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Sansupa, Chakriya, Sara Fareed Mohamed Wahdan, Terd Disayathanoowat e Witoon Purahong. "Identifying Hidden Viable Bacterial Taxa in Tropical Forest Soils Using Amplicon Sequencing of Enrichment Cultures". Biology 10, n.º 7 (22 de junho de 2021): 569. http://dx.doi.org/10.3390/biology10070569.
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This study aims to estimate the proportion and diversity of soil bacteria derived from eDNA-based and culture-based methods. Specifically, we used Illumina Miseq to sequence and characterize the bacterial communities from (i) DNA extracted directly from forest soil and (ii) DNA extracted from a mixture of bacterial colonies obtained by enrichment cultures on agar plates of the same forest soil samples. The amplicon sequencing of enrichment cultures allowed us to rapidly screen a culturable community in an environmental sample. In comparison with an eDNA community (based on a 97% sequence similarity threshold), the fact that enrichment cultures could capture both rare and abundant bacterial taxa in forest soil samples was demonstrated. Enrichment culture and eDNA communities shared 2% of OTUs detected in total community, whereas 88% of enrichment cultures community (15% of total community) could not be detected by eDNA. The enrichment culture-based methods observed 17% of the bacteria in total community. FAPROTAX functional prediction showed that the rare and unique taxa, which were detected with the enrichment cultures, have potential to perform important functions in soil systems. We suggest that enrichment culture-based amplicon sequencing could be a beneficial approach to evaluate a cultured bacterial community. Combining this approach together with the eDNA method could provide more comprehensive information of a bacterial community. We expected that more unique cultured taxa could be detected if further studies used both selective and non-selective culture media to enrich bacteria at the first step.
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Conrad, Cheyenne C., Kim Stanford, Tim A. McAllister, James Thomas e Tim Reuter. "Competition during enrichment of pathogenicEscherichia colimay result in culture bias". FACETS 1, n.º 1 (1 de março de 2017): 114–26. http://dx.doi.org/10.1139/facets-2016-0007.
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Deadly outbreaks and illnesses due to Shiga toxin-producing Escherichia coli (STEC) occur worldwide; however, the cultivation methods required for adequate monitoring and traceback investigations are inefficient at best. Detection of STEC relies heavily on enrichment; yet no standard media or protocols exist. Furthermore, whether enrichment may bias detection of multiple STEC serogroups from complex samples is unknown. Thus, 14 STEC strains of serogroups O157 and the top six non-O157s (O26, O45, O103, O111, O121, and O145) were enriched in pairs for 6–78 h in broth and evaluated by quantitative polymerase chain reaction (qPCR). Here we show that a conventional 6-h enrichment protocol did not result in intra-species culture bias for the isolates tested. However, subsequent enrichments often produced biased cultures, with differences in the qPCR gene copy number ≥2 log10apparent in 12%, 38%, and 52% of competitions after 30, 54, and 78 h of consecutive enrichments, respectively. Some strains were able to prevail and (or) out-compete the opponent strain in 100% of competitions. Our results suggest that culture bias should be considered and (or) evaluated further due to the potential implications during routine pathogen screening and outbreak investigations.
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Kamashwaran, S. R., e Don L. Crawford. "Mechanisms of cadmium resistance in anaerobic bacterial enrichments degrading pentachlorophenol". Canadian Journal of Microbiology 49, n.º 7 (1 de julho de 2003): 418–24. http://dx.doi.org/10.1139/w03-053.
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The mechanisms of heavy-metal resistance used by adapted sulfidogenic and methanogenic enrichments degrading pentachlorophenol in the presence of cadmium (Cd) were studied. The enrichment cultures adapted to and readily tolerated bioavailable Cd concentrations up to 50 ppm while degrading an equal concentration of pentachlorophenol. Both cultures removed >95% of the Cd from solution. Transmission electron micrographs revealed (i) the presence of electron-dense particles surrounding the cells in the sulfidogenic enrichments and (ii) the unusual clumping of cells and the presence of an exopolymer in the methanogenic enrichments. Energy dispersive X-ray analysis showed that the sulfidogenic enrichments removed Cd by extracellular precipitation of cadmium sulfide, while the methanogenic enrichment culture removed Cd by extracellular sequestration of Cd into the exopolymer.Key words: cadmium, pentachlorophenol, sulfidogenic, methanogenic, resistance.
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Duvall, Robert E., Marjut Eklund, Tony T. Tran e Anthony D. Hitchins. "Improved DNA Probe Detection of Listeria monocytogenes in Enrichment Culture After Physical-Chemical Fractionation". Journal of AOAC INTERNATIONAL 89, n.º 1 (1 de janeiro de 2006): 172–79. http://dx.doi.org/10.1093/jaoac/89.1.172.
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Abstract Bacterial detection in foods by nucleic acid probes is limited by microflora competition during selective enrichment. Probe target concentration by extraction and fractionation of enrichments may diminish this limitation. The 1-h AccuProbe chemiluminescent culture identification test for Listeria monocytogenes was used as a model. Its high detection threshold provides a stringent challenge for evaluating enrichmentwork-up protocols. Detection of L. monocytogenes, at 14 colony-forming units/g food,was not consistently possible in 48 h enrichment cultures usingAccuProbe. Concentration by cell sedimentationwas occasionally helpful but the volume of co-sedimented food limited concentration to about 10-fold. To improve concentration, enrichment sedimentswere sonicated or enzymatically lysed to release the probe's target, r-RNA. The RNAwas separated from non-RNA material by extraction with phenol and precipitation with ethanol. Enrichments (250 mL) were concentrated 2500-fold, and the limitation was food RNA volume. A strongly competitive Enterococcus faecium food isolate was used to demonstrate the effect of artificial competition on the kit's ability to detect L. monocytogenes in enrichments. High competitor concentrations repressed the level of the target below the detection threshold, but concentration of r-RNA enabled detection of L. monocytogenes. The effectiveness of this enrichment sample work-up was demonstrated with naturally contaminated hummus.
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Grosser, Robert J., Michael Friedrich, David M. Ward e William P. Inskeep. "Effect of Model Sorptive Phases on Phenanthrene Biodegradation: Different Enrichment Conditions Influence Bioavailability and Selection of Phenanthrene-Degrading Isolates". Applied and Environmental Microbiology 66, n.º 7 (1 de julho de 2000): 2695–702. http://dx.doi.org/10.1128/aem.66.7.2695-2702.2000.
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ABSTRACT The sorption of organic contaminants by natural organic matter (NOM) often limits substrate bioavailability and is an important factor affecting microbial degradation rates in soils and sediments. We hypothesized that reduced substrate bioavailability might influence which microbial assemblages are responsible for contaminant degradation under enrichment culture conditions. Our primary goal was to characterize enrichments in which different model organic solid phases were used to establish a range of phenanthrene bioavailabilities for soil microorganisms. Phenanthrene sorption coefficients (expressed as log KD values) ranged from 3.0 liters kg−1 for Amberlite carboxylic acid cation-exchange resin (AMB) to 3.5 liters kg−1 for Biobeads polyacrylic resin (SM7) and 4.2 liters kg−1 for Biobeads divinyl benzene resin (SM2). Enrichment cultures were established for control (no sorptive phase), sand, AMB, SM7, and SM2 treatments by using two contaminated soils (from Dover, Ohio, and Libby, Mont.) as the initial inocula. The effects of sorption by model phases on the degradation of phenanthrene were evaluated for numerous transfers in order to obtain stable microbial assemblages representative of sorptive and nonsorptive enrichment cultures and to eliminate the effects of the NOM present in the initial inoculum. Phenanthrene degradation rates were similar for each soil inoculum and ranged from 4 to 5 μmol day−1 for control and sand treatments to approximately 0.4 μmol day−1 in the presence of the SM7 sorptive phase. The rates of phenanthrene degradation in the highly sorptive SM2 enrichment culture were insignificant; consequently, stable microbial populations could not be obtained. Bacterial isolates obtained from serial dilutions of enrichment culture samples exhibited significant differences in rates of phenanthrene degradation performed in the presence of SM7, suggesting that enrichments performed in the presence of a sorptive phase selected for different microbial assemblages than control treatments containing solid phase phenanthrene.
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D’Aoust, Jean-Yves, Anne M. Sewell e Paula Greco. "Detection of Salmonella in Dry Foods Using Refrigerated Pre-Enrichment and Enrichment Broth Cultures: Summary of Collaborative Study". Journal of AOAC INTERNATIONAL 77, n.º 6 (1 de novembro de 1994): 1490–91. http://dx.doi.org/10.1093/jaoac/77.6.1490.
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Abstract A collaborative study was conducted to compare the productivity of refrigerated pre-enrichment and enrichment broth cultures with the U.S. Food and Drug Administration culture methods for detection of Salmonella. The refrigerated pre-enrichment and selective enrichment broth culture methods for detection of Salmonella in dry foods have been adopted first action by AOAC INTERNATIONAL.
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WILLIAMS, L. K., L. C. SAIT, T. A. COGAN, F. JØRGENSEN, R. GROGONO-THOMAS e T. J. HUMPHREY. "Enrichment culture can bias the isolation ofCampylobactersubtypes". Epidemiology and Infection 140, n.º 7 (19 de setembro de 2011): 1227–35. http://dx.doi.org/10.1017/s0950268811001877.
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SUMMARYEnrichment culture is often used to isolateCampylobacter. This study compared isolation ofCampylobacterspp. from 119 broiler chicken environments from two farms, using Preston and modified Exeter (mExeter) and modified Bolton (mBolton) enrichments. mExeter was significantly more effective in isolatingCampylobacterspp. from the environmental samples compared to Preston (P<0·001) and mBolton (P<0·04) broths but there was no significant difference between the latter two methods (P>0·05). Enrichment broth type did not affect isolation from chicken faecal or soil and litter samples.C. jejuniwas isolated from significantly more environmental samples using mExeter broth compared to Preston (P<0·01) and mBolton (P<0·003) broths; there was no difference between the latter two methods or between all methods for detection ofC. coli(P>0·05). OnlyC. coliwas isolated from the soil and litter samples and although bothC. jejuniandC. coliwere recovered from the faecal samples there was no effect of using different enrichment broths. The majority of samples where the same species had been isolated yielded the same or closely related genotypes as defined by pulsed-field gel electrophoresis. Isolates recovered using Preston and mBolton broths were less genetically diverse than those from mExeter broth. We conclude that the enrichment method used affects both the number and species ofCampylobacterisolated from naturally contaminated samples.
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Hilyard, Edward J., Joanne M. Jones-Meehan, Barry J. Spargo e Russell T. Hill. "Enrichment, Isolation, and Phylogenetic Identification of Polycyclic Aromatic Hydrocarbon-Degrading Bacteria from Elizabeth River Sediments†". Applied and Environmental Microbiology 74, n.º 4 (21 de dezembro de 2007): 1176–82. http://dx.doi.org/10.1128/aem.01518-07.
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ABSTRACT The diversity of indigenous bacteria in sediments from several sites in the Elizabeth River (Virginia) able to degrade multiple polycyclic aromatic hydrocarbons (PAHs) was investigated by the use of classical selective enrichment and molecular analyses. Enrichment cultures containing naphthalene, phenanthrene, fluoranthene, or pyrene as a sole carbon and energy source were monitored by denaturing gradient gel electrophoresis (DGGE) to detect changes in the bacterial-community profile during enrichment and to determine whether the representative strains present were successfully cultured. The DGGE profiles of the final enrichments grown solely on naphthalene and pyrene showed no clear relationship with the site from which the inoculum was obtained. The enrichments grown solely on pyrene for two sample sites had >80% similarity, which suggests that common pyrene-degrading strains may be present in these sediments. The final enrichments grown on fluoranthene and phenanthrene remained diverse by site, suggesting that these strains may be influenced by environmental conditions. One hundred and one isolates were obtained, comprising representatives of the actinomycetes and alpha-, beta-, and gammaproteobacteria, including seven novel isolates with 16S rRNA gene sequences less than 98% similar to known strains. The ability to degrade multiple PAHs was demonstrated by mineralization of 14C-labeled substrate and growth in pure culture. This supports our hypothesis that a high diversity of bacterial strains with the ability to degrade multiple PAHs can be confirmed by the combined use of classical selective enrichment and molecular analyses. This large collection of diverse PAH-degrading strains provides a valuable resource for studies on mechanisms of PAH degradation and bioremediation.
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MURAKAMI, TAKU. "Filter-Based Pathogen Enrichment Technology for Detection of Multiple Viable Foodborne Pathogens in 1 Day". Journal of Food Protection 75, n.º 9 (1 de setembro de 2012): 1603–10. http://dx.doi.org/10.4315/0362-028x.jfp-12-039.
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Conventional foodborne pathogen assays currently used in the food industry often require long culture enrichments to increase pathogen levels so they can be detected. Even using sensitive real-time PCR assays, culture enrichment at least overnight is necessary especially for detection of pathogens with slow growth rates such as Listeria monocytogenes. To eliminate this cumbersome enrichment step and detect minute amounts of pathogens within 1 day, filter-based pathogen enrichment technology was developed utilizing a unique combination of glass fiber depth filter and porous filter aid materials to efficiently separate pathogens from food homogenates and avoid filter clogging by food particles. After pathogen immobilization in depth filters, only viable pathogens were selectively collected in a small volume of growth medium via microbial multiplication and migration; nonviable pathogens remained inside the filters. By assaying viable pathogens using real-time PCRs, multiple species of foodborne pathogens were detected, including L. monocytogenes, Salmonella enterica, and Escherichia coli O157:H7, at around 1 CFU/ml or 1 CFU/g in various food samples. This filter-based pathogen enrichment technology is a unique bacterial enrichment alternative to the conventional culture enrichment step and can significantly shorten the time necessary to obtain assay results.
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BAILEY, J. S., D. L. FLETCHER e N. A. COX. "Effect of Enrichment Media and Sampling Protocol on Recovery of Listeria monocytogenes". Journal of Food Protection 53, n.º 6 (1 de junho de 1990): 505–7. http://dx.doi.org/10.4315/0362-028x-53.6.505.
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These studies examined the differences in recovery of Listeria monocytogenes from pure culture and in the populations of mixed aerobic microflora from chicken and Brie cheese incubated in University of Vermont (UVM) and Listeria enrichment broth (LEB) enrichment broths for different times and conditions. No significant differences were observed in levels of L. monocytogenes from pure cultures in UVM or LEB on any sampling day. No differences were observed in the levels of mixed microflora from Brie cheese in either UVM or LEB, but from chicken rinse the level of mixed flora competitors was significantly higher on all sampling days in LEB as compared to UVM. No differences were observed between a single enrichment in UVM or LEB for 2 d and a transfer to a secondary enrichment tube after 1 d. Overall, the level of mixed microflora capable of growing in enrichment broths was greater from chicken rinse than from Brie cheese. The ratio of L. monocytogenes to mixed microflora which survived the selective enrichments was most favorable for recovery of L. monocytogenes after 2 d of enrichment.
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Futamata, Hiroyuki, Yayoi Nagano, Kazuya Watanabe e Akira Hiraishi. "Unique Kinetic Properties of Phenol-Degrading Variovorax Strains Responsible for Efficient Trichloroethylene Degradation in a Chemostat Enrichment Culture". Applied and Environmental Microbiology 71, n.º 2 (fevereiro de 2005): 904–11. http://dx.doi.org/10.1128/aem.71.2.904-911.2005.
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ABSTRACT A chemostat enrichment of soil bacteria growing on phenol as the sole carbon source has been shown to exhibit quite high trichloroethylene (TCE)-degrading activities (H. Futamata, S. Harayama, and K. Watanabe, Appl. Environ. Microbiol. 67:4671-4677, 2001). To identify the bacterial populations responsible for the high TCE-degrading activity, a multidisciplinary survey of the chemostat enrichment was conducted by employing molecular-ecological and culture-dependent approaches. Three chemostat enrichment cultures were newly developed under different phenol-loading conditions (0.25, 0.75, and 1.25 g liter−1 day−1) in this study, and the TCE-degrading activities of the enrichments were measured. Among them, the enrichment at 0.75 g liter−1 day−1 (enrichment 0.75) expressed the highest activity. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments detected a Variovorax ribotype as the strongest band in enrichment 0.75; however, it was not a major ribotype in the other samples. Bacteria were isolated from enrichment 0.75 by direct plating, and their 16S rRNA genes and genes encoding the largest subunit of phenol hydroxylase (LmPHs) were analyzed. Among the bacteria isolated, several strains were affiliated with the genus Variovorax and were shown to have high-affinity-type LmPHs. The LmPH of the Variovorax strains was also detected as the major genotype in enrichment 0.75. Kinetic analyses of phenol and TCE degradation revealed, however, that these strains exhibited quite low affinity for phenol compared to other phenol-degrading bacteria, while they showed quite high specific TCE-degrading activities and relatively high affinity for TCE. Owing to these unique kinetic traits, the Variovorax strains can obviate competitive inhibition of TCE degradation by the primary substrate of the catabolic enzyme (i.e., phenol), contributing to the high TCE-degrading activity of the chemostat enrichments. On the basis of physiological information, mechanisms accounting for the way the Variovorax population overgrew the chemostat enrichment are discussed.
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Yoochatchaval, W., S. Kumakura, D. Tanikawa, T. Yamaguchi, M. F. M. Yunus, S. S. Chen, K. Kubota, H. Harada e K. Syutsubo. "Anaerobic degradation of palm oil mill effluent (POME)". Water Science and Technology 64, n.º 10 (1 de novembro de 2011): 2001–8. http://dx.doi.org/10.2166/wst.2011.782.
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The biodegradation characteristics of palm oil mill effluent (POME) and the related microbial community were studied in both actual sequential anaerobic ponds in Malaysia and enrichment cultures. The significant degradation of the POME was observed in the second pond, in which the temperature was 35–37 °C. In this pond, biodegradation of major long chain fatty acids (LCFA), such as palmitic acid (C16:0) and oleic acid (C18:1), was also confirmed. The enrichment culture experiment was conducted with different feeding substrates, i.e. POME, C16:0 and C18:1, at 35 °C. Good recovery of methane indicated biodegradation of feeds in the POME and C16:0 enrichments. The methane production rate of the C18:1 enrichment was slower than other substrates and inhibition of methanogenesis was frequently observed. Denaturing gradient gel electrophoresis (DGGE) analyses indicated the existence of LCFA-degrading bacteria, such as the genus Syntrophus and Syntorophomonas, in all enrichment cultures operated at 35 °C. Anaerobic degradation of the POME under mesophilic conditions was stably processed as compared with thermophilic conditions.
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Sousa, Diana Z., M. Alcina Pereira, Alfons J. M. Stams, M. Madalena Alves e Hauke Smidt. "Microbial Communities Involved in Anaerobic Degradation of Unsaturated or Saturated Long-Chain Fatty Acids". Applied and Environmental Microbiology 73, n.º 4 (8 de dezembro de 2006): 1054–64. http://dx.doi.org/10.1128/aem.01723-06.
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ABSTRACTAnaerobic long-chain fatty acid (LCFA)-degrading bacteria were identified by combining selective enrichment studies with molecular approaches. Two distinct enrichment cultures growing on unsaturated and saturated LCFAs were obtained by successive transfers in medium containing oleate and palmitate, respectively, as the sole carbon and energy sources. Changes in the microbial composition during enrichment were analyzed by denaturing gradient gel electrophoresis (DGGE) profiling of PCR-amplified 16S rRNA gene fragments. Prominent DGGE bands of the enrichment cultures were identified by 16S rRNA gene sequencing. A significant part of the retrieved 16S rRNA gene sequences was most similar to those of uncultured bacteria. Bacteria corresponding to predominant DGGE bands in oleate and palmitate enrichment cultures clustered with fatty acid-oxidizing bacteria withinSyntrophomonadaceaeandSyntrophobacteraceaefamilies. A low methane yield, corresponding to 9 to 18% of the theoretical value, was observed in the oleate enrichment, and acetate, produced according to the expected stoichiometry, was not further converted to methane. In the palmitate enrichment culture, the acetate produced was completely mineralized and a methane yield of 48 to 70% was achieved from palmitate degradation. Furthermore, the oleate enrichment culture was able to use palmitate without detectable changes in the DGGE profile. However, the palmitate-specialized consortia degraded oleate only after a lag phase of 3 months, after which the DGGE profile had changed. Two predominant bands appeared, and sequence analysis showed affiliation with theSyntrophomonasgenus. These bands were also present in the oleate enrichment culture, suggesting that these bacteria are directly involved in oleate degradation, emphasizing possible differences between the degradation of unsaturated and saturated LCFAs.
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Hara-Kudo, Yukiko, Tokuhiro Nishina, Hiroshi Nakagawa, Hirotaka Konuma, Junko Hasegawa e Susumu Kumagai. "Improved Method for Detection of Vibrio parahaemolyticus in Seafood". Applied and Environmental Microbiology 67, n.º 12 (1 de dezembro de 2001): 5819–23. http://dx.doi.org/10.1128/aem.67.12.5819-5823.2001.
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ABSTRACT We have developed a new, effective procedure for detectingVibrio parahaemolyticus in seafoods using enrichment and plating onto a chromogenic agar medium. Samples were cultured in salt Trypticase soy broth, which is a nonselective medium, and then a portion of the culture was cultured with salt polymyxin broth, which is a selective medium for V. parahaemolyticus. This two-step enrichment was more effective than the one-step enrichment in salt polymyxin broth alone. The enrichment cultures were then plated onto a new chromogenic agar containing substrates for beta-galactosidase. TheV. parahaemolyticus colonies developed a purple color on this growth medium that distinguished them from other related bacterial strains. V. parahaemolyticus was isolated more frequently from naturally contaminated seafood samples using the chromogenic agar than thiosulfate citrate bile salts sucrose agar medium, which is currently used for the isolation of V. parahaemolyticus. Our findings suggest that this new enrichment and isolation scheme is more sensitive and accurate for identifying V. parahaemolyticus in seafood samples than previously used methods.
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BOLTON, F. J., A. D. SAILS, A. J. FOX, D. R. A. WAREING e D. L. A. GREENWAY. "Detection of Campylobacter jejuni and Campylobacter coli in Foods by Enrichment Culture and Polymerase Chain Reaction Enzyme-Linked Immunosorbent Assay". Journal of Food Protection 65, n.º 5 (1 de maio de 2002): 760–67. http://dx.doi.org/10.4315/0362-028x-65.5.760.
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A polymerase chain reaction (PCR) assay based on a solution hybridization format with colorimetric end-point detection (PCR ELISA) was investigated for the specific detection of Campylobacter jejuni and Campylobacter coli in food samples following enrichment culture. One hundred fifteen samples of raw meat and offal (poultry, porcine, ovine, and bovine), raw shellfish, and artificially contaminated milk were enriched in blood-free Campylobacter Enrichment Broth for 48 h. Enrichment cultures were subcultured to Campylobacter blood-free selective agar plates, and presumptive isolates were identified by phenotypic methods. DNA was extracted from 1-ml aliquots of the enrichment cultures using a rapid extraction method, and the DNA was used as the template in a PCR ELISA. A comparison of the PCR ELISA with the enrichment culture and subculture to selective agar method showed that the results of 112 of the 115 samples tested were in agreement by both methods. Seventy-one of the various food samples were positive in the PCR ELISA, and 70 samples were positive by culture. The PCR ELISA had a sensitivity of 99% and a specificity of 96%, with a positive predictive value of 97% and a negative predictive value of 98%. The PCR ELISA is a rapid, sensitive, and specific method for the detection of C. jejuni and C. coli in foods following enrichment culture and significantly reduces the time required for their detection.
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Sutherland, Tara D., Irene Horne, Michael J. Lacey, Rebecca L. Harcourt, Robyn J. Russell e John G. Oakeshott. "Enrichment of an Endosulfan-Degrading Mixed Bacterial Culture". Applied and Environmental Microbiology 66, n.º 7 (1 de julho de 2000): 2822–28. http://dx.doi.org/10.1128/aem.66.7.2822-2828.2000.
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ABSTRACT An endosulfan-degrading mixed bacterial culture was enriched from soil with a history of endosulfan exposure. Enrichment was obtained by using the insecticide as the sole source of sulfur. Chemical hydrolysis was minimized by using strongly buffered culture medium (pH 6.6), and the detergent Tween 80 was included to emulsify the insecticide, thereby increasing the amount of endosulfan in contact with the bacteria. No growth occurred in control cultures in the absence of endosulfan. Degradation of the insecticide occurred concomitant with bacterial growth. The compound was both oxidized and hydrolyzed. The oxidation reaction favored the alpha isomer and produced endosulfate, a terminal pathway product. Hydrolysis involved a novel intermediate, tentatively identified as endosulfan monoaldehyde on the basis of gas chromatography-mass spectrometry and chemical derivatization results. The accumulation and decline of metabolites suggest that the parent compound was hydrolyzed to the putative monoaldehyde, thereby releasing the sulfite moiety required for growth. The monoaldehyde was then oxidized to endosulfan hydroxyether and further metabolized to (a) polar product(s). The cytochrome P450 inhibitor, piperonyl butoxide, did not prevent endosulfan oxidation or the formation of other metabolites. These results suggest that this mixed culture is worth investigating as a source of endosulfan-hydrolyzing enzymes for use in enzymatic bioremediation of endosulfan residues.
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Miles, K. I., M. W. D. Wren e S. Benson. "Is enrichment culture necessary for clinical samples?" British Journal of Biomedical Science 63, n.º 2 (janeiro de 2006): 87–88. http://dx.doi.org/10.1080/09674845.2006.11978088.
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Prakapas, Z., M. Denoyelle, C. Dargemont, F. G. Kroese, J. P. Thiery e M. A. Deugnier. "Enrichment and characterization of thymus-repopulating cells in stroma-dependent cultures of rat bone marrow". Journal of Cell Science 104, n.º 4 (1 de abril de 1993): 1039–48. http://dx.doi.org/10.1242/jcs.104.4.1039.
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The bone marrow precursor cells seeding the thymus have been difficult to investigate using fresh bone marrow and in vivo thymus reconstitution assays. We have therefore designed a short-term bone marrow culture system allowing the study of thymus-repopulating cells in the marrow microenvironment. Low-density rat bone marrow cells were grown on pre-established mouse bone marrow stromal cell layers. Cocultured cells were maintained either under steroid-free conditions (Whitlock/Witte-type culture) or in the presence of 10(−7) M hydrocortisone (Dexter-type culture). After 3 days in vitro, the unanchored cell fractions were tested for their ability to colonize and repopulate fetal mouse thymic lobes in vitro. Both fresh low-density cells and Whitlock/Witte-type cultures, but not Dexter-type cultures, gave rise intrathymically to significant numbers of rat donor-type Thy-1.1high CD2+ CD5low CD43+ cells accounting for 50% to 90% of the organ-cultured cells at day 14. Repopulation of fetal mouse thymic lobes by rat Thy-1.1high cells could be used as a readout assay for initiation of thymopoiesis from bone marrow precursor cells, since 90% of the cells were CD3-/low and TCR alpha beta-/low and 15% of the cells co-expressed CD4 and CD8. Dose-response analysis showed that thymus repopulating cells were at least maintained, if not amplified during the 3-day culture period, leading to at least a 10-fold enrichment as compared to unfractionated bone marrow. Unlike fresh low-density cells before culture, short-term Whitlock/Witte-type cultures were depleted in myeloid-restricted precursor cells. In culture, the thymus-repopulating activity was predominantly associated with a 10% lymphoid cell subset which did not express the B-lineage-associated antigens revealed by HIS24 (the rat B220 equivalent) and HIS50 mAbs. We propose that unanchored thymus-repopulating cells enriched in Whitlock/Witte-type cultures may represent lymphoid-restricted, T-cell precursors of the bone marrow capable of emigrating and colonizing the thymus.
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WARREN, B. R., M. E. PARISH e K. R. SCHNEIDER. "Comparison of Conventional Culture Methods and FTA Filtration–Nested PCR for the Detection of Shigella boydii and Shigella sonnei on Tomato Surfaces†". Journal of Food Protection 68, n.º 8 (1 de agosto de 2005): 1606–12. http://dx.doi.org/10.4315/0362-028x-68.8.1606.
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Detection of Shigella boydii UI02 and Shigella sonnei UI05 artificially inoculated onto tomatoes was evaluated using enrichment protocols of the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) and the American Public Health Association's Compendium of Methods for the Microbiological Examination of Food (CMMEF), enrichment in Enterobacteriaceae enrichment (EE) broth supplemented with 1.0 μg/ml novobiocin and incubated at 42°C, and FTA filtration–nested PCR. To assess the effect of natural tomato microflora on recovery, conventional culture enrichments were repeated using rifampin-adapted inocula and enrichment medium supplemented with 50 μg/ml rifampin. The lowest detection levels for S. boydii UI02 were >5.3 × 105 (BAM, CMMEF, and EE broth) and 6.2 CFU per tomato (FTA filtration–nested PCR). For S. sonnei UI05, the lowest detection levels were 1.9 × 101 (BAM), 1.5 × 103 (CMMEF), 1.1 × 101 (EE broth), and 7.4 CFU per tomato (FTA filtration–nested PCR). Natural tomato microflora had a large impact on recovery of S. sonnei UI05 and completely inhibited recovery of S. boydii UI02. EE broth was inhibitory to S. boydii UI02. FTA filtration–nested PCR provided superior detection (P < 0.05) compared with the conventional culture enrichment protocols.
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Suneethi, S., e Kurian Joseph. "Batch culture enrichment of ANAMMOX populations from anaerobic and aerobic seed cultures". Bioresource Technology 102, n.º 2 (janeiro de 2011): 585–91. http://dx.doi.org/10.1016/j.biortech.2010.07.121.
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STERN, NORMAN J., e J. ERIC LINE. "Comparison of Three Methods for Recovery of Campylobacter spp. from Broiler Carcasses". Journal of Food Protection 55, n.º 9 (1 de setembro de 1992): 663–66. http://dx.doi.org/10.4315/0362-028x-55.9.663.
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We compared three enrichment methods, with different sampling times (mid-and endpoint incubation) and various dilutions of enrichment culture for productivity in the isolation of Campylobacter spp. from 50 retail-level chicken carcasses. We subcultured the enrichment cultures onto three Campylobacter spp.-selective media (Campy-BAP, CCDA, Campy-Cefex) to compare yields of the organism. The highest yield (43/50) of Campylobacter spp. from these carcasses was derived by using the 24-h enrichment culture of Hunt & Radle diluted 1:100 before plating onto any of the three selective plating media. When all carcass analyses were combined, Campylobacter spp. was recovered from 49 of 50 broilers. This study indicates the optimum approaches for the recovery of Campylobacter spp., as well as the high incidence of the organism among the broiler carcasses tested.
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Meckenstock, Rainer U., Eva Annweiler, Walter Michaelis, Hans H. Richnow e Bernhard Schink. "Anaerobic Naphthalene Degradation by a Sulfate-Reducing Enrichment Culture". Applied and Environmental Microbiology 66, n.º 7 (1 de julho de 2000): 2743–47. http://dx.doi.org/10.1128/aem.66.7.2743-2747.2000.
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ABSTRACT Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture was studied by substrate utilization tests and identification of metabolites by gas chromatography-mass spectrometry. In substrate utilization tests, the culture was able to oxidize naphthalene, 2-methylnaphthalene, 1- and 2-naphthoic acids, phenylacetic acid, benzoic acid, cyclohexanecarboxylic acid, and cyclohex-1-ene-carboxylic acid with sulfate as the electron acceptor. Neither hydroxylated 1- or 2-naphthoic acid derivatives and 1- or 2-naphthol nor the monoaromatic compounds ortho-phthalic acid, 2-carboxy-1-phenylacetic acid, and salicylic acid were utilized by the culture within 100 days. 2-Naphthoic acid accumulated in all naphthalene-grown cultures. Reduced 2-naphthoic acid derivatives could be identified by comparison of mass spectra and coelution with commercial reference compounds such as 1,2,3,4-tetrahydro-2-naphthoic acid and chemically synthesized decahydro-2-naphthoic acid. 5,6,7,8-Tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. The metabolites identified suggest a stepwise reduction of the aromatic ring system before ring cleavage. In degradation experiments with [1-13C]naphthalene or deuterated D8-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [13C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene, 13C incorporation into the carboxylic group of 2-naphthoic acid was shown, indicating that activation of naphthalene by carboxylation was the initial degradation step. No ring fission products were identified.
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Riley, T. V., J. S. Brazier, Hamimah Hassan, Kathleen Williams e K. D. Phillips. "Comparison of alcohol shock enrichment and selective enrichment for the isolation ofClostridium difficile". Epidemiology and Infection 99, n.º 2 (outubro de 1987): 355–59. http://dx.doi.org/10.1017/s0950268800067832.
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SUMMARYTwo enrichment methods were compared for their ability to recoverClostridium difficilefrom stool samples. One method used selective enrichment in an antibiotic-containing broth followed by detection with a latex particle agglutination (LPA) reagent. The other used enrichment in a non-selective broth following treatment of the specimen with alcohol. With clinical specimens enrichment culture was significantly more successful at detectingC. difficilethan direct plating. Alcohol shock enrichment was twice as effective as direct culture, while selective broth enrichment was three times more effective. The use of LPA for screening selective enrichment broths forC. difficileshould prove a cost-effective measure as only positive broths (about 20%) require subculture for confirmation.
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Muniesa, Maite, Anicet R. Blanch, Francisco Lucena e Juan Jofre. "Bacteriophages May Bias Outcome of Bacterial Enrichment Cultures". Applied and Environmental Microbiology 71, n.º 8 (agosto de 2005): 4269–75. http://dx.doi.org/10.1128/aem.71.8.4269-4275.2005.
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ABSTRACT Enrichment cultures are widely used for the isolation of bacteria in clinical, biotechnological, and environmental studies. However, competition, relative growth rates, or inhibitory effects may alter the outcome of enrichment cultures, causing the phenomenon known as enrichment bias. Bacteriophages are a major component in many microbial systems, and it abounds in natural settings. This abundance means that bacteriophages are likely to be present in many laboratory enrichment cultures. Our hypothesis was that bacteriophages present in the sample might bias the enriched subpopulation, since it can infect and lyse the target bacteria during the enrichment step once the bacteria reach a given density. Here we show that the presence of bacteriophages in Salmonella and Shigella enrichment cultures produced a significant reduction (more than 1 log unit) in the number of these bacteria compared with samples in which bacteriophages had been reduced by filtration through 0.45-μm non-protein-binding membranes. Furthermore, our data indicate that the Salmonella biotypes isolated after the enrichment culture change if bacteriophages are present, thus distorting the results of the analysis.
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Rushton, Michael. "Subsidizing Culture: Taxpayer Enrichment of the Creative Class". Contemporary Sociology: A Journal of Reviews 46, n.º 4 (19 de junho de 2017): 409–10. http://dx.doi.org/10.1177/0094306117714500b.
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van den Berg, Eveline M., Udo van Dongen, Ben Abbas e Mark CM van Loosdrecht. "Enrichment of DNRA bacteria in a continuous culture". ISME Journal 9, n.º 10 (24 de abril de 2015): 2153–61. http://dx.doi.org/10.1038/ismej.2015.26.
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Iovene, Maria Rosaria, Francesca Martora, Francesca Bombace, Fortunato Montella, Chiara Del Vecchio, Michele De Rosa, Virginia D'Oriano, Marilena Galdiero e Mariateresa Vitiello. "A new enrichment diagnostic platform for semen culture". Journal of Microbiological Methods 144 (janeiro de 2018): 168–72. http://dx.doi.org/10.1016/j.mimet.2017.11.018.
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Attaway, Hubert, e Mark Smith. "Reduction of perchlorate by an anaerobic enrichment culture". Journal of Industrial Microbiology 12, n.º 6 (dezembro de 1993): 408–12. http://dx.doi.org/10.1007/bf01569673.
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Kataoka, N., Y. Tokiwa, Y. Tanaka, K. Takeda e T. Suzuki. "Enrichment culture and isolation of slow-growing bacteria". Applied Microbiology and Biotechnology 45, n.º 6 (24 de julho de 1996): 771–77. http://dx.doi.org/10.1007/s002530050761.
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Dietrich, G., e J. Winter. "Anaerobic degradation of chlorophenol by an enrichment culture". Applied Microbiology and Biotechnology 34, n.º 2 (novembro de 1990): 253–58. http://dx.doi.org/10.1007/bf00166791.
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Platen, H., e B. Schink. "Methanogenic degradation of acetone by an enrichment culture". Archives of Microbiology 149, n.º 2 (dezembro de 1987): 136–41. http://dx.doi.org/10.1007/bf00425079.
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Muenks, Carol E., Patrick G. Hogan, Carey-Ann D. Burnham e Stephanie A. Fritz. "Comparing the Yield of Staphylococcus aureus Recovery with Static versus Agitated Broth Incubation". Journal of Pathogens 2018 (26 de julho de 2018): 1–3. http://dx.doi.org/10.1155/2018/1462671.
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Given the lack of standardization of methodologies for microbial recovery from built environments, we sought to compare the yield of Staphylococcus aureus with a broth enrichment method when incubated in agitated versus static conditions. Five unique strains of S. aureus at five different concentrations were cultured to compare direct plating, agitated broth enrichment, and static broth enrichment culture methods. All samples were incubated at 35° in ambient air. The lowest concentration recovered across three replicates and five strains did not differ between culture methods (Fisher’s exact test, p=0.50); notably, recovery of S. aureus was equivalent between static and agitated broth incubation. When broth enrichment was used (both static and agitated), the burden of S. aureus growth was higher (by semiquantitative assessment of 4-quadrant streaking) compared to the direct plating culture method. Optimizing strategies for microbial recovery is essential, particularly in areas of lower biomass, given the paucity of research concerning microbial communities of built environments. The results of this study, in conjunction with other experiments investigating microbiomes of built environments, can help inform protocols for standardizing culturing methods within built environments.
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Herlinda, Siti, Muhamad Darma Utama, Yulia Pujiastuti e Suwandi Suwandi. "KERAPATAN DAN VIABILITAS SPORA BEAUVERIA BASSIANA (BALS.) AKIBAT SUBKULTUR DAN PENGAYAAN MEDIA, SERTA VIRULENSINYA TERHADAP LARVA PLUTELLA XYLOSTELLA (LINN.)". Jurnal Hama dan Penyakit Tumbuhan Tropika 6, n.º 2 (8 de setembro de 2006): 70–78. http://dx.doi.org/10.23960/j.hptt.2670-78.
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Density and viability of spores of Beauveria bassiana (Bals.) Vuill. due to sub-cultures and media enriched, and its virulence against larvae of Plutella xylostella (Linn.) This laboratory research was conducted to determine density and viability of spores of Beauveria bassiana due to sub-cultures and media enriched, and to investigate the fungi virulence againts larvae of Plutella xylostella. B. bassiana was grown in Saborroud Dextrose Broth (SDB) cultures enriched with cricket powder. The results showed that the best sub-culture was the fourth sub-culture enriched with cricket powder, producing 6.05 x 108 spores/ml with 30.7% viability. The highest larval mortality was found in the second sub-culture enriched with cricket powder which was reach 78.33%. The spore density and viability, and virulence of B. bassiana grown in sub-culture without cricket powder enrichment was consistently decreasing from the second sub-culture to the eighth. Sub-cultures enriched with cricket powder could increase density, viability, and virulence of B. bassiana in every sub-culture. Overall, the enrichment with cricket powder up to 0.5% could increase the density, viability, and virulence of B. bassiana.
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Qiu, Yan-Ling, Yuji Sekiguchi, Hiroyuki Imachi, Yoichi Kamagata, I.-Cheng Tseng, Sheng-Shung Cheng, Akiyoshi Ohashi e Hideki Harada. "Identification and Isolation of Anaerobic, Syntrophic Phthalate Isomer-Degrading Microbes from Methanogenic Sludges Treating Wastewater from Terephthalate Manufacturing". Applied and Environmental Microbiology 70, n.º 3 (março de 2004): 1617–26. http://dx.doi.org/10.1128/aem.70.3.1617-1626.2004.
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ABSTRACT The microbial populations responsible for anaerobic degradation of phthalate isomers were investigated by enrichment and isolation of those microbes from anaerobic sludge treating wastewater from the manufacturing of terephthalic acid. Primary enrichments were made with each of three phthalate isomers (ortho-, iso-, and terephthalate) as the sole energy source at 37�C with two sources of anaerobic sludge (both had been used to treat wastewater containing high concentrations of phthalate isomers) as the inoculum. Six methanogenic enrichment cultures were obtained which not only degraded the isomer used for the enrichment but also had the potential to degrade part of other phthalate isomers as well as benzoate with concomitant production of methane, presumably involving strictly syntrophic substrate degradation. Our 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization revealed that the predominant bacteria in the enrichment cultures were affiliated with a recently recognized non-sulfate-reducing subcluster (subcluster Ih) in the group ‘Desulfotomaculum lineage I' or a clone cluster (group TA) in the class delta-Proteobacteria. Several attempts were made to isolate these microbes, resulting in the isolation of a terephthalate-degrading bacterium, designated strain JT, in pure culture. A coculture of the strain with the hydrogenotrophic methanogen Methanospirillum hungatei converted terephthalate to acetate and methane within 3 months of incubation, whereas strain JT could not degrade terephthalate in pure culture. During the degradation of terephthalate, a small amount of benzoate was transiently accumulated as an intermediate, indicative of decarboxylation of terephthalate to benzoate as the initial step of the degradation. 16S rRNA gene sequence analysis revealed that the strain was a member of subcluster Ih of the group ‘Desulfotomaculum lineage I', but it was only distantly related to other known species.
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Rahm, Brian G., Robert M. Morris e Ruth E. Richardson. "Temporal Expression of Respiratory Genes in an Enrichment Culture Containing Dehalococcoides ethenogenes". Applied and Environmental Microbiology 72, n.º 8 (agosto de 2006): 5486–91. http://dx.doi.org/10.1128/aem.00855-06.
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ABSTRACT Multiple reductive dehalogenase (RDase), hydrogenase (H2ase), and other respiration-associated (RA) oxidoreductase genes have been identified in cultured representatives of Dehalococcoides. Although their products are likely to play key roles in the environmentally important process of reductive dechlorination, very little information is available about their regulation and specific functions. Here we show increased expression and temporal variability in the expression of five RDase genes and in the expression of genes for a putative formate dehydrogenase (Fdh) and two H2ases, including a periplasmic [Ni/Fe] H2ase (Hup) and a cytoplasmic [Fe] H2ase (Vhu). mRNA transcripts extracted from tetrachloroethene-dechlorinating mixed cultures corresponding to Fdh, the H2ase Hup, and the RDase targets TceA and DET0162 were expressed most highly, with average levels 34 (± 7.5)-, 23 (± 6.7)-, 16 (± 3.3)-, and 13 (± 3.3)-fold higher, respectively, than that for RNA polymerase (RpoB). H2ase and RA transcripts reached their respective expression maxima within the first 2 h after feeding. RDase transcripts, however, were most highly expressed after 3 h and exhibited greater temporal variability than other transcripts. Comparison with D. ethenogenes strain 195 pure culture expression levels indicated that RDase DET1545 was more highly expressed in mixed cultures, where, on average, its transcript level was sixfold higher than that of RpoB. While the specific functions of several of these gene products remain elusive, the high expression levels and temporal variability reported here suggest that these groups of enzymes are metabolically important for the respiration of chlorinated ethenes in mixed cultures containing Dehalococcoides.
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Liang, Xiaoming, Olivia Molenda, Shuiquan Tang e Elizabeth A. Edwards. "Identity and Substrate Specificity of Reductive Dehalogenases Expressed in Dehalococcoides-Containing Enrichment Cultures Maintained on Different Chlorinated Ethenes". Applied and Environmental Microbiology 81, n.º 14 (1 de maio de 2015): 4626–33. http://dx.doi.org/10.1128/aem.00536-15.
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ABSTRACTMany reductive dehalogenases (RDases) have been identified in organohalide-respiring microorganisms, and yet their substrates, specific activities, and conditions for expression are not well understood. We tested whether RDase expression varied depending on the substrate-exposure history of reductive dechlorinating communities. For this purpose, we used the enrichment culture KB-1 maintained on trichloroethene (TCE), as well as subcultures maintained on the intermediatescis-dichloroethene (cDCE) and vinyl chloride (VC). KB-1 contains a TCE-to-cDCE dechlorinatingGeobacterand severalDehalococcoidesstrains that together harbor many of the known chloroethene reductases. Expressed RDases were identified using blue native polyacrylamide gel electrophoresis, enzyme assays in gel slices, and peptide sequencing. As anticipated but never previously quantified, the RDase fromGeobacterwas only detected transiently at the beginning of TCE dechlorination. TheDehalococcoidesRDase VcrA and smaller amounts of TceA were expressed in the parent KB-1 culture during complete dechlorination of TCE to ethene regardless of time point or amended substrate. TheDehalococcoidesRDase BvcA was only detected in enrichments maintained on cDCE as growth substrates, in roughly equal abundance to VcrA. Only VcrA was detected in subcultures enriched on VC. Enzyme assays revealed that 1,1-DCE, a substrate not used for culture enrichment, afforded the highest specific activity.trans-DCE was substantially dechlorinated only by extracts from cDCE enrichments expressing BvcA. RDase gene distribution indicated enrichment of different strains ofDehalococcoidesas a function of electron acceptor TCE, cDCE, or VC. Each chloroethene reductase has distinct substrate preferences leading to strain selection in mixed communities.
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JACOB, M. E., J. BAI, D. G. RENTER, A. T. ROGERS, X. SHI e T. G. NAGARAJA. "Comparing Real-Time and Conventional PCR to Culture-Based Methods for Detecting and Quantifying Escherichia coli O157 in Cattle Feces". Journal of Food Protection 77, n.º 2 (1 de fevereiro de 2014): 314–19. http://dx.doi.org/10.4315/0362-028x.jfp-13-304.
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Detection of Escherichia coli O157 in cattle feces has traditionally used culture-based methods; PCR-based methods have been suggested as an alternative. We aimed to determine if multiplex real-time (mq) or conventional PCR methods could reliably detect cattle naturally shedding high (≥104 CFU/g of feces) and low (~102 CFU/g of feces) concentrations of E. coli O157. Feces were collected from pens of feedlot cattle and evaluated for E. coli O157 by culture methods. Samples were categorized as (i) high shedders, (ii) immunomagnetic separation (IMS) positive after enrichment, or (iii) culture negative. DNA was extracted pre- and postenrichment from 100 fecal samples from each category (high shedder, IMS positive, culture negative) and subjected to mqPCR and conventional PCR assays based on detecting three genes, rfbE, stx1, and stx2. In feces from cattle determined to be E. coli O157 high shedders by culture, 37% were positive by mqPCR prior to enrichment; 85% of samples were positive after enrichment. In IMS-positive samples, 4% were positive by mqPCR prior to enrichment, while 43% were positive after enrichment. In culture-negative feces, 7% were positive by mqPCR prior to enrichment, and 40% were positive after enrichment. The proportion of high shedder–positive and culture-positive (high shedder and IMS) samples were significantly different from mqPCR-positive samples before and after enrichment (P < 0.01). Similar results were observed for conventional PCR. Our data suggest that mqPCR and conventional PCR are most useful in identifying high shedder animals and may not be an appropriate substitute to culture-based methods for detection of E. coli O157 in cattle feces.
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JIANG, XIUPING, e MICHAEL P. DOYLE. "Optimizing Enrichment Culture Conditions for Detecting Helicobacter pylori in Foods". Journal of Food Protection 65, n.º 12 (1 de dezembro de 2002): 1949–54. http://dx.doi.org/10.4315/0362-028x-65.12.1949.
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The survival and growth of Helicobacter pylori under enrichment conditions in fresh, autoclaved and irradiated ground beef were determined. H. pylori grew in autoclaved ground beef at 37°C under microaerobic conditions in brain heart infusion broth with 7% horse serum at pH 7.3 after 3 to 7 days of lag time but did not grow within 7 days in irradiated (10 kGy) ground beef under the same enrichment conditions. Adjustment of the enrichment broth to pH 5.5 enabled the growth (ca. 2 log10 CFU/ml) of H. pylori within 7 days in the presence of irradiated ground beef and the prolific growth (ca. 3 to 4 log10 CFU/ml) of H. pylori within 3 days in the presence of autoclaved beef. H. pylori in fresh ground beef could not be isolated from enrichment media with antibiotics; however, H. pylori ureA could be detected by polymerase chain reaction (PCR) in such enrichment media after 1 to 3 days of incubation at 37°C. The addition of supplements, i.e., 0.3% mucin, 0.05% ferrous sulfate, and 0.05% sodium pyruvate or 0.008 M urea, or the adjustment of the enrichment broth pH to 5.5 or 4.5 enabled the detection of H. pylori ureA in enrichment media incubated for 1, 2, 3, and/or 7 days at 37°C. H. pylori in sterile milk refrigerated at 4°C at an initial level of 106 CFU/ml was inactivated to an undetectable level within 6 days; however, H. pylori was not detected either by a PCR assay or by the plating of enrichment cultures of 120 raw bovine milk samples.
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Venkateswaran, Kasthuri, e Shigeaki Harayama. "Sequential enrichment of microbial populations exhibiting enhanced biodegradation of crude oil". Canadian Journal of Microbiology 41, n.º 9 (1 de setembro de 1995): 767–75. http://dx.doi.org/10.1139/m95-106.
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The distribution of oil-degrading bacteria in the coastal water and sediments of Hokkaido, Japan, was surveyed. The potential of mixed microbial populations to degrade weathered crude oil was not confined to any ecological components (water or sediment) nor to the sampling stations. One microbial culture that was stable during repeated subculturing degraded 45% of the saturates and 20% of the aromatics present in crude oil in 10 days during the initial screening. The residual hydrocarbons in this culture were extracted by chloroform and dispersed in a fresh seawater-based medium and subsequently inoculated with microorganisms from the first culture. After full growth of the second culture, the residual hydrocarbons were again extracted and dispersed in a fresh medium in which microorganisms from the second culture had been inoculated. This sequential process was carried out six times to enrich those microorganisms that grew on the recalcitrant components of crude oil. After repeated exposure of the residual crude oil to the enriched microorganisms, about 80% of the initially added crude oil was degraded. The cultures obtained after each enrichment cycle were kept, and the degradation of fresh crude oil by the enriched microorganisms was examined. The degradative activity of the enriched cultures increased as the number of enrichment cycles increased. A microbial population that had been selected six times on the residual crude oil could degrade 70% of the saturates and 30% of the aromatics of crude oil. Thus, growth of a microbial population on residual crude oil improved its ability to biodegrade crude oil.Key words: crude oil, biodegradation, sequential enrichment, saturated hydrocarbon, aromatic hydrocarbon.
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Duhamel, Melanie, Kaiguo Mo e Elizabeth A. Edwards. "Characterization of a Highly Enriched Dehalococcoides-Containing Culture That Grows on Vinyl Chloride and Trichloroethene". Applied and Environmental Microbiology 70, n.º 9 (setembro de 2004): 5538–45. http://dx.doi.org/10.1128/aem.70.9.5538-5545.2004.
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ABSTRACT A highly enriched culture that reductively dechlorinates trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), and vinyl chloride (VC) to ethene without methanogenesis is described. The Dehalococcoides strain in this enrichment culture had a yield of (5.6 ± 1.4) × 108 16S rRNA gene copies/μmol of Cl− when grown on VC and hydrogen. Unlike the other VC-degrading cultures described in the literature, strains VS and BAV1, this culture maintained the ability to grow on TCE with a yield of (3.6 ± 1.3) × 108 16S rRNA gene copies/μmol of Cl−. The yields on an electron-equivalent basis measured for the culture grown on TCE and on VC were not significantly different, indicating that both substrates supported growth equally well. PCR followed by denaturing gradient gel electrophoresis, cloning, and phylogenetic analyses revealed that this culture contained one Dehalococcoides 16S rRNA gene sequence, designated KB-1/VC, that was identical (over 1,386 bp) to the sequences of previously described organisms FL2 and CBDB1. A second Dehalococcoides sequence found in separate KB-1 enrichment cultures maintained on cDCE, TCE, and tetrachloroethene was no longer present in the VC-H2 enrichment culture. This second Dehalococcoides sequence was identical to that of BAV1. As neither FL2 nor CBDB1 can dechlorinate VC to ethene in a growth-related fashion, it is clear that current 16S rRNA gene-based analyses do not provide sufficient information to distinguish between metabolically diverse members of the Dehalococcoides group.
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STERN, NORMAN J., e MARK A. MOZOLA. "Methods for Selective Enrichment of Campylobacter spp. from Poultry for Use in Conjunction with DNA Hybridization". Journal of Food Protection 55, n.º 10 (1 de outubro de 1992): 767–70. http://dx.doi.org/10.4315/0362-028x-55.10.767.
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A DNA hybridization test was investigated for application to the detection of Campylobacter spp. in poultry samples. The test chemistry involves solution phase hybridization and detection by means of an enzymatically generated colorimetric endpoint. DNA probes used in the test system are targeted to unique sequences of ribosomal RNA and are specific for Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter fetus subsp. fetus. Initial experiments with pure cultures of C. jejuni established the sensitivity limit of the DNA hybridization assay at approximately 106–7 CFU per sample. Experiments were designed to define optimal conditions for recovery and selective enrichment of Campylobacter spp. from chicken carcasses for use in conjunction with the DNA hybridization assay. Following overnight enrichment, cultures were swabbed onto Campy-Cefex agar plates and allowed to incubate for 24 h. This overnight growth was then suspended and assayed with the DNA probe. The remainder of the overnight enrichment was centrifuged and the resulting pellet was analyzed. Thirty-eight chicken carcasses were assayed for Campylobacter spp. by DNA probe and culture methodology employing culture enrichment and selective plating. Culture procedures isolated Campylobacter spp. from 23 carcasses, while the DNA probe assay detected the organism from 21 carcasses. The DNA probe registered five “false” positives and seven “false” negatives relative to the cultural bacteriologic approach.
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Deshpande, Ruta Suresh, Devi Sundaravadivelu, Pablo Campo, Jorge W. SantoDomingo e Robyn N. Conmy. "Comparative Study on Rate of Biodegradation of Diluted Bitumen and Conventional Oil in Fresh Water". International Oil Spill Conference Proceedings 2017, n.º 1 (1 de maio de 2017): 2256–67. http://dx.doi.org/10.7901/2169-3358-2017.1.2256.
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Abstract 2017-271 In recent years, diluted bitumen (or dilbit) has become an important source of hydrocarbon-based fuel. While information on the degradation of crude oils has been well researched, dilbit degradation has been studied at a much lesser extent. The objective of this study was to compare biodegradation of dilbit with a conventional crude oil (CCO) under various conditions. Two different microcosm experiments were set up, one containing a mixed culture acclimated to dilbit (Kalamazoo River Enrichment, KRC) and the other having a mixed culture enriched on soil contaminated with hydrocarbons (Anderson Ferry Enrichment, AFC). The microcosms were run for 60 d at 25 °C and for 72 days at 5 °C in flasks containing sterile Bushnell Hass broth and naturally dispersed oil. Each flask was inoculated with the KRC and AFC mixed cultures, and rotated on an orbital shaker (200 rpm) at the above stated temperatures. On each sampling day, triplicates were sacrificed to determine the residual hydrocarbon concentration. Additionally, some samples were used to determine the bacterial composition using 16S rRNA gene sequencing analysis. Hydrocarbon analysis (alkanes and PAHs) was performed by gas chromatography/mass spectrometry (GC/MS/MS). Higher degradation rates were achieved at 25 °C as compared to 5 °C. All the enrichments metabolized CCO as well dilbit, but the nature and extent of the degradation was distinct. KRC meso culture was the most effective among all, as it completely removed alkanes and most of the PAHs. AFC enrichment performed differently at the two temperatures; an acclimation period (8 d) was observed at 5 °C while there was no lag at 25 °C. KRC cryo culture as well as AFC culture at both temperatures degraded alkanes completely while they were not able to metabolize heavier fractions of the oil (C2–4 homologues of 3- and 4-ring compounds). All cultures showed the presence of diverse oil degrading bacteria and the differences in their compositions affected the biodegradation. Although dilbit was biodegraded, for all the treatments except AFC at 5 °C, the rate of degradation and the extent of degradation was greater for CCO owing to the higher concentrations of lighter hydrocarbons.
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Yamazaki, Wataru, Masumi Taguchi, Takao Kawai, Kentaro Kawatsu, Junko Sakata, Kiyoshi Inoue e Naoaki Misawa. "Comparison of Loop-Mediated Isothermal Amplification Assay and Conventional Culture Methods for Detection of Campylobacter jejuni and Campylobacter coli in Naturally Contaminated Chicken Meat Samples". Applied and Environmental Microbiology 75, n.º 6 (9 de janeiro de 2009): 1597–603. http://dx.doi.org/10.1128/aem.02004-08.
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ABSTRACT We investigated the efficacy of a loop-mediated isothermal amplification (LAMP) assay for detection of chicken meat samples naturally contaminated with Campylobacter jejuni and Campylobacter coli. A total of 144 Preston enrichment broth cultures from chicken meat samples were assessed by using the LAMP assay and conventional culture methods, which consist of a combination of Preston enrichment culturing and plating onto Butzler and modified charcoal cefoperazone deoxycholate agars. Compared with C. jejuni-C. coli isolation using the conventional culture test, the LAMP results showed 98.5% (67/68) and 97.4% (74/76) sensitivity and specificity, respectively, and the positive and negative predictive values were 97.1% (67/69) and 98.7% (74/75), respectively. The conventional culture test required more than 3 to 4 days to isolate and identify C. jejuni and C. coli in the Preston enrichment cultures. In contrast, the LAMP assay was markedly faster, requiring less than 90 min from the beginning of DNA extraction to final detection and differentiation of C. jejuni and C. coli. In total, the LAMP assay required 23.5 to 25.5 h from the beginning of the enrichment culture to final determination. These results suggest that our LAMP assay is a powerful tool for rapid, sensitive, and practical detection of C. jejuni and C. coli which may facilitate surveillance and control of C. jejuni-C. coli contamination in chicken, as well as investigations of food poisoning incidents caused by these organisms. This is the first report of a highly sensitive and specific LAMP assay to detect and differentiate C. jejuni and C. coli in chicken meat samples.
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Fedio, Willis, George M. Blackstone, Lynne Kikuta-Oshima, Chitra Wendakoon, Timothy H. McGrath e Angelo DePaola. "Rapid Detection of the Vibrio cholerae ctx Gene in Food Enrichments Using Real-Time Polymerase Chain Reaction". Journal of AOAC INTERNATIONAL 90, n.º 5 (1 de setembro de 2007): 1278–83. http://dx.doi.org/10.1093/jaoac/90.5.1278.
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Abstract A real-time polymerase chain reaction (qPCR) assay for the detection of the ctxA gene of toxigenic Vibrio cholerae (Vc) was validated against standard culture techniques. The first experimental phase determined optimal enrichment conditions for detection by culture and qPCR of Vc in shrimp, bottled water, milk, and potato salad. The conditions tested included temperature (35 and 42C), time (6 and 18 h), and effect of shaking (0 and 100 rpm). No definitive trends were found with enrichment temperature or shaking on Vc isolation frequency or detection by qPCR. Generally, Vc was detected by qPCR more frequently than Vc was isolated, but this difference was significant only in the 35C 6 h enrichment without shaking. In the second phase of experiments, shrimp, bottled water, milk, potato salad, and oysters were inoculated with each of 3 toxigenic Vc strains (Latin American O1 strain, an O139 strain, and an O1 strain from the U.S. Gulf Coast) and enriched under static conditions at 42C for 6 and 18 h. Overall, detection frequency of ctx by qPCR was 98 (88/90) and 100 (90/90) after 6 and 18 h enrichments, respectively, while Vc isolation frequency was 87 (78/90) and 83 (75/90) after 6 and 18 h, respectively. Toxigenic Vc can be detected by qPCR within an 8 h work day using the 6 h enrichment procedure, assuming an initial level of at least 12 colony-forming units/g; however, overnight enrichment may be necessary to detect lower levels. These data indicate that the qPCR assay for ctx is a more reliable, sensitive, and rapid alternative to standard Vc culture methods and is applicable to diverse food products.
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Bentham, Richard, Nick McClure e David Catcheside. "Biotreatment of an industrial waste oil condensate". Water Science and Technology 36, n.º 10 (1 de novembro de 1997): 125–29. http://dx.doi.org/10.2166/wst.1997.0374.
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The biotreatment of an industrial waste oil condensate has been investigated. The waste is an oily emulsion resulting from chemical processing and condensation of grease trap wastes and industrial waste oils. The oil consists of a complex mix of hydrocarbons with significant fuel oil and lube oil fractions. Currently this waste is disposed of by incineration. The feasibility of using a biological pretreatment process to remove a significant proportion of the hydrocarbons has been investigated. Enrichment cultures produced a stable bacterial consortium. Flask cultures of this enrichment culture were capable of rapid emulsification of the oil. Within 10 days, 40–50% of the oil waste was degraded. Degradation was monitored using gas chromatographic analysis with flame ionisation detector (GC-FID) and by assessment of microbial dehydrogenase activity using triphenyl tetrazolium chloride (TTC) dye reduction. The enrichment culture consisted of 9 component organisms, 7 Gram negative and one Gram positive organisms. Their degradative abilities in monoculture have been investigated. Degradation of the waste using monocultures was monitored using GC-FID analysis of the Pristane:C17 ratio in the waste. The degradation capability of each of the component organisms in pure culture was similar to that of the consortium.
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He, Zhanfei, Sha Geng, Chaoyang Cai, Shuai Liu, Yan Liu, Yawei Pan, Liping Lou, Ping Zheng, Xinhua Xu e Baolan Hu. "Anaerobic Oxidation of Methane Coupled to Nitrite Reduction by Halophilic Marine NC10 Bacteria". Applied and Environmental Microbiology 81, n.º 16 (5 de junho de 2015): 5538–45. http://dx.doi.org/10.1128/aem.00984-15.
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ABSTRACTAnaerobic oxidation of methane (AOM) coupled to nitrite reduction is a novel AOM process that is mediated by denitrifying methanotrophs. To date, enrichments of these denitrifying methanotrophs have been confined to freshwater systems; however, the recent findings of 16S rRNA andpmoAgene sequences in marine sediments suggest a possible occurrence of AOM coupled to nitrite reduction in marine systems. In this research, a marine denitrifying methanotrophic culture was obtained after 20 months of enrichment. Activity testing and quantitative PCR (qPCR) analysis were then conducted and showed that the methane oxidation activity and the number of NC10 bacteria increased correlatively during the enrichment period. 16S rRNA gene sequencing indicated that only bacteria in group A of the NC10 phylum were enriched and responsible for the resulting methane oxidation activity, although a diverse community of NC10 bacteria was harbored in the inoculum. Fluorescencein situhybridization showed that NC10 bacteria were dominant in the enrichment culture after 20 months. The effect of salinity on the marine denitrifying methanotrophic culture was investigated, and the apparent optimal salinity was 20.5‰, which suggested that halophilic bacterial AOM coupled to nitrite reduction was obtained. Moreover, the apparent substrate affinity coefficients of the halophilic denitrifying methanotrophs were determined to be 9.8 ± 2.2 μM for methane and 8.7 ± 1.5 μM for nitrite.
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Nguyen, Phong D., Sarah T. Hsiao, Priyadharshini Sivakumaran, Shiang Y. Lim e Rodney J. Dilley. "Enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro". American Journal of Physiology-Cell Physiology 303, n.º 12 (15 de dezembro de 2012): C1220—C1228. http://dx.doi.org/10.1152/ajpcell.00449.2011.
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Long-term culture of primary neonatal rat cardiomyocytes is limited by the loss of spontaneous contractile phenotype within weeks in culture. This may be due to loss of contractile cardiomyocytes from the culture or overgrowth of the non-cardiomyocyte population. Using the mitochondria specific fluorescent dye, tetramethylrhodamine methyl ester perchlorate (TMRM), we showed that neonatal rat cardiomyocytes enriched by fluorescence-activated cell sorting can be maintained as contractile cultures for long periods (24-wk culture vs. 2 wk for unsorted cardiomyocytes). Long-term culture of this purified cardiomyocyte (TMRM high) population retained the expression of cardiomyocyte markers, continued calcium cycling, and displayed cyclic electrical activity that could be regulated pharmacologically. These findings suggest that non-cardiomyocyte populations can negatively influence contractility of cardiomyocytes in culture and that by purifying cardiomyocytes, the cultures retain potential as an experimental model for longitudinal studies of cardiomyocyte biology in vitro.
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Sauvage, Justine, Gary H. Wikfors, Xiaoxu Li, Mark Gluis, Nancy Nevejan, Koen Sabbe e Alyssa Joyce. "Effect of pluronic block polymers and N-acetylcysteine culture media additives on growth rate and fatty acid composition of six marine microalgae species". Applied Microbiology and Biotechnology 105, n.º 5 (12 de fevereiro de 2021): 2139–56. http://dx.doi.org/10.1007/s00253-021-11147-8.
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Abstract The efficiency of microalgal biomass production is a determining factor for the economic competitiveness of microalgae-based industries. N-acetylcysteine (NAC) and pluronic block polymers are two compounds of interest as novel culture media constituents because of their respective protective properties against oxidative stress and shear-stress-induced cell damage. Here we quantify the effect of NAC and two pluronic (F127 and F68) culture media additives upon the culture productivity of six marine microalgal species of relevance to the aquaculture industry (four diatoms-Chaetoceros calcitrans, Chaetoceros muelleri, Skeletonema costatum, and Thalassiosira pseudonana; two haptophytes-Tisochrysis lutea and Pavlova salina). Algal culture performance in response to the addition of NAC and pluronic, singly or combined, is dosage- and species-dependent. Combined NAC and pluronic F127 algal culture media additives resulted in specific growth rate increases of 38%, 16%, and 24% for C. calcitrans, C. muelleri, and P. salina, respectively. Enhanced culture productivity for strains belonging to the genus Chaetoceros was paired with an ~27% increase in stationary-phase cell density. For some of the species examined, culture media enrichments with NAC and pluronic resulted in increased omega-3-fatty acid content of the algal biomass. Larval development (i.e., growth and survival) of the Pacific oyster (Crassostrea gigas) was not changed when fed a mixture of microalgae grown in NAC- and F127-supplemented culture medium. Based upon these results, we propose that culture media enrichment with NAC and pluronic F127 is an effective and easily adopted approach to increase algal productivity and enhance the nutritional quality of marine microalgal strains commonly cultured for live-feed applications in aquaculture. Key points • Single and combined NAC and pluronic F127 culture media supplementation significantly enhanced the productivity of Chaetoceros calcitrans and Chaetoceros muelleri cultures. • Culture media enrichments with NAC and F127 can increase omega-3-fatty acid content of algal biomass. • Microalgae grown in NAC- and pluronic F127-supplemented culture media are suitable for live-feed applications.
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Xie, Xuan, Rafael Nóbrega e Martin Pšenička. "Spermatogonial Stem Cells in Fish: Characterization, Isolation, Enrichment, and Recent Advances of In Vitro Culture Systems". Biomolecules 10, n.º 4 (22 de abril de 2020): 644. http://dx.doi.org/10.3390/biom10040644.
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Spermatogenesis is a continuous and dynamic developmental process, in which a single diploid spermatogonial stem cell (SSC) proliferates and differentiates to form a mature spermatozoon. Herein, we summarize the accumulated knowledge of SSCs and their distribution in the testes of teleosts. We also reviewed the primary endocrine and paracrine influence on spermatogonium self-renewal vs. differentiation in fish. To provide insight into techniques and research related to SSCs, we review available protocols and advances in enriching undifferentiated spermatogonia based on their unique physiochemical and biochemical properties, such as size, density, and differential expression of specific surface markers. We summarize in vitro germ cell culture conditions developed to maintain proliferation and survival of spermatogonia in selected fish species. In traditional culture systems, sera and feeder cells were considered to be essential for SSC self-renewal, in contrast to recently developed systems with well-defined media and growth factors to induce either SSC self-renewal or differentiation in long-term cultures. The establishment of a germ cell culture contributes to efficient SSC propagation in rare, endangered, or commercially cultured fish species for use in biotechnological manipulation, such as cryopreservation and transplantation. Finally, we discuss organ culture and three-dimensional models for in vitro investigation of fish spermatogenesis.
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Bobek, Vladimir, Martin Cegan e Katarina Kolostova. "Circulating tumour cells in patients with urothelial tumours: Enrichment and in vitro culture". Canadian Urological Association Journal 8, n.º 9-10 (22 de outubro de 2014): 715. http://dx.doi.org/10.5489/cuaj.1978.
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Introduction: Results of clinical trials have demonstrated that circulating tumour cells (CTCs) are frequently detected in patients with urothelial tumours. The monitoring of CTCs has the potential to improve therapeutic management at an early stage and also to identify patients with increased risk of tumour progression or recurrence before the onset of clinically detected metastasis. In this study, we report a new effectively simplified methodology for a separation and in vitro culturing of viable CTCs from peripheral blood.Method: We include patients diagnosed with 3 types of urothelial tumours (prostate cancer, urinary bladder cancer, and kidney cancer). A size-based separation method for viable CTC - enrichment from unclothed peripheral blood has been introduced (MetaCell, Ostrava, Czech Republic). The enriched CTCs fraction was cultured directly on the separation membrane, or transferred from the membrane and cultured on any plastic surface or a microscopic slide.Results: We report a successful application of a CTCs isolation procedure in patients with urothelial cancers. The CTCs captured on the membrane are enriched with a remarkable proliferation potential. This has enabled us to set up in vitro cell cultures from the viable CTCs unaffected by any fixation buffers, antibodies or lysing solutions. Next, the CTCs were cultured in vitro for a minimum of 10 to 14 days to enable further downstream analysis (e.g., immunohistochemistry).Conclusion: We demonstrated an efficient CTCs capture platform, based on a cell size separation principle. Furthermore, we report an ability to culture the enriched cells – a critical requirement for post-isolation cellular analysis.