determine whether exposure to exogenous physiological concentrations of caffeine influence contractility, Ca2+ handling, and fatigue development in isolated single Xenopus laevis skeletal muscle fibers. After isolation, two identical contractile periods (separated by 60-min rest) were conducted in each single myofiber (n = 8) at 20°C. During the first contractile period, four fibers were perfused with a noncaffeinated Ringer solution, while the other four fibers were perfused with a caffeinated (70 μM) Ringer solution. The order was reversed for the second contractile period. The single myofibers were stimulated during each contractile period at increasing frequencies (0.16, 0.20, 0.25, 0.33, 0.50, and 1.0 tetanic contractions/s), with each stimulation frequency lasting 2 min until fatigue ensued, defined in this study as a fall in tension development to 66% of maximum. Tension development and free cytosolic [Ca2+] (fura-2 fluorescence spectroscopy) were simultaneously measured. There was no significant difference in the peak force generation, time to fatigue, cytosolic Ca2+ levels, or relaxation times between the noncaffeinated and caffeinated trials. These results demonstrate that physiological levels of caffeine have no significant effect on Xenopus single myofiber contractility, Ca2+ handling, and fatigue development, and suggest that any ergogenic effects of physiological levels of caffeine on muscle performance during contractions of moderate to high intensity are likely related to factors extraneous to the muscle fiber. PMID:19261915

  18. Two classes of single-stranded regions evident in deproteinized preparations of replicating DNA isolated from mammalian cells

    SciTech Connect

    Stewart, B.W.; Kavallaris, M.; Catchpoole, D.; Norris, M.D. )

    1991-02-01

    In DNA isolated from proliferating human lymphoblastoid CCRF-CEM cells which had been pulse-labeled by exposure to (3H)thymidine for periods from 30 s to 10 min, single-stranded regions were analyzed by caffeine-gradient elution from benzoylated DEAE-cellulose. Two classes of structural defect were evident. Some replicating DNA exhibited single-stranded regions of approximately 200 nucleotides, while most newly incorporated radioactivity was associated with DNA containing single-stranded regions from 900 to approximately 4000 nucleotides. The distribution of thymidine-derived radioactivity did not suggest sequential or preferential labeling of these DNA fractions as the incorporation time was varied. The findings may be correlated with recent proposals regarding the structural basis of eukaryotic DNA replication.

  19. Lead Speciation in Microorganisms.

    PubMed

    Stewart, Theodora J

    2017-04-10

    The biogeochemical cycles of lead (Pb) have been largely affected by anthropogenic activities as a result of its high natural abundance and use over the centuries [1]. At sites more strongly impacted by urbanization [2] and mining [3], Pb is found at high nano to low micromolar concentrations in surface waters, and can be significantly higher in soil and sediment [4]. Microorganisms are found everywhere and their responses to Pb exposure can range from resistant to highly sensitive [5, 6]. These varying levels of toxicity can be attributed to the cellular handling of Pb, making it important to understand the role of intracellular Pb speciation for more accurate toxicity predictions.

  20. Laboratory Identification of Leptotrichia Species Isolated From Bacteremia Patients at a Single Institution.

    PubMed

    Cho, Eun Hye; Park, Kyung Sun; Yang, Mina; Song, Dong Joon; Huh, Hee Jae; Ki, Chang Seok; Lee, Nam Yong

    2017-05-01

    We describe the laboratory identification of Leptotrichia species from clinical isolates collected over a six-year period. Five isolates from blood cultures were identified as Leptotrichia species. Gram stain showed large, fusiform, gram-negative or -variable bacilli. Identification based on biochemical testing was unsuccessful; however, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry proved to be a useful tool for identifying Leptotrichia species to the genus level. Species level identification was successfully achieved by using 16S ribosomal RNA gene sequencing. © The Korean Society for Laboratory Medicine.

  1. Percutaneous Coronary Intervention of Hidden Coronary Artery-Unusual Type of Isolated Single Coronary Artery

    PubMed Central

    Patil, Shivanand; Ramalingam, Rangaraj; Manjunath, Cholenahally Nanjappa; Subramanyam, Kasamsetty

    2016-01-01

    Single coronary artery is a rare congenital coronary artery anomaly, the incidence of which is 0.024-0.066% as described in literature. Report of cases having single coronary artery along with acute myocardial infarction are scanty and reports of percutaneous intervention in such a situation are even fewer, technically challenging and potentially cataclysmic. As single coronary artery supplies the entire myocardium, occlusion of this can result in significant ischemic insult, resulting in severe biventricular dysfunction. Percutaneous Coronary Intervention (PCI) of single coronary artery is technically challenging and carries high risk which may be equated to left main intervention. We report a rare interesting case of L1 variety of single coronary artery which presented as acute inferoposterior myocardial infarction with successful rescue PCI to Left Circumflex Artery (LCx). PMID:27656488

  2. Use of antibody gene library for the isolation of specific single chain antibodies by ampicillin-antigen conjugates.

    PubMed

    Neumann-Schaal, Meina; Messerschmidt, Katrin; Grenz, Nicole; Heilmann, Katja

    2013-03-01

    Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage display and ribosome display or different cell-surface display formats. We describe a new method which allows the selection of Escherichia coli cells producing the required single chain antibody by cultivation in presence of ampicillin conjugated to the antigen of interest. The method utilizes the neutralization of the conjugate by the produced single chain antibody which is secreted to the periplasm. Therefore, a new expression system based on the pET26b vector was designed and a library was constructed. The method was successfully established first for the selection of E. coli BL21 Star (DE3) cells expressing a model single chain antibody (anti-fluorescein) by a simple selection assay on LB-agar plates. Using this selection assay, we could identify a new single chain antibody binding biotin by growing E. coli BL21 Star (DE3) containing the library in presence of a biotin-ampicillin conjugate. In contrast to methods as molecular or cell surface display our selection system applies the soluble single chain antibody molecule and thereby avoids undesired effects, e.g. by the phage particle or the yeast fusion protein. By selecting directly in an expression strain, production and characterization of the selected single chain antibody is possible without any further cloning or transformation steps.

  3. The effect of isolated valgus moments on ACL strain during single-leg landing: A simulation study

    PubMed Central

    Shin, Choongsoo S.; Chaudhari, Ajit M.; Andriacchi, Thomas P.

    2009-01-01

    Valgus moments on the knee joint during single-leg landing have been suggested as a risk factor for anterior cruciate ligament (ACL) injury. The purpose of this study was to test the influence of isolated valgus moment on ACL strain during single-leg landing. Physiologic levels of valgus moments from an in vivo study of single-leg landing were applied to a three-dimensional dynamic knee model, previously developed and tested for ACL strain measurement during simulated landing. The ACL strain, knee valgus angle, tibial rotation, and medial collateral ligament (MCL) strain were calculated and analyzed. The study shows that the peak ACL strain increased nonlinearly with increasing peak valgus moment. Subjects with naturally high valgus moments showed greater sensitivity for increased ACL strain with increased valgus moment, but ACL strain plateaus below reported ACL failure levels when the applied isolated valgus moment rises above the maximum values observed during normal cutting activities. In addition, the tibia was observed to rotate externally as the peak valgus moment increased due to bony and soft-tissue constraints. In conclusion, knee valgus moment increases peak ACL strain during single-leg landing. However, valgus moment alone may not be sufficient to induce an isolated ACL tear without concomitant damage to the MCL, because coupled tibial external rotation and increasing strain in the MCL prevent proportional increases in ACL strain at higher levels of valgus moment. Training that reduces the external valgus moment, however, can reduce the ACL strain and thus may help athletes reduce their overall ACL injury risk. PMID:19100550

  4. Microorganism lipid droplets and biofuel development.

    PubMed

    Liu, Yingmei; Zhang, Congyan; Shen, Xipeng; Zhang, Xuelin; Cichello, Simon; Guan, Hongbin; Liu, Pingsheng

    2013-12-01

    Lipid droplet (LD) is a cellular organelle that stores neutral lipids as a source of energy and carbon. However, recent research has emerged that the organelle is involved in lipid synthesis, transportation, and metabolism, as well as mediating cellular protein storage and degradation. With the exception of multi-cellular organisms, some unicellular microorganisms have been observed to contain LDs. The organelle has been isolated and characterized from numerous organisms. Triacylglycerol (TAG) accumulation in LDs can be in excess of 50% of the dry weight in some microorganisms, and a maximum of 87% in some instances. These microorganisms include eukaryotes such as yeast and green algae as well as prokaryotes such as bacteria. Some organisms obtain carbon from CO2 via photosynthesis, while the majority utilizes carbon from various types of biomass. Therefore, high TAG content generated by utilizing waste or cheap biomass, coupled with an efficient conversion rate, present these organisms as bio-tech 'factories' to produce biodiesel. This review summarizes LD research in these organisms and provides useful information for further LD biological research and microorganism biodiesel development.

  5. Geographical Differences Associated with Single-Nucleotide Polymorphisms (SNPs) in Nine Gene Targets among Resistant Clinical Isolates of Mycobacterium tuberculosis

    PubMed Central

    Hoshide, Matt; Qian, Lishi; Rodrigues, Camilla; Warren, Rob; Victor, Tommie; Evasco, Henry B.; Tupasi, Thelma; Crudu, Valeriu

    2014-01-01

    Alternative diagnostic methods, such as sequence-based techniques, are necessary for increasing the proportion of tuberculosis cases tested for drug resistance. Despite the abundance of data on drug resistance, isolates can display phenotypic resistance but lack any distinguishable markers. Furthermore, because resistance-conferring mutations develop under antibiotic pressure, different drug regimens could favor unique single-nucleotide polymorphisms (SNPs) in different geographical regions. A total of 407 isolates were collected from four geographical regions with a high prevalence of drug-resistant tuberculosis (India, Moldova, the Philippines, and South Africa). The “hot spot” or promoter sequences of nine genes (rpoB, gyrA, gyrB, katG, inhA promoter, ahpC promoter, eis promoter, rrs, and tlyA) associated with resistance to four types of antibiotics (rifampin, isoniazid, fluoroquinolones, and aminoglycosides) were analyzed for markers. Four genes contributed largely to resistance (rpoB, gyrA, rrs, and katG), two genes contributed moderately to resistance (the eis and inhA promoters), and three genes contributed little or no resistance (gyrB, tlyA, and the ahpC promoter) in clinical isolates. Several geographical differences were found, including a double mutation in rpoB found in 37.1% of isolates from South Africa, the C→T mutation at position −12 of the eis promoter found exclusively in 60.6% of isolates from Moldova, and the G→A mutation at position −46 of the ahpC promoter found only in India. These differences in polymorphism frequencies emphasize the uniqueness of isolates found in different geographical regions. The inclusion of several genes provided a moderate increase in sensitivity, and elimination of the examination of other genes might increase efficiency. PMID:23784122

  6. Immediate primary anastomosis for isolated oesophageal atresia: A single-centre experience

    PubMed Central

    Uygun, Ibrahim; Zeytun, Hikmet; Otcu, Selcuk

    2015-01-01

    Background: Isolated oesophageal atresia without tracheo-oesophageal fistula represents a major challenge for most paediatric surgeons. Here, we present our experience with six neonates with isolated oesophageal atresia who successfully underwent immediate primary anastomosis using multiple Livaditis circular myotomy. Materials and Methods: All six neonates were gross type A isolated oesophageal atresia (6%), from among 102 neonates with oesophageal atresia, treated between January 2009 and December 2013. Five neonates were female; one was male. The mean birth weight was 2300 (range 1700-3100) g. Results: All six neonates successfully underwent immediate primary anastomosis using multiple myotomies (mean 3; range 2-4) within 10 (median 3) days after birth. The gap under traction ranged from 6 to 7 cm. One neonate died of a major cardiac anomaly. Another neonate was lost to follow-up after being well for 3 months. Three anastomotic strictures were treated with balloon dilatation, and four anastomotic leaks were treated conservatively. The mean duration of follow-up was 33 months. Conclusions: To treat isolated oesophageal atresia, an immediate primary anastomosis can be achieved using multiple myotomies. Although, this approach is associated with high complication rates, as are other similar approaches, these complications can be overcome. PMID:26712295

  7. Rapid isolation of nuclei from living immune cells by a single centrifugation through a multifunctional lysis gradient.

    PubMed

    Poglitsch, Marko; Katholnig, Karl; Säemann, Marcus D; Weichhart, Thomas

    2011-10-28

    Due to their low protein content and limited nuclear detergent stability, primary human immune cells such as monocytes or T lymphocytes represent a great challenge for standard nuclear isolation protocols. Nuclei clumping during the multiple centrifugation steps or contamination of isolated nuclei with cytoplasmic proteins due to membrane lysis is a frequently observed problem. Here we describe a versatile and novel method for the isolation of clean and intact nuclei from primary human monocytes, which can be applied for virtually any cell type. Living cells were applied on an iso-osmolar discontinuous iodixanol-based density gradient including a detergent-containing lysis layer. Mild cell lysis as well as efficient washing of the nuclei was performed during the course of one single low g-force centrifugation step. The isolation procedure, which we call lysis gradient centrifugation (LGC), results in the recovery of 90-95% of highly pure nuclei. This easy and highly reproducible procedure allows an effective preparation of nuclei and the cytoplasm in only 15 min with the ability to handle as little as one million cells per sample and easy parallel processing of multiple samples.

  8. Microorganisms having enhanced resistance to acetate and methods of use

    DOEpatents

    Brown, Steven D; Yang, Shihui

    2014-10-21

    The present invention provides isolated or genetically modified strains of microorganisms that display enhanced resistance to acetate as a result of increased expression of a sodium proton antiporter. The present invention also provides methods for producing such microbial strains, as well as related promoter sequences and expression vectors. Further, the present invention provides methods of producing alcohol from biomass materials by using microorganisms with enhanced resistance to acetate.

  9. Clostridiumm ljungdahlii, an anaerobic ethanol and acetate producing microorganism

    DOEpatents

    Gaddy, J.L.; Clausen, E.C.

    1992-12-22

    A newly discovered microorganism was isolated in a biologically pure culture and designated Clostridium ljungdahlii, having the identifying characteristics of ATCC No. 49587. Cultured in an aqueous nutrient medium under anaerobic conditions, this microorganism is capable of producing ethanol and acetate from CO and H[sub 2]O and/or CO[sub 2] and H[sub 2] in synthesis gas. Under optimal growth conditions, the microorganism produces acetate in preference to ethanol. Conversely, under non-growth conditions, ethanol production is favored over acetate. 3 figs.

  10. Clostridiumm ljungdahlii, an anaerobic ethanol and acetate producing microorganism

    DOEpatents

    Gaddy, James L.; Clausen, Edgar C.

    1992-01-01

    A newly discovered microorganism was isolated in a biologically pure culture and designated Clostridium ljungdahlii, having the identifying characteristics of ATCC No. 49587. Cultured in an aqueous nutrient medium under anaerobic conditions, this microorganism is capable of producing ethanol and acetate from CO and H.sub.2 O and/or CO.sub.2 and H.sub.2 in synthesis gas. Under optimal growth conditions, the microorganism produces acetate in preference to ethanol. Conversely, under non-growth conditions, ethanol production is favored over acetate.

  11. Efficient single muscle fiber isolation from alcohol-fixed adult muscle following β-galactosidase staining for satellite cell detection.

    PubMed

    Verma, Mayank; Asakura, Atsushi

    2011-01-01

    Staining for β-galactosidase activity for whole tissues, sections, and cells is a common method to detect expression of β-galactosidase reporter transgene as well as senescence-dependent β-galactosidase activity. Choice of fixatives is a critical step for detection of β-galactosidase activity, subsequent immunostaining, and enzymatic digestion of tissue to dissociate cells. In this report, the authors examined several aldehyde and alcohol fixatives in mouse skeletal muscle tissues for their efficiency at improving detection of β-galactosidase activity as well as detection by immunostaining. In addition, fixatives were also analyzed for their efficiency for collagenase digestion to isolate single muscle fibers on postfixed β-galactosidase-stained whole skeletal muscle tissues. The results show that fixing cells with isopropanol yields the greatest reliability and intensity in both β-galactosidase staining as well as double staining for β-galactosidase activity and antibodies. In addition, isopropanol and ethanol, but not glutaraldehyde or paraformaldehyde, allow for the isolation of single muscle fibers from the diaphragm and tibialis anterior muscles following postfixed β-galactosidase staining. Using this method, it is possible to identify the amount of cells that occupy the satellite cell compartment in single muscle fibers prepared from any muscle tissues, including tibialis anterior muscle and diaphragm.

  12. Slowing of velocity during isotonic shortening in single isolated smooth muscle cells. Evidence for an internal load

    PubMed Central

    1990-01-01

    In single smooth muscle cells, shortening velocity slows continuously during the course of an isotonic (fixed force) contraction (Warshaw, D.M. 1987. J. Gen. Physiol. 89:771-789). To distinguish among several possible explanations for this slowing, single smooth muscle cells were isolated from the gastric muscularis of the toad (Bufo marinus) and attached to an ultrasensitive force transducer and a length displacement device. Cells were stimulated electrically and produced maximum stress of 144 mN/mm2. Cell force was then reduced to and maintained at preset fractions of maximum, and cell shortening was allowed to occur. Cell stiffness, a measure of relative numbers of attached crossbridges, was measured during isotonic shortening by imposing 50-Hz sinusoidal force oscillations. Continuous slowing of shortening velocity was observed during isotonic shortening at all force levels. This slowing was not related to the time after the onset of stimulation or due to reduced isometric force generating capacity. Stiffness did not change significantly over the course of an isotonic shortening response, suggesting that the observed slowing was not the result of reduced numbers of cycling crossbridges. Furthermore, isotonic shortening velocity was better described as a function of the extent of shortening than as a function of the time after the onset of the release. Therefore, we propose that slowing during isotonic shortening in single isolated smooth muscle cells is the result of an internal load that opposes shortening and increases as cell length decreases. PMID:2121895

  13. Conformational change in an isolated single synthetic polymer chain on a mica surface observed by atomic force microscopy.

    PubMed

    Kumaki, Jiro; Hashimoto, Takeji

    2003-04-23

    The random coil conformation of an isolated conventional synthetic polymer chain was clearly imaged by atomic force microscopy (AFM). The sample used was a poly(styrene)-block-poly(methyl methacrylate) diblock copolymer. A very dilute solution of the copolymer with benzene was spread on a water surface. The structure thus formed on water was subsequently transferred and deposited onto mica at various surface pressures and observed under AFM. The AFM images obtained with films deposited at a low surface pressure (<0.1 mN/m) showed a single polystyrene (PS) block chain aggregated into a single PS particle with a single poly(methyl methacrylate) (PMMA) block chain emanating from the particle. Immediately after the deposition, the single PMMA block chain aggregated to form a condensed monolayer around the polystyrene particles. However, after exposing the deposited film to highly humid air for 1 day, the PMMA chains spread out so that the single PMMA block chain could be identified as a random coil on the substrate. The thin water layer formed on the mica substrate in humid air may enable the PMMA block chain to be mobilized on the substrate, leading to the conformational rearrangement from the condensed monolayer conformation to an expanded and elongated coil. The elongation of the PMMA chain was highly sensitive to the humidity; the maximum elongation was obtained at 79% relative humidity. The elongation was a slow process and took about 20 h.

  14. Retrospective analysis of mortality and Candida isolates of 75 patients with candidemia: a single hospital experience

    PubMed Central

    Hirano, Ryuichi; Sakamoto, Yuichi; Kudo, Kumiko; Ohnishi, Motoki

    2015-01-01

    The mortality rate for candidemia is approximately 30%–60%. However, prognostic factors in patients with candidemia have not yet been elucidated in detail. The aim of the present study was to analyze prognostic factors for candidemia using the mortality rate and Candida isolates of patients with candidemia. Seventy-five patients with candidemia were analyzed between January 2007 and December 2013. The main outcome of this study was the 30-day mortality rate after the diagnosis of candidemia. The acute physiology and chronic health evaluation II score (APACHE II score) was measured in 34 patients (45.3%). Odds ratios (ORs) for death due to candidemia were analyzed using a multivariate stepwise logistic regression analysis. Twenty (26.6%) patients died within 30 days of being diagnosed with candidemia. Non-survivors had a significantly higher APACHE II score (n=7, mean; 18.9±4.5) than that of survivors (n=27, mean; 14.0±5.0). Advanced age (OR =1.1, 95% confidence interval =1.01–1.23, P=0.04) was a significant risk factor for a high mortality rate, whereas removal of a central venous catheter (OR =0.03, 95% confidence interval =0.002–0.3, P=0.01) was associated with a lower mortality rate. Seventy-six Candida spp. were isolated from blood cultures: Candida albicans 28 (36.8%), Candida parapsilosis 23 (30.2%), Candida guilliermondii 16 (21.0%), Candida glabrata four (5.2%), Candida tropicalis two (2.6%), and Candida spp. three (3.9%) that could not be identified. C. parapsilosis was the most frequently isolated species in younger patients (<65 years), whereas C. albicans was the most frequently isolated in elderly patients (≥65 years). Physicians who treat candidemia need to consider removing the central venous catheter and pay attention to the general condition of patients, particularly that of elderly patients. PMID:26185460

  15. Isolation of single cells for protein therapeutics using microwell selection and Surface Plasmon Resonance imaging.

    PubMed

    Abali, F; Stevens, M; Tibbe, A G J; Terstappen, L W M M; van der Velde, P N; Schasfoort, R B M

    2017-08-15

    Here the feasibility is demonstrated that by combining Surface Plasmon Resonance Imaging (SPRi) and self-sorting microwell technology product secretion of individual cells can be monitored. Additionally isolation of the selected cells can be performed by punching the cells from the microwells using coordinates of the positions of microwells obtained with SPRi. Cells of interest can be retrieved sterile from the microwell array for further cultivation. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. Proteolysis in hyperthermophilic microorganisms

    DOE PAGES

    Ward, Donald E.; Shockley, Keith R.; Chang, Lara S.; ...

    2002-01-01

    Proteases are found in every cell, where they recognize and break down unneeded or abnormal polypeptides or peptide-based nutrients within or outside the cell. Genome sequence data can be used to compare proteolytic enzyme inventories of different organisms as they relate to physiological needs for protein modification and hydrolysis. In this review, we exploit genome sequence data to compare hyperthermophilic microorganisms from the euryarchaeotal genus Pyrococcus , the crenarchaeote Sulfolobus solfataricus , and the bacterium Thermotoga maritima . An overview of the proteases in these organisms is given based on those proteases that have been characterized and on putativemore » proteases that have been identified from genomic sequences, but have yet to be characterized. The analysis revealed both similarities and differences in the mechanisms utilized for proteolysis by each of these hyperthermophiles and indicated how these mechanisms relate to proteolysis in less thermophilic cells and organisms.« less

  17. Gravitaxis in unicellular microorganisms

    NASA Astrophysics Data System (ADS)

    Häder, D.-P.

    1999-01-01

    Orientation of organisms with respect to the gravitational field of the Earth has been studied for more than 100 years in a number of unicellular microorganisms including flagellates and ciliates. Several hypotheses have been developed how the weak stimulus is perceived. Intracellular statoliths have been found to be involved in gravitaxis of Loxodes, while no specialized organelles have been detected in other ciliates, e.g. Paramecium. Also in the slime mold Physarum no specialized gravireceptors have been identified yet. In the flagellate Euglena gracilis the whole cell body, which is denser than the surrounding medium, seems to act as a statolith pressing onto the lower membrane where it activates mechanosensitive ion channels. Similar results were obtained for the ciliate Paramecium. In contrast to the flagellate Euglena, several ciliates have been found to show gravikinesis, which is defined as a dependence of the swimming velocity on the direction of movement in the gravity field.

  18. Molecular typing of isolates of the fish pathogen, Flavobacterium columnare, by single-strand conformation polymorphism analysis.

    PubMed

    Olivares-Fuster, Oscar; Shoemaker, Craig A; Klesius, Phillip H; Arias, Covadonga R

    2007-04-01

    Flavobacterium columnare intraspecies diversity was revealed by analyzing the 16S rRNA gene and the 16S-23S internal spacer region (ISR). Standard restriction fragment length polymorphism (RFLP) of these sequences was compared with single-strand conformation polymorphism (SSCP). Diversity indexes showed that both 16S-SSCP and ISR-SSCP improved resolution (D>or=0.9) when compared with standard RFLP. ISR-SSCP offered a simpler banding pattern than 16S-SSCP while providing high discrimination between isolates. SSCP analysis of rRNA genes proved to be a simple, rapid, and cost-effective method for routine fingerprinting of F. columnare.

  19. Isolated, well-defined organovanadium(iii) on silica: Single-site catalyst for hydrogenation of alkenes and alkynes

    DOE PAGES

    Sohn, H.; Camacho-Bunquin, J.; Langeslay, R. R.; ...

    2017-05-03

    Well-defined, isolated, single-site organovanadium(III) catalyst on SiO2 [(SiO2)V(Mes)(THF)] were synthesized via surface organometallic chemistry, and fully characterized using a combination of analytical and spectroscopic techniques (EA, ICP, 1H NMR, TGA-MS, EPR, XPS, DR-UV/Vis, UV-Raman, DRIFTS, XAS). The catalysts exhibit unprecedented reactivity in liquid- and gas-phase alkene/alkyne hydrogenation. Catalyst poisoning experiments revealed that 100% of the V sites are active for hydrogenation.

  20. Assessment of microorganisms from Indonesian Oil Fields

    SciTech Connect

    Kadarwati, S.; Udiharto, M.; Rahman, M.; Jasjfi, E.; Legowo, E.H.

    1995-12-31

    Petroleum resources have been the mainstay of the national development in Indonesia. However, resources are being depleted after over a century of exploitation, while the demand continues to grow with the rapid economic development of the country. In facing the problem, EOR has been applied in Indonesia, such as the steamflooding project in Duri field, but a more energy efficient technology would be preferable. Therefore, MEOR has been recommended as a promising solution. Our study, aimed at finding indigenous microorganisms which can be developed for application in MEOR, has isolated microbes from some oil fields of Indonesia. These microorganisms have been identified, their activities studied, and the effects of their metabolisms examined. This paper describes the research carried out by LEMIGAS in this respect, giving details on the methods of sampling, incubation, identification, and activation of the microbes as well as tests on the effects of their metabolites, with particular attention to those with potential for application in MEOR.

  1. Solubilization of Australian lignites by microorganisms

    SciTech Connect

    Catcheside, D.E.A.; Mallett, K.J.; Cox, R.E.

    1988-01-01

    Australia has substantial lignite deposits, particularly in the Latrobe Valley in Victoria where 4.10/sup 10/ tons are accessible with available technologies. The authors have investigated the susceptibility of these coal to solubilization by microorganisms, including species additional to those already identified as active on North American lignites. The data presented here show that acid oxidized lignites from the Latrobe Valley are solubilized by each of seven species of microorganisms previously found to be active on Leonardite and oxidized North American lignites. These are the wood rot fungi: Trametes versicolor, Poria placenta and Phanerochaete chrysosporium, the lignin degrading prokaryote Streptomyces viridosporus and three fungi isolated from lignite in Mississippi: Candida ML-13, Cunninghamelia YML-1 and Penicillium waksmanii.

  2. Sequence-Independent, Single-Primer Amplification Next-Generation Sequencing of Hantaan Virus Cell Culture-Based Isolates.

    PubMed

    Song, Dong Hyun; Kim, Won-Keun; Gu, Se Hun; Lee, Daesang; Kim, Jeong-Ah; No, Jin Sun; Lee, Seung-Ho; Wiley, Michael R; Palacios, Gustavo; Song, Jin-Won; Jeong, Seong Tae

    2017-02-08

    Hantaan virus (HTNV), identified in the striped field mouse (Apodemus agrarius), belongs to the genus Hantavirus of the family Bunyaviridae and contains tripartite RNA genomes, small (S), medium (M), and large (L) segments. HTNV is a major causative for hemorrhagic fever with renal syndrome (HFRS) with fatality rates ranging from 1% to 15% in the Republic of Korea (ROK) and China. Defining of HTNV whole-genome sequences and isolation of the infectious particle play a critical role in the characterization and preventive and therapeutic strategies of hantavirus outbreaks. Next-generation sequencing (NGS) provides an advanced tool for massive genomic sequencing of viruses. However, the isolation of viral infectious particles is a huge obstacle to investigate and develop anti-virals for hantaviruses. Here, we report 12 HTNV isolates from lung tissues of the striped field mouse in the highly HFRS-endemic areas. Sequence-independent, single-primer amplification (SISPA) NGS was attempted to recover the genomic sequences of HTNV isolates. The nucleotide sequence of HTNV S, M, and L segments were covered up to 99.4-100%, 97.5-100%, and 95.6-99.8%, respectively, based on the full length of the prototype HTNV 76-118. The whole-genome sequencing of HTNV isolates was accomplished by additional reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification cDNA ends (RACE) PCR. In conclusion, this study will lead to the attempt and usage of SISPA NGS technologies to delineate the whole-genome sequence of hantaviruses, providing a new era of viral genomics for the surveillance, trace, and disease risk management of HFRS incidents.

  3. Cationic Mn4 single-molecule magnet with a sterically isolated core.

    PubMed

    Heroux, Katie J; Quddusi, Hajrah M; Liu, Junjie; O'Brien, James R; Nakano, Motohiro; del Barco, Enrique; Hill, Stephen; Hendrickson, David N

    2011-08-15

    The synthesis, structure, and magnetic properties of a ligand-modified Mn(4) dicubane single-molecule magnet (SMM), [Mn(4)(Bet)(4)(mdea)(2)(mdeaH)(2)](BPh(4))(4), are presented, where the cationic SMM units are significantly separated from neighboring molecules in the crystal lattice. There are no cocrystallized solvate molecules, making it an ideal candidate for single-crystal magnetization hysteresis and high-frequency electron paramagnetic resonance studies. Increased control over intermolecular interactions in such materials is a crucial factor in the future application of SMMs.

  4. Label-free isolation of a prostate cancer cell among blood cells and the single-cell measurement of drug accumulation using an integrated microfluidic chip

    PubMed Central

    Khamenehfar, A.; Beischlag, T. V.; Russell, P. J.; Ling, M. T. P.; Nelson, C.; Li, P. C. H.

    2015-01-01

    Circulating tumor cells (CTCs) are found in the blood of patients with cancer. Although these cells are rare, they can provide useful information for chemotherapy. However, isolation of these rare cells from blood is technically challenging because they are small in numbers. An integrated microfluidic chip, dubbed CTC chip, was designed and fabricated for conducting tumor cell isolation. As CTCs usually show multidrug resistance (MDR), the effect of MDR inhibitors on chemotherapeutic drug accumulation in the isolated single tumor cell is measured. As a model of CTC isolation, human prostate cancer cells were mixed with mouse blood cells and the label-free isolation of the tumor cells was conducted based on cell size difference. The major advantages of the CTC chip are the ability for fast cell isolation, followed by multiple rounds of single-cell measurements, suggesting a potential assay for detecting the drug responses based on the liquid biopsy of cancer patients. PMID:26594265

  5. Pseudomonas aeruginosa clinical and environmental isolates constitute a single population with high phenotypic diversity

    PubMed Central

    2014-01-01

    Background Pseudomonas aeruginosa is an opportunistic pathogen with a high incidence of hospital infections that represents a threat to immune compromised patients. Genomic studies have shown that, in contrast to other pathogenic bacteria, clinical and environmental isolates do not show particular genomic differences. In addition, genetic variability of all the P. aeruginosa strains whose genomes have been sequenced is extremely low. This low genomic variability might be explained if clinical strains constitute a subpopulation of this bacterial species present in environments that are close to human populations, which preferentially produce virulence associated traits. Results In this work, we sequenced the genomes and performed phenotypic descriptions for four non-human P. aeruginosa isolates collected from a plant, the ocean, a water-spring, and from dolphin stomach. We show that the four strains are phenotypically diverse and that this is not reflected in genomic variability, since their genomes are almost identical. Furthermore, we performed a detailed comparative genomic analysis of the four strains studied in this work with the thirteen previously reported P. aeruginosa genomes by means of describing their core and pan-genomes. Conclusions Contrary to what has been described for other bacteria we have found that the P. aeruginosa core genome is constituted by a high proportion of genes and that its pan-genome is thus relatively small. Considering the high degree of genomic conservation between isolates of P. aeruginosa from diverse environments, including human tissues, some implications for the treatment of infections are discussed. This work also represents a methodological contribution for the genomic study of P. aeruginosa, since we provide a database of the comparison of all the proteins encoded by the seventeen strains analyzed. PMID:24773920

  6. A new method to evaluate the biocontrol potential of single spore isolates of fungal entomopathogens

    PubMed Central

    Posada, Francisco J.; Vega, Fernando E.

    2005-01-01

    Fifty Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) strains isolated from the coffee berry borer were used to develop a novel screening method aimed at selecting strains with the highest biocontrol potential. The screening method is based on percent insect mortality, average survival time, mortality distribution, percent spore germination, fungal life cycle duration, and spore production on the insect. Based on these parameters, only 11 strains merited further study. The use of a sound scientific protocol for the selection of promising fungal entomopathogens should lead to more efficient use of time, labor, and financial resources in biological control programs. PMID:17119619

  7. A new method to evaluate the biocontrol potential of single spore isolates of fungal entomopathogens.

    PubMed

    Posada, Francisco J; Vega, Fernando E

    2005-12-06

    Fifty Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) strains isolated from the coffee berry borer were used to develop a novel screening method aimed at selecting strains with the highest biocontrol potential. The screening method is based on percent insect mortality, average survival time, mortality distribution, percent spore germination, fungal life cycle duration, and spore production on the insect. Based on these parameters, only 11 strains merited further study. The use of a sound scientific protocol for the selection of promising fungal entomopathogens should lead to more efficient use of time, labor, and financial resources in biological control programs.

  8. [Granulomatous diseases and pathogenic microorganism].

    PubMed

    Inoue, Yoshikazu; Suga, Moritaka

    2008-02-01

    Granuloma formation is a chronic inflammatory reaction where macrophage system and other inflammatory cells are involved. After some antigen exposure and processing, T cells, macrophages, epithelioid cells, and giant cell are activated, and granulomas are formed. Granuloma is considered as a defense mechanism against antigens, which stay in the organs without inactivation. Granulomas including fibroblasts extra-cellular matrix surround and isolate the antigens. Granulomas are classified to noninfectious granulomas and infectious granulomas. However recent studies revealed pathogenic microorganism are suspected to be a cause of granuloma in non-inflammatory diseases. Balance between pathogenic microorganisms and defense mechanisms of the host might be important in the special immunologic reaction. In some cases, it is hard to clearly classify infectious and noninfectious granulomas. Recently, Eishi et al. reported that latent infection of Propionibacterium acnes might be cause of sarcoidosis. Several hypersensitivity pneumonias are considered to be caused by exogenous microorganisms. The symposium was organized to know and clarify the new mechanisms of non-infectious granulomatous lung diseases and pathogenic microorganisms. This report is a summary of a symposium entitled "Granulomatous Diseases and Pathogenic Microorganism", organized in the 82nd Japanese Society for Tuberculosis (president Dr. Mitsunori Sakatani, M.D.). 1. Imaging of Granulomatous Lung Diseases: Masanori AKIRA (Department of Radiology, National Hospital Organization Kinki-chuo Chest Medical Center) High-resolution computed tomography (HRCT) is a useful tool in the evaluation of parenchymal changes in patients with a granulomatous lung disease. In sarcoidosis, the HRCT findings include small, well-defined nodules in relation to lymphatic roots, lymph node enlargement, and middle or upper lobe predominance. The appearances of subacute hypersensitivity pneumonitis include ill-defined centrilobular

  9. Somatic FGFR and TWIST mutations are not a common cause of isolated nonsyndromic single suture craniosynostosis.

    PubMed

    Anderson, Peter J; Cox, Timothy C; Roscioli, Tony; Elakis, George; Smithers, Lisa; David, David J; Powell, Barry

    2007-03-01

    Pathogenic mutations in FGFR2 and TWIST genes are detected in the majority of individuals with Crouzon, Pfeiffer, Apert, and Saethre-Chotzen syndromes. In contrast, mutations have been identified rarely in cases of nonsyndromic, single suture craniosynostosis. Recently, two studies confirming somatic mosaicism with local expression of an FGFR mutation have been reported. This study investigates whether somatic mosaicism could account for nonsyndromic, single suture craniosynostosis. Eight individuals with single suture craniosynostosis who were negative for known mutations in FGFR1-3 and TWIST after screening in their leucocyte DNA were tested for the presence of pathogenic mutations in suture cell-derived DNA. Five had sagittal synostosis, two had metopic synostosis, and the other unicoronal synostosis. Osteoprogenitor cells from surgically excised fusing sutures and an adjacent open suture were cultured. DNA from the cultured cells grown to passage 3 was then examined for underlying FGFR and TWIST mutations. No mutations within the exons of the FGFR or TWIST genes studied were identified in any suture cells. This study found no evidence to support the notion that mosaicism for FGFR or TWIST mutations, normally associated with syndromal forms of craniosynostosis, occur in single suture craniosynostosis. Thus, any underlying genetic defects must occur in regions outside those normally implicated in syndromal craniosynostosis, or this disorder could arise as a consequence of some other epigenetic modification.

  10. A multispectral sorting device for isolating single wheat kernels with high protein content

    USDA-ARS?s Scientific Manuscript database

    Automated sorting of single wheat kernels according to protein content was demonstrated using two novel multispectral sorting devices with different spectral ranges; 470-1070 nm (silicone based detector) and 910nm-1550 nm (InGaAs based detector). The multispectral data were acquired by rapidly (~12...

  11. [Characterization and determination of antibiotic resistance profiles of a single clone Acinetobacter baumannii strains isolated from blood cultures].

    PubMed

    Karagöz, Alper; Baran, Irmak; Aksu, Neriman; Acar, Sümeyra; Durmaz, Rıza

    2014-10-01

    were susceptible to TG and COL. The resistance rates of the environmental isolates to SCF, AMK, GEN, NT, LVF, TET and SXT were determined as 57.1%, 85.7%, 85.7%, 28.8%, 28.6%, 85.7% and 57.1%, respectively. PFGE analysis done by the use of ApaI enzyme revealed the presence of one major clone. Dendogram analysis indicated that environmental and clinical isolates were in the same clone indicating that the outbreak was possibly originated from the same internal ICUs. Our data emphasized that multidrug resistant A.baumannii isolates were quite common in our hospital, and enviromental cross-contamination throughout the year was confirmed by molecular methods. Despite the precautions such as continous education on effective hand washing, use of gloves and hospital cleaning, established in our hospital, this single clonal spread was attributed to staff shortage and poor adherence to infection control rules. In conclusion, for the prevention of dissemination of multidrug resistant A.baumannii strains and control of nosocomial infections, infection control strategies should be established and strict compliance to these rules should be provided.

  12. Effects of beneficial microorganisms on lowland rice development.

    PubMed

    Nascente, Adriano Stephan; de Filippi, Marta Cristina Corsi; Lanna, Anna Cristina; de Sousa, Thatyane Pereira; de Souza, Alan Carlos Alves; da Silva Lobo, Valácia Lemes; da Silva, Gisele Barata

    2017-09-19

    Microorganisms can promote plant growth by increasing phytomass production, nutrient uptake, photosynthesis rates, and grain yield, which can result in higher profits for farmers. However, there is limited information available about the physiological characteristics of lowland rice after treatment with beneficial microorganisms in the tropical region. This study aimed to determine the effects of different beneficial microorganisms and various application forms on phytomass production, gas exchange, and nutrient contents in the lowland rice cultivar 'BRS Catiana' in a tropical region. The experiment was performed under greenhouse conditions utilizing a completely randomized design and a 7 × 3 + 1 factorial scheme with four replications. The treatments consisted of seven microorganisms, including the rhizobacterial isolates BRM 32113, BRM 32111, BRM 32114, BRM 32112, BRM 32109, and BRM 32110 and Trichoderma asperellum pooled isolates UFRA-06, UFRA-09, UFRA-12, and UFRA-52, which were applied using three different methods (microbiolized seed, microbiolized seed + soil drenched with a microorganism suspension at 7 and 15 days after sowing (DAS), and microbiolized seed + plant spraying with a microorganism suspension at 7 and 15 DAS) with a control (water). The use of microorganisms can provide numerous benefits for rice in terms of crop growth and development. The microorganism types and methods of application positively and differentially affected the physiological characteristics evaluated in the experimental lowland rice plants. Notably, the plants treated with the bioagent BRM 32109 on the seeds and on seeds + soil produced plants with the highest dry matter biomass, gas exchange rate, and N, P, Fe, and Mg uptake. Therefore, our findings indicate strong potential for the use of microorganisms in lowland rice cultivation systems in tropical regions. Currently, an additional field experiment is in its second year to validate the beneficial result reported

  13. The ‘single big cryoballoon’ technique for acute pulmonary vein isolation in patients with paroxysmal atrial fibrillation: a prospective observational single centre study

    PubMed Central

    Chun, Kyoung-Ryul Julian; Schmidt, Boris; Metzner, Andreas; Tilz, Roland; Zerm, Thomas; Köster, Ilka; Fürnkranz, Alexander; Koektuerk, Buelent; Konstantinidou, Melanie; Antz, Matthias; Ouyang, Feifan; Kuck, Karl Heinz

    2009-01-01

    Aims Cryothermal energy (CTE) ablation via a balloon catheter (Arctic Front, Cryocath™) represents a novel technology for pulmonary vein isolation (PVI). However, balloon-based PVI approaches are associated with phrenic nerve palsy (PNP). We investigated whether ‘single big cryoballoon’-deployed CTE lesions can (i) achieve acute electrical PVI without left atrium (LA) imaging and (ii) avoid PNP in patients with paroxysmal atrial fibrillation (PAF). Methods and results After double transseptal punctures, one Lasso catheter and a big 28 mm cryoballoon catheter using a steerable sheath were inserted into the LA. PV angiography and ostial Lasso recordings from all PVs were obtained. Selective PV angiography was used to evaluate balloon to LA–PV junction contact. CTE ablation lasted 300 s, and the PN was paced during freezing at right-sided PVs. Twenty-seven patients (19 males, mean age: 56 ± 9 years, LA size: 42 ± 5 mm) with PAF (mean duration: 6.6 ± 5.7 years) were included. PVI was achieved in 97/99 PVs (98%). Median (Q1; Q3) procedural, balloon, and fluoroscopy times were 220 min (190; 245), 130 min (90; 170), and 50 min (42; 69), respectively. Three transient PNP occurred after distal PV ablations. No PV stenosis occurred. Total median (Q1; Q3) follow-up time was 271 days (147; 356), and 19 of 27 patients (70%) remained in sinus rhythm (3-month blanking period). Conclusion Using the single big cryoballoon technique, almost all PVs (98%) could be electrically isolated without LA imaging and may reduce the incidence of PNP as long as distal ablation inside the septal PVs is avoided. PMID:19109353

  14. Gene Expression in Single Cells Isolated from the CWR-R1 Prostate Cancer Cell Line and Human Prostate Tissue Based on the Side Population Phenotype

    PubMed Central

    Gangavarapu, Kalyan J; Miller, Austin; Huss, Wendy J

    2016-01-01

    Defining biological signals at the single cell level can identify cancer initiating driver mutations. Techniques to isolate single cells such as microfluidics sorting and magnetic capturing systems have limitations such as: high cost, labor intense, and the requirement of a large number of cells. Therefore, the goal of our current study is to identify a cost and labor effective, reliable, and reproducible technique that allows single cell isolation for analysis to promote regular laboratory use, including standard reverse transcription PCR (RT-PCR). In the current study, we utilized single prostate cells isolated from the CWR-R1 prostate cancer cell line and human prostate clinical specimens, based on the ATP binding cassette (ABC) transporter efflux of dye cycle violet (DCV), side population assay. Expression of four genes: ABCG2; Aldehyde dehydrogenase1A1 (ALDH1A1); androgen receptor (AR); and embryonic stem cell marker, Oct-4, were determined. Results from the current study in the CWR-R1 cell line showed ABCG2 and ALDH1A1 gene expression in 67% of single side population cells and in 17% or 100% of non-side population cells respectively. Studies using single cells isolated from clinical specimens showed that the Oct-4 gene is detected in only 22% of single side population cells and in 78% of single non-side population cells. Whereas, AR gene expression is in 100% single side population and non-side population cells isolated from the same human prostate clinical specimen. These studies show that performing RT-PCR on single cells isolated by FACS can be successfully conducted to determine gene expression in single cells from cell lines and enzymatically digested tissue. While these studies provide a simple yes/no expression readout, the more sensitive quantitative RT-PCR would be able to provide even more information if necessary. PMID:27785389

  15. Single Cell Fluorescence Action Spectra for the Targeted Study and Isolation of Phytoplankton Within Complex Assemblages

    NASA Astrophysics Data System (ADS)

    Thompson, A. W.

    2016-02-01

    Discerning the diversity, abundance, and functional role of distinct phytoplankton groups is essential to the study of aquatic systems. The diversity and ecological roles of phytoplankton are still understudied relative to their importance in the ocean system. Here, we present a new flow cytometry method that uses up to five excitation colors to determine the relative fluorescence action spectra of phytoplankton within complex assemblages, thus leveraging the precise and high-throughput capabilities of flow cytometry and the unique combinations of photosynethetic pigments in phylogenetically related groups of phytoplankton. We tested the method on cultivated Synechococcus of known pigment composition and genotype then applied the method to a natural phytoplankton assemblage from several coastal and open ocean environments. We determined the relative fluorescence action spectra of up to 8 distinct phytoplankton populations. By coupling multi-laser flow cytometry to cell sorting we demonstrated that natural phytoplankton populations with similar relative fluorescence action spectra belonged to the same taxonomic classes based on 18S rRNA gene phylogeny. Based on these results, we suggest that multi-laser flow cytometry could be applied to enumeration and isolation of Class-level genotypically distinct phytoplankton groups for improved genome assembly from metagenomes, metabolic analysis including nutrient uptake, and targeted isolation for cultivation.

  16. Discrimination of Bacillus anthracis from closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microchips

    DOEpatents

    Bavykin, Sergei G.; Mirzabekov, Andrei D.

    2007-10-30

    The present invention is directed to a novel method of discriminating a highly infectious bacterium Bacillus anthracis from a group of closely related microorganisms. Sequence variations in the 16S and 23S rRNA of the B. cereus subgroup including B. anthracis are utilized to construct an array that can detect these sequence variations through selective hybridizations. The identification and analysis of these sequence variations enables positive discrimination of isolates of the B. cereus group that includes B. anthracis. Discrimination of single base differences in rRNA was achieved with a microchip during analysis of B. cereus group isolates from both single and in mixed probes, as well as identification of polymorphic sites. Successful use of a microchip to determine the appropriate subgroup classification using eight reference microorganisms from the B. cereus group as a study set, was demonstrated.

  17. Single nucleotide repeat analysis of B. anthracis isolates in Canada through comparison of pyrosequencing and Sanger sequencing.

    PubMed

    Hahn, Kristen R; Janzen, Timothy W; Thomas, Matthew C; Shields, Michael J; Goji, Noriko; Valle, Edith; Amoako, Kingsley K

    2014-03-14

    Several technology platforms have been developed to resolve the phylogenetic placement of B. anthracis. However, these methods lack the resolution to identify differences between closely related strains within an outbreak due to the highly clonal nature of B. anthracis. Single Nucleotide Repeats (SNRs) are a type of rapidly evolving genetic marker that can be used to track epidemiological distribution in the event of an outbreak. Four SNR targets were used to detect and type 35 B. anthracis isolates in our collection; 18 from across Canada obtained between 1972 and 2005 and 17 from the 2006 Anthrax outbreak in north eastern Saskatchewan. A control sequence was developed for pyrosequencing which yielded consistent and accurate reads of SNRs. However, when DNA from the isolates was tested using pyrosequencing the results were inconsistent and did not reflect the number of SNRs obtained by Sanger sequencing. The SNR numbers derived from the Sanger sequencing show two of the four SNR loci could provide information on subtype, whereas the other two were not discriminatory. There is variation in SNRs between strains isolated from different outbreaks, the subset of 2006 outbreak strains showed very little difference in SNR number, and thus suggests low diversity among the strains sampled from the same outbreak.

  18. Physiology of isolated anomalous pulmonary venous connection of a single pulmonary vein as determined by cardiac magnetic resonance imaging.

    PubMed

    Dyme, Joshua L; Prakash, Ashwin; Printz, Beth F; Kaur, Avnit; Parness, Ira A; Nielsen, James C

    2006-07-01

    The physiology of isolated partially anomalous pulmonary venous connection of a single pulmonary vein has yet to be fully characterized. This study assessed the magnitude of the left-to-right shunt and right ventricular (RV) dilation from a single anomalous pulmonary vein using cardiac magnetic resonance imaging. Subjects with >1 anomalous pulmonary vein or associated lesions, including atrial septal defects, were excluded. In the 6 subjects identified, the median pulmonary-to-systemic flow ratio was 1.55 (range 1.3 to 1.6). The mean RV end-diastolic volume indexed to body surface area in the subjects was significantly larger than in a normal reference cohort (108 +/- 16 vs 78 +/- 18 cm(3)/m(2), p = 0.0009) and greater than the upper limit of normal in all 6 subjects. Older age did not correlate with increased magnitude of shunting (r = 0.3, p = 0.5), but increased age did correlate with RV end-diastolic volume indexed to body surface area (r = 0.96, p = 0.01). Isolated partially anomalous pulmonary venous connection with only 1 vein connecting anomalously results in a modest left-to-right shunt and mild RV dilation.

  19. Brief communication genotyping of Burkholderia pseudomallei revealed high genetic variability among isolates from a single population group

    PubMed Central

    Zueter, Abdelrahman Mohammad; Rahman, Zaidah Abdul; Yean, Chan Yean; Harun, Azian

    2015-01-01

    Burkholderia pseudomallei is a soil dwelling Gram-negative bacteria predominates in Southeast Asia zone and the tropical part of Australia. Genetic diversity has been explored among various populations and environments worldwide. To date, little data is available on MLST profiling of clinical B. pseudomallei isolates in peninsular Malaysia. In this brief report, thirteen culture positive B. pseudomallei cases collected from a single population of Terengganu state in the Western Peninsular Malaysia and were confirmed by In-house TTS1-PCR. Isolates were subjected for multi-locus sequence typing (MLST) to explore their genotypic diversity and to investigate for possible clonal clustering of a certain sequence type. Patient’s clinical information was examined to investigate for clinical correlation among the different genotypes. In spite of small sample set, MLST results indicated predictive results; considerable genotypic diversity, predominance and novelty among B. pseudomallei collected over a single geographically-located population in Malaysia. Massive genotypic heterogeneity was observed; 8 different sequence types with predominance of sequence type 54 and discovery of two novel sequence types. However, no clear pathogenomic or organ tropism clonal relationships were predicted. PMID:26417404

  20. Microorganisms in honey.

    PubMed

    Snowdon, J A; Cliver, D O

    1996-08-01

    Knowledge of the moisture and temperature conditions influencing growth of microorganisms in honey has long been used to control the spoilage of honey. However, the need for additional microbiological data on honey will increase as new technologies for, and uses of honey develop. Microorganisms in honey may influence quality or safety. Due to the natural properties of honey and control measures in the honey industry, honey is a product with minimal types and levels of microbes. Microbes of concern in post-harvest handling are those that are commonly found in honey (i.e., yeasts and spore-forming bacteria), those that indicate the sanitary or commercial quality of honey (i.e., coliforms and yeasts), and those that under certain conditions could cause human illness. Primary sources of microbial contamination are likely to include pollen, the digestive tracts of honey bees, dust, air, earth and nectar, sources which are very difficult to control. The same secondary (after-harvest) sources that influence any food product are also sources of contamination for honey. These include air, food handlers, cross-contamination, equipment and buildings. Secondary sources of contamination are controlled by good manufacturing practices. The microbes of concern in honey are primarily yeasts and spore-forming bacteria. Total plate counts from honey samples can vary from zero to tens of thousands per gram for no apparent reason. Most samples of honey contain detectable levels of yeasts. Although yeast counts in many honey samples are below 100 colony forming units per gram (cfu/g), yeasts can grow in honey to very high numbers. Standard industry practices control yeast growth. Bacterial spores, particularly those in the Bacillus genus, are regularly found in honey. The spores of C. botulinum are found in a fraction of the honey samples tested-normally at low levels. No vegetative forms of disease-causing bacterial species have been found in honey. Bacteria do not replicate in honey

  1. Analysis of population structures of viral isolates using single-strand conformation polymorphism method.

    PubMed

    Delaunay, Agnès; Rolland, Mathieu; Jacquot, Emmanuel

    2009-01-01

    The analysis of viral populations requires the use of techniques that describe characteristics of individuals. The single-strand conformation polymorphism (SSCP) makes possible the identification of genetic differences between viral sequences and constitutes an alternative to the expensive and time-consuming cloning and sequencing strategies. Applied to small genomic regions (from 100 to 500 bases in length), SSCP patterns could describe, under appropriate experimental conditions, single nucleotide variations in the studied sequence. The different steps of a complete SSCP procedure, from sampling to pattern analysis (including nucleic acid extraction, RT-PCR amplification, double-stranded DNA quantification, polyacrylamide gel preparation, electrophoresis conditions, and staining procedures), are described using a region (500 bases) of the barley yellow dwarfvirus-PAV (BYDV-PAV, Luteovirus) genome as molecular target.

  2. Portable automatic bioaerosol sampling system for rapid on-site detection of targeted airborne microorganisms.

    PubMed

    Usachev, Evgeny V; Pankova, Anna V; Rafailova, Elina A; Pyankov, Oleg V; Agranovski, Igor E

    2012-10-26

    Bioaerosols could cause various severe human and animal diseases and their opportune and qualitative precise detection and control is becoming a significant scientific and technological topic for consideration. Over the last few decades bioaerosol detection has become an important bio-defense related issue. Many types of portable and stationary bioaerosol samplers have been developed and, in some cases, integrated into automated detection systems utilizing various microbiological techniques for analysis of collected microbes. This paper describes a personal sampler used in conjunction with a portable real-time PCR technique. It was found that a single fluorescent dye could be successfully used in multiplex format for qualitative detection of numerous targeted bioaerosols in one PCR tube making the suggested technology a reliable "first alert" device. This approach has been specifically developed and successfully verified for rapid detection of targeted microorganisms by portable PCR devices, which is especially important under field conditions, where the number of microorganisms of interest usually exceeds the number of available PCR reaction tubes. The approach allows detecting targeted microorganisms and triggering some corresponding sanitary and quarantine procedures to localize possible spread of dangerous infections. Following detailed analysis of the sample under controlled laboratory conditions could be used to exactly identify which particular microorganism out of a targeted group has been rapidly detected in the field. It was also found that the personal sampler has a collection efficiency higher than 90% even for small-sized viruses (>20 nm) and stable performance over extended operating periods. In addition, it was found that for microorganisms used in this project (bacteriophages MS2 and T4) elimination of nucleic acids isolation and purification steps during sample preparation does not lead to the system sensitivity reduction, which is extremely

  3. Shallow-trench-isolation bounded single-photon avalanche diodes in commercial deep submicron CMOS technologies

    NASA Astrophysics Data System (ADS)

    Finkelstein, Hod

    This dissertation describes the first single-photon detection device to be manufactured in a commercial deep-submicron CMOS technology. It also describes novel self-timed peripheral circuits which optimize the performance of the new device. An extension of the new device for dual-color single-photon detection is investigated. Finally, an area- and power-efficient method for single-photon frequency upconversion is presented, analyzed, and experimentally examined. Single-photon avalanche diodes have been used in diverse applications, including three-dimensional laser radar, three-dimensional facial mapping, fluorescence-correlation techniques and time-domain tomography. Due to the high electric fields which these devices must sustain, they have traditionally been manufactured in custom processes, severely limiting their speed and the ability to integrate them in high-resolution imagers. By utilizing a process module originally designed to enhance the performance of CMOS transistors, we achieve highly planar junctions in an area-efficient manner. This results in SPADs exhibiting high fill factors, small pitch and ultrafast operation. Device miniaturization is accompanied by excessive noise, which was shown to emanate from trapped avalanche charges. Due to the fast recharging of the device, these charges are released in a subsequent charged phase of the device, causing correlated after-pulses. We present electrostatic and electrical simulation results, as well as a comprehensive characterization of the new device. We also show for the first time that by utilizing the two junctions included in the device, we can selectively detect photons of different wavelengths in the same pixel, as is desirable in cross-correlation experiments. This dissertation also describes an efficient new method for single-photon frequency upconversion. This is desirable for applications including quantum-key distribution and high-resolution near-infrared imaging. The new technique is based on

  4. Social evolution theory for microorganisms.

    PubMed

    West, Stuart A; Griffin, Ashleigh S; Gardner, Andy; Diggle, Stephen P

    2006-08-01

    Microorganisms communicate and cooperate to perform a wide range of multicellular behaviours, such as dispersal, nutrient acquisition, biofilm formation and quorum sensing. Microbiologists are rapidly gaining a greater understanding of the molecular mechanisms involved in these behaviours, and the underlying genetic regulation. Such behaviours are also interesting from the perspective of social evolution - why do microorganisms engage in these behaviours given that cooperative individuals can be exploited by selfish cheaters, who gain the benefit of cooperation without paying their share of the cost? There is great potential for interdisciplinary research in this fledgling field of sociomicrobiology, but a limiting factor is the lack of effective communication of social evolution theory to microbiologists. Here, we provide a conceptual overview of the different mechanisms through which cooperative behaviours can be stabilized, emphasizing the aspects most relevant to microorganisms, the novel problems that microorganisms pose and the new insights that can be gained from applying evolutionary theory to microorganisms.

  5. Polysaccharides from Extremophilic Microorganisms

    NASA Astrophysics Data System (ADS)

    Nicolaus, B.; Moriello, V. Schiano; Lama, L.; Poli, A.; Gambacorta, A.

    2004-02-01

    Several marine thermophilic strains were analyzed for exopolysaccharide production. The screening process revealed that a significant number of thermophilic microorganisms were able to produce biopolymers, and some of them also revealed interesting chemical compositions. We have identified four new polysaccharides from thermophilic marine bacteria, with complex primary structures and with different repetitive units: a galacto-mannane type from strain number 4004 and mannane type for the other strains. The thermophilic Bacillus thermantarcticus produces two exocellular polysaccharides (EPS 1, EPS 2) that give the colonies a typical mucous character. The exopolysaccharide fraction was produced with all substrates assayed, although a higher yield 400 mg liter-1 was obtained with mannose as carbon and energy source. NMR spectra confirmed that EPS 1 was a heteropolysaccharide of which the repeating unit was constituted by four different α-D-mannoses and three different β-D-glucoses. It seems to be close to some xantan polymers. EPS 2 was a mannan. Four different α-D-mannoses were found as the repeating unit. Production and chemical studies of biopolymers produced by halophilic archaea, Haloarcula species were also reported.

  6. A single-step method for RNA isolation from tropical crops in the field

    PubMed Central

    Breitler, J.-C.; Campa, C.; Georget, F.; Bertrand, B.; Etienne, H.

    2016-01-01

    The RNAzol RT reagent was used to provide pure RNA from human cells. We develop a protocol using RNAzol RT reagent to extract pure RNA from plants tissues and demonstrate that this RNA extraction method works not only at room temperature but also at elevated temperatures and provides the simplest and most effective single-step method to extract pure and undegraded RNA directly from tropical plants in the field. RNA extraction directly in a complex field environment opens up the way for studying gene-environment interactions at transcriptome level to decipher the complex regulatory network involved in multiple-stress responses. PMID:27922073

  7. Beyond the single-atom response in isolated attosecond-pulse generation

    SciTech Connect

    Altucci, Carlo; Velotta, Raffaele; Tosa, Valer

    2007-06-15

    It is demonstrated that three-dimensional propagation effects essentially influence attosecond-pulse generation by few-cycle, carrier-envelope-phase stabilized laser pulses used in a polarization-gating configuration. The rapidly changing polarization status gives rise to electron trajectories even longer than those observed with linearly polarized light, but the off-axis contributions and the propagation effects can efficiently act as a filter to produce a single attosecond pulse. It is also found that the attosecond beams can have a significant spatial divergence.

  8. Resistance of soil microorganisms to starvation.

    NASA Technical Reports Server (NTRS)

    Chen, M.; Alexander, M.

    1972-01-01

    Most groups of soil microorganisms died when exposed to prolonged starvation in a carbon-free solution, but the relative abundance of Bacillus and actinomycetes increased with time. Certain nonspore-forming bacteria also persisted. The ability of individual soil isolates to endure starvation in solution was not correlated with their glycogen content or rate of endogenous respiration. However, cells of the resistant populations were rich in poly-beta-hydroxybutyrate, whereas the starvation-susceptible bacteria generally contained little of this substance. Poly-beta-hydroxybutyrate was used rapidly in cells deprived of exogenous sources of carbon.

  9. Temperature response of Antarctic cryptoendolithic photosynthetic microorganisms

    NASA Technical Reports Server (NTRS)

    Ocampo-Friedmann, R.; Meyer, M. A.; Chen, M.; Friedmann, E. I.

    1988-01-01

    Growth responses to temperatures between 12.5 [degrees] C and 25 degrees C were determined for five photosynthetic microorganisms isolated from the Ross Desert cryptoendolithic community. Among eukaryotic algae, two strains of Trebouxia sp. have an upper temperature limit of 20 degrees C, and two strains of Hemichloris antarctica of 25 degrees C. The cyanobacterium Chroococcidiopsis sp., in contrast, grows at temperatures above 25 degrees C. These and earlier studies suggest that the eukaryotic algae of the Antarctic cryptoendolithic community have an upper temperature limit near 25 degrees C.

  10. Temperature response of Antarctic cryptoendolithic photosynthetic microorganisms

    NASA Technical Reports Server (NTRS)

    Ocampo-Friedmann, R.; Meyer, M. A.; Chen, M.; Friedmann, E. I.

    1988-01-01

    Growth responses to temperatures between 12.5 [degrees] C and 25 degrees C were determined for five photosynthetic microorganisms isolated from the Ross Desert cryptoendolithic community. Among eukaryotic algae, two strains of Trebouxia sp. have an upper temperature limit of 20 degrees C, and two strains of Hemichloris antarctica of 25 degrees C. The cyanobacterium Chroococcidiopsis sp., in contrast, grows at temperatures above 25 degrees C. These and earlier studies suggest that the eukaryotic algae of the Antarctic cryptoendolithic community have an upper temperature limit near 25 degrees C.

  11. Toolbox for Antibiotics Discovery from Microorganisms.

    PubMed

    Fisch, Katja M; Schäberle, Till F

    2016-09-01

    Microorganisms produce a vast array of biologically active metabolites. Such compounds are applied by humans to positively influence their health and, therefore, natural products serve as drug leads for pharmaceutical and medicinal chemistry. In this minireview, tools for the discovery and the production of potential drug leads are explained. A snapshot is provided, starting from the isolation of new producer strains, across genomic mining of (meta)genomes to identify biosynthetic gene clusters corresponding to natural products, toward heterologous expression to produce potential drug leads. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Resistance of soil microorganisms to starvation.

    NASA Technical Reports Server (NTRS)

    Chen, M.; Alexander, M.

    1972-01-01

    Most groups of soil microorganisms died when exposed to prolonged starvation in a carbon-free solution, but the relative abundance of Bacillus and actinomycetes increased with time. Certain nonspore-forming bacteria also persisted. The ability of individual soil isolates to endure starvation in solution was not correlated with their glycogen content or rate of endogenous respiration. However, cells of the resistant populations were rich in poly-beta-hydroxybutyrate, whereas the starvation-susceptible bacteria generally contained little of this substance. Poly-beta-hydroxybutyrate was used rapidly in cells deprived of exogenous sources of carbon.

  13. ANTIMICROBIAL RESISTANCE OF ESCHERICHIA COLI STRAINS ISOLATED FROM URINE AT OUTPATIENT POPULATION: A SINGLE LABORATORY EXPERIENCE

    PubMed Central

    Vranic, Sabina Mahmutovic; Uzunovic, Aida

    2016-01-01

    Objectives: The aim of this study was to examine antimicrobial resistance of Escherichia coli strains isolated from urine in outpatient population. Material and methods: We performed a retrospective study for tree months period, between January 1st and March 31st, 2015, at the Department of Microbiology and Parasitology, Faculty of Medicine, University of Sarajevo. We determined the E. coli antimicrobial resistance in 556 first urine samples from outpatient population of Hrasno community in Sarajevo, Bosnia and Herzegovina. E. coli is the most frequent agent causing urinary tract infections in outpatients as well. The standard methods of descriptive statistics were performed for data analysis. Results: We observed the highest antimicrobial resistance of E. coli for ampicillin (82,79%), followed by trimethoprim-sulfamethoxazole (40,86%), nalidixic acid (19,35%), cephazolin (7,52%), nitrofurantoin (5,37%), gentamicin (2,15%) and ciprofloxacin (4,30%). Conclusions: The results of study showed that E. coli has the highest resistance to ampicillin and trimethoprim-sulfamethoxazole in outpatient population of Hrasno community. PMID:27147918

  14. Single-channel current recordings of acetylcholine receptors in electroplax isolated from the Electrophorus electricus Main and Sachs' electric organs.

    PubMed

    Pasquale, E B; Udgaonkar, J B; Hess, G P

    1986-01-01

    Extensive chemical kinetic measurements of acetylcholine receptor-controlled ion translocation in membrane vesicles isolated from the electroplax of Electrophorus electricus have led to the proposal of a minimum model which accounts for the activation, desensitization, and voltage-dependent inhibition of the receptor by acetylcholine, suberyldicholine, and carbamoylcholine. Comparison of chemical kinetic measurements of the dynamic properties of the acetylcholine receptor in vesicles with the properties of the receptor in cells obtained from the same organ and animal have been hampered by an inability to make the appropriate measurements with Electrophorus electricus electroplax cells. Here we report a method for exposing and cleaning the surface of electroplax cells obtained from both the Main electric organ and the organ of Sachs and the results of single-channel current recordings which have now become possible. The single-channel current recordings were made in the presence of either carbamoylcholine or suberyldicholine, as a function of temperature and transmembrane voltage. Both the channel open times and the single-channel conductance were measured. The data were found to be consistent with the model based on chemical kinetic measurements using receptor-rich membrane vesicles prepared from the Main electric organ of E. electricus.

  15. Isolating single primary rat hippocampal neurons & astrocytes on ultra-thin patterned parylene-C/silicon dioxide substrates.

    PubMed

    Unsworth, Charles P; Delivopoulos, Evangelos; Gillespie, Trudi; Murray, Alan F

    2011-04-01

    We report here the patterning of primary rat neurons and astrocytes from the postnatal hippocampus on ultra-thin parylene-C deposited on a silicon dioxide substrate, following observations of neuronal, astrocytic and nuclear coverage on strips of different lengths, widths and thicknesses. Neuronal and glial growth was characterized 'on', 'adjacent to' and 'away from' the parylene strips. In addition, the article reports how the same material combination can be used to isolate single cells along thin tracks of parylene-C. This is demonstrated with a series of high magnification images of the experimental observations for varying parylene strip widths and thicknesses. Thus, the findings demonstrate the possibility to culture cells on ultra-thin layers of parylene-C and localize single cells on thin strips. Such work is of interest and significance to the Neuroengineering and Multi-Electrode Array (MEA) communities, as it provides an alternative insulating material in the fabrication of embedded micro-electrodes, which can be used to facilitate single cell stimulation and recording in capacitive coupling mode. Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. Analysing one isolated single walled carbon nanotube in the near-field domain with selective nanovolume Raman spectroscopy.

    PubMed

    Atalay, Han; Lefrant, Serge

    2004-09-01

    In this paper, we describe a new method to the selective nanovolume analysing of one isolated single walled carbon nanotube (SWNT). This concept is based on actually available imaging micro-spectrometry systems for working in near-field domain combined with a stigmatic solid immersion lens. This combination of different analytical methods, and modified and configured equipment entitles us to expand the functionality toward a three-dimensional (3D) nanovolume Raman mapping and photoluminescence intensity with a possible discrimination in polarization, as well as photoluminescence decaytime constant mapping with their unique combination. Subsequently, selective spectra can be acquired from the same location on the samples. By spectrally selecting a SWNT, we registered the spatial distribution of the emitted photons in x, y, z vectors to determine the position of a SWNT in the near-field domain. For the SWNTs that are localized with an accuracy better than 18 nm in the x, y and <1 nm in the z directions, we demonstrate an analytical sensitivity close to a single nanotube with unity throughput. This near-field capability is applied to resolve local variations unambiguously in the Raman spectrum along one single SWNT. Finally, in this paper, we report what we believe to be the first evidence of Raman mapping and 3D real optical imaging of carbon nanotubes with near-field resolution.

  17. Fibrinogenolytic and fibrinolytic activity in oral microorganisms.

    PubMed Central

    Wikström, M B; Dahlén, G; Linde, A

    1983-01-01

    Samples were taken from blood accumulated in dental alveoli after surgical removal of mandibular third molars, from subgingival plaque of teeth with advanced periodontal destructions, from teeth with infected necrotic pulps, and from subjects suffering from angular cheilitis. Of the microorganisms subcultured from these samples, 116 strains were assayed for enzymes degrading fibrinogen and fibrin. Enzymes degrading fibrinogen were assayed with the thin-layer enzyme assay cultivation technique. This assay involves the cultivation of microorganisms on culture agars applied over fibrinogen-coated polystyrene surfaces. Enzymes degrading fibrin were assayed with both a plate assay and a tube assay, in which fibrin was mixed with a microbial culture medium. Microorganisms degrading fibrinogen or fibrin or both were isolated from all sampling sites. Activity was mainly detected in strains of Actinomyces, Bacteroides, Fusobacterium, Peptococcus, Propionibacterium, and Staphylococcus aureus. Most Fusobacterium strains degraded fibrinogen only. Enzymes degrading fibrinogen as well as enzymes degrading fibrin via activation of plasminogen were revealed in strains of Clostridium, S. aureus, and Streptococcus pyogenes. It was generally found that fibrinogen was degraded by more strains than was fibrin, which indicates that different proteases may be involved. PMID:6345573

  18. Stress-tolerant P-solubilizing microorganisms.

    PubMed

    Vassilev, N; Eichler-Löbermann, B; Vassileva, M

    2012-08-01

    Drought, high/low temperature, and salinity are abiotic stress factors accepted as the main reason for crop yield losses in a world with growing population and food price increases. Additional problems create nutrient limitations and particularly low P soil status. The problem of phosphate fertilizers, P plant nutrition, and existing phosphate bearing resources can also be related to the scarcity of rock phosphate. The modern agricultural systems are highly dependent on the existing fertilizer industry based exclusively of this natural, finite, non-renewable resource. Biotechnology offers a number of sustainable solutions that can mitigate these problems by using plant beneficial, including P-solubilizing, microorganisms. This short review paper summarizes the current and future trends in isolation, development, and application of P-solubilizing microorganisms in stress environmental conditions bearing also in mind the imbalanced cycling and unsustainable management of P. Special attention is devoted to the efforts on development of biotechnological strategies for formulation of P-solubilizing microorganisms in order to increase their protection against adverse abiotic factors.

  19. 40 CFR 725.420 - Recipient microorganisms.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Recipient microorganisms. 725.420... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS General Exemptions for New Microorganisms § 725.420 Recipient microorganisms. The following recipient microorganisms are eligible for either...

  20. 40 CFR 725.420 - Recipient microorganisms.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Recipient microorganisms. 725.420... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS General Exemptions for New Microorganisms § 725.420 Recipient microorganisms. The following recipient microorganisms are eligible for either...

  1. 40 CFR 725.420 - Recipient microorganisms.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Recipient microorganisms. 725.420... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS General Exemptions for New Microorganisms § 725.420 Recipient microorganisms. The following recipient microorganisms are eligible for either...

  2. 40 CFR 725.420 - Recipient microorganisms.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Recipient microorganisms. 725.420... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS General Exemptions for New Microorganisms § 725.420 Recipient microorganisms. The following recipient microorganisms are eligible for either...

  3. 40 CFR 725.420 - Recipient microorganisms.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Recipient microorganisms. 725.420... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS General Exemptions for New Microorganisms § 725.420 Recipient microorganisms. The following recipient microorganisms are eligible for either...

  4. An isolated single L-II type coronary artery anomaly: A rare coronary anomaly

    PubMed Central

    Ermis, Emrah; Demirelli, Selami; Korkmaz, Ali Fuat; Sahin, Bingul Dilekci; Kantarci, Abdulmecit

    2015-01-01

    Summary The incidence of congenital artery anomalies is 0.2–1.4%, and most are benign. Single coronary artery (SCA) anomalies are very rare. The right coronary artery (RCA) originating from the left coronary system is one such SCA anomaly, and the risk of sudden cardiac death (SCD) increases if it courses between the pulmonary artery and aorta and coexists with other congenital heart diseases. Additionally, coursing of the RCA between the great vessels increases the risk of atherosclerosis. We herein present the case of a 57 year-old man who was admitted to our cardiology outpatient clinic and diagnosed with an SCA anomaly in which the RCA arose from the left main coronary artery (LMCA) and coursed between the pulmonary artery and aorta. However a critical stenosis was not detected in imaging techniques, and myocardial perfusion scintigraphic evidence of ischaemia was found in a small area. Therefore, he was managed with conservative medical therapy. PMID:26668781

  5. Photochemistry and infrared spectrum of single-bridged diborane(5) anion isolated in solid argon

    NASA Astrophysics Data System (ADS)

    Liu, Meng-Chen; Chen, Hui-Fen; Huang, Wei-Jie; Chin, Chih-Hao; Chen, Sian-Cong; Huang, Tzu-Ping; Wu, Yu-Jong

    2016-08-01

    Three-center two-electron bonds are important for understanding electron-deficient molecules. To examine such a molecule, we produced a diborane(5) anion with a single-bridged structure upon electron bombardment during matrix deposition of Ar containing a small proportion of diborane(6). The diborane(5) anion was destroyed upon photolysis at 180, 220, 385, and 450 nm, but not at 532 nm. Moreover, the possible formation of neutral diborane(5) was observed upon photolysis at 385 and 450 nm, whereas neutral diborane(3) was observed upon photolysis at 180 and 220 nm. The observed line wavenumbers, relative intensities, and isotopic ratios of the diborane(5) anion agreed satisfactorily with those predicted by density functional theory calculations at the B3LYP/aug-cc-pVTZ level of theory. Thus, this method produced the boron hydride anion of interest with few other fragments, which enabled us to clearly identify the IR spectrum of the diborane(5) anion.

  6. Single Nanotube Spectral Imaging To Determine Molar Concentrations of Isolated Carbon Nanotube Species.

    PubMed

    Galassi, Thomas V; Jena, Prakrit V; Roxbury, Daniel; Heller, Daniel A

    2017-01-17

    Electronic and biological applications of carbon nanotubes can be highly dependent on the species (chirality) of nanotube, purity, and concentration. Existing bulk methods, such as absorbance spectroscopy, can quantify sp(2) carbon based on spectral bands, but nanotube length distribution, defects, and carbonaceous impurities can complicate quantification of individual particles. We present a general method to relate the optical density of a photoluminescent nanotube sample to the number of individual nanotubes. By acquiring 3-dimensional images of nanotubes embedded in a gel matrix with a reducing environment, we quantified all emissive nanotubes in a volume. Via spectral imaging, we assessed structural impurities and precisely determined molar concentrations of the (8,6) and (9,4) nanotube species. We developed an approach to obtain the molarity of any structurally enriched semiconducting single-walled carbon nanotube preparation on a per-nanotube basis.

  7. Inward rectification in freshly isolated single smooth muscle cells of the rabbit jejunum.

    PubMed Central

    Benham, C D; Bolton, T B; Denbigh, J S; Lang, R J

    1987-01-01

    1. Single smooth muscle cells were obtained by collagenase digestion of longitudinal muscle of rabbit jejunum. Membrane potential or current under voltage clamp was recorded by patch-clamp technique in the whole-cell recording mode at 22-25 degrees C. 2. At a membrane potential of -50 mV small hyperpolarizing current pulses produced electrotonic potentials which asymptotically approached a steady-state size and did not 'sag'. Stronger hyperpolarization resulted in a 'sag' of the electrotonic potential. Under voltage clamp an inward current, i, was activated in the range -60 to -110 mV. 3. This current had an equilibrium potential of -24.5 +/- 3.5 mV which was shifted negatively by reducing the sodium concentration, and positively by raising the potassium concentration of the bathing solution. 4. This current was blocked by caesium (1 mM) but less affected by barium ions up to 10 mM. 5. The time course of i upon stepping into its activation range was accelerated by increasing negativity and following an initial short delay could be described by a single exponential with a time constant in the range 60 s(-60 mV) to 1 s(-130 mV). 6. It is concluded that these jejunal cells possess a current to which sodium and potassium ions make a contribution which is responsible for the inward rectification they show upon hyperpolarization. This current is activated in a range which would allow it to contribute to the slow potential changes shown by longitudinal jejunal muscle. PMID:2443653

  8. Microencapsulated 3-Dimensional Sensor for the Measurement of Oxygen in Single Isolated Pancreatic Islets

    PubMed Central

    Khalil, Gamal; Sweet, Ian R.; Shen, Amy Q.

    2012-01-01

    Background Oxygen consumption reflects multiple processes in pancreatic islets including mechanisms contributing to insulin secretion, oxidative stress and viability, providing an important readout in studies of islet function, islet viability and drug testing. Due to the scarcity, heterogeneity, and intrinsic kinetic properties of individual islets, it would be of great benefit to detect oxygen consumption by single islets. We present a novel method we have developed to image oxygen in single islets. Methodology/Principal Findings Using a microfluidics system, individual islets and a fluorescent oxygen-sensitive dye were encased within a thin alginate polymer layer. Insulin secretion by the encapsulated islets was normal. Fluorescent signal from the encased dye, detected using a standard inverted fluorescence microscope and digital camera, was stable and proportional to the amount of oxygen in the media. When integrated into a perifusion system, the sensing system detected changes in response to metabolic substrates, mitochondrial poisons, and induced-oscillations. Glucose responses averaged 30.1±7.1% of the response to a metabolic inhibitor (cyanide), increases were observed in all cases (n = 6), and the system was able to resolve changes in oxygen consumption that had a period greater than 0.5 minutes. The sensing system operated similarly from 2–48 hours following encapsulation, and viability and function of the islets were not significantly affected by the encapsulation process. Conclusions/Significance An oxygen-dependent dye situated around and within a pancreatic islet encapsulated by a thin layer of alginate was sensitive to changes in oxygen consumption, and was not harmful to the function or viability of islets over the course of two days. The microcapsule-based sensing method is particularly suited to assessing the effects of compounds (dose responses and time courses) and chronic changes occurring over the course of days. The approach should be

  9. Autecology of microorganisms of typical Ecuador biotopes.

    PubMed

    Tashyrev, O B; Pidgorskyi, V S; Toro, Miguel Naranjo; Gualoto, Miguel; Gladka, G V; Tashyreva, H O; Rokitko, P V; Romanovskaya, V A

    2014-01-01

    34 strains of aerobic chemoorganotrophic microorganisms were isolated from 23 soil and plant samples selected from highland biotopes of Ecuador-Andes massif (Papallacta, 4020 m), ash at the foot of the volcano Tungurahua, mountainous jungle (La Favorita, 1600 m), as well as in humid tropic botanical garden (state Puyo, 950 m). In mountain jungle samples the high number of bacteria--10(5)-10(7) CFU/g of sample were represented by 2-5 morphotypes. In highland (4020 m) samples the bacterial counts made from 10(2) to 10(7) CFU/g of sample. The current study describes resistance of isolated strains to high salinity, UV radiation and toxic metal ions. The majority of isolated strains were halotolerant. Isolates from volcanic ash showed high resistance level to UV radiation--LD99,99 made 1000-1440 J/m2; resistance level for isolates from the soil of Puyo Botanical Garden and isolates from rock lichen (Papallacta) LD99,99 made 1160 and 800 J/m2 respectively. Strains isolated from mountain jungle (La Favorita) showed lower UV-resistance. In highland biotopes of Ecuador occurred bacteria resistant to toxic metal ions. The highest resistance to Hg2+ was shown by isolate of lichen from mountain jungle, the maximal growth concentration was 0.025 g/L; to Cr(VI)--by isolate from lichen rock massif--3,0 g/L. Correlation between metal-resistance, halotolerace and UV resistance for studied strains was not detected, probably because of different microbial cell damage/repair mechanisms under the action of these factors.

  10. [Molecular karyotyping of eukaryotic microorganisms].

    PubMed

    Nasonova, E S

    2012-01-01

    In many fungi and protists small size and weak morphological differentiation of chromosomes embarrass the study of karyotypes using microscopical tools. Molecular karyotyping based on the fractionation of intact chromosomal DNAs by pulsed field gel electrophoresis (PFGE) provides an alternative approach to the analysis of chromosomal sets in such organisms. To assign the bands observed in PFGE gel to the individual chromosomes the following methods of chromosome identification are applied: densitometric analysis of the bands; Southern hybridization with chromosome- and telomere-specific probes, which often is combined with comparative karyotyping of a series of strains with pronounced size polymorphism of chromosomes; comparison of the patterns of restriction fragments of chromosomal DNAs fractioned by KARD 2-D PFGE; comparison with the strains with well-studied interchromosomal rearrangements. Besides estimation of the number and the size of chromosomes, molecular karyotyping allows assessment of haploid genome size and ploidy level, study of genome dynamics, identification of chromosomal rearrangements and associated chromosomal polymorphism. The analysis of karyotype and dynamics of the genomes is important for the study of intra- and interspecial variability, investigation of the chromosome evolution in closely related species and elaboration of the models of speciation. The comparison of molecular karyotypes among isolates of different origin is of great practical importance for clinical diagnostics and for agricultural microbiology. In this review we discuss: 1) the methods of karyotyping and their application to the analysis of chromosomal sets in eukaryotic microorganisms; 2) the specificity of the methods used for extraction and fractionation of intact chromosomal DNAs; 3) the reasons for difficulties in interpretation of molecular karyotypes and the ways of their overcoming; 4) fields of application of molecular karyotyping; 5) the definition of

  11. Single-center experience in the management of spontaneous isolated abdominal aortic dissection.

    PubMed

    Böckler, Dittmar; Bianchini Massoni, Claudio; Geisbüsch, Philipp; Hakimi, Maani; von Tengg-Kobligk, Hendrik; Hyhlik-Dürr, Alexander

    2016-03-01

    This study aims to report the management of patients with spontaneous isolated dissection of the abdominal aorta (sIAAD). A cohort of 18 consecutive patients (12 male, mean age 58 years) with sIAAD was treated between 1990 and 2009. Dissection was asymptomatic in ten and symptomatic in eight patients. Retrospective data analysis from patient charts was performed. Follow-up included clinical examination, ultrasound, and/or CT-angiography. Mean follow-up was 54 months (range 1-211). In total, eight out of 18 received invasive treatment. All asymptomatic patients initially underwent conservative treatment and surveillance. Spontaneous false lumen thrombosis occurred in four (40 %), and three patients showed relevant aneurysmatic progression and underwent elective invasive treatment (open n = 2, endovascular n = 1), representing a crossover rate of 30 %. Late mortality was 20 % (n = 2) in this group. In symptomatic patients, five underwent urgent treatment due to persistent abdominal or back pain (n = 4) or contained rupture (n = 1); one was treated for claudication. The remaining two patients presented with irreversible spinal cord ischemia and were treated conservatively. Three patients were treated by open surgery and three by endovascular interventions (two stentgrafts, one Palmaz XXL stent). Early and late morbidity and mortality was 0 % in this group. There were no reinterventions The majority of patients with sIADD require invasive treatment, with EVAR being the preferable treatment option today. In asymptomatic IADD, primary surveillance is justifiable, but close surveillance due to expansion is necessary.

  12. CCAST: a model-based gating strategy to isolate homogeneous subpopulations in a heterogeneous population of single cells.

    PubMed

    Anchang, Benedict; Do, Mary T; Zhao, Xi; Plevritis, Sylvia K

    2014-07-01

    A model-based gating strategy is developed for sorting cells and analyzing populations of single cells. The strategy, named CCAST, for Clustering, Classification and Sorting Tree, identifies a gating strategy for isolating homogeneous subpopulations from a heterogeneous population of single cells using a data-derived decision tree representation that can be applied to cell sorting. Because CCAST does not rely on expert knowledge, it removes human bias and variability when determining the gating strategy. It combines any clustering algorithm with silhouette measures to identify underlying homogeneous subpopulations, then applies recursive partitioning techniques to generate a decision tree that defines the gating strategy. CCAST produces an optimal strategy for cell sorting by automating the selection of gating markers, the corresponding gating thresholds and gating sequence; all of these parameters are typically manually defined. Even though CCAST is optimized for cell sorting, it can be applied for the identification and analysis of homogeneous subpopulations among heterogeneous single cell data. We apply CCAST on single cell data from both breast cancer cell lines and normal human bone marrow. On the SUM159 breast cancer cell line data, CCAST indicates at least five distinct cell states based on two surface markers (CD24 and EPCAM) and provides a gating sorting strategy that produces more homogeneous subpopulations than previously reported. When applied to normal bone marrow data, CCAST reveals an efficient strategy for gating T-cells without prior knowledge of the major T-cell subtypes and the markers that best define them. On the normal bone marrow data, CCAST also reveals two major mature B-cell subtypes, namely CD123+ and CD123- cells, which were not revealed by manual gating but show distinct intracellular signaling responses. More generally, the CCAST framework could be used on other biological and non-biological high dimensional data types that are

  13. Isolation and optimization of camelid single-domain antibodies: Dirk Saerens’ work on nanobodies

    PubMed Central

    Saerens, Dirk

    2010-01-01

    It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from all other species, these special antibodies are devoid of light chains, and are composed of a heavy chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma genes. An immune response is raised in these HCAbs following a classical immunization protocol. These HCAbs are easily purified from serum, and their antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. The antigen binding site of the dromedary HCAb comprises one single domain, referred to as VHH or nanobody (Nb), therefore, a strategy was designed to clone the Nb repertoire of an immunized dromedary and to select the Nb with specificity for our target antigens. The monoclonal Nb is produced well in bacteria, is very stable and highly soluble, and it binds the antigen with high affinity and specificity. Currently, the recombinant Nb has been developed successfully for research purposes, as a probe in biosensors, to diagnose infections, or to treat diseases such as cancer or trypanosomiasis. PMID:21537479

  14. Isolation of an Asymmetric RNA Uncoating Intermediate for a Single-Stranded RNA Plant Virus

    PubMed Central

    Bakker, Saskia E.; Ford, Robert J.; Barker, Amy M.; Robottom, Janice; Saunders, Keith; Pearson, Arwen R.; Ranson, Neil A.; Stockley, Peter G.

    2012-01-01

    We have determined the three-dimensional structures of both native and expanded forms of turnip crinkle virus (TCV), using cryo-electron microscopy, which allows direct visualization of the encapsidated single-stranded RNA and coat protein (CP) N-terminal regions not seen in the high-resolution X-ray structure of the virion. The expanded form, which is a putative disassembly intermediate during infection, arises from a separation of the capsid-forming domains of the CP subunits. Capsid expansion leads to the formation of pores that could allow exit of the viral RNA. A subset of the CP N-terminal regions becomes proteolytically accessible in the expanded form, although the RNA remains inaccessible to nuclease. Sedimentation velocity assays suggest that the expanded state is metastable and that expansion is not fully reversible. Proteolytically cleaved CP subunits dissociate from the capsid, presumably leading to increased electrostatic repulsion within the viral RNA. Consistent with this idea, electron microscopy images show that proteolysis introduces asymmetry into the TCV capsid and allows initial extrusion of the genome from a defined site. The apparent formation of polysomes in wheat germ extracts suggests that subsequent uncoating is linked to translation. The implication is that the viral RNA and its capsid play multiple roles during primary infections, consistent with ribosome-mediated genome uncoating to avoid host antiviral activity. PMID:22306464

  15. Isolating the Unique Effects of the Unified Protocol Treatment Modules Using Single Case Experimental Design.

    PubMed

    Sauer-Zavala, Shannon; Cassiello-Robbins, Clair; Conklin, Laren R; Bullis, Jacqueline R; Thompson-Hollands, Johanna; Kennedy, Katherine A

    2017-03-01

    The Unified Protocol (UP) for the Transdiagnostic Treatment of Emotional Disorders is a cognitive-behavioral intervention designed to treat the range of anxiety, depressive, and related disorders. Thus far, the UP treatment modules have only been studied when they are delivered in their entirety and presented in a standard sequence. To personalize the presentation of the UP modules for a given patient's presentation (e.g., providing the modules in a varied order, dropping irrelevant modules), it is first necessary to establish that each module leads to change in the skill it is designed to promote, and that these changes can occur in the absence of the other modules. Using a multiple baseline design in accordance with the single-case reporting guidelines in behavioral interventions (SCRIBE), eight patients with heterogeneous emotional disorders were randomly assigned to a 1- or 3-week baseline assessment phase followed by four sessions of one of four UP modules (psychoeducation, emotional awareness, cognitive flexibility, and countering emotional behaviors). Results provide preliminary support for the notion that each UP module under study leads to change in its associated skill in the absence of the other modules (five of eight patients demonstrated reliable change in the module-specific skill). In addition, exploratory analyses suggest that the emotion awareness training and cognitive flexibility modules appeared to exhibit change specific to their associated skills, psychoeducation, and countering emotional behaviors demonstrated somewhat more broad-based change across skills.

  16. Real-Time PCR for Dihydrofolate Reductase Gene Single-Nucleotide Polymorphisms in Plasmodium vivax Isolates

    PubMed Central

    Brega, Sara; de Monbrison, Frédérique; Severini, Carlo; Udomsangpetch, Rachanee; Sutanto, Inge; Ruckert, Paul; Peyron, François; Picot, Stéphane

    2004-01-01

    Mutations in the dhfr gene of Plasmodium vivax (pvdhfr) are associated with resistance to the antifolate antimalarial drugs. Polymorphisms in the pvdhfr gene were assessed by hybridization probe technology on the LightCycler instrument with 134 P. vivax-infected blood samples from Turkey (n = 24), Azerbaijan (n = 39), Thailand (n = 16), Indonesia (n = 53), and travelers (n = 19). Double mutations (S58R and S117N) or quadruple mutations (F57L/I, S58R, T61M, and S117N) in the pvdhfr genes were found in all Thai samples (100%). pvdhfr mutant-type alleles were significantly more common in samples from travelers (42%) than in those from patients from Indonesia (5%). Surprisingly, the pvdhfr single-mutation allele (S117N) was identified at a high frequency in parasites from Turkey and Azerbaijan (71 and 36%, respectively), where sulfadoxine-pyrimethamine is not recommended for the treatment of P. vivax malaria by the World Health Organization and the Malaria National Programs. PMID:15215112

  17. Perinatal outcome in cases of isolated single umbilical artery and its effects on neonatal cord blood gas indices.

    PubMed

    Doğan, S; Özyüncü, Ö; Atak, Z; Turgal, M

    2014-10-01

    Abstract We aimed to evaluate perinatal outcomes and blood gas indices of isolated single umbilical artery (SUA). In this retrospective study, 134 fetuses with SUA were evaluated. A total of 77 (57.4%) fetuses with isolated SUA (iSUA) were compared with maternal age-matched 95 fetuses with double umbilical arteries (DUA), with respect to pregnancy outcome and umbilical cord blood gas indices. The incidence of SUA in our cohort was 1.04%. Patients with iSUA had lower neonatal birth weight compared with the control group (2,635 ± 972 g vs 2,991 ± 669 g; p = 0.009). Small for gestational age (SGA) frequency was higher in the group of iSUA when compared with fetuses with DUA (17.4% vs 5.4%; p = 0.03). Regarding the umbilical cord blood gas, the parameters were comparable between the two groups. SUA has a favourable obstetric outcome as long as the accompanying malformations are ruled out. Furthermore, neonatal cord blood gas parameters in fetuses with iSUA do not differ from fetuses with DUA.

  18. Design-Filter Selection for H2 Control of Microgravity Isolation Systems: A Single-Degree-of-Freedom Case Study

    NASA Technical Reports Server (NTRS)

    Hampton, R. David; Whorton, Mark S.

    2000-01-01

    Many microgravity space-science experiments require active vibration isolation, to attain suitably low levels of background acceleration for useful experimental results. The design of state-space controllers by optimal control methods requires judicious choices of frequency-weighting design filters. Kinematic coupling among states greatly clouds designer intuition in the choices of these filters, and the masking effects of the state observations cloud the process further. Recent research into the practical application of H2 synthesis methods to such problems, indicates that certain steps can lead to state frequency-weighting design-filter choices with substantially improved promise of usefulness, even in the face of these difficulties. In choosing these filters on the states, one considers their relationships to corresponding design filters on appropriate pseudo-sensitivity- and pseudo-complementary-sensitivity functions. This paper investigates the application of these considerations to a single-degree-of-freedom microgravity vibration-isolation test case. Significant observations that were noted during the design process are presented. along with explanations based on the existent theory for such problems.

  19. Isolation and characterization of recombinant single chain fragment variable anti-idiotypic antibody specific to Aspergillus fumigatus membrane protein.

    PubMed

    Krishnaswamy, Senthilkumar; Kabir, M Enamul; Rahman, M Mamunur; Miyamoto, Masahiko; Furuichi, Yasuhiro; Komiyama, Tadazumi

    2011-03-07

    Aspergillus fumigatus causes the highly lethal form of invasive aspergillosis (IA). In the present study to develop a novel anti-fungal drug for protection against invasive disease, we identified a single chain fragment variable (scFv) antibody (scFv AF1) by panning against A. fumigatus membrane fraction (AMF) or HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) as antigen. The key step was elution of bound phages with phosphate buffered saline (PBS) at pH 7.0 containing AMF. The specificity of soluble scFv AF1 antibody to antigens was verified by ELISA, which specifically binds to both AMF and nmAb-KT. After nucleotide sequencing, clone expression and purification by HisTrap HP affinity column, scFv AF1 showed in vitro anti-fungal activity against A. fumigatus. By SPR analysis it showed high binding affinity to nmAb-KT (K(d)=5.22×10(-11) M). The method used to isolate scFv AF1 was a new method and we believe that it will be applicable to isolate the specific scFv against any kind of membrane protein of yeast or fungus.

  20. Terrestrial microorganisms at an altitude of 20,000 m in Earth's atmosphere

    USGS Publications Warehouse

    Griffin, Dale W.

    2004-01-01

    A joint effort between the U.S. Geological Survey's (USGS) Global Desert Dust and NASA's Stratospheric and Cosmic Dust Programs identified culturable microbes from an air sample collected at an altitude of 20,000 m. A total of 4 fungal (Penicillium sp.) and 71 bacteria colonyforming units (70 colonies of Bacillus luciferensis believed to have originated from a single cell collected at altitude and one colony of Bacillus sphaericus) were enumerated, isolated and identified using a morphological key and 16S rDNA sequencing respectively. All of the isolates identified were sporeforming pigmented fungi or bacteria of terrestrial origin and demonstrate that the presence of viable microorganisms in Earth's upper atmosphere may not be uncommon.

  1. Single Cell Proteomics Using Frog (Xenopus laevis) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content.

    PubMed

    Sun, Liangliang; Dubiak, Kyle M; Peuchen, Elizabeth H; Zhang, Zhenbin; Zhu, Guijie; Huber, Paul W; Dovichi, Norman J

    2016-07-05

    Single cell analysis is required to understand cellular heterogeneity in biological systems. We propose that single cells (blastomeres) isolated from early stage invertebrate, amphibian, or fish embryos are ideal model systems for the development of technologies for single cell analysis. For these embryos, although cell cleavage is not exactly symmetric, the content per blastomere decreases roughly by half with each cell division, creating a geometric progression in cellular content. This progression forms a ladder of single-cell targets for the development of successively higher sensitivity instruments. In this manuscript, we performed bottom-up proteomics on single blastomeres isolated by microdissection from 2-, 4-, 8-, 16-, 32-, and 50-cell Xenopus laevis (African clawed frog) embryos. Over 1 400 protein groups were identified in single-run reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry from single balstomeres isolated from a 16-cell embryo. When the mass of yolk-free proteins in single blastomeres decreased from ∼0.8 μg (16-cell embryo) to ∼0.2 μg (50-cell embryo), the number of protein group identifications declined from 1 466 to 644. Around 800 protein groups were quantified across four blastomeres isolated from a 16-cell embryo. By comparing the protein expression among different blastomeres, we observed that the blastomere-to-blastomere heterogeneity in 8-, 16-, 32-, and 50-cell embryos increases with development stage, presumably due to cellular differentiation. These results suggest that comprehensive quantitative proteomics on single blastomeres isolated from these early stage embryos can provide valuable insights into cellular differentiation and organ development.

  2. Efficient isolation of soluble intracellular single-chain antibodies using the twin-arginine translocation machinery

    PubMed Central

    Fisher, Adam; DeLisa, Matthew P.

    2008-01-01

    One of the most commonly used recombinant antibody formats is the single-chain variable fragment (scFv) that consists of the antibody variable heavy chain connected to the variable light chain by a flexible linker. Since disulfide bonds are often necessary for scFv folding, it can be challenging to express scFvs in the reducing environment of the cytosol. Thus, we sought to develop a method for antigen-independent selection of scFvs that are stable in the reducing cytosol of bacteria. To this end, we applied a recently developed genetic selection for protein folding and solubility based on the quality control feature of the Escherichia coli twin-arginine translocation (Tat) pathway (Fisher et al., 2006 Protein Sci). This selection employs a tripartite sandwich fusion of a protein-of-interest with an N-terminal Tat-specific signal peptide and C-terminal TEM1 β-lactamase, thereby coupling antibiotic resistance with Tat pathway export. Here, we adapted this assay to develop intrabody selection after Tat export (ISELATE), a high-throughput selection strategy for the identification of solubility-enhanced scFv sequences. Using ISELATE for three rounds of laboratory evolution, it was possible to evolve a soluble scFv from an insoluble parental sequence. We also show that ISELATE enables focusing of an scFv library in soluble sequence space prior to functional screening and thus can be used to increase the likelihood of finding functional intrabodies. Finally, the technique was used to screen a large repertoire of naïve scFvs for clones that conferred significant levels of soluble accumulation. In these ways, we show that the Tat quality control mechanism can be harnessed for molecular evolution of scFvs that are soluble in the reducing cytoplasm of E. coli. PMID:18992254

  3. Isolation of human single chain variable fragment antibodies against specific sperm antigens for immunocontraceptive development

    PubMed Central

    Samuel, A.S.; Naz, R.K.

    2008-01-01

    BACKGROUND Contraceptive vaccines can provide valuable alternatives to current methods of contraception. We describe here the development of sperm-reactive human single chain variable fragment (scFv) antibodies of defined sperm specificity for immunocontraception. METHODS Peripheral blood leukocytes (PBL) from antisperm antibody-positive immunoinfertile and vasectomized men were activated with human sperm antigens in vitro, and the complementary DNA prepared and PCR-amplified using primers based on all the variable regions of heavy and light chains of immunoglobulins. The scFv repertoire was cloned into pCANTAB5E vector to create a human scFv antibody library. RESULTS Panning of the library against specific sperm antigens yielded several clones, and the four strongest reactive were selected for further analysis. These clones had novel sequences with unique complementarity-determining regions. ScFv antibodies were expressed, purified and analyzed for human sperm reactivity and effect on human sperm function. AFA-1 and FAB-7 scFv antibodies both reacted with fertilization antigen-1 antigen, but against different epitopes. YLP20 antibody reacted with the expected human sperm protein of 48 ± 5 kDa. The fourth antibody, AS16, reacted with an 18 kDa sperm protein and seems to be a human homologue of the mouse monoclonal recombinant antisperm antibody that causes sperm agglutination. All these antibodies inhibited human sperm function. CONCLUSIONS This is the first study to report the use of phage display technology to obtain antisperm scFv antibodies of defined antigen specificity. These antibodies will find clinical applications in the development of novel immunocontraceptives, and specific diagnostics for immunoinfertility. PMID:18372255

  4. Single nucleotide polymorphism isolated from a novel EST dataset in garden asparagus (Asparagus officinalis L.).

    PubMed

    Mercati, Francesco; Riccardi, Paolo; Leebens-Mack, Jim; Abenavoli, Maria Rosa; Falavigna, Agostino; Sunseri, Francesco

    2013-04-01

    Single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSR) are abundant and evenly distributed co-dominant molecular markers in plant genomes. SSRs are valuable for marker assisted breeding and positional cloning of genes associated traits of interest. Although several high throughput platforms have been developed to identify SNP and SSR markers for analysis of segregant plant populations, breeding in garden asparagus (Asparagus officinalis L.) has been limited by a low content of such markers. In this study massively parallel GS-FLX pyro-sequencing technology (454 Life Sciences) has been used to sequence and compare transcriptome from two genotypes: a rust tolerant male (1770) and a susceptible female (G190). A total of 122,963 and 99,368 sequence reads, with an average length of 245.7bp, have been recovered from accessions 1770 and 190 respectively. A computational pipeline has been used to predict and visually inspect putative SNPs and SSR sequences. Analysis of Gene Ontology (GO) slim annotation assignments for all assembled uniscripts indicated that the 24,403 assemblies represent genes from a broad array of functions. Further, over 1800 putative SNPs and 1000 SSRs were detected. One hundred forty-four SNPs together with 60 selected SSRs were validated and used to develop a preliminary genetic map by using a large BC(1) population, derived from 1770 and G190. The abundance of SNPs and SSRs provides a foundation for the development of saturated genetic maps and their utilization in assisted asparagus breeding programs. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  5. Screening of biosurfactants from cloud microorganisms

    NASA Astrophysics Data System (ADS)

    Sancelme, Martine; Canet, Isabelle; Traikia, Mounir; Uhliarikova, Yveta; Capek, Peter; Matulova, Maria; Delort, Anne-Marie; Amato, Pierre

    2015-04-01

    The formation of cloud droplets from aerosol particles in the atmosphere is still not well understood and a main source of uncertainties in the climate budget today. One of the principal parameters in these processes is the surface tension of atmospheric particles, which can be strongly affected by trace compounds called surfactants. Within a project devoted to bring information on atmospheric surfactants and their effects on cloud droplet formation, we focused on surfactants produced by microorganisms present in atmospheric waters. From our unique collection of microorganisms, isolated from cloud water collected at the Puy-de-Dôme (France),1 we undertook a screening of this bank for biosurfactant producers. After extraction of the supernatants of the pure cultures, surface tension of crude extracts was determined by the hanging drop technique. Results showed that a wide variety of microorganisms are able to produce biosurfactants, some of them exhibiting strong surfactant properties as the resulting tension surface decreases to values less then 35 mN.m-1. Preliminary analytical characterization of biosurfactants, obtained after isolation from overproducing cultures of Rhodococcus sp. and Pseudomonas sp., allowed us to identify them as belonging to two main classes, namely glycolipids and glycopeptides. 1. Vaïtilingom, M.; Attard, E.; Gaiani, N.; Sancelme, M.; Deguillaume, L.; Flossmann, A. I.; Amato, P.; Delort, A. M. Long-term features of cloud microbiology at the puy de Dôme (France). Atmos. Environ. 2012, 56, 88-100. Acknowledgements: This work is supported by the French-USA ANR SONATA program and the French-Slovakia programs Stefanik and CNRS exchange.

  6. Medical Significance of Microorganisms in Spacecraft Environment

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L.; Ott, C. Mark

    2007-01-01

    Microorganisms can spoil food supplies, contaminate drinking water, release noxious volatile compounds, initiate allergic responses, contaminate the environment, and cause infectious diseases. International acceptability limits have been established for bacterial and fungal contaminants in air and on surfaces, and environmental monitoring is conducted to ensure compliance. Allowable levels of microorganism in water and food have also been established. Environmental monitoring of the space shuttle, the Mir, and the ISS have allowed for some general conclusions. Generally, the bacteria found in air and on interior surfaces are largely of human origin such as Staphylococcus spp., Micrococcus spp. Common environmental genera such as Bacillus spp. are the most commonly isolated bacteria from all spacecraft. Yeast species associated with humans such as Candida spp. are commonly found. Aspergillus spp., Penicillium spp., and Cladosporium spp. are the most commonly isolated filamentous fungi. Microbial levels in the environment differ significantly depending upon humidity levels, condensate accumulation, and availability of carbon sources. However, human "normal flora" of bacteria and fungi can result in serious, life-threatening diseases if human immunity is compromised. Disease incidence is expected to increase as mission duration increases.

  7. Bioremediation of trinitrotolulene by a ruminal microorganism

    SciTech Connect

    Lee, Taejin; Williamson, K.J.; Craig, A.M.

    1995-10-01

    2,4,6-trinitrotoluene (TNT) has been widely used for the production of explosives because of its low boiling point, high stability, low impact sensitivity, and safe manufacture. More than 1,100 military fac