Chapter 6 Flow Cytometric Analysis, Using Rhodamine 123, of Micrococcus luteus at Low Growth Rate in Chemostat Culture Hazel M. Davey, Arseny S. Kaprelyants and Douglas B. Kell Introduction In microbiology it is often necessary to determine the number of viable cells in a sample or culture of interest. This is usually achieved by plating out the sample (diluted as required) on to an agar plate (Postgate 1969; Hattori 1988). There are several problems associated with this technique, the greatest of which is the length of time required to obtain the results. For some slowly growing organisms (e.g. Mycobacteria) it may take in excess of a week to determine how many cells were "viable" in the original sample, and even when the sample contains fast-growing organisms and the plates are incubated under optimal growth conditions a minimum of overnight growth is usually required before the resulting colonies can be counted. For some clinical specimens even an overnight incubation may be too long to be of use and consequently many alternatives to plate counts have been proposed in order to decrease the time required to determine numbers of viable cells (Harris and Kell 1985). Many of these so-called rapid methods involve the use of dyes to stain the cells. These include the DNA stain acridine orange (McPeters et a!. 1991) that is used on the (rather doubtful) assumption that nucleic acids are degraded rapidly following cell death, such that "viable" cells are stained differently from "dead" ones (see Back and Kroll 1991). Other viability stains such as methylene blue exploit the ability of the intact membrane of viable cells to exclude the dye (Jones 1987; Stoicheva et a!. 1989). Dye exclusion and DNA-staining methods rely mainly on microscopic examina- tion of the stained cells to assess viability, and for this reason they are slow to perform and are prone to subjective error. Plate counts, although usually considered to be a measure of viability, actually indicate only how many of the cells can replicate under the condi- tions provided for growth, which are likely to differ from those in the original sample (Roszak and Colwell 1987). Viability staining meanwhile provides information on how many of the cells can exclude the dye (i.e. how many of the cells have intact cell membranes). However, there is abundant D. Lloyd (ed.), Flow Cytometry in Microbiology © Springer-Verlag London 1993