Journal of Rapid Methods and Automation in Microbiology最新文献

{"title":"DEVELOPMENT OF A SPOT-TITER CULTURE ASSAY FOR QUANTIFYING BACTERIA AND VIRAL INDICATORS","authors":"N.K. BECK,&nbsp;K. CALLAHAN,&nbsp;S.P. NAPPIER,&nbsp;H. KIM,&nbsp;M.D. SOBSEY,&nbsp;J.S. MESCHKE","doi":"10.1111/j.1745-4581.2009.00182.x","DOIUrl":"10.1111/j.1745-4581.2009.00182.x","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> ABSTRACT</h3>\u0000 \u0000 <p> <i>The spread-plate and double agar layer (DAL) methods are common for the enumeration of bacteria and viral indicators (bacteriophages). However, they may become cumbersome in large matrix experiments or when the titer of the organism varies by several orders of magnitude. A bacterial spot-titer assay has been available for decades but has not been adapted to bacteriophages and has rarely been applied to the analysis of environmental samples. In this study, a spot-titer culture-based method was investigated for bacteria and bacteriophages. The method involves spot-plating replicate 10-µl volumes of several sample dilutions on a single plate, incubating, and counting colonies or plaques. Parallel assays of laboratory cultures and environmentally isolated organisms show that the spot-titer method is equally straightforward and statistically comparable to the spread-plate and DAL methods (</i>R<i>² = 0.989 for laboratory strains and</i> R<i>² = 0.972 for environmental samples), while more cost- and labor-efficient.</i></p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> PRACTICAL APPLICATIONS</h3>\u0000 \u0000 <p>The spread-plate and double agar layer (DAL) methods currently used for enumeration of bacteria and viral indicators, may become labor- and resource-intensive (culture media, plates, technician time and incubator space) in large matrix experiments, which are often needed in the laboratory to evaluate environmental conditions. The spot-titer method has several advantages over the spread-plate and DAL methods: (1) it requires less time to dispense spots than to spread the microbe; (2) it uses fewer materials (15–20% of the laboratory supplies as the traditional methods); (3) it requires less effort; and (4) since the sample is distributed in distinct spots, colony/plaque counting is faster and less labor intensive. The spot-titer method was found to economize resources without sacrificing accuracy or precision, and is a practical method for routine use in large matrix experiments (e.g., survival or disinfection studies) and enumeration of high-titer environmental samples.</p>\u0000 </section>\u0000 </div>","PeriodicalId":50067,"journal":{"name":"Journal of Rapid Methods and Automation in Microbiology","volume":"17 4","pages":"455-464"},"PeriodicalIF":0.0,"publicationDate":"2009-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1745-4581.2009.00182.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"63426651","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"COMPARATIVE ANALYSIS OF METHODS FOR THE PURIFICATION OF DNA FROM LOW-BIOMASS SAMPLES BASED ON TOTAL YIELD AND CONSERVED MICROBIAL DIVERSITY","authors":"MYRON T. LA DUC,&nbsp;SHARIFF OSMAN,&nbsp;KASTHURI VENKATESWARAN","doi":"10.1111/j.1745-4581.2009.00153.x","DOIUrl":"10.1111/j.1745-4581.2009.00153.x","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> ABSTRACT</h3>\u0000 \u0000 <p> <i>Despite advances in the specificity and sensitivity of molecular biological technologies, the efficient recovery of DNA from low-biomass samples remains extremely challenging. Optimal methods to purify biomolecules from such environments should (1) achieve the greatest total yield and (2) reflect the true microbial diversity of the sample. These attributes were assessed from five DNA purification regimes: a standard-manual procedure, MoBio Ultraclean and Promega Wizard kits, and an automated Axcyte AutoLyser method with and without bead-beating agitation. A homogenous mixture of known concentrations of nine distinct bacterial lineages isolated from low-biomass environments was prepared and suitable aliquots of subsamples were processed in parallel. DNA products from each of these methods were then subjected to polymerase chain reaction (PCR), quantitative PCR and 16S rRNA clone-library analysis. The Axcyte AutoLyser outperformed all other purification regimes examined. This automated method consistently both yielded the highest concentration of PCR-amplifiable DNA, and reported species composition most consistent with the starting solution.</i> </p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> PRACTICAL APPLICATIONS</h3>\u0000 \u0000 <p>This communication carefully examines the effectiveness of common DNA purification regimes as well as an automated method. Comparative analyses convincingly demonstrate that the different methods not only result in different recovery of genomic DNA, but more importantly, different estimations of microbial diversity in the sample. This report will hopefully inspire investigators from various industries (pharmaceutical, ecological, medical, semiconductor, etc.) who find themselves in the initial phases of large-scale studies to devote a significant effort into optimizing sample extraction protocols to achieve the most accurate information.</p>\u0000 </section>\u0000 </div>","PeriodicalId":50067,"journal":{"name":"Journal of Rapid Methods and Automation in Microbiology","volume":"17 3","pages":"350-368"},"PeriodicalIF":0.0,"publicationDate":"2009-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1745-4581.2009.00153.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"63424995","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A NEW FLUOROGENIC ASSAY FOR MONITORING AND DETERMINING PLANKTONIC AND BIOFILM FORMS OF PSEUDOMONAS AERUGINOSA VIABLE COUNT IN VITRO","authors":"WALID F. ELKHATIB,&nbsp;AYMAN M. NOREDDIN","doi":"10.1111/j.1745-4581.2009.00156.x","DOIUrl":"10.1111/j.1745-4581.2009.00156.x","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> ABSTRACT</h3>\u0000 \u0000 <p> <i>A new method was developed to rapidly monitor the</i> Pseudomonas aeruginosa <i>viable counts using alamar blue (AB). The 96-well microtiter plates were used to perform the assay. This procedure is based on fluorogenic measurement as a result of reduction of nonfluorescent AB to red fluorescent form by the viable cells of</i> P. aeruginosa. <i>The correlation between conventional plate count and fluorogenic AB method was highly satisfactory for quantification of planktonic (</i>R² = <i>0.9487) and biofilm cells of</i> P. aeruginosa<i> (</i>R² = <i>0.9296).</i></p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> PRACTICAL APPLICATIONS</h3>\u0000 \u0000 <p>The new fluorogenic method can rapidly monitor <i>Pseudomonas aeruginosa</i> counts <i>in vitro</i> with a high correlation with the conventional plating method. The results indicate that fluorogenic method requires much shorter time (2 h) than the conventional plate count (24 h), is a more cost-effective way, quite amenable to high throughput, and continuous monitoring of <i>P. aeruginosa</i> viability is achievable in the kinetic <i>in vitro</i> models without interference with the cell viability.</p>\u0000 </section>\u0000 </div>","PeriodicalId":50067,"journal":{"name":"Journal of Rapid Methods and Automation in Microbiology","volume":"17 3","pages":"304-314"},"PeriodicalIF":0.0,"publicationDate":"2009-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1745-4581.2009.00156.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"63425172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A NOVEL METHOD FOR ASSESSMENT OF 16S RRNA GENE COPY NUMBER IN BACTERIAL GENOMES BY PULSED-FIELD GEL ELECTROPHORESIS AND PCR AMPLIFICATION","authors":"YONGHUI ZENG,&nbsp;NIANZHI JIAO","doi":"10.1111/j.1745-4581.2009.00165.x","DOIUrl":"10.1111/j.1745-4581.2009.00165.x","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> ABSTRACT</h3>\u0000 \u0000 <p> <i>16S rRNA gene (</i>rrn<i>) copy number in bacterial genomes is indicative of ecological strategies of bacteria and is critical for quantification of bacterial abundance in mixed populations using</i> rrn-<i>based approaches. For accurate assessment of</i> rrn <i>copies, a novel technical strategy by means of pulsed-field gel electrophoresis and polymerase chain reaction amplification analysis was introduced. Experimental and</i> in silico <i>analysis on a test bacterial culture</i> Caulobacter crescentus <i>proved it to be simple, effective, accurate and a good alternative to traditional time-consuming methods.</i></p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> PRATICAL APPLICATIONS</h3>\u0000 \u0000 <p>This method can be used for routine determination of gene copy number in most bacteria whose full genome sequences are not available. Moreover, the pulsed-field gel electrophoresis bands containing a target gene fragment can be determined and therefore constructing an expected fragments oriented genomic library is possible.</p>\u0000 </section>\u0000 </div>","PeriodicalId":50067,"journal":{"name":"Journal of Rapid Methods and Automation in Microbiology","volume":"17 3","pages":"274-279"},"PeriodicalIF":0.0,"publicationDate":"2009-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1745-4581.2009.00165.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"63425468","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"SUITABILITY OF INTERGENIC SPACER OR INTERNAL TRANSCRIBED SPACER MICROSATELLITE-PRIMED PCR FOR THE IDENTIFICATION OF RHIZOCTONIA SOLANI AND SOME PHYTOFUNGI","authors":"K.A. ABD-ELSALAM,&nbsp;J.-R. GUO,&nbsp;M.A. MOSLEM,&nbsp;A.H. BAHKALI,&nbsp;J.-A. VERREET","doi":"10.1111/j.1745-4581.2009.00178.x","DOIUrl":"10.1111/j.1745-4581.2009.00178.x","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> ABSTRACT</h3>\u0000 \u0000 <p> <i>The intergenic spacer (IGS) region or internal transcribed spacer (ITS) region were used in pair-combinations with microsatellite-primed polymerase chain reaction (MP-PCR) primers to establish whether additional polymorphisms can be yielded. A total of 24</i> Rhizoctonia solani <i>isolates representing 13 anstomosis groups and 9 different fungal species isolate were recovered from different areas and hosts. Forty different primer combinations were tested for their ability to provide discrete bands and individual isolates' readily interpretable and reproducible IGS<b>/</b>ITS-MP-PCR profiles. Both approaches produced highly reproducible and complex genomic fingerprints, with fragments ranging in size from 100 to 2,000 bp (IGS-MP-PCR) and 50 to 2,000 bp (ITS-MP-PCR). MP-PCR markers yielded more bands than IGS<b>/</b>ITS-MP-PCR because of their higher redundancy in the fungal genome. The number of fragments generated by both techniques varied according to the fungal species and also with the primer combination used. Each primer used could differentiate all of the fungal isolates examined in this study. The profiles generated were identical and reproducible between repeated PCR experiments.</i></p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> PRACTICAL APPLICATIONS</h3>\u0000 \u0000 <p>Combining the intergenic spacer/internal transcribed spacer-microsatellite-primed polymerase chain reaction technique with microsatellite–detection assay allows the rapid and specific detection of <i>Rhizoctonia solani</i> anastomosis groups and different phytopathogenic fungi. The utility of this approach stems from its simplicity and reproducibility, the high number of polymorphisms revealed, the very small amounts of DNA needed, rapidity, and ease of performance. The improved technique will present valuable information on the role of some phytopathogenic fungi and <i>R. solani</i> in agriculturally important plant diseases.</p>\u0000 </section>\u0000 </div>","PeriodicalId":50067,"journal":{"name":"Journal of Rapid Methods and Automation in Microbiology","volume":"17 3","pages":"383-397"},"PeriodicalIF":0.0,"publicationDate":"2009-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1745-4581.2009.00178.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"63426531","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DETERMINATION OF INDICATOR BACTERIA IN PHARMACEUTICAL SAMPLES BY MULTIPLEX PCR","authors":"S. FARAJNIA,&nbsp;M. HASSAN,&nbsp;S. HALLAJ NEZHADI,&nbsp;L. MOHAMMADNEJAD,&nbsp;M. MILANI,&nbsp;F. LOTFIPOUR","doi":"10.1111/j.1745-4581.2009.00154.x","DOIUrl":"10.1111/j.1745-4581.2009.00154.x","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> ABSTRACT</h3>\u0000 \u0000 <p> <i>Rapid and sensitive detection techniques for indicator pathogens are important in pharmaceutical industry. However, common detection methods rely on bacterial culture in combination with biochemical tests, a process that typically takes 5–6 days to complete. Thus, the aim of this study was to develop a multiplex polymerase chain reaction</i> (<i>mPCR) assay for simultaneous detection and identification of four indicator pathogenic bacteria in a single reaction. Specific primers for indicator bacteria, namely</i> Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa<i>and</i> Salmonella, <i>were applied to allow simultaneous detection of them, and the sensitivity and specificity of each primer pairs were determined. In the mPCR with mixed DNA samples, specific bands for corresponding bacteria were simultaneously detected. Agarose gel electrophoresis of PCR products revealed 100% specificity of mPCR with single bands in the expected sizes. Low levels of microbial contamination less than 10 cfu per milliliter or gram of product were detected using mPCR assay. The detection of all four indicator pathogenic bacteria were completed in less than 8 h with this novel mPCR method, whereas the conventional United States Pharmacopeia methods and uniplex PCR required 5–6 days and 27 h for completion, respectively. Using mPCR assay, the microbial quality control of nonsterile pharmaceutical products can be performed in a cost-effective and timely manner in pharmaceutical industry.</i></p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> PRACTICAL APPLICATIONS</h3>\u0000 \u0000 <p>Detection of pathogenic indicatiors of <i>Escherichia coli</i>, <i>Staphylococcus aureus</i>, <i>Salmonella</i> and <i>Pseudomonas aeruginosa</i> is one of the mandatory tests in microbial quality of nonsterile pharmaceutical products; therefore, rapid and sensitive detection of the contaminations is of great importance for product release. According to the results of the present study, simultaneous detection of low levels of four major potential pathogenic bacteria in pharmaceutical finished products can be performed using mPCR in a cost-effective and timely manner, and upon these properties of the mPCR assay it could have potential applications in pharmaceutical industry.</p>\u0000 </section>\u0000 </div>","PeriodicalId":50067,"journal":{"name":"Journal of Rapid Methods and Automation in Microbiology","volume":"17 3","pages":"328-338"},"PeriodicalIF":0.0,"publicationDate":"2009-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1745-4581.2009.00154.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"63425119","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DIRECT FLUORESCENT ANTIBODY-DIRECT VIABLE COUNT AND POLYMERASE CHAIN REACTION DETECTION LIMIT FOR THE IDENTIFICATION OF VIBRIO CHOLERAE O1 IN MUSSELS (MYTILUS EDULIS)","authors":"S.R. PERESSUTTI,&nbsp;V. JURQUIZA,&nbsp;S. GONZÁLEZ-FRAGA,&nbsp;M. PICHEL,&nbsp;N. BINSZTEIN,&nbsp;M. COSTAGLIOLA","doi":"10.1111/j.1745-4581.2009.00175.x","DOIUrl":"10.1111/j.1745-4581.2009.00175.x","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> ABSTRACT</h3>\u0000 \u0000 <p>Vibrio cholerae <i>O1 is natural to the aquatic environment and can cause gastrointestinal infections when it is consumed from contaminated bivalves. Under unfavorable conditions, this bacterium enters into a viable but nonculturable state. Immunofluorescence and polymerase chain reaction (PCR) methods were a useful alternative for detecting this microorganism without a pre-enrichment step. We investigated the detection limit of the direct fluorescent antibody (DFA)-direct viable count (DVC) and PCR techniques for the identification of</i> V. cholerae <i>O1 in mussel (</i>Mytilus edulis<i>) samples. When 10³ cfu/mL</i> V. cholerae <i>O1 were inoculated in samples, 10²–10³ bacteria mL⁻¹ were determined by immunofluorescence tests and 67% of the samples were positive by PCR assay. No significant difference (</i>T <i>statistic value = 6.5,</i> P = <i>0.2049) between DFA and DFA-DVC procedures was observed. No presence of endogenous</i> V. cholerae <i>O1 was detected</i>.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> PRACTICAL APPLICATIONS</h3>\u0000 \u0000 <p>Vibrios are considered the major cause of identifiable illness and death from shellfish consumption. In Argentina, the viable but nonculturable (VBNC) forms of <i>Vibrio cholerae</i> O1 were identified in samples of water and plankton. Because of these facts, it is relevant to research the presence of <i>V. cholerae</i> O1 in aquatic bivalves. Immunofluorescence and polymerase chain reaction methods are a useful alternative to traditional enrichment testing for detecting both culturable and VBNC forms of <i>V. cholerae</i> O1. In this work, it was demonstrated that these methods were sensitive and efficient for detecting <i>V. cholerae</i> O1 in mussels without a pre-enrichment step. Moreover, they can be a useful tool for the rapid detection of this pathogen in the seafood industry.</p>\u0000 </section>\u0000 </div>","PeriodicalId":50067,"journal":{"name":"Journal of Rapid Methods and Automation in Microbiology","volume":"17 3","pages":"291-303"},"PeriodicalIF":0.0,"publicationDate":"2009-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1745-4581.2009.00175.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"63425887","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PERSISTENCE OF SALMONELLA SPP. ON CHICKEN SKIN AFTER EXPOSURE TO AN ITALIAN MARINADE*","authors":"THOMAS P. OSCAR,&nbsp;MANPREET SINGH","doi":"10.1111/j.1745-4581.2009.00176.x","DOIUrl":"10.1111/j.1745-4581.2009.00176.x","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> ABSTRACT</h3>\u0000 \u0000 <p> <i>A series of experiments with chicken skin was undertaken to determine the effect of an Italian marinade on persistence of</i> Salmonella <i>spp. during refrigerated storage and marinating. Chicken skin was inoculated with 0.4 to 3.7 log of multiple antibiotic resistant strains of</i> Salmonella <i>Typhimurium (</i>n = <i>3), Kentucky (</i>n = <i>1) or Hadar (</i>n = <i>1). Chicken skin was then exposed to the Italian marinade for 4 or 24 h at 6C to simulate normal marinating conditions of consumers. The persistence of</i> Salmonella <i>spp. on chicken skin was reduced (</i>P &lt; <i>0.05) by the Italian marinade with a greater reduction observed at 24 h than at 4 h of marinating. As expected, the persistence during marinating increased as a function of the initial number of</i> Salmonella <i>inoculated. In general, the effect of the Italian marinade on persistence was similar among the five strains of</i> Salmonella <i>tested.</i></p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> PRACTICAL APPLICATIONS</h3>\u0000 \u0000 <p>Marinades contain organic acids (acetic, lactic) that decrease meat pH and help to reduce or eliminate pathogenic bacteria, such as <i>Salmonella</i>, from the product. Inclusion of other ingredients in marinades, such as salts and spices that have antimicrobial properties, helps to further reduce or prevent persistence of <i>Salmonella</i>. Results of this study with an Italian-style marinade indicate that consumers should marinate chicken in the refrigerator for 24 h rather than 4 h to maximize the benefit of the Italian marinade on reducing the risk of exposure to <i>Salmonella</i> that might contaminate and persist on chicken purchased in the retail marketplace.</p>\u0000 </section>\u0000 </div>","PeriodicalId":50067,"journal":{"name":"Journal of Rapid Methods and Automation in Microbiology","volume":"17 3","pages":"369-382"},"PeriodicalIF":0.0,"publicationDate":"2009-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1745-4581.2009.00176.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"63426091","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"FLUORESCENT MEASUREMENTS OF DNA, RNA AND PROTEINS TO PERFORM COMPARATIVE ANALYSES OF MICROBIAL COMMUNITIES FROM THE ENVIRONMENTS","authors":"M. CARMEN PORTILLO,&nbsp;JUAN M. GONZALEZ","doi":"10.1111/j.1745-4581.2009.00179.x","DOIUrl":"10.1111/j.1745-4581.2009.00179.x","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> ABSTRACT</h3>\u0000 \u0000 <p> <i>Simple and rapid methods for the quantification of DNA, RNA and proteins using specific fluorescent dyes are proposed for the comparison and monitoring of microbial communities from the environment. The purpose of this study was the use of straightforward</i> in situ <i>methods which voided the need for preservation of samples and the risk of potential degradation and quantitative changes during transportation. Aside from this, methods used to obtain information on environmental microbial communities are generally time-consuming and present certain difficulty above all when working on solid substrates such as soils and rocks. New generation fluorescent dyes that bind specifically to DNA, RNA and proteins allow simple and rapid estimates of these biomolecules in crude environmental samples.</i></p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> PRACTICAL APPLICATIONS</h3>\u0000 \u0000 <p>Monitoring the metabolic state of microbial communities on different substrates and environments is a requirement for comparing samples and assessing the participation of microorganisms in a variety of processes. Solid substrates are not easily analyzed by microscopic techniques and they require long processing times and tedious work. Aside from this, only a minor fraction (&lt;1%) of microorganisms in most natural environments can be cultured in standard microbiological media (Ward <i>et al.</i> 1990). Other studies using incorporation of labeled substrates to approach activity rates or biomolecule extractions represent complex and long procedures during environmental studies.In order to evaluate microbial communities in a variety of substrates and environments, rapid and simple methods are proposed by measuring DNA, RNA and/or proteins using specific fluorescent dyes, without a need for prior purification, from crude solid samples.</p>\u0000 </section>\u0000 </div>","PeriodicalId":50067,"journal":{"name":"Journal of Rapid Methods and Automation in Microbiology","volume":"17 3","pages":"398-410"},"PeriodicalIF":0.0,"publicationDate":"2009-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1745-4581.2009.00179.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"63426590","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MULTIPLEX DETECTION OF ESCHERICHIA COLI AND SALMONELLA ENTERITIDIS BY USING QUANTUM DOT-LABELED ANTIBODIES","authors":"FAHRIYE CEYDA DUDAK,&nbsp;İSMAİL HAKKI BOYACI","doi":"10.1111/j.1745-4581.2009.00155.x","DOIUrl":"10.1111/j.1745-4581.2009.00155.x","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> ABSTRACT</h3>\u0000 \u0000 <p> <i>In this study, we demonstrated the simultaneous detection of</i> Escherichia coli <i>and</i> Salmonella enteritidis, <i>by coupling immunomagnetic separation (IMS) with quantum dots (QDs) labeling. QDs having different emission wavelengths were conjugated with anti-</i>E. coli <i>and anti-</i>Salmonella <i>antibodies. QD–antibody conjugates were used to label immunomagnetically separated bacteria and the fluorescence intensities were measured for enumerations of both species. The concentrations of primary antibodies used in IMS, the ratio of QDs to antibodies during the conjugation and the concentration of QD–antibody conjugates used in labeling were optimized to enhance the sensitivity of the assay. After labeling bacteria with QDs, the quenching observed between bead–bacteria complex and QDs was eliminated by separating QDs from the complex using sodium dodecyl sulfate solution. The fluorescence intensities due to the capturing of different concentrations of bacteria were measured and the working ranges were found to be 5 × 10² to 5 × 10⁵ cfu/mL for</i> E. coli <i>and 4 </i>×<i> 10² to 4 </i>×<i> 10⁵ cfu/mL for</i> S. enteritidis<i>.</i></p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> PRACTICAL APPLICATIONS</h3>\u0000 \u0000 <p>In this study, antibody-conjugated multicolor quantum dots (QDs) were used for simultaneous detection of <i>Escherichia coli</i> and <i>Salmonella enteritidis</i>. The results of this study indicate that QD labels can be used in multiplex, rapid and selective detection of bacteria with detection limits comparable with those of many novel methods in cases where the assay conditions are optimized. Furthermore, the assay can be modified for the simultaneous detection of more than two species through using QD labels having different emission wavelengths.</p>\u0000 </section>\u0000 </div>","PeriodicalId":50067,"journal":{"name":"Journal of Rapid Methods and Automation in Microbiology","volume":"17 3","pages":"315-327"},"PeriodicalIF":0.0,"publicationDate":"2009-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1745-4581.2009.00155.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"63425139","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}