Direct enumeration of injured Escherichia coli cells harvested onto membrane filters

Abstract

Four methods testing either biosynthesis capacity (Direct Viable Counts), respiratory activity (CTC and XTT reduction assays), or membrane integrity (LIVE/DEAD® BacLight™ VIABILITY kit) were performed to enumerate E. coli injured cells previously harvested onto 0.2- μm black membranes. Some of them remained viable but were no longer culturable when exposed to hyperosmotic or oxidative stresses. The damaged cellular functions were dependent on the stress applied, and the results provided by these methods (except the XTT reduction method which is not sensitive enough), gave information on cell behaviour to one stress, and on the different cellular targets of this stress. The osmotic shock in artificial seawater led to moderate plasma membrane damage (0.8 log reduction in counts determined with the LIVE/DEAD® BacLight™ VIABILITY kit after 5 days in seawater), and to similar reductions of respiration and elongation capacities (2.7 log reduction of CTC counts, and 2.6 log reduction of DVC counts, respectively). After a 30-min oxidative stress induced by peracetic acid (8 mg l −1), the plasma membranes remained intact (no reduction of LIVE/DEAD® BacLight™ VIABILITY counts), and respiration was less impaired than elongation capacity (2.1 log reduction of CTC counts and 3.1 log reduction of DVC counts, respectively).