Assessment ofE. coli andSalmonella viability and starvation by confocal laser microscopy and flow cytometry using rhodamine 123, DiBAC4(3), propidium iodide, and CTC

López-Amorós, R; Castel, Susanna; Comas-Riu, Jaume; Vives-Rego, Josep

doi:10.1002/(sici)1097-0320(19971201)29:4<298::aid-cyto6>3.0.co;2-6

Cited by 107 publications

(92 citation statements)

References 43 publications

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“…The stock (concentration not given by the manufacturer) was diluted 1:10 in sterile (0.22-m-filtered) distilled water, 2 l was added to the cell suspension, and the mixture was incubated for 10 min in the dark. PI, a fluorescent nucleic acid dye, was used to stain cells with compromised membrane integrity (9,18). The stock (1-mg ml Ϫ1 solution in water; Molecular Probes) was diluted in sterile distilled water and added to the cell suspension at 10 g ml Ϫ1 (final concentration) or 5 g ml Ϫ1 , and the mixture was incubated for 30 min in the dark.…”

Section: Microorganismsmentioning

confidence: 99%

Use of Flow Cytometry To Follow the Physiological States of Microorganisms in Cider Fermentation Processes

Herrero

Quirós

Garcı́a

et al. 2006

Appl Environ Microbiol

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Analysis of microbiological samples from fermentation processes in the beverage industry (beer, wine, and cider) by traditional indirect, culture-based standard methods is timeconsuming, and the methods do not produce direct information about the physiological state of the microorganisms. Moreover, the plate-counting method detects only cells able to form colonies under the conditions of the medium that is used, ignoring the presence of cells that do not form colonies but are nevertheless metabolically active (6). As is known, standard laboratory culture media rarely resemble natural environmental conditions (32). The application of flow cytometry (FC) with fluorescent dyes is faster and more direct and has made it possible to distinguish stages beyond the classical definition of viability generally demonstrated by culturing (24). FC can be used to quantitatively measure the optical characteristics of cells as they pass, in single file, into a focused beam of light (for a review, see reference 33). As particles pass through the beam, three parameters are measured: forward light scatter, side angle light scatter, and fluorescence at selected wavelengths. Light scatter is related to cell mass, structure, surface properties, and the optical density of the internal medium. A variety of fluorescent dyes may be used to detect structural or functional cellular properties. In this way, different cellular functions, such as reproductive growth, metabolic activity, and membrane integrity, can be detected, allowing a definition of the term viable-but-not-culturable cells (VBNC) (24) (other authors have called them active but nonculturable). Previous studies have reported that there are two major adaptations that cells undergo during the formation of VBNC states: cell wall toughening and DNA condensation (29). The detection of metabolic activity provides presumptive evidence of reproductive growth by the demonstration of enzyme activity, such as esterases, along with membrane integrity. In the absence of metabolic activity, it is still possible to determine membrane integrity by either dye retention or dye exclusion, the latter using propidium iodide (PI). Cells without an intact membrane cannot maintain or generate the electrochemical gradient that generates the membrane potential and can be considered dead cells (24). The use of FC techniques was reported for the "at-line" study of Escherichia coli fermentations (14), which detected a considerable drop in cell viability (about 20%) during the latter stages of small-scale (5-liter), well-mixed fedbatch fermentations.Flow-cytometric applications using different dyes have been compared to standard methods to assess yeast cultures in baking, wine making, cider making, and brewing (1,5,7,8,10,11,17,19). The technique has also been used to study the membrane integrity of ethanol-stressed Oenococcus oeni cells (9). Most studies of FC applications have focused mainly on microorganims as pure cultures, and only a small number have analyzed mixed populations over time (30).The purp...

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Section: Microorganismsmentioning

confidence: 99%

Use of Flow Cytometry To Follow the Physiological States of Microorganisms in Cider Fermentation Processes

Herrero

Quirós

Garcı́a

et al. 2006

Appl Environ Microbiol

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“…Moreover, the RNA expression level depends on the physiological status of the cell, making accurate measurements of bacterial numbers difficult. Another strategy is the use of dyes for microscopic differentiation between viable and dead cells (15,13,20,29). Although these methods are well established, quantification by extrapolating counts of a limited number of bacteria is problematic and cannot be applied on a routine basis.…”

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confidence: 99%

Selective Removal of DNA from Dead Cells of Mixed Bacterial Communities by Use of Ethidium Monoazide

Nocker¹,

Camper

2006

Appl Environ Microbiol

289

210

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The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A promising and easy-to-use alternative utilizing the DNA-intercalating dye ethidium monoazide bromide (EMA) was published recently. This chemical is known to penetrate only into "dead" cells with compromised cell membrane integrity. Subsequent photoinduced crosslinking was reported to inhibit PCR amplification of DNA from dead cells. We provide evidence here that in addition to inhibition of amplification, most of the DNA from dead cells is actually lost during the DNA extraction procedure, probably together with cell debris which goes into the pellet fraction. Exposure of bacteria to increasing stress and higher proportions of dead cells in defined populations led to increasing loss of genomic DNA. Experiments were performed using Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium as model pathogens and using real-time PCR for their quantification. Results showed that EMA treatment of mixed populations of these two species provides a valuable tool for selective removal of DNA of nonviable cells by using conventional extraction protocols. Furthermore, we provide evidence that prior to denaturing gradient gel electrophoresis, EMA treatment of a mature mixed-population drinking-water biofilm containing a substantial proportion of dead cells can result in community fingerprints dramatically different from those for an untreated biofilm. The interpretation of such fingerprints can have important implications in the field of microbial ecology.Whether bacterial cells are dead or alive is an important question with many implications for monitoring of food and water safety and for analysis of the sterility of pharmaceutical drugs. Due to the relatively long persistence of DNA after cell death, in the range of several days to 3 weeks (10, 14), quantitative analysis of total DNA can lead to a substantial overestimation of the presence of living microorganisms and the accompanying pathogenic threats. The lack of differentiation between DNA from viable and dead bacterial cells is therefore a major obstacle to broad-range application of DNA-based molecular diagnostics (10, 12). The most commonly used strategy to overcome this difficulty is to focus on the presence of the rapidly degrading RNA instead of the stable DNA (16,27,28,31). Due to its rapid turnover, the detection of RNA is far more indicative of the presence of viable cells (1, 2). Nevertheless, working with RNA is technically demanding, and RNA is prone to contamination with RNA-degrading enzymes, resulting in problems of reproducibility. Moreover, the RNA express...

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“…For instance, propidium iodide (PI) has a polarity and penetrates only cells with compromised plasma membranes (18). In this study, dead bacterial cells were detected by staining with PI (2 g/ml; Sigma) for 5 min at room temperature (approximately 25°C) (32).…”

mentioning

confidence: 99%

“…There are many fluorescent dyes which can indicate loss of membrane integrity, which, in turn, can often act as an indicator of cell viability (11,17,18). For instance, propidium iodide (PI) has a polarity and penetrates only cells with compromised plasma membranes (18).…”

mentioning

confidence: 99%

Improved Direct Viable Count Procedure for Quantitative Estimation of Bacterial Viability in Freshwater Environments

Yokomaku

Yamaguchi

Nasu

2000

Appl Environ Microbiol

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Assessment ofE. coli andSalmonella viability and starvation by confocal laser microscopy and flow cytometry using rhodamine 123, DiBAC4(3), propidium iodide, and CTC

Cited by 107 publications

References 43 publications

Use of Flow Cytometry To Follow the Physiological States of Microorganisms in Cider Fermentation Processes

Use of Flow Cytometry To Follow the Physiological States of Microorganisms in Cider Fermentation Processes

Selective Removal of DNA from Dead Cells of Mixed Bacterial Communities by Use of Ethidium Monoazide

Improved Direct Viable Count Procedure for Quantitative Estimation of Bacterial Viability in Freshwater Environments

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