Conclusions

The rapid cytological estimation of true mi-crobial viability is extremely difficult (if not impossible in principle), not least because of the problems in defining viability in microbial cells. Despite the difficulties mentioned above, the view to which the authors of this unit subscribe (Kell et al., 1988) is that only culturabil-ity can provide a gold standard for positive viability. Although the flow cytometric approach has much to offer for the determination of mi-crobial viability, it must be emphasized that no single stain nor even cocktail is likely to be a universal indicator of viability, especially if we require that its interpretation reflect our ability to induce the cells to divide (see Table 11.3.3).

A cell that is killed by exposure to environmental extremes such as heat, pH, and so forth is likely to be very different from a cell that is killed by exposure to an antibiotic or other chemical, and different again from a cell that dies (loses culturability) owing to a lack of nutrients in its environment. Indeed, there is increasing evidence that a reversible loss of culturability may be both widespread and of adaptive significance (Mukamolova et al., 2003). Thus, the flow cytometric properties of a cell and the distribution of dye uptake within a population will depend on how the cells die, and more generally on their entire physiological state and its history.

The exploitation of the sorting capability of flow cytometers permits the design of experiments that carefully evaluate the applicability of so-called viability stains, and the authors strongly recommend that others adopt this approach. In many situations, it is desirable to know both the percentage viability and the viable count (number of viable cells in a unit volume). For these purposes, instruments that allow determination of absolute cell concentration are of particular value.

In conclusion, although there are as yet no perfect stains, careful protocol development currently allows valuable information to be obtained regarding specific problems. In the case of organisms that have not been exposed to excessive stress (e.g., laboratory cultures under normal conditions and in some cases clinical samples), substantial progress is being made towards the rapid and routine flow cytometric assessment of microbial viability or vitality.

Literature Cited

Adams, M.R. and Hope, C.F.A. (eds.). 1989. Rapid Methods in Food Microbiology. Elsevier/North-Holland, Amsterdam.

Aeschbacher, M., Reinhardt, C.A., andZbinden, G. 1986. A rapid cell membrane permeability test using fluorescent dyes and flow cytometry. Cell Biol. Toxicol. 2:247-255.

Amann, R.I., Ludwig, W., and Schleifer, K.H. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59:143-169.

Auger, J., Leonce, S., Jouannet, P., and Ronot, X. 1993. Flow cytometric sorting of living, highly motile human spermatozoa based on evaluation of their mitochondrial activity. J. Histochem. Cy-tochem. 41:1247-1251.

Back, J.P. and Kroll, R.G. 1991. The differential fluorescence of bacteria stained with acridine orange and the effects of heat. J. Appl. Bacteriol. 71:51-58.

Balajee, S.A. and Marr, K.A. 2002. Conidial viability assay for rapid susceptibility testing of Aspergillus species. J. Clin. Microbiol. 40:27412745.

Barer, M.R., Kaprelyants, A.S., Weichart, D.H., Harwood, C.R., and Kell, D.B. 1998. Microbial stress and culturability: Conceptual and operational domains. Microbiology. 144:20092010.

Beck, P. and Huber, R. 1997. Detection of cell viability in cultures of hyperthermophiles. FEMS Microbiol. Lett. 148:11-14.

Ben Amor, K., Breeuwer, P., Verbaarschot, P., Rombouts, F.M., Akkermans, A.D.L., De Vos, W.M., and Abee, T. 2002. Multiparametric flow cytometry and cell sorting for the assessment of viable, injured, and dead bifidobacterium cells during bile salt stress. Appl. Environ. Microbiol. 68:5209-5216.

Bergersen, O., Ronning, O., Helleman, A.L., Vek-terud, K., and Gjelsnes, O. 1995. Isolation and labelling of micro-organisms for use in flow cy-tometry. 1995. NORDFOOD conference, Turku (Abo), Finland.

Berglund, D.L., Taffs, R.E., and Robertson, N.P. 1987. A rapid analytical technique for flow cyto-metric analysis of cell viability using calcofluor white M2R. Cytometry. 8:421-426.

Bohmer, R.M. 1985. Flow cytometric detection of a two-step cell death induced by hyperthermia. Cytometry. 6:215-218.

Braux, A.S., Minet, J., TamanaiShacoori, Z., Riou, G., and Cormier, M. 1997. Direct enumeration of injured Escherichia coli cells harvested onto membrane filters. J. Microbiol. Methods. 31:1-8.

Breeuwer, P., Drocourt, J.L., Rombouts, F.M., and Abee, T. 1994. Energy-dependent, carrier-mediated extrusion of carboxyfluorescein from Saccharomyces cerevisiae allows rapid assessment of cell viability by flow cytometry. Appl. Environ. Microbiol. 60:1467-1472.

Breeuwer, P., Drocourt, J.L., Bunschoten, N., Zwi-etering, M.H., Rombouts, F.M., and Abee, T. 1995. Characterization of uptake and hydrolysis of fluorescein diacetate and carboxyfluorescein diacetate by intracellular esterases in Saccha-romyces cerevisiae, which result in accumulation of fluorescent product. Appl. Environ. Mi-crobiol. 61:1614-1619.

Buchmeier, N.A. and Libby, S.J. 1997. Dynamics of growth and death within a Salmonella typhimurium population during infection of macrophages. Can. J. Microbiol. 43:29-34.

Button, D.K., Schut, F., Quang, P., Martin, R., and Robertson, B.R. 1993. Viability and isolation of marine bacteria by dilution culture—theory, procedures, and initial results. Appl. Environ. Microbiol. 59:881-891.

Chen, L.B. 1988. Mitochondrial membrane potential in living cells. Ann. Rev. Cell Biol. 4:155181.

Chen, L.B., Summerhayes, I.C., Johnson, L.V., Walsh, M.L., Bernal, S.D., and Lampidis, T.J. 1982. Probing mitochondria in living cells with rhodamine 123. Cold Spring Harbor Symp. Quant. Biol. 46:141-155.

Clarke, R.G. and Pinder, A.C. 1998. Improved detection of bacteria by flow cytometry using a combination of antibody and viability markers. J. Appl. Microbiol. 84:577-584.

Comas, J. and Vives-Rego, J. 1998. Enumeration, viability and heterogeneity in Staphylococcus aureus cultures by flow cytometry. J. Microbiol. Methods. 32:45-53.

Darzynkiewicz, Z., Staiano-Coico, L., and Melamed, M.R. 1981. Increased mitochondrial uptake of rhodamine 123 during lymphocyte stimulation. Proc. Natl. Acad. Sci. U.S.A. 78:2383-2387.

Davey, H.M. and Kell, D.B. 1996. Flow cytome-try and cell sorting of heterogeneous microbial populations—the importance of single-cell analyses. Microbiol. Rev. 60:641-696.

Davey, H.M. and Kell, D.B. 1999. A portable flow cytometer for the detection and identification of microorganisms. In NATO Advanced Research Workshop on Rapid Methods for Monitoring the Environment for Biological Hazards. (P.J. Stopa, ed.). In press. Plenum, Warsaw, Poland.

Davey, H.M., Kaprelyants, A.S., and Kell, D.B. 1993. Flow cytometric analysis, using rhodamine 123, of Micrococcus luteus at low growth rate in chemostat culture. In Flow Cy-tometry in Microbiology. (D. Lloyd, ed.) pp. 8393. Springer-Verlag, London.

Decamp, O., Rajendran, N., Nakano, H., and Nair, G.B. 1997. Estimation of the viability of Vibrio cholerae 0139 by assessing cell membrane integrity. Microbios. 92:83-89.

Deere, D., Porter, J., Edwards, C., and Pickup, R. 1995. Evaluation of the suitability of bis-(1,3-dibutylbarbituric acid) trimethine oxonol, (diBA-C4(3)-), for the flow cytometric assessment of bacterial viability. FEMS Microbiol. Lett. 130:165-169.

Deere, D., Shen, J., Vesey, G., Bell, P., Bissinger, P., and Veal, D. 1998. Flow cytometry and cell sorting for yeast viability assessment and cell selection. Yeast. 14:147-160.

Diaper, J.P. and Edwards, C. 1994a. Flow cyto-metric detection of viable bacteria in compost. FEMS Microbiol. Ecol. 14:213-220.

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