Direct In Situ Viability Assessment of Bacteria in Probiotic Dairy Products Using Viability Staining in Conjunction with Confocal Scanning Laser Microscopy

Auty, Mark A.E.; Gardiner, Gillian E.; McBrearty, S.; O’Sullivan, Eleanor; Mulvihill, Daniel M.; Collins, John; Fitzgerald, Gerald F.; Stanton, Catherine; Ross, R. Paul

doi:10.1128/aem.67.1.420-425.2001

Cited by 178 publications

(119 citation statements)

References 37 publications

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“…To further investigate the dissociation of the RT-qPCR counts and CFU counts for C. perfringens at the later stages of culture as described above, we determined the viable cell counts under conditions in which starved C. perfringens cultures were unable to form colonies. We used the SYTO9-PI double staining method, which has been reported to be able to differentiate live and dead bacteria based on differences in plasma membrane permeability (3,15). The number of live cells stained only with SYTO9 remained 10 8 throughout the test period, while the CFU counts decreased markedly, demonstrating that most of the bacteria that lost the ability to form colonies on an agar plate were still alive and maintained their cell membrane integrity and that the numbers of cells in the population that could be detected were nearly equal to those detected by RT-qPCR (Fig.…”

Section: Resultsmentioning

confidence: 99%

Sensitive Quantitative Detection of Commensal Bacteria by rRNA-Targeted Reverse Transcription-PCR

Matsuda

Tsuji

Asahara

et al. 2007

Appl Environ Microbiol

260

232

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A sensitive rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) method was developed for exact and sensitive enumeration of subdominant bacterial populations. Using group-or species-specific primers for 16S or 23S rRNA, analytical curves were constructed for Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Clostridium perfringens, and Pseudomonas aeruginosa, and the threshold cycle value was found to be linear up to an RNA amount of 10 ؊3 cell per RT-PCR. The number of bacteria in culture was determined by RT-qPCR, and the results correlated well with the CFU count over the range from 10 0 to 10 5 CFU. The bacterial counts obtained by RT-qPCR were the same as the CFU counts irrespective of the growth phase in vitro, except for C. perfringens during starvation periods; the viable cell counts obtained by using a combination of 4,6-diamidino-2-phenylindole (DAPI) staining and SYTO9-propidium iodide double staining were in good agreement with the RT-qPCR counts rather than with the CFU counts. The RT-qPCR method could detect endogenous Enterobacteriaceae and P. aeruginosa in feces of hospitalized patients (n ‫؍‬ 38) at a level of 10 3 cells per g of feces, and for enumeration of S. aureus or P. aeruginosa spiked into human peripheral blood, the lower detection limit for RT-qPCR quantification of the bacteria was 2 cells per ml of blood, suggesting that this method was equivalent to the conventional culture method. As only 5 h was needed for RT-qPCR quantification, we suggest that rRNA-targeted RT-qPCR assays provide a sensitive and convenient system for quantification of commensal bacteria and for examining their possible invasion of a host.For almost a century, culture techniques have been recognized as the "gold standards" for determining viable bacterial counts. As the human fecal flora has been reported to consist of approximately 400 bacterial species (12, 35) and these species are present at a concentration of 10 11 viable microorganisms per g of contents (42), conventional culture techniques for enumeration of different populations involve the use of selective microbiological media, followed by isolation of pure cultures and the use of confirmatory biochemical tests. Recently, a number of molecular methods based on immunological and genotypic techniques have been developed (41,48). In analyses of the gut microflora, a number of molecular methods have been used in place of cultivation-based techniques. Techniques such as the clone library method (42, 46), denaturing gradient gel electrophoresis (13), and terminal restriction fragment length polymorphism (31, 36) allow analysis of predominant bacteria that are difficult to culture. The fluorescent in situ hybridization method (18,43) and the quantitative PCR (qPCR) method with rRNA-targeted oligonucleotide probes or primers have also been used as culture-independent methods. Among these, PCR methods targeting mainly well-conserved 16S rRNA genes have prevailed for rapid quantification of bacteria and are recognized as having two advant...

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Section: Resultsmentioning

confidence: 99%

Sensitive Quantitative Detection of Commensal Bacteria by rRNA-Targeted Reverse Transcription-PCR

Matsuda

Tsuji

Asahara

et al. 2007

Appl Environ Microbiol

260

232

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“…Scanning electron microscopy, as well as fluorescence and light microscopy, have been used to examine microbial diversity in cheese (27,52), although these techniques provide only nonspecific information. Recently, confocal microscopy has been also suggested as a tool for studying the viability of bacteria in situ in food (10,48). However, 16S rRNA-FISH analysis has the advantage of potentially providing both specific and nonspecific detection in a single step.…”

Section: Discussionmentioning

confidence: 99%

Bacterial Community Structure and Location in Stilton Cheese

Ercolini

Hill

Dodd

2003

Appl Environ Microbiol

244

135

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The microbial diversity occurring in Stilton cheese was evaluated by 16S ribosomal DNA analysis with PCR-denaturing gradient gel electrophoresis. DNA templates for PCR experiments were directly extracted from the cheese as well as bulk cells harvested from a variety of viable-count media. The variable V3 and V4-V5 regions of the 16S genes were analyzed. Closest relatives of Lactococcus lactis, Enterococcus faecalis, Lactobacillus plantarum, Lactobacillus curvatus, Leuconostoc mesenteroides, Staphylococcus equorum, and Staphylococcus sp. were identified by sequencing of the DGGE fragments. Fluorescently labeled oligonucleotide probes were developed to detect Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides in fluorescence in situ hybridization (FISH) experiments, and their specificity for the species occurring in the community of Stilton cheese was checked in FISH experiments carried out with reference cultures. The combined use of these probes and the bacterial probe Eub338 in FISH experiments on Stilton cheese sections allowed the assessment of the spatial distribution of the different microbial species in the dairy matrix. Microbial colonies of bacteria showed a differential location in the different parts of the cheese examined: the core, the veins, and the crust. Lactococci were found in the internal part of the veins as mixed colonies and as single colonies within the core. Lactobacillus plantarum was detected only underneath the surface, while Leuconostoc microcolonies were homogeneously distributed in all parts observed. The combined molecular approach is shown to be useful to simultaneously describe the structure and location of the bacterial flora in cheese. The differential distribution of species found suggests specific ecological reasons for the establishment of sites of actual microbial growth in the cheese, with implications of significance in understanding the ecology of food systems and with the aim of achieving optimization of the fermentation technologies as well as preservation of traditional products.

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“…PI and SYTO 9 have been used for the direct enumeration of physiologically active bacteria in drinking water (Boulos et al, 1999) and also in other fields of bacteriological research (Lebaron et al, 1998;Auty et al, 2001). For ).…”

Section: Discussionmentioning

confidence: 99%

Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting

Tebaldi¹,

Peters²,

Souza³

et al. 2010

Trop. plant pathol.

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Flow cytometric analysis of immuno-stained cells (immuno-FCM) was compared to immunofluorescence microscopy (IF) and dilution plating on a semi-selective medium, for quantitative detection of Xanthomonas axonopodis pv. phaseoli (Xap) in bean seed extracts. Cell concentrations of Xap between 10 3 -10 7 CFU/mL were added to healthy bean seed extracts. A flow cytometry sorting procedure was developed to separate immuno-stained Xap cells from crude seed extracts and confirming by PCR. FCM was evaluated for direct viable counting (DVC) of Xap using combinations of propidium iodide (PI) and carboxy fluorescein diacetate (cFDA) or PI and SYTO 9 and also the combination of immuno-FCM and PI. Dilution plating and IF allowed detection of Xap in bean seed extracts in a range of 10 3 -10 6 CFU/mL and immuno-FCM from 10 4 -10 6 CFU/mL. Sorted cells could be detected in crude seed extracts by PCR without further extraction. FCM also allowed quantification of viable cells of Xap after DVC procedures; the red fluorescent dye propidium iodide was used to identify dead cells in combination with the green fluorescent dyes cFDA or SYTO 9, these identifying live cells. The combination of immuno-FCM and PI could be more promising and reliable to detect this pathogen in seeds. Key words: seed pathology, flow sorting, PCR-amplification, viability probes, immunofluorescence, bacteria. RESUMO Detecção de Xanthomonas axonopodis pv. phaseoli em sementes de feijão usando citometria de fluxo em combinação com anticorpo e sondas fluorescentes de viabilidadeA combinação do uso do citômetro de fluxo (FCM) e de anticorpo policlonal (imuno-FCM) foi comparada à microscopia de imunofluorescência (IF) e ao plaqueamento em meio de cultura semi-seletivo, para a detecção de Xanthomonas axonopodis pv. phaseoli (Xap) em sementes de feijão. Concentrações de Xap variando de 10 3 a 10 7 CFU/mL foram adicionados aos extratos de sementes. Um método de separação pelo citômetro de fluxo foi desenvolvido para a detecção de Xap em extratos de semente e posterior confirmação por PCR. Para avaliação da viabilidade das células foram usadas sondas fluorescentes, iodeto de propídio (PI)/carboxi diacetato de fluoresceína (cFDA) e PI/SYTO 9 e também, a combinação de imuno-FCM e PI. Em meio semi-seletivo e IF foram detectadas 10 3 -10 6 UFC/mL e no FCM 10 4 -10 6 UFC/mL, em extratos de sementes artificialmente infestados. Xap somente foi detectada em extratos de sementes por PCR, após o processo de separação pelo FCM. Foi possível pelo FCM a quantificação e identificação de células viáveis (verde fluorescente) e células mortas (vermelho fluorescente) de Xap, pelas sondas cFDA/SYTO 9 e PI, respectivamente. A combinação de immuno-FCM e PI poderá ser uma técnica promissora e segura para a detecção deste patógeno em sementes. Palavras-chave: patologia de sementes, PCR, sondas de viabilidade, imuno-fluorescência, bactéria. INTRODUCTIONXanthomonas axonopodis pv. phaseoli (Smith) Vauterin, Hoste, Kersters & Swinges (Xap) is a seedtransmitted plant-pathogenic bacterium w...

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Direct In Situ Viability Assessment of Bacteria in Probiotic Dairy Products Using Viability Staining in Conjunction with Confocal Scanning Laser Microscopy

Cited by 178 publications

References 37 publications

Sensitive Quantitative Detection of Commensal Bacteria by rRNA-Targeted Reverse Transcription-PCR

Sensitive Quantitative Detection of Commensal Bacteria by rRNA-Targeted Reverse Transcription-PCR

Bacterial Community Structure and Location in Stilton Cheese

Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting

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