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Sample records for single isolated microorganisms

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

    PubMed Central

    Rohde, Robert A.; Price, P. Buford

    2007-01-01

    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 CO2, O2, N2, CO, and CH4 diffusing through the ice lattice. We show that there is an adequate supply of diffusing molecules throughout deep glacial ice to sustain metabolism for >105 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 μl with the protein mapper and in 1.9 μl with the methanogen mapper. With such precise localization, it should be possible to extract single cells for molecular identification. PMID:17940052

  2. Microorganism characterization by single particle mass spectrometry.

    PubMed

    Russell, Scott C

    2009-01-01

    In recent years a major effort by several groups has been undertaken to identify bacteria by mass spectrometry at the single cell level. The intent of this review is to highlight the recent progress made in the application of single particle mass spectrometry to the analysis of microorganisms. A large portion of the review highlights improvements in the ionization and mass analysis of bio-aerosols, or particles that contain biologically relevant molecules such as peptides or proteins. While these are not direct applications to bacteria, the results have been central to a progression toward single cell mass spectrometry. Developments in single particle matrix-assisted laser desorption/ionization (MALDI) are summarized. Recent applications of aerosol laser desorption/ionization (LDI) to the analysis of single microorganisms are highlighted. Successful applications of off-line and on-the-fly aerosol MALDI to microorganism detection are discussed. Limitations to current approaches and necessary future achievements are also addressed. PMID:18949817

  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 OF MICROORGANISMS FOR BIOLOGICAL DETOXIFICATION OF LIGNOCELLULOSIC HYDROLYSATES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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-hydrox...

  5. Complete nitrification by a single microorganism.

    PubMed

    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

    2015-12-24

    Nitrification is a two-step process where ammonia is first oxidized to nitrite by ammonia-oxidizing bacteria and/or archaea, and subsequently to nitrate by nitrite-oxidizing bacteria. Already described by Winogradsky in 1890, this division of labour between the two functional groups is a generally accepted characteristic of the biogeochemical nitrogen cycle. 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 microorganisms. 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 the 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 of 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

  6. Isolated microorganisms from Iranian grapes and its derivatives

    PubMed Central

    Maulani, S; Hosseini, SM; Elikaie, A; Mirnurollahi, SM

    2012-01-01

    Background and Objectives The objective of this study was to monitor the microorganisms isolated from grapes and its derivative traditional products produced in Iran. Material and Methods Four kinds of grapes cultivated summer of 2010 in vineyard of Takestan and also grape derived products from Shahrod, Hamedan and Takestan were used for this study. The samples were cultured in specific media to isolate the microorganisms that might grow on or pollute the products. Results Species of bacteria and fungi isolated from 4 kinds of grapes cultivated in Takestan graveyards and also from 2 kinds of derived traditional products; grape sap and sour grape (abe-ghure locally named), were taken from Takestan, Shahrod and Hamedan cities. Also, bacteria Bacillus spp., Micrococcus spp., Clostridium spp., and fungus of Penicillium spp., and Aspergillus spp. were isolated. Conclusion The isolated bacteria were common microorganisms that grow in soil or in the organic fertilizer and may appear from the environments that samples were collected. These bacteria were not pathogenic to human. The fungus isolated from the grapes may harm humans as they produce toxin. The results suggested that bacterial diversity on grapes and its derived traditional products are expected to be monitored and described in all Iranian graveyards as Iran has been known as one of the world's biggest grape producers. PMID:22783457

  7. 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

  8. 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

  9. 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. PMID:12908085

  10. The isolation of microorganisms capable of phenol degradation.

    PubMed

    Przybulewska, Krystyna; Wieczorek, Andrzej; Nowak, Andrzej; Pochrzaszcz, Magdalena

    2006-01-01

    The results of a study on the composition of microflora settling the pilot biofilter bed that purifies the exhausting gases from a cable factory's coil-wire varnishing division are presented in this study. The ability of isolated bacterial strains to biodegrade phenol was also evaluated using culture media of various compositions. Phenol was introduced into the medium at the following concentrations: 0.25, 0.5, 0.75 and 1 g x dm(-3). In addition, air in desiccators, where micro-organisms grew, was saturated with phenol. The isolated microorganisms were graded by the phenol decomposition rate using gas chromatography. The beds of biofilters utilized in industry appeared to be the source of microorganisms capable of degrading phenol. The most active were: Rhodococcus rhodochrous, Gordonia sputi, Pseudomonas putida. Their mixture showed higher degradation activity than the particular isolates. Isolated and identified bacteria metabolized phenol at high rate (about 14 to 42 g x m(-3) x h(-3)). PMID:16878606

  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. PMID:22806048

  12. Organochlorinated pesticide degrading microorganisms isolated from contaminated soil.

    PubMed

    Lovecka, Petra; Pacovska, Iva; Stursa, Petr; Vrchotova, Blanka; Kochankova, Lucie; Demnerova, Katerina

    2015-01-25

    Degradation of selected organochlorinated pesticides (γ-hexachlorocyclohexane - γ-HCH, dichlorodiphenyltrichloroethane - DDT, hexachlorobenzene - HCB) by soil microorganisms was studied. Bacterial strains isolated from contaminated soil from Klatovy-Luby, Hajek and Neratovice, Czech Republic, capable of growth on the selected pesticides were isolated and characterised. These isolates were subjected to characterisation and identification by MS MALDI-TOF of whole cells and sequence analysis of 16S rRNA genes. The isolates were screened by gas chromatography for their ability to degrade the selected pesticides. Some isolates were able to degrade pesticides, and the formation of degradation products (γ-pentachlorocyclohexane (γ-PCCH), dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD)) observed in liquid culture confirmed their degradation capability. The isolates and DNA samples isolated from the contaminated soil were also screened for the bphA1 gene (encoding biphenyl-2,3-dioxygenase, the first enzyme in the PCB degradation pathway) and its occurrence was demonstrated. The isolates were also screened for the presence of linA, encoding dehydrochlorinase, the first enzyme of the HCH degradation pathway. The linA gene could not be found in any of the tested isolates, possibly due to the high specificity of the primers used. The isolates with the most effective degradation abilities could be used for further in situ bioremediation experiments with contaminated soil. PMID:25094051

  13. 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. PMID:27199953

  14. 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

  15. 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.

  16. Coaggregation occurs between microorganisms isolated from different environments.

    PubMed

    Stevens, Michael R E; Luo, Ting L; Vornhagen, Jay; Jakubovics, Nicholas S; Gilsdorf, Janet R; Marrs, Carl F; Møretrø, Trond; Rickard, Alexander H

    2015-11-01

    Coaggregation, the specific recognition and adherence of different microbial species, is thought to enhance biofilm formation. To date, no studies have focused on the ability of microorganisms isolated from a broad range of environments to coaggregate with each other and it is unclear whether coaggregation promotes the transmission of microorganisms between environmental niches. We aimed to evaluate the coaggregation ability of 29 bacteria and one fungus, isolated from a range of different environments, and to characterize the cell-surface polymers that mediate coaggregation between selected pairs. Strains were categorized as belonging to one of the four microbial archetypes: aquatic, broad environment, human opportunistic pathogen or human oral. A total of 23 of the 30 strains (77%) coaggregated with at least one other and 21/30 (70%) coaggregated with strains belonging to other archetypes. Nasopharyngeal bacteria belonging to the human opportunistic pathogen archetype showed the least number of coaggregations, and five Haemophilus influenzae strains did not coaggregate. Protease and sugar treatments indicated that coaggregation between strains of different archetypes was often mediated by lectin-saccharide interactions (9 of 15 evaluated pairs). In conclusion, coaggregation can occur between taxonomically disparate species isolated from discrete environments. We propose that these organisms be labeled as 'cross-environment coaggregating organisms'. The ability to coaggregate may aid species to colonize non-indigenous biofilms. PMID:26475462

  17. 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. PMID:24459833

  18. 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

  19. IDENTIFICATION OF Pseudomonas spp. AS AMOEBA-RESISTANT MICROORGANISMS IN ISOLATES OF Acanthamoeba

    PubMed Central

    Maschio, Vinicius José; 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. PMID:25651331

  20. 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

  1. 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. PMID:26559553

  2. 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%. PMID:24722403

  3. 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.

  4. 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.

  5. In vitro susceptibility of high virulence microorganisms isolated in heart valve banking.

    PubMed

    Villalba, R; Solis, F; Fornés, G; Jimenez, A; Eisman, M; González, Ana I; Linares, M J; Casal, M; Gómez Villagrán, J L

    2012-08-01

    Storage preparation of human heart valves for implants generally includes incubation in an antimicrobial disinfection solution and cryopreservation. Changes in patterns of microorganisms susceptibility to antibiotics is a variable process of that promote its inefficiency. The aim of this study has been an evaluation of in vitro susceptibility of high virulence microorganisms isolated in our tissue bank for 14 years in order to evaluate the efficiency, and to promote changes for further antibiotics mixtures as well. Data presented in this study show that microorganisms isolates in valve banking display susceptibility patterns similar to those shown in other clinical circumstances, and the most commonly used antibiotics regimes are useful to date. An antibiotic cocktail containing aminoglicoside in addition to ciprofloxacin and vancomycin is an efficient mixture to be used in valve banking. Further studies will be necessary for monitoring patterns changes of in vitro susceptibility of microbiological isolates in tissue banking. PMID:22618487

  6. Isolation of microorganisms capable of degrading isoquinoline under aerobic conditions

    SciTech Connect

    Aislabie, J.; Rothenburger, S.; Atlas, R.M. )

    1989-12-01

    Isoquinoline-degrading microbial cultures were isolated from oil- and creosote-contaminated soils. The establishment of initial enrichment cultures required the use of emulsified isoquinoline. Once growth on isoquinoline was established, isoquinoline emulsification was no longer required for utilization of isoquinoline as the sole source of carbon and nitrogen by these cultures. An isoquinoline-degrading Acinetobacter strain was isolated from one of the enrichment cultures. The degradation of isoquinoline was accompanied by the accumulation of a red cell-associated pigment and of 1-hydroxyisoquinoline, which was further degraded to unknown intermediary ring-cleavage products and carbon dioxide.

  7. 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.

  8. 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

  9. 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

  10. Diversity of Microorganisms Isolated from the Soil Sample surround Chroogomphus rutilus in the Beijing Region

    PubMed Central

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

    2011-01-01

    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. PMID:21448282

  11. 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. PMID:11131025

  12. Isolation of a microorganism capable of degrading poly-(L-lactide).

    PubMed

    Ikura, Yoko; Kudo, Toshiaki

    1999-10-01

    The isolation of poly-(L-lactide) (PLA)-degrading microorganisms was investigated. A PLA-degrading actinomycete, strain No. 3118, was isolated and tentatively identified as a member of the genus Amycolatopsis. The optimum conditions for degradation of PLA were 43 degrees C at about pH 7 in a mineral salt medium with a low concentration of organic nutrients (0.002% yeast extract). The original shape of PLA film (Mw=2.3x10(5) after sterilization, 20 &mgr;m thick) disappeared within 2 weeks. Lactic acid was detected after the film was incubated with culture supernatant. PMID:12501367

  13. 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

  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. PMID:16489498

  15. 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. PMID:15156037

  16. Isolation and screening of biopolymer-degrading microorganisms from northern Thailand.

    PubMed

    Penkhrue, Watsana; Khanongnuch, Chartchai; Masaki, Kazuo; Pathom-Aree, Wasu; Punyodom, Winita; Lumyong, Saisamorn

    2015-09-01

    Forty agricultural soils were collected from Chiang Mai and Lampang provinces in northern Thailand. Bacteria, actinomycetes and fungi were isolated and screened for their ability to degrade polylactic acid (PLA), polycaprolactone (PCL) and poly(butylene succinate) (PBS) by the agar diffusion method. Sixty-seven actinomycetes, seven bacteria and five fungal isolates were obtained. The majority of actinomycetes were Streptomyces based on morphological characteristic, chemotaxonomy and 16S rRNA gene data. Seventy-nine microorganisms were isolated from 40 soil samples. Twenty-six isolates showed PLA-degradation (32.9 %), 44 isolates showed PBS-degradation (55.7 %) and 58 isolates showed PCL-degradation (73.4 %). Interestingly, 16 isolates (20.2 %) could degrade all three types of bioplastics used in this study. The Amycolatopsis sp. strain SCM_MK2-4 showed the highest enzyme activity for both PLA and PCL, 0.046 and 0.023 U/mL, respectively. Moreover, this strain produced protease, esterase and lipase on agar plates. Approximately, 36.7 % of the PLA film was degraded by Amycolatopsis sp. SCM_MK2-4 after 7 days of cultivation at 30 °C in culture broth. PMID:26135516

  17. 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.

  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. Exploring the multiple biotechnological potential of halophilic microorganisms isolated from two Argentinean salterns.

    PubMed

    Nercessian, Débora; Di Meglio, Leonardo; De Castro, Rosana; Paggi, Roberto

    2015-11-01

    The biodiversity and biotechnological potential of microbes from central Argentinean halophilic environments have been poorly explored. Salitral Negro and Colorada Grande salterns are neutral hypersaline basins exploded for NaCl extraction. As part of an ecological analysis of these environments, two bacterial and seven archaeal representatives were isolated, identified and examined for their biotechnological potential. The presence of hydrolases (proteases, amylases, lipases, cellulases and nucleases) and bioactive molecules (surfactants and antimicrobial compounds) was screened. While all the isolates exhibited at least one of the tested activities or biocompounds, the species belonging to Haloarcula genus were the most active, also producing antimicrobial compounds against their counterparts. In general, the biosurfactants were more effective against olive oil and aromatic compounds than detergents (SDS or Triton X-100). Our results demonstrate the broad spectrum of activities with biotechnological potential exhibited by the microorganisms inhabiting the Argentinean salterns and reinforce the importance of screening pristine extreme environments to discover interesting/novel bioactive molecules. PMID:26369649

  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. PMID:16522539

  2. 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.

  3. Phylogenetic and Physiological Diversity of Microorganisms Isolated from a Deep Greenland Glacier Ice Core

    PubMed Central

    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. PMID:14711643

  4. 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

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

    PubMed

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

    2015-12-01

    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

  6. [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. PMID:20695223

  7. Use of continuous culture to screen for lipase-producing microorganisms and interesterification of butter fat by lipase isolates.

    PubMed

    Pabai, F; Kermasha, S; Morin, A

    1996-05-01

    The continuous cultivation technique was used to investigate the screening for lipase-producing microorganisms from four commercial starters suitable for the degradation of domestic wastes. Using this technique, three strains of lipase-producing bacteria were isolated and identified: Pantoea agglomerans (BB96CC1, BB168CC2) and Pseudomonas fluorescens (BW96CC1). In addition, butter fat induced more lipase production when present in the growth medium. Interesterification of butter fat triacylglycerols by enzymatic extracts of the isolated strains of microorganisms resulted in an appreciable interesterification yield, implying that hydrolysis was suppressed and interesterification of butter fat triacylglycerols was maximized in a microemulsion free-cosurfactant system. PMID:8640605

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. Technologies for Single-Cell Isolation.

    PubMed

    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

  13. Transformation of indole by methanogenic and sulfate-reducing microorganisms isolated from digested sludge

    SciTech Connect

    Shanker, R.; Bollag, J.M. )

    1990-01-01

    In the present study, mineralization of an aromatic N-heterocyclic molecule, indole, by microorganisms present in anaerobically digested sewage sludge was examined. The first step in indole mineralization was the formation of a hydroxylated intermediate, oxindole. The rate of transformation of indole to oxindole and its subsequent disappearance was dependent on the concentration of inoculum and indole and the incubation temperature. Methanogenesis appeared to be the dominant process in the mineralization of indole in 10% digested sludge even in the presence of high concentrations of sulfate. Enrichment of the digested sludge with sulfate as an electron acceptor allowed the isolation of a metabolically stable mixed culture of anaerobic bacteria which transformed indole to oxindole and acetate, and ultimately to methane and carbon dioxide. This mixed culture exhibited a predominance of sulfate-reducers over methanogens with more than 75% of the substrate mineralized to carbon dioxide. The investigation demonstrates that indole can be transformed by both methanogenic and sulfate-reducing microbial populations.

  14. 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. PMID:23762760

  15. 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. PMID:26283066

  16. Biodegradation of commercial gasoline (24% ethanol added) in liquid medium by microorganisms isolated from a landfarming site.

    PubMed

    Oliveira, Núbia M; Bento, Fátima M; Camargo, Flávio A O; Knorst, Aline Jéssica; Dos Santos, Anai Loreiro; Pizzolato, Tania M; Peralba, Maria do Carmo R

    2011-01-01

    Isolation of soil microorganisms from a landfarming site with a 19-year history of petrochemical residues disposal was carried out. After isolation, the bacteria behavior in mineral medium with 1% commercial gasoline (24% ethanol) was evaluated. Parameters employed for microorganism evaluation and selection of those with the greatest degradation potential were: microbial growth; biosurfactant generation and compound reduction in commercial gasoline. Starting from bacteria that presented the best degradation results, consortiums formed by 4 distinct microorganisms were formed. A microbial growth in the presence of commercial gasoline was observed and, for most of the bacteria, degradations of compounds such as benzene, toluene and xylenes (BTX) as well as biosurfactant production was observed. Ethanol was partially degraded by the bacterial isolates although the data does not allow affirming that it was degraded preferentially to the aromatic hydrocarbons investigated. The analyzed consortiums present an efficiency of 95% to 98% for most of the commercial gasoline compounds and a preferential attack to ethanol under the essay condition was not observed within 72 h. PMID:21104499

  17. 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

    2015-10-01

    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. PMID:26231417

  18. 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. PMID:27043057

  19. In vitro characterization of the digestive stress response and immunomodulatory properties of microorganisms isolated from smear-ripened cheese.

    PubMed

    Adouard, Nadège; Foligné, Benoît; Dewulf, Joëlle; Bouix, Marielle; Picque, Daniel; Bonnarme, Pascal

    2015-03-16

    Thirty-six microorganisms (twenty-one bacteria, twelve yeasts and three fungi) were isolated from surface-ripened cheeses and subjected to in vitro digestive stress. The approach mimicked gastric and/or duodenal digestion. Lactobacillus rhamnosus GG, Escherichia coli Nissle 1917 and Saccharomyces boulardii were used as reference strains. We studied the microorganisms grown separately in culture medium and then included (or not) in a rennet gel. The microorganisms' immunomodulatory abilities were also assessed by profiling cytokine induction in human peripheral blood mononuclear cells (PBMCs). The loss of viability was less than 1 log CFU/mL for yeasts under all conditions. In contrast, Gram-negative bacteria survived gastric and/or duodenal stress well but most of the Gram-positive bacteria were more sensitive (especially to gastric stress). Inclusion of sensitive Gram-positive bacteria in rennet gel dramatically improved gastric survival, when compared with a non-included cultured (with a 4 log CFU/mL change in survival). However, the rennet gel did not protect the bacteria against duodenal stress. The PBMC cytokine assay tests showed that the response to yeasts was usually anti-inflammatory, whereas the response to bacteria varied from one strain to another. PMID:25589362

  20. 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. PMID:14649709

  1. 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.

  2. [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. PMID:15122392

  3. 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.

  4. 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.

  5. 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

  6. 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. PMID:23874571

  7. 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

  8. Antimicrobial activity of pure platelet-rich plasma against microorganisms isolated from oral cavity

    PubMed Central

    2013-01-01

    Background Autologous platelet concentrates (PCs) have been extensively used in a variety of medical fields to promote soft and hard tissue regeneration. The significance behind their use lies in the abundance of growth factors in platelets α-granules that promotes wound healing. In addition, antibacterial properties of PCs against various bacteria have been recently pointed out. In this study, the antimicrobial effect of pure platelet-rich plasma (P-PRP) was evaluated against oral cavity microorganisms such as Enterococcus faecalis, Candida albicans, Streptococcus agalactiae, Streptococcus oralis and Pseudomonas aeruginosa. Blood samples were obtained from 17 patients who underwent oral surgery procedures involving the use of P-PRP. The antibacterial activity of P-PRP, evaluated as the minimum inhibitory concentration (MIC), was determined through the microdilution twofold serial method. Results P-PRP inhibited the growth of Enterococcus faecalis, Candida albicans, Streptococcus agalactiae and Streptococcus oralis, but not of Pseudomonas aeruginosa strains. Conclusions P-PRP is a potentially useful substance in the fight against postoperative infections. This might represent a valuable property in adjunct to the enhancement of tissue regeneration. PMID:23442413

  9. Anaerobic liquefaction/solubilization of coal by microorganisms and isolated enzymes

    SciTech Connect

    Scott, C.D.; Faison, B.D.; Woodward, C.A.

    1991-01-01

    Biocatalytic systems utilizing either living organisms or modified enzymes have been shown to enhance the liquefaction (products are liquid at ambient conditions) or solubilization of coal under anaerobic conditions. Microbial tests have been carried out in aqueous media with organisms isolated from outcropping of coal or from premium coal samples. Some of these isolates have been shown to grow on coal as the only carbon source and to produce small quantities of oxychemicals such as acetate or ethanol. Reducing enzymes, such as hydrogenase and cytochrome C, can be chemically modified to increase solubilization in organic solvents by attaching less polar chemicals, such as phenyl groups or polyethylene glycol, to the free amino groups on the enzymes. These biocatalysts have been shown to degrade model compounds and enhance the solubilization of coal in organic solvents under a hydrogen atmosphere. The resulting product is a relatively light hydrocarbon mixture with reasonably high volatility. 5 refs., 6 figs., 4 tabs.

  10. 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.

  11. 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. PMID:25004759

  12. [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

  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. PMID:22414193

  14. Enhanced degradation in soil of the herbicide EPTC and determination of its degradative pathway by an isolated soil microorganism

    SciTech Connect

    Ankumah, R.O.

    1988-01-01

    A series of experiments was conducted to examine the ability of Ohio soils to develop enhanced degradation of the herbicide EPTC (s-ethyl N,N-dipropyl carbamothiaote) and to determine its metabolism by an isolated soil microorganism. Three soils selected to obtain an range in pH, texture, and organic carbon were treated with EPTC for 4 consecutive applications (6 weeks between applications). EPTC concentrations as measured by gas chromatography, decreased 80% or more one week after the second application in all three soils. Metabolism of unlabelled and labelled EPTC by an isolated soil microbe was followed by GC/MS and TLC/LSC analysis, respectively. Rapid decrease in 14-C activity in the organic fraction corresponded with rapid {sup 14}CO{sub 2} evolution and transient increase in 14-C activity in the aqueous fraction. Four metabolites were observed in the TLC analysis. Two were identified as EPTC-sulfoxide and N-depropyl EPTC with N-depropyl EPTC being confirmed by GC/MS analysis. The availability of different pathways for EPTC metabolism by soil microbes after repeated applications to the soil results in its very rapid degradation and loss of efficacy.

  15. 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. PMID:27136610

  16. 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.

  17. 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.

  18. Micro-organic single crystalline phototransistors of 7,7,8,8-tetracyanoquinodimethane and tetrathiafulvalene

    NASA Astrophysics Data System (ADS)

    Jiang, Hui; Yang, Xianjin; Cui, Zhenduo; Liu, Yongchang; Li, Hongxiang; Hu, Wenping

    2009-03-01

    Classical p-type and n-type organic single crystals, tetrathiafulvalene (TTF) and 7,7,8,8-tetracyanoquinodimethane (TCNQ), are introduced to investigate photoswitch and phototransistor. High photoresponsivity, low persistent conductivity, and response reversibility can be found in single crystalline TCNQ, while TTF has large persistent conductivity when the light is switched on and off. It is probably attributed to different band gaps and the compactness of molecular packing. Single crystalline TCNQ combines light detection, switching, signal amplification in a single device and realization of multiple functions which exhibit a very promising potential for the fabrication of organic photoelectric devices.

  19. Single Clinical Isolates from Acute Uncomplicated Urinary Tract Infections Are Representative of Dominant In Situ Populations

    PubMed Central

    Willner, Dana; Low, Serene; Steen, Jason A.; George, Narelle; Nimmo, Graeme R.; Schembri, Mark A.; Hugenholtz, Philip

    2014-01-01

    ABSTRACT Urinary tract infections (UTIs) are one of the most commonly acquired bacterial infections in humans, and uropathogenic Escherichia coli strains are responsible for over 80% of all cases. The standard method for identification of uropathogens in clinical laboratories is cultivation, primarily using solid growth media under aerobic conditions, coupled with morphological and biochemical tests of typically a single isolate colony. However, these methods detect only culturable microorganisms, and characterization is phenotypic in nature. Here, we explored the genotypic identity of communities in acute uncomplicated UTIs from 50 individuals by using culture-independent amplicon pyrosequencing and whole-genome and metagenomic shotgun sequencing. Genus-level characterization of the UTI communities was achieved using the 16S rRNA gene (V8 region). Overall UTI community richness was very low in comparison to other human microbiomes. We strain-typed Escherichia-dominated UTIs using amplicon pyrosequencing of the fimbrial adhesin gene, fimH. There were nine highly abundant fimH types, and each UTI sample was dominated by a single type. Molecular analysis of the corresponding clinical isolates revealed that in the majority of cases the isolate was representative of the dominant taxon in the community at both the genus and the strain level. Shotgun sequencing was performed on a subset of eight E. coli urine UTI and isolate pairs. The majority of UTI microbial metagenomic sequences mapped to isolate genomes, confirming the results obtained using phylogenetic markers. We conclude that for the majority of acute uncomplicated E. coli-mediated UTIs, single cultured isolates are diagnostic of the infection. PMID:24570371

  20. 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

  1. [Hemoglobin, from microorganisms to man: a single structural motif, multiple functions].

    PubMed

    Wajcman, Henri; Kiger, Laurent

    2002-12-01

    Haemoglobins from unicellular organisms, plants or animals, share a common structure, which results from the folding, around the heme group, of a polypeptide chain made from 6-8 helices. Nowadays, deciphering the genome of several species allows one to draw the evolutionary tree of this protein going back to 1800 millions of years, at a time when oxygen began to accumulate in the atmosphere. This permits to follow the evolution of the ancestral gene and of its product. It is likely that, only in complex multicellular species, transport and storage of oxygen became the main physiological function of this molecule. In addition, in unicellular organisms and small invertebrates, it is likely that the main function of this protein was to protect the organism from the toxic effect of O2, CO and NO*. The very high oxygen affinity of these molecules, leading them to act rather as a scavenger as an oxygen carrier, supports this hypothesis. Haemoglobins from microorganisms, which may probably be the closest vestiges to the ancestral molecules, are divided into three families. The first one is made from flavohaemoglobins, a group of chimerical proteins carrying a globin domain and an oxido-reduction FAD-dependant domain. The second corresponds to truncated haemoglobins, which are hexacoordinated with very high oxygen-affinity molecules, 20-40 residues shorter than classical haemoglobins. The third group is made from bacterial haemoglobins such as that of Vitreoscilla. Some specific structural arrangements in the region surrounding the heme are cause of their high oxygen affinity. In plants, two types of haemoglobins are present (non-symbiotic and symbiotic), that arose from duplication of an ancestral vegetal gene. Non-symbiotic haemoglobins, which are probably the oldest, are scarcely distributed within tissues having high energetic consumption. Conversely, symbiotic haemoglobins (also named leghaemoglobins) are present at a high concentration (mM) mostly in the rhizomes of

  2. 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. PMID:21046953

  3. 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.

  4. Automated single cell isolation from suspension with computer vision

    PubMed Central

    Ungai-Salánki, Rita; Gerecsei, Tamás; Fürjes, Péter; Orgovan, Norbert; Sándor, Noémi; Holczer, Eszter; Horvath, Robert; Szabó, Bálint

    2016-01-01

    Current robots can manipulate only surface-attached cells seriously limiting the fields of their application for single cell handling. We developed a computer vision-based robot applying a motorized microscope and micropipette to recognize and gently isolate intact individual cells for subsequent analysis, e.g., DNA/RNA sequencing in 1–2 nanoliters from a thin (~100 μm) layer of cell suspension. It can retrieve rare cells, needs minimal sample preparation, and can be applied for virtually any tissue cell type. Combination of 1 μm positioning precision, adaptive cell targeting and below 1 nl liquid handling precision resulted in an unprecedented accuracy and efficiency in robotic single cell isolation. Single cells were injected either into the wells of a miniature plate with a sorting speed of 3 cells/min or into standard PCR tubes with 2 cells/min. We could isolate labeled cells also from dense cultures containing ~1,000 times more unlabeled cells by the successive application of the sorting process. We compared the efficiency of our method to that of single cell entrapment in microwells and subsequent sorting with the automated micropipette: the recovery rate of single cells was greatly improved. PMID:26856740

  5. Automated single cell isolation from suspension with computer vision.

    PubMed

    Ungai-Salánki, Rita; Gerecsei, Tamás; Fürjes, Péter; Orgovan, Norbert; Sándor, Noémi; Holczer, Eszter; Horvath, Robert; Szabó, Bálint

    2016-01-01

    Current robots can manipulate only surface-attached cells seriously limiting the fields of their application for single cell handling. We developed a computer vision-based robot applying a motorized microscope and micropipette to recognize and gently isolate intact individual cells for subsequent analysis, e.g., DNA/RNA sequencing in 1-2 nanoliters from a thin (~100 μm) layer of cell suspension. It can retrieve rare cells, needs minimal sample preparation, and can be applied for virtually any tissue cell type. Combination of 1 μm positioning precision, adaptive cell targeting and below 1 nl liquid handling precision resulted in an unprecedented accuracy and efficiency in robotic single cell isolation. Single cells were injected either into the wells of a miniature plate with a sorting speed of 3 cells/min or into standard PCR tubes with 2 cells/min. We could isolate labeled cells also from dense cultures containing ~1,000 times more unlabeled cells by the successive application of the sorting process. We compared the efficiency of our method to that of single cell entrapment in microwells and subsequent sorting with the automated micropipette: the recovery rate of single cells was greatly improved. PMID:26856740

  6. 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.

  7. Isolation and Characterization of Single Cells from Zebrafish Embryos.

    PubMed

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

    2016-01-01

    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

  8. 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. PMID:25791233

  9. 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.

  10. 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

  11. 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. PMID:25461068

  12. Magnetic microfluidic system for isolation of single cells

    NASA Astrophysics Data System (ADS)

    Mitterboeck, Richard; Kokkinis, Georgios; Berris, Theocharis; Keplinger, Franz; Giouroudi, Ioanna

    2015-06-01

    This paper presents the design and realization of a compact, portable and cost effective microfluidic system for isolation and detection of rare circulating tumor cells (CTCs) in suspension. The innovative aspect of the proposed isolation method is that it utilizes superparamagnetic particles (SMPs) to label CTCs and then isolate those using microtraps with integrated current carrying microconductors. The magnetically labeled and trapped CTCs can then be detected by integrated magnetic microsensors e.g. giant magnetoresistive (GMR) or giant magnetoimpedance (GMI) sensors. The channel and trap dimensions are optimized to protect the cells from shear stress and achieve high trapping efficiency. These intact single CTCs can then be used for additional analysis, testing and patient specific drug screening. Being able to analyze the CTCs metastasis-driving capabilities on the single cell level is considered of great importance for developing patient specific therapies. Experiments showed that it is possible to capture single labeled cells in multiple microtraps and hold them there without permanent electric current and magnetic field.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. 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.

  15. Isolating single stranded DNA using a microfluidic dialysis device

    PubMed Central

    Sheng, Yixiao

    2013-01-01

    Isolating a particular strand of DNA from a double stranded DNA duplex is an important step in aptamer generation as well as many other biotechnology applications. Here we describe a microfluidic, flow-through, dialysis device for isolating single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA). The device consists of two channels fabricated in polydimethylsiloxane (PDMS) separated by a track etched polycarbonate membrane (800 nm pore size). To isolate ssDNA, dual-biotin labelled dsDNA was immobilized onto streptavidin-coated polystyrene beads. Alkaline treatment was used to denature dsDNA, releasing the non-biotinylated ssDNA. In the flow-through dialysis device the liberated ssDNA was able to cross the membrane and was collected in an outlet channel. The complementary sequence bound to the bead was unable to cross the membrane and was directed to a waste channel. The effect of NaOH concentration and flow rate on purity and yield were compared. >95% ssDNA purity was achieved at 25mM NaOH. However, lower flow rates were necessary to achieve ssDNA yields approaching the 50% theoretical maximum of the concurrent-flow device. Under optimized conditions the microfluidic isolation achieved even higher purity ssDNA than analogous manual procedures. PMID:24213273

  16. Isolation of amylolytic, xylanolytic, and cellulolytic microorganisms extracted from the gut of the termite Reticulitermes santonensis by means of a micro-aerobic atmosphere.

    PubMed

    Tarayre, Cédric; Brognaux, Alison; Bauwens, Julien; Brasseur, Catherine; Mattéotti, Christel; Millet, Catherine; Destain, Jacqueline; Vandenbol, Micheline; Portetelle, Daniel; De Pauw, Edwin; Eric, Haubruge; Francis, Frédéric; Thonart, Philippe

    2014-05-01

    The aim of this work was to isolate enzyme-producing microorganisms from the tract of the termite Reticulitermes santonensis. The microorganisms were extracted from the guts and anaerobic (CO₂ or CO₂/H₂) and micro-aerobic atmospheres were used to stimulate growth. Three different strategies were tried out. First, the sample was spread on Petri dishes containing solid media with carboxymethylcellulose, microcrystalline cellulose or cellobiose. This technique allowed us to isolate two bacteria: Streptomyces sp. strain ABGxAviA1 and Pseudomonas sp. strain ABGxCellA. The second strategy consisted in inoculating a specific liquid medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. The samples were then spread on Petri dishes with the same specific medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. This led to the isolation of the mold Aspergillus sp. strain ABGxAviA2. Finally, the third strategy consisted in heating the first culture and spreading samples on agar plates containing rich medium. This led to the isolation of the bacterium Bacillus subtilis strain ABGx. All those steps were achieved in controlled atmospheres. The four enzyme-producing strains which were isolated were obtained by using a micro-aerobic atmosphere. Later, enzymatic assays were performed on the four strains. Streptomyces sp. strain ABGxAviA1 was found to produce only amylase, while Pseudomonas sp. strain ABGxCellA was found to produce β-glucosidase as well. Aspergillus sp. strain ABGxAviA2 showed β-glucosidase, amylase, cellulase, and xylanase activities. Finally, B. subtilis strain ABGx produced xylanase and amylase. PMID:24353041

  17. Phylogenetic relationships among subsurface microorganisms

    SciTech Connect

    Nierzwicki-Bauer, S.A.

    1991-01-01

    This report summarizes the progress made from 6/90--3/91 toward completion of our project, Phylogenetic Relationships among subsurface microorganisms. 16S rRNA was sequenced, and based on the sequence the SMCC isolates were assigned to preliminary groups. Microorganisms were obtained at various depths at the Savannah River Site, including the Surface (0 m), Congaree (91 m), and Middendorf (244 m, 259 m, 265 m). Sequence data from four isolates from the Congaree formation indicate these microorganisms can be divided into Pseudomonas spp. or Acinetobacter spp. Three 16S rRNA probes were synthesized based on sequence data. The synthesized probes were tested through in situ hybridization. Optimal conditions for in situ hybridization were determined. Because stability of RNA-DNA hybrids is dependent on hybridization stringency, related organisms can be differentiated using a single probe under different strigencies. The results of these hybridizations agree with results obtained by Balkwill and Reeves using restriction fragment length polymorphism analysis. The RNA content of a cell reflects its metabolic state. Cells which are starved for four days are not detectable with the homologous 16S rRNA probe. However, within 15 minutes of refeeding, detectable rRNA appeared. This suggests that organisms which are undetectable in environmental samples due to starvation may be detectable after addition of nutrients. Stepwise addition of specific nutrients could indicate which nutrients are rate limiting for growth. Preliminary experiments with soil samples from the Hanford Site indicate indigenous microorganisms can be detected by oligionucleotide probes. Further, using multiple probes based on universal sequences increases the number of organisms detected. Double label experiments, using a rhodamine-labelled oligionucleotide probe with free coumarin succinimidyl ester will allow simultaneous detection of total bacteria and specific 16S rRNA containing bacteria. 4 tabs. (MHB)

  18. 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

    2013-01-01

    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

  19. 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

  20. Detection and Isolation of Ultrasmall Microorganisms from a 120,000-Year-Old Greenland Glacier Ice Core

    PubMed Central

    Miteva, Vanya I.; Brenchley, Jean E.

    2005-01-01

    The abundant microbial population in a 3,043-m-deep Greenland glacier ice core was dominated by ultrasmall cells (<0.1 μm3) that may represent intrinsically small organisms or starved, minute forms of normal-sized microbes. In order to examine their diversity and obtain isolates, we enriched for ultrasmall psychrophiles by filtering melted ice through filters with different pore sizes, inoculating anaerobic low-nutrient liquid media, and performing successive rounds of filtrations and recultivations at 5°C. Melted ice filtrates, cultures, and isolates were analyzed by scanning electron microscopy, flow cytometry, cultivation, and molecular methods. The results confirmed that numerous cells passed through 0.4-μm, 0.2-μm, and even 0.1-μm filters. Interestingly, filtration increased cell culturability from the melted ice, yielding many isolates related to high-G+C gram-positive bacteria. Comparisons between parallel filtered and nonfiltered cultures showed that (i) the proportion of 0.2-μm-filterable cells was higher in the filtered cultures after short incubations but this difference diminished after several months, (ii) more isolates were obtained from filtered (1,290 isolates) than from nonfiltered (447 isolates) cultures, and (iii) the filtration and liquid medium cultivation increased isolate diversity (Proteobacteria; Cytophaga-Flavobacteria-Bacteroides; high-G+C gram-positive; and spore-forming, low-G+C gram-positive bacteria). Many isolates maintained their small cell sizes after recultivation and were phylogenetically novel or related to other ultramicrobacteria. Our filtration-cultivation procedure, combined with long incubations, enriched for novel ultrasmall-cell isolates, which is useful for studies of their metabolic properties and mechanisms for long-term survival under extreme conditions. PMID:16332755

  1. 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.

  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. Fermented corn flour poisoning in rural areas of China. III. Isolation and identification of main toxin produced by causal microorganisms.

    PubMed

    Hu, W J; Chen, X M; Meng, H D; Meng, Z H

    1989-03-01

    Flavotoxin A was isolated from Pseudomonas cocovenenans subsp. farinofermentans culture in semisolid potato-dextrose-agar medium, which was isolated from fermented corn meal that had caused food poisoning outbreaks in China. The isolation, purification, and chemical structure of this toxin were studied. The NMR spectra, the uv spectra, and molar extinction coefficients, and the mass spectra of Flavotoxin A are in good agreement with those reported for bongkrekic acid. Therefore, Flavotoxin A and bongkrekic acid are the same organic chemical compound; the molecular formula is C28H38O7. The oral LD50 of the purified Flavotoxin A in mice was 3.16 mg/kg (1.53-6.15 mg/kg). The existence of bongkrekic acid in toxic fermented corn samples collected during food poisoning outbreaks was also confirmed. It is concluded that bongkrekic acid has played an important role in the outbreaks of fermented corn poisoning. PMID:2590494

  4. Isolation, identification and characterisation of the dominant microorganisms of kule naoto: the Maasai traditional fermented milk in Kenya.

    PubMed

    Mathara, Julius Maina; Schillinger, Ulrich; Kutima, Phillip Museve; Mbugua, Samuel K; Holzapfel, Wilhelm H

    2004-08-01

    From 22 samples of kule naoto, the traditional fermented milk products of the Maasai in Kenya, 300 lactic acid bacterial strains were isolated and phenotypically characterised by their ability to ferment different carbohydrates and by additional biochemical tests. Lactic acid bacteria (LAB), especially the genus Lactobacillus, followed by Enterococcus, Lactococcus and Leuconostoc, dominated the microflora of these samples. The major Lactobacillus species was Lactobacillus plantarum (60%), with a lower frequency of isolation for Lactobacillus fermentum, Lactobacillus paracasei and Lactobacillus acidophilus. Most strains produced enzymes such as beta-galactosidase and peptidases, which are of relevance to cultured dairy product processing, and exhibited similar patterns of enzymatic activity between species. Enterobacteriaceae could not be detected in 15 out of 22 samples (detection level 10(2)/ml). Conversely, yeasts (detection level 10(1)/ml) were detected in those samples in which Enterobacteriaceae were not found. The pH values of all these samples were < 4.5. PMID:15246238

  5. 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.

  6. Autonomous Pattern Formation of Micro-organic Cell Density with Optical Interlink between Two Isolated Culture Dishes.

    PubMed

    Ozasa, Kazunari; Lee, Jeesoo; Song, Simon; Hara, Masahiko; Maeda, Mizuo

    2015-01-01

    Artificial linking of two isolated culture dishes is a fascinating means of investigating interactions among multiple groups of microbes or fungi. We examined artificial interaction between two isolated dishes containing Euglena cells, which are photophobic to strong blue light. The spatial distribution of swimming Euglena cells in two micro-aquariums in the dishes was evaluated as a set of new measures: the trace momentums (TMs). The blue light patterns next irradiated onto each dish were deduced from the set of TMs using digital or analogue feedback algorithms. In the digital feedback experiment, one of two different pattern-formation rules was imposed on each feedback system. The resultant cell distribution patterns satisfied the two rules with an and operation, showing that cooperative interaction was realized in the interlink feedback. In the analogue experiment, two dishes A and B were interlinked by a feedback algorithm that illuminated dish A (B) with blue light of intensity proportional to the cell distribution in dish B (A). In this case, a distribution pattern and its reverse were autonomously formed in the two dishes. The autonomous formation of a pair of reversal patterns reflects a type of habitat separation realized by competitive interaction through the interlink feedback. According to this study, interlink feedback between two or more separate culture dishes enables artificial interactions between isolated microbial groups, and autonomous cellular distribution patterns will be achieved by correlating various microbial species, despite environmental and spatial scale incompatibilities. The optical interlink feedback is also useful for enhancing the performance of Euglena-based soft biocomputing. PMID:25622016

  7. 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)

  8. 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). PMID:26749799

  9. 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.

  10. Degradation of olestra, a non caloric fat replacer, by microorganisms isolated from activated sludge and other environments.

    PubMed

    Lee, D M; Ventullo, R M

    1996-06-01

    Olestra is a non-caloric fat substitute consisting of fatty acids esterified to sucrose. Previous work has shown that olestra is not metabolized in the gut and is excreted unmodified in human feces. To better understand the fate of olestra in engineered and natural environments, aerobic bacteria and fungi that degrade olestra were enriched from sewage sludges, soils and municipal solid waste compost not previously exposed to olestra. Various mixed and pure cultures were obtained from these sources which were able to utilize olestra as a sole carbon and energy source. The fastest growing enrichment was obtained from activated sludge and later yielded an olestra-degrading pure culture of Pseudomonas aeruginosa. This mixed culture extensively degraded both 14C-fatty acid labeled olestra and 14C-sucrose labeled olestra during 8 days of incubation. Longer-term incubation with pure cultures of P.aeruginosa demonstrated that > 98% of 14C-sucrose labeled olestra and > 72% of 14C-fatty acid labeled olestra was mineralized to CO2 after 69 days. These results indicate that olestra degraders are present in environments not previously exposed to olestra and that olestra can serve as a sole carbon and energy source. Furthermore, a common bacterial species was isolated from activated sludge and shown to have the ability to degrade olestra. PMID:8782396

  11. Ventilator associated pneumonia in a medical intensive care unit: Microbial aetiology, susceptibility patterns of isolated microorganisms and outcome

    PubMed Central

    Goel, Varun; Hogade, Sumati A; Karadesai, SG

    2012-01-01

    Background: Ventilator-associated pneumonia (VAP) is a common complication of ventilatory support for patients with acute respiratory failure and is associated with increased morbidity and mortality. Aim of the Study: The present study was undertaken to do quantitative cultures of aerobic bacteria, perform the antibiotic susceptibility testing from the endotracheal aspirates and clinical outcome of the clinically suspected patients of VAP. Methods: A prospective study was performed over a period of one year in a tertiary care hospital, enrolling patients on mechanical ventilation (MV) for ≥48 hr. Endotracheal aspirates (ETA) were collected from patients with suspected VAP, and direct gram's stain criteria was used to accept the sample. Quantitative cultures of ETA were performed with the threshold for microbiological diagnosis of VAP was taken as ≥105 colony forming units (cfu)/ml. Results: Out of 53 cases, 2 (3.77%) were polymicrobial. Multidrug resistant bacteria, mainly Acinetobacter baumannii 49.09% (27/55) and Pseudomonas aeruginosa 30.91% (17/55) were the most common pathogens isolated. Metallo-beta lactamases (MBLs) was produced by 47.06% (8/17) of Pseudomonas aeruginosa and 62.96% (17/27) of Acinetobacter baumannii. Conclusion: The bacteriological approach for the management of VAP helps the clinicians in choosing the appropriate antibiotics. This study showed that quantitative cultures of endotracheal aspirate at a cutoff point of 105 cfu/ml is one of the alternative to bronchoscopy in the diagnosis of clinically suspected ventilator associated pneumonia. PMID:23325941

  12. Precipitation of phosphate minerals by microorganisms isolated from a fixed-biofilm reactor used for the treatment of domestic wastewater.

    PubMed

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

    2014-04-01

    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

  13. 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

  14. Evaluation of Epidemiological Cut-Off Values Indicates that Biocide Resistant Subpopulations Are Uncommon in Natural Isolates of Clinically-Relevant Microorganisms

    PubMed Central

    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. PMID:24466194

  15. 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

  16. 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.

  17. 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. PMID:20881166

  18. Genotyping of Toxoplasma gondii Isolates with 15 Microsatellite Markers in a Single Multiplex PCR Assay ▿

    PubMed Central

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

    2010-01-01

    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. PMID:20881166

  19. Relaxation paths for single modes of vibrations in isolated molecules

    NASA Astrophysics Data System (ADS)

    Papoular, R.

    2006-06-01

    A numerical simulation of vibrational excitation of molecules was devised and used to excite computational models of common molecules into a prescribed, pure, normal vibration mode in the ground electronic state, with varying, controllable energy content. The redistribution of this energy (either non-chaotic or irreversible IVR) within the isolated, free molecule is then followed in time with a view to determining the coupling strength between modes. This work was triggered by the need to predict the general characters of the infrared spectra to be expected from molecules in interstellar space, after being excited by photon absorption or reaction with a radical. It is found that IVR from a pure normal mode is very 'restricted' indeed at energy contents of one mode quantum or so. However, as this is increased, or when the excitation is localized, our approach allows us to isolate, describe and quantify a number of interesting phenomena, known to chemists and in nonlinear mechanics, but difficult to demonstrate experimentally: frequency dragging, mode locking or quenching or, still, instability near a potential surface crossing, the first step to generalized chaos as the energy content per mode is increased.

  20. 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

  1. 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).

  2. A Microfluidic Platform for High-throughput Single-cell Isolation and Culture.

    PubMed

    Lin, Ching-Hui; Chang, Hao-Chen; Hsu, Chia-Hsien

    2016-01-01

    Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency. PMID:27341146

  3. 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

  4. Assessment of the geographic origins of pinewood nematode isolates via single nucleotide polymorphism in effector genes.

    PubMed

    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

  5. Exploring the Potential of Stable Isotope (Resonance) Raman Microspectroscopy and Surface-Enhanced Raman Scattering for the Analysis of Microorganisms at Single Cell Level.

    PubMed

    Kubryk, Patrick; Kölschbach, Janina S; Marozava, Sviatlana; Lueders, Tillmann; Meckenstock, Rainer U; Niessner, Reinhard; Ivleva, Natalia P

    2015-07-01

    Raman microspectroscopy is a prime tool to characterize the molecular and isotopic composition of microbial cells. However, low sensitivity and long acquisition times limit a broad applicability of the method in environmental analysis. In this study, we explore the potential, the applicability, and the limitations of stable isotope Raman microspectroscopy (SIRM), resonance SIRM, and SIRM in combination with surface-enhanced Raman scattering (SERS) for the characterization of single bacterial cells. The latter two techniques have the potential to significantly increase sensitivity and decrease measurement times in SIRM, but to date, there are no (SERS-SIRM) or only a limited number (resonance SIRM) of studies in environmental microbiology. The analyzed microorganisms were grown with substrates fully labeled with the stable isotopes (13)C or (2)H and compounds with natural abundance of atomic isotopes ((12)C 98.89% or (1)H 99.9844%, designated as (12)C or (1)H, respectively). Raman bands of bacterial cell compounds in stable isotope-labeled microorganisms exhibited a characteristic red-shift in the spectra. In particular, the sharp phenylalanine band was found to be an applicable marker band for SIRM analysis of the Deltaproteobacterium strain N47 growing anaerobically on (13)C-naphthalene. The study of G. metallireducens grown with (13)C- and (2)H-acetate showed that the information on the chromophore cytochrome c obtained by resonance SIRM at 532 nm excitation wavelength can be successfully complemented by whole-organism fingerprints of bacteria cells achieved by regular SIRM after photobleaching. Furthermore, we present here for the first time the reproducible SERS analysis of microbial cells labeled with stable isotopes. Escherichia coli strain DSM 1116 cultivated with (12)C- or (13)C-glucose was used as a model organism. Silver nanoparticles synthesized in situ were applied as SERS media. We observed a reproducible red-shift of an adenine-related marker band

  6. 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. PMID:25771302

  7. A convenient, optimized pipeline for isolation, fluorescence microscopy and molecular analysis of live single cells

    PubMed Central

    2014-01-01

    Background Heterogeneity within cell populations is relevant to the onset and progression of disease, as well as development and maintenance of homeostasis. Analysis and understanding of the roles of heterogeneity in biological systems require methods and technologies that are capable of single cell resolution. Single cell gene expression analysis by RT-qPCR is an established technique for identifying transcriptomic heterogeneity in cellular populations, but it generally requires specialized equipment or tedious manipulations for cell isolation. Results We describe the optimization of a simple, inexpensive and rapid pipeline which includes isolation and culture of live single cells as well as fluorescence microscopy and gene expression analysis of the same single cells by RT-qPCR. We characterize the efficiency of single cell isolation and demonstrate our method by identifying single GFP-expressing cells from a mixed population of GFP-positive and negative cells by correlating fluorescence microscopy and RT-qPCR. Conclusions Single cell gene expression analysis by RT-qPCR is a convenient means for investigating cellular heterogeneity, but is most useful when correlating observations with additional measurements. We demonstrate a convenient and simple pipeline for multiplexing single cell RT-qPCR with fluorescence microscopy which is adaptable to other molecular analyses. PMID:24834016

  8. 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. PMID:20045215

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

    PubMed

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

    2015-12-01

    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. PMID:26674422

  10. 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

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

    PubMed

    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

  12. 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

  13. Growth and Characterization of Isolated Single Wall Carbon Nanotubes using Liquid Precursors

    NASA Astrophysics Data System (ADS)

    Choi, Young Chul; Parker, Allen; Kesker, Gayatri; Luo, J.; Rao, A. M.

    2004-03-01

    Isolated single wall carbon nanotubes (SWNTs) were prepared on quartz and oxidized silicon substrates using chemical vapor deposition (CVD) in which a liquid precursor, such as xylene, was used as the carbon source. The density of isolated SWNTs was controlled by adjusting the concentration of iron (III) nitrate nonahydrate/2-propanol solution which provided the Fe seed catalyst particles. Micro-Raman measurements using the 785 nm excitation showed tangential bands around 1590 cm-1. The radial breathing mode (RBM) peaks ranged from 150 - 240 cm-1 and the estimated tube diameters are in good agreement with those obtained from atomic force microscope (AFM) images. Our synthesis technique facilitates controlled doping of isolated SWNTs with nitrogen and is achieved by mixing acetonitrile with xylene. Isolated nitrogen doped SWNTs are useful in making stable TUBEFETs.

  14. 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.

  15. 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.

  16. 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.

  17. Plasmids in group JK coryneform bacteria isolated in a single hospital.

    PubMed Central

    Kerry-Williams, S. M.; Noble, W. C.

    1986-01-01

    Investigation of 39 JK-type coryneform isolates from patients at a single hospital revealed that 23 possessed plasmids, which formed six groups on restriction endonuclease analysis. Four of the groups were associated with production of similar bacteriocin-like substances, and shared a minimum of 6.4 kilobase pairs of DNA. These plasmids, found in isolates from different patients, provide strong direct evidence that person-to-person transmission of JK bacteria had occurred within the hospital. Images Fig. 1 PMID:3023480

  18. Distribution of tropomyosin isoforms in different types of single fibers isolated from bovine skeletal muscles.

    PubMed

    Oe, M; Ojima, K; Nakajima, I; Chikuni, K; Shibata, M; Muroya, S

    2016-08-01

    To clarify the relationship between myosin heavy chain (MyHC) isoforms and tropomyosin (TPM) isoforms in single fibers, 64 single fibers were isolated from each of bovine three muscles (masseter, semispinalis and semitendinosus). mRNA expressions of MyHC and TPM isoforms were analyzed by real-time PCR. All single fibers from the masseter expressed MyHC-slow. The fibers from the semispinalis expressed both MyHC-slow and 2a. The fibers from the semitendinosus expressed MyHC-slow, 2a and 2x. TPM-1 and TPM-2 were co-expressed in 2a and 2x type fibers, and TPM-2 and TPM-3 were co-expressed in slow type fibers. The expression pattern of TPM isoforms in each fiber type was similar between fibers isolated from different muscles. These results suggest that TPM-1 and TPM-3 isoforms correspond to the function of 2a or 2x type fibers and slow type fibers, respectively, with TPM-2 in common. Furthermore, the patterns of MyHC and TPM isoform combinations did not vary among single fibers isolated from the individual muscles examined. PMID:27105153

  19. Coupled dynamic analysis of a single gimbal control moment gyro cluster integrated with an isolation system

    NASA Astrophysics Data System (ADS)

    Luo, Qing; Li, Dongxu; Jiang, Jianping

    2014-01-01

    Control moment gyros (CMGs) are widely used as actuators for attitude control in spacecraft. However, micro-vibrations produced by CMGs will degrade the pointing performance of high-sensitivity instruments on-board the spacecraft. This paper addresses dynamic modelling and performs an analysis on the micro-vibration isolation for a single gimbal CMG (SGCMG) cluster. First, an analytical model was developed to describe both the coupled SGCMG cluster and the multi-axis isolation system that can express the dynamic outputs. This analytical model accurately reflects the mass and inertia properties, the gyroscopic effects and flexible modes of the coupled system, which can be generalized for isolation applications of SGCMG clusters. Second, the analytical model was validated using MSC.NASTRAN software based on the finite element technique. The dynamic characteristics of the coupled system are affected by the mass distribution and the gyroscopic effects of the SGCMGs. The gyroscopic effects produced by the rotary flywheel will stiffen or soften several of the structural modes of the coupled system. In addition, the gyroscopic effect of each SGCMG can interact with or counteract that of others, which induce vibration modes coupled together. Finally, the performance of the passive isolation was analysed. It was demonstrated that the gyroscopic effects should be considered in isolation studies on SGCMG clusters; otherwise, the isolation performance will be underestimated if they are ignored.

  20. 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. PMID:26130851

  1. 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.

  2. Controllable optical phase shift over one radian from a single isolated atom.

    PubMed

    Jechow, A; Norton, B G; Händel, S; Blūms, V; Streed, E W; Kielpinski, D

    2013-03-15

    Fundamental optics such as lenses and prisms work by applying phase shifts of several radians to incoming light, and rapid control of such phase shifts is crucial to telecommunications. However, large, controllable optical phase shifts have remained elusive for isolated quantum systems. We have used a single trapped atomic ion to induce and measure a large optical phase shift of 1.3±0.1 radians in light scattered by the atom. Spatial interferometry between the scattered light and unscattered illumination light enables us to isolate the phase shift in the scattered component. The phase shift achieves the maximum value allowed by atomic theory over the accessible range of laser frequencies, pointing out new opportunities in microscopy and nanophotonics. Single-atom phase shifts of this magnitude open up new quantum information protocols, in particular long-range quantum phase-shift-keying cryptography. PMID:25166534

  3. A single centrifugation method for isolating fat droplets from cells and tissues

    PubMed Central

    Harris, Lydia-Ann L. S.; Shew, Trevor M.; Skinner, James R.; Wolins, Nathan E.

    2012-01-01

    Fat droplets (FDs) have important roles in cellular energy regulation. Isolating FDs from either cells or tissue continues to be important for studying these organelles. Here, we describe a procedure wherein whole homogenates of cultured cells or tissue are fractionated with a single centrifugation step in a standard microcentrifuge. This procedure reproducibly yields three fractions highly enriched in either FDs, soluble cellular components, or sedimentable organelles/membranes. PMID:22327205

  4. Isolated single coronary artery presenting as acute coronary syndrome: case report and review.

    PubMed

    Mahapatro, Anil K; Patro, A Sarat K; Sujatha, Vipperala; Sinha, Sudhir C

    2014-06-01

    Congenital single coronary artery is commonly associated with complex congenital heart diseases and manifests in infancy or childhood. But isolated single coronary artery is a rare congenital anomaly which can present as acute coronary syndrome in adults. The aim of the work is to discuss on isolated single coronary artery in two adults presenting as acute coronary syndrome. The first case underwent coronary angiography (CAG) through right radial route, but switched over to femoral for confirmation of diagnosis and due to radial spasm. An aortic root angiogram was done to rule out presence of any other coronary ostia. It revealed a single coronary artery originating from right sinus of valsalva. After giving rise to posterior descending artery branch at crux, it continued in the atrioventricular groove to the anterior basal surface of the heart and traversed as anterior descending artery. There was no atheromatous occlusive stenosis. This is R-I type single coronary artery as per Lipton classification. In the second case, angiography was completed through right radial route. It revealed a single coronary artery arising from right aortic sinus. Anterior descending and circumflex branch were originating from proximal common trunk of the single coronary artery and supplying the left side of the heart. The right coronary artery has diffuse atheromatous disease without significant stenosis in any major branch. This is R-III C type as per Lipton classification. A coronary anomaly of both origin and course is very rare. It may be encountered in adults evaluated for atherosclerotic coronary heart disease. Knowledge and understanding of anatomical types of this congenital anomaly will reduce time, anxiety, complications during CAG and cardiac surgery. PMID:25075168

  5. 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

  6. Isolation of functional single domain antibody by whole cell immunization: implications for cancer treatment.

    PubMed

    Baral, Toya Nath; Murad, Yanal; Nguyen, Thanh-Dung; Iqbal, Umar; Zhang, Jianbing

    2011-08-31

    Carcinoembryonic antigen related cell adhesion molecule (CEACAM) 6 is over-expressed in different types of cancer cells. In addition, it has also been implicated in some infectious diseases. Targeting this molecule by an antibody might have applications in diverse tumor models. Single domain antibody (sdAb) is becoming very useful format in antibody engineering as potential tools for treating acute and chronic disease conditions such as cancer for both diagnostic as well as therapeutic application. Generally, sdAbs with good affinity are isolated from an immune library. Discovery of a new target antigen would require a new immunization with purified antigen which is not always easy. In this study, we have isolated, by phage display, an sdAb against CEACAM6 with an affinity of 5 nM from a llama immunized with cancer cells. The antibody has good biophysical properties, and it binds to the cells expressing the target antigen. Furthermore, it reduces cancer cells proliferation in vitro and shows an excellent tumor targeting in vivo. This sdAb could be useful in diagnosis as well as therapy of CEACAM6 expressing tumors. Finally, we envisage it would be feasible to isolate good sdAbs against other interesting tumor associated antigens from this library. Therefore, this immunization method could be a general strategy for isolating sdAbs against any surface antigen without immunizing the animal with the antigen of interest each time. PMID:21741385

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

    PubMed

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

    2015-05-15

    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

  8. 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. PMID:22470372

  9. 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

  10. 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. PMID:24290622

  11. Low Temperature Scanning Tunneling Spectroscopy of isolated Mn12-Ph Single Molecule Magnets

    NASA Astrophysics Data System (ADS)

    Reaves, K.; Han, P.; Iwaya, K.; Hitosugi, T.; Packwood, D.; Katzgraber, H. G.; Zhao, H.; Dunbar, K. R.; Kim, K.; Teizer, W.

    2015-03-01

    We study Mn12O12(C6H5COO)16(H2O)4 (Mn12-Ph) single-molecule magnets on a Cu(111) surface using scanning tunneling microscopy and scanning tunneling spectroscopy at cryogenic temperatures (T < 6K). We report the observation of Mn12-Ph in isolation and in thin films, deposited through in situ vacuum spray deposition onto clean Cu(111). The tunneling current of isolated Mn12-Ph, normalized with respect to the Cu background, shows a strong bias voltage dependence within the molecular interior. The qualitative features of these I vs.V curves differ by spatial location in several intriguing ways (e.g. fixed junction impedance with increasing bias voltages). We explore these normalized I vs. V curves and present a phenomenological explanation for the observed behaviors, corresponding to the physical and electronic structure within the molecule. Funding from WPI-AIMR.

  12. 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

  13. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes

    PubMed Central

    Parks, Donovan H.; Imelfort, Michael; Skennerton, Connor T.; Hugenholtz, Philip; Tyson, Gene W.

    2015-01-01

    Large-scale recovery of genomes from isolates, single cells, and metagenomic data has been made possible by advances in computational methods and substantial reductions in sequencing costs. Although this increasing breadth of draft genomes is providing key information regarding the evolutionary and functional diversity of microbial life, it has become impractical to finish all available reference genomes. Making robust biological inferences from draft genomes requires accurate estimates of their completeness and contamination. Current methods for assessing genome quality are ad hoc and generally make use of a limited number of “marker” genes conserved across all bacterial or archaeal genomes. Here we introduce CheckM, an automated method for assessing the quality of a genome using a broader set of marker genes specific to the position of a genome within a reference genome tree and information about the collocation of these genes. We demonstrate the effectiveness of CheckM using synthetic data and a wide range of isolate-, single-cell-, and metagenome-derived genomes. CheckM is shown to provide accurate estimates of genome completeness and contamination and to outperform existing approaches. Using CheckM, we identify a diverse range of errors currently impacting publicly available isolate genomes and demonstrate that genomes obtained from single cells and metagenomic data vary substantially in quality. In order to facilitate the use of draft genomes, we propose an objective measure of genome quality that can be used to select genomes suitable for specific gene- and genome-centric analyses of microbial communities. PMID:25977477

  14. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes.

    PubMed

    Parks, Donovan H; Imelfort, Michael; Skennerton, Connor T; Hugenholtz, Philip; Tyson, Gene W

    2015-07-01

    Large-scale recovery of genomes from isolates, single cells, and metagenomic data has been made possible by advances in computational methods and substantial reductions in sequencing costs. Although this increasing breadth of draft genomes is providing key information regarding the evolutionary and functional diversity of microbial life, it has become impractical to finish all available reference genomes. Making robust biological inferences from draft genomes requires accurate estimates of their completeness and contamination. Current methods for assessing genome quality are ad hoc and generally make use of a limited number of "marker" genes conserved across all bacterial or archaeal genomes. Here we introduce CheckM, an automated method for assessing the quality of a genome using a broader set of marker genes specific to the position of a genome within a reference genome tree and information about the collocation of these genes. We demonstrate the effectiveness of CheckM using synthetic data and a wide range of isolate-, single-cell-, and metagenome-derived genomes. CheckM is shown to provide accurate estimates of genome completeness and contamination and to outperform existing approaches. Using CheckM, we identify a diverse range of errors currently impacting publicly available isolate genomes and demonstrate that genomes obtained from single cells and metagenomic data vary substantially in quality. In order to facilitate the use of draft genomes, we propose an objective measure of genome quality that can be used to select genomes suitable for specific gene- and genome-centric analyses of microbial communities. PMID:25977477

  15. Isolation and Epitope Mapping of Staphylococcal Enterotoxin B Single-Domain Antibodies

    PubMed Central

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

    2014-01-01

    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. PMID:24949641

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

    PubMed Central

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

    2014-01-01

    Background 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. Aim To develop a single-step protocol to isolate vesicles from human body fluids. Methods 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. Results 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. Conclusions 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. PMID:25279113

  17. 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

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

    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.

  19. A droplet microfluidic approach to single-stream nucleic acid isolation and mutation detection

    PubMed Central

    Shin, Dong Jin; Zhang, Yi

    2014-01-01

    In this work, a droplet microfluidic platform for genetic mutation detection from crude biosample is described. Single-stream integration of nucleic acid isolation and amplification is realized on a simple fluidic cartridge. Subsequent DNA melting curve is employed with signal normalizing algorithm to differentiate heterozygous K-ras codon 12 c.25G>A mutant from the wildtype. This technique showcases an alternative to modular bench-top approaches for genetic mutation screening, which is of interest to decentralized diagnostic platforms. PMID:25386112

  20. 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.

  1. Isolation of single stranded DNA related to the transcriptional activity of animal cells.

    PubMed Central

    Tapiero, H; Leibowitch, S A; Shaool, D; Monier, M N; Harel, J

    1976-01-01

    Single stranded DNA (s.s.DNA) comprising 1-2% of the total nuclear DNA was isolated by an improved method of hydroxyapatite chromatography from native nuclear DNA3 of embryonic chick cells, labeled for several cell generations with 3H-thymidine. Small quantities of 3H-DNA were annealed with a large excess of unlabeled DNA or polysomal RNA from chick embryos. Hybridization kinetics (monitored by the use of SI nuclease digestion, hydroxyapatite chromatography and thermalfusion), indicated that s.s.DNA belongs to the non repetitious fraction of the cell genome. One third represents DNA sequences engaged in the transcription of messenger RNA's. PMID:944919

  2. Isolation of dimorphic chloroplasts from the single-cell C4 species Bienertia sinuspersici

    PubMed Central

    2012-01-01

    Three terrestrial plants are known to perform C4 photosynthesis without the dual-cell system by partitioning two distinct types of chloroplasts in separate cytoplasmic compartments. We report herein a protocol for isolating the dimorphic chloroplasts from Bienertia sinuspersici. Hypo-osmotically lysed protoplasts under our defined conditions released intact compartments containing the central chloroplasts and intact vacuoles with adhering peripheral chloroplasts. Following Percoll step gradient purification both chloroplast preparations demonstrated high homogeneities as evaluated from the relative abundance of respective protein markers. This protocol will open novel research directions toward understanding the mechanism of single-cell C4 photosynthesis. PMID:22394490

  3. Identification of a single-nucleocapsid baculovirus isolated from Clanis bilineata tsingtauica (Lepidoptera: Sphingidae).

    PubMed

    Wang, Liqun; Yi, Jianping; Zhu, Shanying; Li, Bing; Chen, Yan; Shen, Weide; Wang, Wenbing

    2008-01-01

    A nucleopolyhedrovirus isolated from infected larvae of Clanis bilineata tsingtauica was characterized. Electron microscopical studies on the ultrastructure of C. bilineata nucleopolyhedrovirus (ClbiSNPV) occlusion bodies (OBs) showed several virions (up to 16) with a single nucleocapsid packaged within a single viral envelope. The diameter of the OBs was 0.77-1.7 mum with a mean of 1.13 +/- 0.19 mum. The complete sequence of the ClbiSNPV polyhedrin (polh) gene contained 741 nucleotides, predicting a protein of 246 amino acids. Phylogenetic analyses using the complete sequence of the polh genes indicated that ClbiSNPV clusters with Group II NPVs. This is the first record of a baculovirus from C. bilineata. PMID:18584114

  4. 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. PMID:25933619

  5. 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

    2016-09-01

    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. PMID:27479540

  6. 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

  7. 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…

  8. Prevalence of drug resistance and culture-positive rate among microorganisms isolated from patients with ocular infections over a 4-year period

    PubMed Central

    Shimizu, Yusuke; Toshida, Hiroshi; Honda, Rio; Matsui, Asaki; Ohta, Toshihiko; Asada, Yousuke; Murakami, Akira

    2013-01-01

    Purpose To investigate the microbial isolates from patients with ocular infections and the trend in the emergence of levofloxacin-resistant strains over the past four years from 2006 to 2009 retrospectively. Patients and methods The subjects were 242 patients with ocular infections or traumas treated in our hospital including outpatients, inpatients, and emergency room patients. Most of them needed urgent care presenting with eye complaints, traumas, or decreased vision. Clinical samples were obtained from discharges, corneal, conjunctival tissues or vitreous fluid or aqueous humor, and cultured. Items for assessment included the patient’s age, the diagnosis, the prevalence of isolated bacteria, and the results of susceptibility tests for levofloxacin (LVFX) cefamezin (CEZ), gentamicin (GM) and vancomycin. This information was obtained from the patients’ medical records. Results There were 156 male patients and 86 female patients who were aged from 2 months old to 94 years old and mean age was 56.8 ± 24.2 years. Of the 242 patients, 78 (32.2%) had positive cultures. The culture-positive rate was significantly higher in male patients than female in total (P = 0.002) and in patients with corneal perforation (P = 0.005). Corneal perforation was the highest culture-positive rate (60.0%), followed by orbital cellulitis (56.5%), blepharitis (50.0%), dacryoadenitis (45.5%), conjunctivitis (38.2%), infectious corneal ulcer (28.5%) and endophthalmitis (24.7%). LVFX-resistant strains accounted for 40 out of a total of 122 strains (32.8%), and the minimum inhibitory concentration (MIC) was significantly higher in LVFX and GM compared with the other antibiotics. There were no vancomycin-resistant strains. Conclusion Attention should be paid to a possible future increase of strains with resistance to LVFX, as commonly prescribed ocular antibiotics bring emergence of resistant bacteria. Although no vancomycin-resistant strains were isolated this drug should be reserved as

  9. 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

  10. 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

  11. Isolation of Clostridium thermocellum auxotrophs

    SciTech Connect

    Mendez, B.S.; Gomez, R.F.

    1982-02-01

    The conversion of biomass of fuels and chemical feedstocks by microbial fermentation offers the potential of solving two of today's important problems: waste accumulation and exhaustion of fossil fuels. Microorganisms with the capabilities of converting biomass components such as cellulos and hemicellulose to chemicals and fuels in a single step are of particular interest. One such microorganism is Clostridium thermocellum, a thermophilic anaerobe which degrades cellulose to ethanol and organic acids. For efficient industrial use, the cellulolytic capacity of this strain must be improved by genetic means. Spontaneous and UV irradiation-induced auxotrophic mutants of Clostridium thermocellum, an anaerobic cellulolytic thermophile, were isolated after penicillin enrichment in a chemically defined medium.

  12. Isolation and Characterization of a Novel Single-Stranded RNA Virus Infectious to a Marine Fungoid Protist, Schizochytrium sp. (Thraustochytriaceae, Labyrinthulea)

    PubMed Central

    Takao, Yoshitake; Nagasaki, Keizo; Mise, Kazuyuki; Okuno, Tetsuro; Honda, Daiske

    2005-01-01

    Thraustochytrids are cosmopolitan osmoheterotrophic microorganisms that play important roles as decomposers, producers of polyunsaturated fatty acids, and pathogens of mollusks, especially in coastal ecosystems. SssRNAV, a novel single-stranded RNA (ssRNA) virus infecting the marine fungoid protist Schizochytrium sp. (Labyrinthulea, Thraustochytriaceae) was isolated from the coastal water of Kobe Harbor, Japan, in July 2000, and its basic characteristics were examined. The virus particle is icosahedral, lacks a tail, and is ca. 25 nm in diameter. SssRNAV formed crystalline arrays and random assemblies within the cytoplasm of host cells, and it was also concentrated along the intracellular membrane structures. By means of one-step growth experiments, the lytic cycle and the burst size were estimated to be <8 h and 5.8 × 103 to 6.4 × 104 infectious units per host cell, respectively. SssRNAV had a single molecule of ssRNA that was approximately 10.2 kb long, three major proteins (37, 34, and 32 kDa), and two minor proteins (80 and 18 kDa). Although SssRNAV was considered to have some similarities with invertebrate viruses belonging to the family Dicistroviridae based on its partial nucleotide sequence, further genomic analysis is required to determine the detailed classification and nomenclature of SssRNAV. Our results indicate that viral infection is one of the significant factors controlling the dynamics of thraustochytrids and provide new insights into understanding the ecology of these organisms. PMID:16085844

  13. Single-Cell Isolation of Circulating Tumor Cells from Whole Blood by Lateral Magnetophoretic Microseparation and Microfluidic Dispensing.

    PubMed

    Kim, Jinho; Cho, Hyungseok; Han, Song-I; Han, Ki-Ho

    2016-05-01

    This paper introduces a single-cell isolation technology for circulating tumor cells (CTCs) using a microfluidic device (the "SIM-Chip"). The SIM-Chip comprises a lateral magnetophoretic microseparator and a microdispenser as a two-step cascade platform. First, CTCs were enriched from whole blood by the lateral magnetophoretic microseparator based on immunomagnetic nanobeads. Next, the enriched CTCs were electrically identified by single-cell impedance cytometer and isolated as single cells using the microshooter. Using 200 μL of whole blood spiked with 50 MCF7 breast cancer cells, the analysis demonstrated that the single-cell isolation efficiency of the SIM-Chip was 82.4%, and the purity of the isolated MCF7 cells with respect to WBCs was 92.45%. The data also showed that the WBC depletion rate of the SIM-Chip was 2.5 × 10(5) (5.4-log). The recovery rates were around 99.78% for spiked MCF7 cells ranging in number from 10 to 90. The isolated single MCF7 cells were intact and could be used for subsequent downstream genetic assays, such as RT-PCR. Single-cell culture evaluation of the proliferation of MCF7 cells isolated by the SIM-Chip showed that 84.1% of cells at least doubled in 5 days. Consequently, the SIM-Chip could be used for single-cell isolation of rare target cells from whole blood with high purity and recovery without cell damage. PMID:27093098

  14. Antimicrobial metabolites from marine microorganisms.

    PubMed

    Habbu, Prasanna; Warad, Vijayanand; Shastri, Rajesh; Madagundi, Smita; Kulkarni, Venkatrao H

    2016-02-01

    Marine ecological niches have recently been described as "particularly promising" sources for search of new antimicrobials to combat antibiotic-resistant strains of pathogenic microorganisms. Marine organisms are excellent sources for many industrial products, but they are partly explored. Over 30 000 compounds have been isolated from marine sources. Bacteria, fungi, and cyanobacteria obtained from various marine sources secret several industrially useful bioactive compounds, possessing antibacterial, antifungal, and antimycobacterial activities. Sustainable cultivation methods for promising marine organisms and biotechnological processes for selected compounds can be developed, along with the establishment of biosensors for monitoring the target compounds. The semisynthetic modifications of marine-based bioactive compounds produce their new derivatives, structural analogs and mimetics that could serve as novel lead compounds against resistant pathogens. The present review focuses on promising antimicrobial compounds isolated from marine microbes from 1991-2013. PMID:26968676

  15. 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

  16. 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. PMID:26216949

  17. 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

  18. 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

  19. Phenotypic and Genotypic Analysis of Clostridium difficile Isolates: a Single-Center Study

    PubMed Central

    Zhou, Yanjiao; Burnham, Carey-Ann D.; Hink, Tiffany; Chen, Lei; Shaikh, Nurmohammad; Wollam, Aye; Sodergren, Erica; Weinstock, George M.; Tarr, Phillip I.

    2014-01-01

    Clostridium difficile infections (CDI) are a growing concern in North America, because of their increasing incidence and severity. Using integrated approaches, we correlated pathogen genotypes and host clinical characteristics for 46 C. difficile infections in a tertiary care medical center during a 6-month interval from January to June 2010. Multilocus sequence typing (MLST) demonstrated 21 known and 2 novel sequence types (STs), suggesting that the institution's C. difficile strains are genetically diverse. ST-1 (which corresponds to pulsed-field gel electrophoresis strain type NAP1/ribotype 027) was the most prevalent (32.6%); 43.5% of the isolates were binary toxin gene positive, of which 75% were ST-1. All strains were ciprofloxacin resistant and metronidazole susceptible, and 8.3% and 13.0% of the isolates were resistant to clindamycin and tetracycline, respectively. The corresponding resistance loci, including potential novel mutations, were identified from the whole-genome sequencing (WGS) of the resistant strains. Core genome single nucleotide polymorphisms (SNPs) determining the phylogenetic relatedness of the 46 strains recapitulated MLST types and provided greater interstrain differentiation. The disease severity was greatest in patients infected with ST-1 and/or binary gene-positive strains, but genome-wide SNP analysis failed to provide additional associations with CDI severity within the same STs. We conclude that MLST and core genome SNP typing result in the same phylogenetic grouping of the 46 C. difficile strains collected in a single hospital. WGS also has the capacity to differentiate those strains within STs and allows the comparison of strains at the individual gene level and at the whole-genome level. PMID:25275005

  20. 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.

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

    PubMed

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

    2015-01-01

    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 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. PMID:26167808

  2. 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.

  3. 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.

  4. Faraday isolator based on a TSAG single crystal with compensation of thermally induced depolarization inside magnetic field

    NASA Astrophysics Data System (ADS)

    Snetkov, Ilya; Palashov, Oleg

    2015-04-01

    A Faraday isolator based on a terbium scandium aluminum garnet (TSAG) single crystal with compensation of thermally induced depolarization inside magnetic field was demonstrated. An isolation ratio of 32 dB at 350 W cw laser radiation power was achieved. Thermally induced depolarization and thermal lens were studied and compared with similar thermal effects arising in the widely used terbium gallium garnet crystal (TGG) for the first time.

  5. Quest for Microorganisms Existing at High Atmosphere and Space

    NASA Astrophysics Data System (ADS)

    Yokobori, S.; Yang, Y.; Sugino, T.; Kawaguchi, Y.; Itahashi, S.; Fujisaki, K.; Fushimi, H.; Hasegawa, S.; Hashimoto, H.; Hayashi, N.; Imai, E.; Itoh, T.; Kawai, H.; Kobayashi, K.; Marumo, K.; Mita, H.; Nakagawa, K.; Narumi, I.; Okudaira, K.; Shimada, H.; Tabata, M.; Takahashi, Y.; Yabuta, H.; Yamashita, M.; Yano, H.; Yoshida, S.; Yoshimura, Y.; Yamagishi, A.

    2010-04-01

    We have tested effects of various factors in space environment on survivability of Deinococcus spp. including our newly isolated species at high altitude. In "Tanpopo" mission, we are planning to expose microorganisms such as deinococcal species.

  6. 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

  7. 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.

  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. 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

  10. 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

  11. 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.

  12. 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.

  13. Association Between Isolated Single Umbilical Artery and Perinatal Outcomes: A Meta-Analysis

    PubMed Central

    Xu, Yajuan; Ren, Lidan; Zhai, Shanshan; Luo, Xiaohua; Hong, Teng; Liu, Rui; Ran, Limin; Zhang, Yingying

    2016-01-01

    Background To evaluate the association between the isolated single umbilical artery (iSUA) and perinatal outcomes, including pregnancy outcomes and perinatal complications. Material/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. PMID:27130891

  14. 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

    2016-01-01

    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. PMID:27130891

  15. 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

  16. A simple and efficient method for isolation of a single Eimeria oocyst from poultry litter using a micromanipulator

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The chicken, which is the host for seven species of Eimeria, typically is infected simultaneously by multiple Eimeria species and the oocysts of coccidia are excreted in the feces. A prerequisite for investigation of individual Eimeria species is to isolate a single oocyst from fecal samples. A nov...

  17. Isolating scattering resonances of an air-filled spherical shell using iterative, single-channel time reversal.

    PubMed

    Waters, Zachary J; Dzikowicz, Benjamin R; Simpson, Harry J

    2012-01-01

    Iterative, single-channel time reversal is employed to isolate backscattering resonances of an air-filled spherical shell in a frequency range of 0.5-20 kHz. Numerical simulations of free-field target scattering suggest improved isolation of the dominant target response frequency in the presence of varying levels of stochastic noise, compared to processing returns from a single transmission and also coherent averaging. To test the efficacy of the technique in a realistic littoral environment, monostatic scattering experiments are conducted in the Gulf of Mexico near Panama City, Florida. The time reversal technique is applied to returns from a hollow spherical shell target sitting proud on a sandy bottom in 14 m deep water. Distinct resonances in the scattering response of the target are isolated, depending upon the bandwidth of the sonar system utilized. PMID:22280594

  18. Isolation and characterization of a novel single-stranded RNA Virus infectious to a marine fungoid protist, Schizochytrium sp. (Thraustochytriaceae, Labyrinthulea).

    PubMed

    Takao, Yoshitake; Nagasaki, Keizo; Mise, Kazuyuki; Okuno, Tetsuro; Honda, Daiske

    2005-08-01

    Thraustochytrids are cosmopolitan osmoheterotrophic microorganisms that play important roles as decomposers, producers of polyunsaturated fatty acids, and pathogens of mollusks, especially in coastal ecosystems. SssRNAV, a novel single-stranded RNA (ssRNA) virus infecting the marine fungoid protist Schizochytrium sp. (Labyrinthulea, Thraustochytriaceae) was isolated from the coastal water of Kobe Harbor, Japan, in July 2000, and its basic characteristics were examined. The virus particle is icosahedral, lacks a tail, and is ca. 25 nm in diameter. SssRNAV formed crystalline arrays and random assemblies within the cytoplasm of host cells, and it was also concentrated along the intracellular membrane structures. By means of one-step growth experiments, the lytic cycle and the burst size were estimated to be <8 h and 5.8 x 10(3) to 6.4 x 10(4) infectious units per host cell, respectively. SssRNAV had a single molecule of ssRNA that was approximately 10.2 kb long, three major proteins (37, 34, and 32 kDa), and two minor proteins (80 and 18 kDa). Although SssRNAV was considered to have some similarities with invertebrate viruses belonging to the family Dicistroviridae based on its partial nucleotide sequence, further genomic analysis is required to determine the detailed classification and nomenclature of SssRNAV. Our results indicate that viral infection is one of the significant factors controlling the dynamics of thraustochytrids and provide new insights into understanding the ecology of these organisms. PMID:16085844

  19. Radiation resistence of microorganisms from radiation sterilization processing environments

    NASA Astrophysics Data System (ADS)

    Sabovljev, Svetlana A.; Žunić, Zora S.

    The radiation resistance of microorganisms was examined on the samples of dust collected from the radiation sterilization processing environments including assembly, storage, and sterilization plant areas. The isolation of radiation resistant strains was performed by irradiation with screening doses ranging from 10 to 35 kGy and test pieces containing 10 6 to 10 8 CFU in dried serum-broth, representing 100 to 5000 colonies of primary cultures of microorganisms from 7 different sites. In an examination of 16900 colonies of aerobic microorganisms from 3 hygienically controlled production sites and 4 uncontrolled ones, 30 strains of bacteria were isolated. Of those 15 were classified as genus Bacillus, 9 as Micrococcus and 6 as Sarcina. All of the 15 strains of Gram positive sporeforming aerobic rods exhibited an exponential decrease in the surviving fraction as a function of dose, indicating that the inactivation of spores of aerobic rods is a consequence of a single energy deposition into the target. All strains were found to be moderately resistant to radiation with D-6 values (dose required to reduce survival to 6 log cycles) between 18 and 26 kGy. All of the isolated Gram positive cocci showed inactivation curves having a shoulder, indicating that different processes are involved in the inactivation of these cells, e.g. accumulation of sublethal lesions, or final repair capacity of potential lethal lesions. Moderate radiation resistance was observed in 13 strains with D-6 values between 16 to 30 kGy. Two slow-growing, red pigmented strains tentatively classified as genus Micrococcus isolated from uncontrolled sites (human dwellings) were exceptionally resistant with D-6 more than 45 kGy. For hygienically controlled sites, Gram positive spereforming rods composed two thirds of the resistant microflora, while Gram positive cocci comprised one third. For hygienically uncontrolled sites this ratio was reversed. An assumption is made that one isolated strain has grown

  20. 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)

  1. Common antimicrobial resistance phenotypes and genotypes of fecal Escherichia coli isolates from a single family over a 6-month period.

    PubMed

    Al-Dweik, Manar R; Shehabi, Asem A

    2009-06-01

    This study investigated the antimicrobial resistance phenotypes and genotypes among fecal Escherichia coli isolates from the members of a single Jordanian family over a 6-month period. A total of 55 (51%) E. coli isolates were resistant to >2, and 21 (19%) to >3 of the 14 tested antimicrobial agents, respectively. The highest resistance rates were observed to tetracycline (42%), followed by coamoxyclav and cotrimoxazole (32%), gentamicin (31%), and nalidixic acid (27%). Sixteen out of 21 (76%) multiresistant E. coli isolates (resistant to >3 drugs) transferred most of their resistance markers in vitro to E. coli K12. Five out of the six family members were colonized with E. coli carrying one or two of the two common plasmid sizes (54.3 and 13.2 kb). Ten of these isolates (48%) were positive for class 1 integron genes and harbored four tet (A) and five tet (B) genes, respectively, but all were negative for tet (39). The genetic diversity of E. coli isolates using randomly amplified polymorphic DNA-PCR demonstrated 13 major clusters of genotype groups, and most of the isolates (63%) belonged to one genotype group. This study indicates that all six family members are colonized with fecal E. coli isolates exhibiting a common number of antimicrobial resistance phenotypes and at least one prevalent genotype. PMID:19432518

  2. Patient isolation in multichannel bioelectric recordings by digital transmission through a single optical fiber.

    PubMed

    MettingVanRijn, A C; Kuiper, A P; Linnenbank, A C; Grimbergen, C A

    1993-03-01

    A design for patient isolation in 64-channel ECG recordings is presented. Small dimensions of the isolated section and the use of an optical fiber as the only connection between the isolated section and the grounded section of the measurement system ensured a minimal capacitance between the patient and the environment. The consistent low-power design of the isolated section resulted in a power consumption of 210 mW, which enabled a 10 h continuous operating time of the battery powered isolated section. The system handles 64 signals with a dynamic range of 75 dB. Analog to digital conversion is performed in the isolated section with a sample rate of 1 kHz per channel. The receiver interfaces to a commercially available DMA board for a standard personal computer. PMID:8335335

  3. 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. PMID:24865686

  4. Isolation of a Highly Thermal Stable Lama Single Domain Antibody Specific for Staphylococcus aureus Enterotoxin B

    PubMed Central

    2011-01-01

    Background Camelids and sharks possess a unique subclass of antibodies comprised of only heavy chains. The antigen binding fragments of these unique antibodies can be cloned and expressed as single domain antibodies (sdAbs). The ability of these small antigen-binding molecules to refold after heating to achieve their original structure, as well as their diminutive size, makes them attractive candidates for diagnostic assays. Results Here we describe the isolation of an sdAb against Staphyloccocus aureus enterotoxin B (SEB). The clone, A3, was found to have high affinity (Kd = 75 pM) and good specificity for SEB, showing no cross reactivity to related molecules such as Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin D (SED), and Shiga toxin. Most remarkably, this anti-SEB sdAb had an extremely high Tm of 85°C and an ability to refold after heating to 95°C. The sharp Tm determined by circular dichroism, was found to contrast with the gradual decrease observed in intrinsic fluorescence. We demonstrated the utility of this sdAb as a capture and detector molecule in Luminex based assays providing limits of detection (LODs) of at least 64 pg/mL. Conclusion The anti-SEB sdAb A3 was found to have a high affinity and an extraordinarily high Tm and could still refold to recover activity after heat denaturation. This combination of heat resilience and strong, specific binding make this sdAb a good candidate for use in antibody-based toxin detection technologies. PMID:21933444

  5. 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.

  6. 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.

  7. 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).

  8. Complete genome assemblies for two single-chromosome Vibrio cholerae isolates, strains 1154-74 (serogroup O49) and 10432-62 (serogroup O27)

    DOE PAGESBeta

    Johnson, Shannon Lyn; Khiani, A.; Bishop-Lilly, K. A.; Chapman, C.; Patel, M.; Verratti, K.; Teshima, Hazuki; Munk, A. C.; Bruce, David Carlton; Han, C. S.; et al

    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.

  9. Genome sequences of ten Salmonella enterica serovars isolated from a single dairy farm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. ...

  10. Isolated hydatid cyst in a single moiety of an incomplete duplex kidney.

    PubMed

    Priyadarshi, Vinod; Mishra, Shwetank; Bera, Malay Kumar; Pal, Dilip Kumar

    2015-01-01

    Isolated hydatid cyst of kidney is very rare. Hydatid cyst of a duplex renal system is even more rare. We report a 13-year old girl with duplex system of right kidney with isolated hydatid cyst in upper moiety. Right nephrectomy was done to cure the condition. PMID:25628991

  11. 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.

  12. Single-pinhole diffraction of few-cycle isolated attosecond pulses with a two-color field

    NASA Astrophysics Data System (ADS)

    Shaoyi, Wang; Dan, Han; Kegong, Dong; Yuchi, Wu; Fang, Tan; Bin, Zhu; Quanping, Fan; Leifeng, Cao; Yuqiu, Gu

    2016-03-01

    The spatio-temporal characterization of an isolated attosecond pulse is investigated theoretically in a two-color field. Our results show that a few-cycle isolated attosecond pulse with the center wavelength of 16 nm can be generated effectively by adding a weak controlling field. Using the split and delay units, the isolated attosecond pulse can be split to the two same ones, and then single-pinhole diffractive patterns of the two pulses with different delays can be achieved. The diffractive patterns depend severely on the periods of the attosecond pulses, which can be helpful to obtain temporal information of the coherent sources. Project supported by the National Science Instruments Major Project of China (Grant No. 2012YQ130125), the National Natural Science Foundation of China (Grant Nos. 11405159, 11375161, and 11174259), and the Science and Technology on Plasma Physics Laboratory at CAEP (Grant No. 9140C680302130C68242).

  13. 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.

  14. The stimulus interval-tension relation in enzymatically isolated single myocytes of the frog heart.

    PubMed Central

    Cecchi, G; Colomo, F; Poggesi, C; Tesi, C

    1992-01-01

    1. Apparatus for recording the small tensions developed by electrically stimulated single intact myocytes of frog heart is described. A laser-light optoelectronic transducer was used. The compliance of the force probes was 10-20 nm/nN, with a frequency response of 600-900 Hz in Ringer solution. The myocyte shortening during an ordinary twitch contraction was no greater than 1% of the slack length. The overall sensitivity of the transducer system was 5-10 mV/nN, with a total noise of 0.5-1 nN peak to peak. The experiments were performed at 20-23 degrees C on either atrial or ventricular myocytes at 2.15-2.2 microns sarcomere length, in 1 mM-Ca2+ Ringer solution. 2. Isoprenaline (5 microM), increases in external Ca2+ concentration ([Ca2+]o), and shortening of stimulus interval potentiated the myocyte twitch tension. The dependence of twitch characteristics on these inotropic interventions for all the atrial and ventricular myocytes tested was comparable to that of multicellular preparations under similar experimental conditions. This implies that the enzymatic isolation procedure had not altered the physiological properties of the myocytes. 3. The stimulus interval-tension relation for premature twitches of atrial and ventricular myocytes showed (i) a very steep rising phase in the region of intervals just longer than 0.52 and 0.66 s (the duration of the mechanical refractoriness in atrial or ventricular cells), (ii) a peak, at intervals of 0.7-0.8 s, where the twitch tension was strongly potentiated compared to that of the controls, and (iii) as the stimulus interval was further increased, a progressive return to the control level. The stimulus interval-tension relation for steady-state conditions exhibited similar characteristics. 4. The degree of tension potentiation by isoprenaline was greater in the controls than in the earliest test twitches. The result was that the stimulus interval-tension relations for isoprenaline-treated myocytes showed a gentler rise and

  15. Biodegradation of bisphenol A with diverse microorganisms from river sediment.

    PubMed

    Peng, Yu-Huei; Chen, Ya-Jou; Chang, Ying-Jie; Shih, Yang-hsin

    2015-04-01

    The wide distribution of bisphenol A (BPA) in the environment is problematic because of its endocrine-disrupting characteristics and toxicity. Developing cost-effective remediation methods for wide implementation is crucial. Therefore, this study investigated the BPA biodegradation ability of various microorganisms from river sediment. An acclimated microcosm completely degraded 10 mg L(-1) BPA within 28 h and transformed the contaminant into several metabolic intermediates. During the degradation process, the microbial compositions fluctuated and the final, predominant microorganisms were Pseudomonas knackmussii and Methylomonas clara. From the original river sediment, we isolated four distinct strains, which deplete the BPA over 7-9 days. They were all genetically similar to P. knackmussii. The degradation ability of mixed strains was higher than that of single strain but was far less than that of the microbial consortium. The novel BPA degradation ability of P. knackmussii and its role in the decomposing microcosm were first demonstrated. Our results revealed that microbial diversity plays a crucial role in pollutant decomposition. PMID:25590822

  16. 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.

  17. 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

  18. High-throughput de novo screening of receptor agonists with an automated single-cell analysis and isolation system.

    PubMed

    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 × 10(6) 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

  19. Isolation and characterization of a single-stranded DNA virus infecting the marine diatom Chaetoceros sp. strain SS628-11 isolated from western Japan.

    PubMed

    Kimura, Kei; Tomaru, Yuji

    2013-01-01

    Diatoms are significant organisms for primary production in the earth's aquatic environment. Hence, their dynamics are an important focus area in current studies. Viruses are a great concern as potential factors of diatom mortality, along with other physical, chemical, and biological factors. We isolated and characterized a new diatom virus (Csp07DNAV) that lyses the marine planktonic diatom Chaetoceros sp. strain SS628-11. This paper examines the physiological, morphological, and genomic characteristics of Csp07DNAV. The virus was isolated from a surface water sample that was collected at Hiroshima Bay, Japan. It was icosahedral, had a diameter of 34 nm, and accumulated in the nuclei of host cells. Rod-shaped virus particles also coexisted in the host nuclei. The latent period and burst size were estimated to be <12 h and 29 infectious units per host cell, respectively. Csp07DNAV had a closed circular single-stranded DNA genome (5,552 nucleotides), which included a double-stranded region and 3 open reading frames. The monophyly of Csp07DNAV and other Bacilladnavirus group single-stranded DNA viruses was supported by phylogenetic analysis that was based on the amino acid sequence of each virus protein. On the basis of these results, we considered Csp07DNAV to be a new member of the genus Bacilladnavirus. PMID:24358139

  20. 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

  1. Microorganism lipid droplets and biofuel development

    PubMed Central

    Liu, Yingmei; Zhang, Congyan; Shen, Xipeng; Zhang, Xuelin; Cichello, Simon; Guan, Hongbin; Liu, Pingsheng

    2013-01-01

    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. [BMB Reports 2013; 46(12): 575-581] PMID:24355300

  2. 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.

  3. Microorganisms having enhanced resistance to acetate and methods of use

    SciTech Connect

    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.

  4. 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.

  5. 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.

  6. 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.

  7. A snapshot of the predominant single nucleotide polymorphism cluster groups of Mycobacterium tuberculosis clinical isolates in Delhi, India.

    PubMed

    Varma-Basil, Mandira; Narang, Anshika; Chakravorty, Soumitesh; Garima, Kushal; Gupta, Shraddha; Kumar Sharma, Naresh; Giri, Astha; Zozio, Thierry; Couvin, David; Hanif, Mahmud; Bhatnagar, Anuj; Menon, Balakrishnan; Niemann, Stefan; Rastogi, Nalin; Alland, David; Bose, Mridula

    2016-09-01

    Several attempts have been made to associate phylogenetic differences among Mycobacterium tuberculosis strains to variations in the clinical outcome of the disease and to drug resistance. We genotyped 139 clinical isolates of M. tuberculosis obtained from patients of pulmonary tuberculosis in North Delhi region. The isolates were analyzed using nine Single nucleotide polymorphism (SNP) markers, spoligotyping and MIRU-VNTRs; and the results were correlated with their drug susceptibility profile. Results of SNP cluster group (SCG) analysis (available for 138 isolates) showed that the most predominant cluster was SCG 3a, observed in 58.7% (81/138) of the isolates with 44.4% (36/81) of these being drug susceptible, while 16% (13/81) were multidrug resistant (MDR). Of the ancestral cluster SCG 1 observed in 19.5% (27/138) of the isolates, 14.8% (4/27) were MDR while 44.4% (12/27) were drug susceptible. SCG 2 formed 5.79% (8/138) of the isolates and 50% (4/8) of these were multidrug resistant (MDR). Spoligotyping subdivided the strains into 45 shared types (n = 125) and 14 orphan strains. The orphan strains were mostly associated with SCG 3a or SCG 1, reflecting the principal SCGs found in the Indian population. SCG 1 and SCG 2 genotypes were concordant with the East African Indian (EAI) and Beijing families respectively. Central Asian (CAS) clade and its sublineages were predominantly associated with SCG 3a. No consistent association was seen between the SCGs and Harlem, T or X clades. The 15 loci MIRU-VNTR typing revealed 123/136 isolates to be unclustered, while 13 isolates were present in 6 clusters of 2-3 isolates each. However, correlating the cluster analysis with patient details did not suggest any evidence of recent transmission. In conclusion, though our study revealed the preponderance of SCG 1 and 3a in the M. tuberculosis population circulating in the region, the diversity of strains highlights the changes occurring within lineages and reemphasizes the

  8. 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

  9. 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. PMID:23453960

  10. Proton-transfer mechanism for dispersed decay kinetics of single molecules isolated in potassium hydrogen phthalate.

    PubMed

    Bott, Eric D; Riley, Erin A; Kahr, Bart; Reid, Philip J

    2009-08-25

    The excited-state decay kinetics of single 2',7'-dichlorofluorescein (DCF) molecules oriented and overgrown within crystals of potassium acid phthalate (KAP) are reported. Time-correlated single-photon counting measurements (TCSPC) of 56 DCF molecules in KAP reveal that single-exponential decay is exhibited by roughly half of the molecules. The remainder demonstrates complex excited-state decay kinetics that are well fit by a stretched exponential function consistent with dispersed kinetics. Histograms of single-molecule luminescence energies revealed environmental fluctuations and distinct chemical species. The TCSPC results are compared to Monte Carlo simulations employing a first-passage model for excited-state decay. Agreement between experiment and theory, on both bulk and single-molecule levels, suggests that a subset of the DCF molecules in KAP experience fluctuations in the surrounding environment that modify the energy barrier to proton transfer leading to dispersed kinetics. PMID:19658424

  11. Isolation of Salmonella Enteritidis PT 30 from a single almond orchard over a 4-year period

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In 2001, Salmonella Enteritidis phage type (PT) 30 was isolated from drag swabs of 17 61-ha almond orchards on three farms linked to an outbreak of salmonellosis associated with consumption of raw almonds. The objective of this study was to evaluate the long-term persistence of Salmonella Enteritidi...

  12. 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

  13. 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.

  14. 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.

  15. Proteolysis in hyperthermophilic microorganisms

    DOE PAGESBeta

    Ward, Donald E.; Shockley, Keith R.; Chang, Lara S.; Levy, Ryan D.; Michel, Joshua K.; Conners, Shannon B.; Kelly, Robert M.

    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

  16. Gravitaxis in unicellular microorganisms.

    PubMed

    Hader, 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. PMID:11542630

  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. High Isolation Single-Pole Four-Throw RF MEMS Switch Based on Series-Shunt Configuration

    PubMed Central

    Khaira, Navjot

    2014-01-01

    This paper presents a novel design of single-pole four-throw (SP4T) RF-MEMS switch employing both capacitive and ohmic switches. It is designed on high-resistivity silicon substrate and has a compact area of 1.06 mm2. The series or ohmic switches have been designed to provide low insertion loss with good ohmic contact. The pull-in voltage for ohmic switches is calculated to be 7.19 V. Shunt or capacitive switches have been used in each port to improve the isolation for higher frequencies. The proposed SP4T switch provides excellent RF performances with isolation better than 70.64 dB and insertion loss less than 0.72 dB for X-band between the input port and each output port. PMID:24711730

  19. Isolation and detection of single molecules on paramagnetic beads using sequential fluid flows in microfabricated polymer array assemblies.

    PubMed

    Kan, Cheuk W; Rivnak, Andrew J; Campbell, Todd G; Piech, Tomasz; Rissin, David M; Mösl, Matthias; Peterça, Andrej; Niederberger, Hans-Peter; Minnehan, Kaitlin A; Patel, Purvish P; Ferrell, Evan P; Meyer, Raymond E; Chang, Lei; Wilson, David H; Fournier, David R; Duffy, David C

    2012-03-01

    We report a method for isolating individual paramagnetic beads in arrays of femtolitre-sized wells and detecting single enzyme-labeled proteins on these beads using sequential fluid flows in microfabricated polymer array assemblies. Arrays of femtolitre-sized wells were fabricated in cyclic olefin polymer (COP) using injection moulding based on DVD manufacturing. These arrays were bonded to a complementary fluidic structure that was also moulded in COP to create an enclosed device to allow delivery of liquids to the arrays. Enzyme-associated, paramagnetic beads suspended in aqueous solutions of enzyme substrate were delivered fluidically to the array such that one bead per well was loaded by gravity. A fluorocarbon oil was then flowed into the device to remove excess beads from the surface of the array, and to seal and isolate the femtolitre-sized wells containing beads and enzyme substrate. The device was then imaged using standard fluorescence imaging to determine which wells contained single enzyme molecules. The analytical performance of this device as the detector for digital ELISA compared favourably to the standard method, i.e., glass arrays mechanically sealed against a silicone gasket; prostate specific antigen (PSA) could be detected from 0.011 pg mL(-1) up to 100 pg mL(-1). The use of an enclosed fluidic device to isolate beads in single-molecule arrays offers a multitude of advantages for low-cost manufacturing, ease of automation, and instrument development to enable applications in biomarker validation and medical diagnosis. PMID:22179487

  20. Synergistic Degradation of Linuron by a Bacterial Consortium and Isolation of a Single Linuron-Degrading Variovorax Strain

    PubMed Central

    Dejonghe, Winnie; Berteloot, Ellen; Goris, Johan; Boon, Nico; Crul, Katrien; Maertens, Siska; Höfte, Monica; De Vos, Paul; Verstraete, Willy; Top, Eva M.

    2003-01-01

    The bacterial community composition of a linuron-degrading enrichment culture and the role of the individual strains in linuron degradation have been determined by a combination of methods, such as denaturing gradient gel electrophoresis of the total 16S rRNA gene pool, isolation and identification of strains, and biodegradation assays. Three strains, Variovorax sp. strain WDL1, Delftia acidovorans WDL34, and Pseudomonas sp. strain WDL5, were isolated directly from the linuron-degrading culture. In addition, subculture of this enrichment culture on potential intermediates in the degradation pathway of linuron (i.e., N,O-dimethylhydroxylamine and 3-chloroaniline) resulted in the isolation of, respectively, Hyphomicrobium sulfonivorans WDL6 and Comamonas testosteroni WDL7. Of these five strains, only Variovorax sp. strain WDL1 was able to use linuron as the sole source of C, N, and energy. WDL1 first converted linuron to 3,4-dichloroaniline (3,4-DCA), which transiently accumulated in the medium but was subsequently degraded. To the best of our knowledge, this is the first report of a strain that degrades linuron further than the aromatic intermediates. Interestingly, the rate of linuron degradation by strain WDL1 was lower than that for the consortium, but was clearly increased when WDL1 was coinoculated with each of the other four strains. D. acidovorans WDL34 and C. testosteroni WDL7 were found to be responsible for degradation of the intermediate 3,4-DCA, and H. sulfonivorans WDL6 was the only strain able to degrade N,O-dimethylhydroxylamine. The role of Pseudomonas sp. strain WDL5 needs to be further elucidated. The degradation of linuron can thus be performed by a single isolate, Variovorax sp. strain WDL1, but is stimulated by a synergistic interaction with the other strains isolated from the same linuron-degrading culture. PMID:12620840

  1. 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.

  2. Isolation and Assessment of Single Long-Term Reconstituting Hematopoietic Stem Cells from Adult Mouse Bone Marrow.

    PubMed

    Kent, David G; Dykstra, Brad J; Eaves, Connie J

    2016-01-01

    Hematopoietic stem cells with long-term repopulating activity can now be routinely obtained at purities of 40% to 50% from suspensions of adult mouse bone marrow. Here we describe robust protocols for both their isolation as CD45(+) EPCR(+) CD150(+) CD48(-) (ESLAM) cells using multiparameter cell sorting and for tracking their clonal growth and differentiation activity in irradiated mice transplanted with single ESLAM cells. The simplicity of these procedures makes them attractive for characterizing the molecular and biological properties of individual hematopoietic stem cells with unprecedented power and precision. © 2016 by John Wiley & Sons, Inc. PMID:27532815

  3. 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

  4. Complete Genome Sequence of Mycobacterium avium, Isolated from Commercial Domestic Pekin Ducks (Anas platyrhynchos domestica), Determined Using PacBio Single-Molecule Real-Time Technology.

    PubMed

    Song, Xiao-Heng; Chen, Hong-Xi; Zhou, Wang-Shu; Wang, Jiang-Bo; Liu, Ma-Feng; Wang, Ming-Shu; Cheng, An-Chun; Jia, Ren-Yong; Chen, Shun; Sun, Kun-Feng; Yang, Qiao; Wu, Ying; Chen, Xiao-Yue; Zhu, De-Kang

    2016-01-01

    Mycobacterium avium is an important pathogenic bacterium in birds and has never, to our knowledge, reported to be isolated from domestic ducks. We present here the complete genome sequence of a virulent strain of Mycobacterium avium, isolated from domestic Pekin ducks for the first time, which was determined by PacBio single-molecule real-time technology. PMID:27587804

  5. Complete Genome Sequence of Mycobacterium avium, Isolated from Commercial Domestic Pekin Ducks (Anas platyrhynchos domestica), Determined Using PacBio Single-Molecule Real-Time Technology

    PubMed Central

    Song, Xiao-Heng; Chen, Hong-Xi; Zhou, Wang-Shu; Wang, Jiang-Bo; Liu, Ma-Feng; Wang, Ming-Shu; Cheng, An-Chun; Jia, Ren-Yong; Chen, Shun; Sun, Kun-Feng; Yang, Qiao; Wu, Ying; Chen, Xiao-Yue

    2016-01-01

    Mycobacterium avium is an important pathogenic bacterium in birds and has never, to our knowledge, reported to be isolated from domestic ducks. We present here the complete genome sequence of a virulent strain of Mycobacterium avium, isolated from domestic Pekin ducks for the first time, which was determined by PacBio single-molecule real-time technology. PMID:27587804

  6. [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. PMID:25492652

  7. Generation of an intense single isolated attosecond pulse by use of two-colour waveform control

    NASA Astrophysics Data System (ADS)

    Zeng, Bin; Yu, Yongli; Chu, Wei; Yao, Jinping; Fu, Yuxi; Xiong, Hui; Xu, Han; Cheng, Ya; Xu, Zhizhan

    2009-07-01

    We theoretically demonstrate the generation of an intense single attosecond pulse by superposing a weak sub-harmonic pulse upon a sine-waveform few-cycle driving pulse. By use of a sine-waveform few-cycle pulse instead of its traditionally used cosine waveform counterpart, we show that efficient tunnel ionization for generating electrons which can revisit their parent ion with high kinetic energy can occur only once in the few-cycle laser field, leading to an increase of efficiency by nearly two orders of magnitude in single attosecond pulse generation as compared with the use of a cosine-waveform field.

  8. Understanding the role of nitrogen dissimilation in soil microorganisms

    NASA Astrophysics Data System (ADS)

    Roco, C. A.; Bakken, L. R.; Bergaust, L. L.; Frostegård; Shapleigh, J. P.; Yavitt, J. B.

    2011-12-01

    Uncertainty about the fate of nitrate in ecosystems has led to increased interest in soil nitrogen (N) transformations and microbial biogeochemistry of N. Microorganisms can utilize nitrate by either assimilatory or dissimilatory processes. The best studied dissimilatory processes are nitrate reduction to ammonium and denitrification, both of which are thought to occur under low O2 conditions. While there is an appreciation that denitrifying bacteria are diverse, the activity of each enzyme in the pathway is viewed more uniformly, in that all are presumed to have activity that is inversely correlated with O2 levels. However, the first step of denitrification, dissimilatory reduction of nitrate to nitrite, can occur at O2 concentrations that are high enough to repress downstream reduction of nitrite to gaseous products. To explore this in more detail, we tested for aerobic nitrate reduction (ANR) activity in a range of agricultural, wetland and forest soils located near Ithaca, New York. ANR was found in some environments, as evidenced by nitrite production in samples provided with both nitrate and a carbon source but not in controls. We next undertook a screen to isolate bacteria capable of ANR on an oxidized carbon source, succinate. Bacteria capable of ANR were surprisingly easy to isolate, as this phenotype was present in 10-15% of the isolates. 16S rDNA sequencing showed that the isolates included both gram negative and gram positive bacteria, although the majority were proteobacteria. The ANR isolates were tested for anoxic growth and less then 20% were able to grow under anoxic conditions as denitrifiers. To confirm the ANR phenotype, we measured the level of O2 present when nitrate reduction was first detected in two of the isolates using a robotic gas sampler. The O2 levels detected during ANR were higher than levels associated with the onset of nitrite reduction, since nitrite production began between 84% to 22% of atmospheric O2. Production of gaseous

  9. 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

  10. 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