The viability of Clavibacter michiganensis subsp. michiganensis (Cmm) was determined by measuring the intracellular pH (pH(in)) as a viability parameter. This was based on the observation that growth of Cmm was inhibited at pH 5.5 and below Therefore, viable cells should maintain their pH(in) above this pH value. The pH(in) of Cmm was determined using the fluorescent probe 5(and 6-)-carboxyfluorescein succinimidyl ester (cFSE). The pH(in) of Cmm cells exposed to acid treatments was determined using fluorescence spectrofluorometry, and for cells exposed to elevated temperatures, the pH(in) was determined using fluorescence spectrofluorometry and flow cytometry (FCM). A good correlation was found between the presence of a pH gradient and the number of colony-forming units (cfu) observed in plate counts. However, with the spectrofluorometry technique, the analysis is based on the whole cell population and the detection sensitivity of this technique is rather low, i.e., cell numbers of at least 10(7) cfu ml(-1) are needed for the analysis. Using FCM heated-treated and non-treated Cmm cells could be distinguished based on the absence and presence of a pH gradient, respectively. The major advantage of FCM is its high sensitivity, allowing analysis of microbial populations even at low numbers, i.e., 10(2)-10(3) cfu ml(-1).