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.

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