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

Adrien Ducret,Sam Dukan

doi:10.1002/mbo3.62

Abstract

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.

Highlights

In environmental microbiology, discrimination among viable or dead and active or inactive cells in a microbial community is a vital question to address issues on ecological microbiology, food safety, drinking water quality, infections of pathogens, or efficacy of disinfectants and antibiotics
Ability of cells to grow in selective media and form colonies on nutrient agar plates is routinely used as a retrospective criterion for the detection of viable bacteria
As cell elongation implies that the cell (1) has an intact membrane, (2) is metabolically active, and (3) is able to grow in the presence of nutrients, it is commonly assumed that the direct viable count (DVC) procedure is one of the most integrative procedures to estimate the cell viability at single-cell level, up to date

Summary

Introduction

Instead of observing only a specific metabolic activity of individual cells (Kogure et al 1979; Joux and Lebaron 1997), methods such as the direct viable count (DVC) procedure have been developed to assess the ability of cells to divide. As cell elongation implies that the cell (1) has an intact membrane, (2) is metabolically active, and (3) is able to grow in the presence of nutrients, it is commonly assumed that the DVC procedure is one of the most integrative procedures to estimate the cell viability at single-cell level, up to date.

Results

Conclusion