Temperature-induced changes in bacterial physiology as determined by flow cytometry

Flow cytometry was employed to measure membrane integrity and potential, esterase activity, intracellular pH and production of superoxides in four bacterial strains when challenged with a temperature stress. The physiology of the bacterial strains is being studied in order to understand their behaviour and resistance under extreme conditions (such as temperature). This information is of potential usefulness in studies of bioremediation. Suspensions of Ralstonia metallidurans, Escherichia coli, Shewanella oneidensis and Deinococcus radiodurans were submitted to a 1-hour temperature stress (-170, -80, -20, 4, 15, 28, 37, 45, 50, 60 or 70 °C). Cell membrane permeability (propidium iodide) and potential (rhodamine-123, 3,3'-dihexyloxacarbocyanine iodide), intracellular esterase activity (fluorescein diacetate), production of reactive oxygen species (hydroethidine) and intracellular pH (carboxy-flurorescein diacetate succinimidyl ester (5(6)) were assessed to evaluate the physiological state and the overall fitness of individual bacterial cells under temperature stress. The four bacterial strains exhibited varying staining intensities. For the four bacterial strains, the physiological status was not affected at 4 and 37 °C in comparison with 28 °C, which was taken as the reference temperature. Moderate physiological damage was observed at 45 °C. Membrane permeability and potential, esterase activity, intracellular pH and production of reactive oxygen species were increased to high levels in all four strains after freezing (-170, -80 and -20 °C) or heat (50, 60 and 70 °C) treatments. In conclusion, it is apparent that a range of significant physiological alterations occurs after temperature stress and that fluorescent staining methods coupled with flow cytometry are useful for monitoring the changes induced not only by temperature stress but also other stresses like oxidative stress, radiation, pressure and pH that are extensively studied in our laboratories.