ASSESSMENT OF METABOLIC-ACTIVITY OF SINGLE BACTERIAL-CELLS - NEW DEVELOPMENTS IN MICROCOLONY AND DEHYDROGENASE ASSAYS

Some bacteria lose culturability in natural environments but retain me asurable metabolic activity and are thus considered viable. Several te chniques have been proposed to determine the activity of nonculturable cells. Due to the considerable physiological heterogeneity of bacteri al populations in the environment, it is imperative to apply methods w hich measure cellular activity at the single cell level. This review f ocuses on two promising methods: the microcolony assay and the respira tion assay based on reduction of 5-cyano-2,3-ditolyl tetrazolium chlor ide (CTC). In the microcolony assay, viable cells are identified by th eir ability to perform a limited number of cell divisions and this app roach is thus related to conventional culture techniques. Some recent methodological developments of the technique aiming at improving the i ncubation conditions and the detection of microcolonies are presented. Results obtained by the microcolony technique are used to introduce i ts advantages and limitations. The CTC-reduction assay determines a ce ntral cellular metabolic activity, but does not measure cell growth. R esults of studies using this assay are presented, and it is emphasized that great care should be taken to optimize assay conditions for the studied organisms. Finally, the results obtained by different viabilit y assays are compared. For a specific bacterium, several assays, addre ssing different aspects of cell metabolism, can provide comparable res ults suggesting that they provide meaningful viability estimates. On t he other hand, the use of viability assays on complex indigenous popul ations may be ambiguous.