Physiological significance of the cytometric distribution of fluorescent yeasts after viability staining

Bouchez, J.C.; Cornu, Marie; Danzart, M.; Leveau, Jean-Yves; Duchiron, Francis; Bouix, Marielle

doi:10.1002/bit.20054

Cited by 12 publications

(9 citation statements)

References 41 publications

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“…However, it has been reported that FDA is not an ideal fluorescent substrate, as bacteria retain fluorescence poorly, resulting in weaker staining and increased background (33). da Silveira et al (9) reported the use of the derivative 5(6)-carboxyfluorescein diacetate (cF) for staining O. oeni cells, with improved retention ability (33), although it should be noted that the cleavage product of this dye can be actively extruded from S. cerevisiae (4,10).…”

Section: Resultsmentioning

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: Resultsmentioning

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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“…In contrast, the products cleaved from its hydrophobic derivates such as diacetate of carboxifluorescein (CFDA) are better retained within intact cells, although it has been reported that the cleavage product of this dye can be actively extruded from Saccharomyces cerevisiae [62,116]. Besides difficulties with dye uptake, some staining problems arise from dye extrusion [117,118]. Calcein-AM stands out as indicator of cell viability due to its superior cell retention and the relative insensitivity of its fluorescence to pH in the physiological range.…”

Section: Metabolic Activitymentioning

confidence: 97%

“…Besides beer production monitoring [152,227], FC has been applied to follow up wine [118,232,233] and cider fermentation processes [23,127,236]. The method was validated by comparing with other viability analysis techniques such as hemocytometry and plating [237,238].…”

Section: Saccharomyces Cerevisiaementioning

confidence: 99%

Application of flow cytometry to industrial microbial bioprocesses

Dı́az

Herrero

Garcı́a

et al. 2010

Biochemical Engineering Journal

243

186

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“…These uses include indirect assessment of size distribution in the population and study of its statistical characteristics [13][14][15]30], development of protocols to distinguish between normal and arrested fermentations [4], validation of yeast population growth models [5,27] as well as monitoring of microbial evolution during the transition from exponential to stationary phases [1]. In flow cytometry, the incident light scattered from one cell is collected at two different angles: a narrow forward angle (forward scatter) and approximately a right angle from the light beam (side scatter).…”

Section: Introductionmentioning

confidence: 99%

Differences in stationary-phase cells of a commercial Saccharomyces cerevisiae wine yeast grown in aerobic and microaerophilic batch cultures assessed by electric particle analysis, light diffraction and flow cytometry

Portell

Gisbert

Carbó

et al. 2010

J Ind Microbiol Biotechnol

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We applied electric particle analysis, light diffraction and flow cytometry to obtain information on the morphological changes during the stationary phase of Saccharomyces cerevisiae. The reported analyses of S. cerevisiae populations were obtained under two different conditions, aerobic and microaerophilic, at 27°C. The samples analysed were taken at between 20 and 50 h from the beginning of culture. To assist in the interpretation of the observed distributions a complexity index was used. The aerobically grown culture reached significantly greater cell density. Under these conditions, the cell density experienced a much lower reduction (3%) compared with the microaerophilic conditions (30%). Under aerobic conditions, the mean cell size determined by both electric particle analysis and light diffraction was lower and remained similar throughout the experiment. Under microaerophilic conditions, the mean cell size determined by electric particle analysis decreased slightly as the culture progressed through the stationary phase. Forward and side scatter distributions revealed two cell subpopulations under both growth conditions. However, in the aerobic growing culture the two subpopulations were more separated and hence easier to distinguish. The distributions obtained with the three experimental techniques were analysed using the complexity index. This analysis suggested that a complexity index is a good descriptor of the changes that take place in a yeast population in the stationary phase, and that it aids in the discussion and understanding of the implications of these distributions obtained by these experimental techniques.

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Physiological significance of the cytometric distribution of fluorescent yeasts after viability staining

Cited by 12 publications

References 41 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

Application of flow cytometry to industrial microbial bioprocesses

Differences in stationary-phase cells of a commercial Saccharomyces cerevisiae wine yeast grown in aerobic and microaerophilic batch cultures assessed by electric particle analysis, light diffraction and flow cytometry

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