Exploring the frontier between life and death in Escherichia coli: evaluation of different viability markers in live and heat- or UV-killed cells

A number of methods have been proposed to assess the viability of cells wit hout culture. Each method is based on criteria that reflect different level s of cellular integrity or functionality. As a consequence, the interpretat ion of viability is often ambiguous. The purposes of this work were to eval uate the capacity of current viability markers to distinguish between live and dead Escherichia coli K-12 cells. Methods that assess 'viability' by th e demonstration of metabolic activities (esterase activity, active electron transport chain, transport of glucose), cellular integrity (membrane integ rity, presence of nucleic acids) or the building up of cellular material (c ell elongation) have been evaluated in live and UV- or heat-killed cells. W ith live cells, viability markers detected cells in counts similar to the c olony count. However, these so-called viability markers could stain dead ce lls for some time after the lethal treatment. For the UV-killed cells, resi dual activities were detected even after 48 h of storage at 20 degreesC. Ho wever, for heat-treated cells, these activities disappeared within hours af ter heat treatment. Only a combination of fluorescence in situ hybridizatio n with rRNA probes and cell elongation in response to nutrients (in the pre sence of an inhibitor of cell division) had the ability to differentiate li ve from dead cells. Problems in the definition of a viable but nonculturabl e state are in part due to the lack of a clear definition of bacterial deat h. We consider death as an irreversible state where no growth, cell elongat ion or protein synthesis may occur. (C) 2000 Editions scientifiques et medi cales Elsevier SAS.