INTRODUCTION In nature, many bacteria manage to proliferate in very extreme environments such as polar (arctic and Antarctic) regions or deep in the sea, acidic natural springs of hot water or deserts. Such organisms are able to tolerate rough conditions and are often submitted to quick changes of the environment. Temperature, among the various environ- mental factors that have an impact on the viability of microor- ganisms, is of particular interest since it affects immediate- ly the inner part as well as the membrane of the cells. There- fore, understanding the physiological changes induced by temperature stress is of importance and could reveal strate- gies to increase microbial tolerance. Traditional microbiological techniques known to follow bacterial proliferation and viability have various disadvan- tages. First, dry-cell weight and optical density (OD) meas- urements give no information on the physiological and meta- bolic state of the bacteria. Furthermore, OD measurements do not take into account bacterial cell size or background medium composition that is know to particularly affect the OD. In addition, dry-cell weight data can only be obtained a long time after sampling and the provided data do not per- mit analysing the non-lethal stress situations under which the bacteria were submitted (Hewitt et al., 1999). Most impor- tantly, those methods imply population (bulk) measure- ments assuming that bacterial populations are homoge- neous with regard to their physiological and metabolic states. Other techniques commonly available in traditional microbi- ology are the dilution plating and the manual cell counts. The gold standard for proof of cell viability states that a cell is viable when it has been shown to proliferate. However, this definition assumes that all the viable cells was, not is, able to reproduce and does not take into account i) the nutrient requirement, ii) the potential for long lag phases and iii) the growth medium composition, all these factors can lead to dif- ferences of up to several logs per millilitre (Nebe-von-Caron et al., 2000; Stephens et al., 2000) or that stressed, dam- ages or “viable but nonculturable” bacteria can be hidden. The dilution plating method depends on postsampling growth (usually by using nonselective media) and results can take up to 12 hours before being obtained and analysed, which does not allow anymore changes in the process control. Finally, manual counts are fatiguing to accomplish. Flow cytometry allows the rapid analysis of large num- bers of cells, enabling the evaluation of the size and content of individual cells at the rapid rate of about 1000 cells s -1 . The multiparametric ability of flow cytometry and the increas- ing availability of fluorescent probes have been demon- strated in eukaryotic cells. Since the 1980s, various studies using rapid flow cytometry methods have been conducted to determine susceptibilities of external perturbations. Amongst Annals of Microbiology, 55 (1) 73-80 (2005) Temperature-induced changes in bacterial physiology as determined by flow cytometry Sara BAATOUT*, Patrick DE BOEVER, Max MERGEAY Laboratory of Microbiology and Radiobiology, Belgian Nuclear Research Centre, SCK•CEN, Boeretang 200, B-2400 Mol, Belgium Abstract - Flow cytometry was employed to measure membrane integrity and potential, esterase activity, intracellular pH and pro- duction 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 diac- etate 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 sta- tus 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 oxy- gen 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. Key words: flow cytometry, Ralstonia metallidurans, Escherichia coli, Shewanella oneidensis, Deinococcus radiodurans, membrane per- meability, membrane potential, esterase activity, intracellular pH, superoxide anion production, heat stress, freeze-thawing. * Corresponding Author. Phone: +32-14 33 27 29; Fax: +32-14 31 47 93; E-mail: sarah.baatout@sckcen.be