Flow cytometry analysis of cell viability of Lactobacillus reuteri during growth under different conditions

Abstract: Lactobacillus reuteri is a probiotic bacterium which when consumed or administered in certain recommended dosages can provide a number of health benefits to the consumer. To have an effect on the consumer, these bacteria must survive until they reach the gut. Process conditions during production of the cells determine their ability to adapt to subsequent environmental shifts during storage and administration. In this project, different conditions during shake flask cultivation were studied to determine the best conditions with respect to growth rate and cell viability as determined by flow cytometry. Specifically, the effect of growth temperature, agitation and supplementation of citrate and acetate to MRS medium was studied. 44°C gave the highest growth rate and cell titers, as well as a survivability of 96% of the cells during the first 8 hours of fermentation. Agitation of the cells in a shake flask incubator was found to be inhibitory for growth as well as detrimental to the survivability of the cells. In addition, a mixture of CFDA (carboxy fluorescein diacetate) and PI (propidium iodide) as fluorescent dyes to determine cell viability were benchmarked to SYBR green and PI. It was found that CFDA can be used instead of SYBR green to reach comparable results. The best assay conditions were to use an OD620 of 0.1, 10 mM CFDA, and 1.5 mM PI in a PBS buffer.