Single-cell analysis of cell viability after a biocide treatment unveils an absence of positive correlation between two commonly used viability markers

Discrimination among viable/active or dead/inactive cells in a microbial community is a vital question to address issues on ecological microbiology or microbiological quality control. It is commonly assumed that metabolically active cells (ChemchromeV6 [CV6] procedure) correspond to viable cells (direct viable count procedure [DVC]), although this assumption has never been demonstrated and is therefore a matter of debate. Indeed, simultaneous determination of cell viability and metabolic activity has never been performed on the same cells. Here, we developed a microfluidic device to investigate the viability and the metabolic activity of Escherichia coli cells at single-cell level. Cells were immobilized in a flow chamber in which different solutions were sequentially injected according to different scenarios. By using time-lapse microscopy combined with automated tracking procedures, we first successfully assessed the ability of cells to divide and their metabolic activity at single-cell level. Applying these two procedures on the same cells after a hypochlorous acid (HOCl) treatment, we showed that the ability of cells to divide and their metabolic activity were anticorrelated. These results indicate that the relation between CV6 uptake and cell viability may be partially incorrect. Care must be taken in using the terms “CV6-positive” and “viable” synonymously.