Flow cytometry has become a valuable tool in aquatic and environmental microbiology that combines direct and rapid assays to determine numbers, cell size distribution and additional biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population or community. Flow cytometry exhibits three unique technical properties of high potential to study the microbiology of aquatic systems: (i) its tremendous velocity to obtain and process data; (ii) the sorting capacity of some cytometers, which allows the transfer of specific populations or even single cells to a determined location, thus allowing further physical, chemical, biological or molecular analysis; and (iii) high-speed multiparametric data acquisition and multivariate data analysis. Flow cytometry is now commonly used in aquatic microbiology, although the application of cell sorting to microbial ecology and quantification of heterotrophic nanoflagellates and viruses is still under development. The recent development of laser scanning cytometry also provides a new way to further analyse sorted cells or cells recovered on filter membranes or slides. The main infrastructure limitations of flow cytometry are: cost, need for skilled and well-trained operators, and adequate refrigeration systems for high-powered lasers and cell sorters. The selection and obtaining of the optimal fluorochromes, control microorganisms and validations for a specific application may sometimes be difficult to accomplish.

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Bacterial diversity and activity were simultaneously investigated by microbial ecological and molecular biological methods along an estuarine gradient from the Rhone River to the Mediterranean Sea. Following a Lagrangian strategy, we sampled plume, frontal and marine layers. The sampled estuarine gradient exhibited large changes both in physico-chemical and in microbiological characteristics. Bacterial abundances and activities showed a more drastic decrease in the low salinity range of the gradient than expected from simple dilution models, indicating that an important fraction of freshwater bacteria disappeared in the mixing area. High specific activities, in particular for leucine, in the marine end-part of the gradient suggested important bacterial protein synthesis, which may be a sign of an active survival strategy for bacterial communities subjected to oligotrophic conditions. Bacterial genetic diversity of the sampled estuarine area, as estimated by the number of DNA-derived denaturing gradient gel electrophoresis (DGGE) bands, was high (13 to 55 bands) compared to that reported in other aquatic ecosystems. This high diversity may be the consequence of the interface position of estuaries. The proportion of active populations was estimated using the ratio of DGGE bands derived from RNA and DNA. This ratio was lower in Rhone water than in marine water, indicating that only a part of the constitutive populations were active, while the activity was distributed within a larger fraction of populations in the marine assemblage. Very few DGGE bands detected in freshwater samples were also detected in the marine end-part of the gradient, suggesting that a very limited number of freshwater bacteria could survive under marine conditions. Detection of these freshwater populations from RNA might indicate that these bacteria were able to synthesize different stress proteins as the result of a survival strategy or that these bacteria were able to maintain metabolic activity under marine conditions. The structure of marine communities was strongly affected by decreasing salinity. However, it seems that the decrease of DNAderived bands may simply have been the consequence of the mixing of marine and freshwater. No obvious relationship between genetic richness and activity changes was observed. This lack of a relationship may be the consequence of a very short residence time of water in the mixing area studied.

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The use of flow cytometry in microbiology allows rapid characterization of cells from a nonhomogeneous population. A method based on flow cytometry to assess the effects of lethal agents and the bacterial survival in starved cultures through the use of membrane potential-sensitive dyes and a nucleic acid marker is presented. The use of propidium iodide, rhodamine, and oxonol has facilitated the differentiation of cells of Escherichia coli and Salmonella typhimurium of various states of vitality following various treatments (heat, sonication, electroporation, and incubation with gramicidin) and during starvation in artificial seawater. The fluorescence intensity is directly correlated with viable cell counts for rhodamine 123 labelling, whereas oxonol and propidium iodide labelling is inversely correlated with viable counts. The distribution of rhodamine and oxonol uptake during starvation-survival clearly indicates that single-species starved bacteria are heterogeneous populations, and flow cytometry can be a fundamental tool for quantifying this heterogeneity.