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

Abstract

During the intake of contaminated water, for diarrheal disease to occur, Vibrio cholerae 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 Vibrio cholerae 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.

Attenuation of quorum sensing system and virulence in Vibrio cholerae by phytomolecules

“…The bacterial cell viability was determined using the propidium iodide staining method as described by Singh and Barnard (2021) with some modifications. Briefly, V. cholerae strains VC287 and N16961 were grown in the presence of quercetin and naringenin (50 μg/ml) at 37°C for 24 h. The overnight grown culture was diluted 500 times with LB broth to an OD 600nm = 1.…”

Opportunities in optical and electrical single-cell technologies to study microbial ecosystems

“…Applications of microbial flow cytometry have been around since the late 1970s (Paau et al, 1977), and have mostly been used to assess microbial quantities (Wang et al, 2010;Frossard et al, 2016;Chodkowski and Shade, 2017;Van Nevel et al, 2017;Gryp et al, 2021). However, phenotypic attributes of cells, including size, intracellular complexity, macromolecular composition, viability, vitality, and other properties based on scattered light or fluorescent signals of single cells in a heterogeneous population can be measured in high-throughput as well (Sieracki et al, 1999;Müller and Nebe-Von-Caron, 2010;Hatzenpichler et al, 2020;Singh and Barnard, 2021). It is important to note that certain properties can be obtained directly without further staining, while others require staining procedures.…”

Raman cell sorting for single-cell research

“…This technique serves not only for cell counting but also for an extensive array of studies delving into the extraction of biological information through single-cell data. High-throughput detection of properties such as cell size, intracellular complexity, and macromolecular composition is feasible ( Sieracki et al, 1999 ; Hatzenpichler et al, 2020 ; Singh and Barnard, 2021 ). The measurement of these phenotypic properties relies on the cell labeling technique used ( Müller and Nebe-von-Caron, 2010 ; Hatzenpichler et al, 2020 ; Rubbens and Props, 2021 ).…”