A Possible Flow Cytometry-Based Viability and Vitality Assessment Protocol for Pathogenic O1 and O139 Postexposure to Simulated Gastric Fluid.

During the intake of contaminated water, for diarrheal disease to occur, must survive through the bactericidal digestive secretion of gastric fluid during passage through the stomach. Determining the viability of these bacteria is challenging, with the standard cultivation methods for viability being time-consuming and unable to culture cells that may still function accordingly. This study assessed the use of enzyme action and membrane integrity as alternatives for determining vitality and viability, respectively, in gastric acid-stressed pathogenic O1 and O139, using fluorescent probes thiazole orange (TO) for viability based on membrane integrity, carboxyfluorescein diacetate (CFDA) with acetoxymethyl ester (AM) for vitality based on metabolic activity, and propidium iodide (PI) for cell death/damage due to loss of membrane integrity, with flow cytometry. Simulated gastric fluid-treated bacterial cells were labelled with blends of TO+PI and CFDA-AM+PI, and these stained cells were separated into heterologous populations based on their fluorescence signal. The gastric acid exposed cells presented with high green fluorescence signals after staining with the metabolic probe CFDA-AM, which indicated intact (live) cells due to being metabolically active, whereas when the same cells were stained with the DNA probe (TO), these appeared to be in a "stressed state" due to loss of membrane integrity. Damaged cells (dead cells) showed high red fluorescence levels after staining with PI probe. The use of flow cytometry with fluorescent probes is a favorable method for evaluating the vitality and viability of bacteria when cells are labelled with a combination of CFDA-AM+PI.