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Various dyes were assessed for their ability to discriminate between viable and non-viable bacteria. Two methods of killing were employed: by heat treatment or by gramicidin treatment. Staining was carried out in two ways; by staining directly in the medium or by washing cells prior to staining in buffer. Carbocyanine and rhodamine 123 dyes only exhibited small changes in fluorescence between viable and non-viable populations of bacteria. Both oxonol dye (bis 1,3-dibutylbarbituric acid trimethine oxonol) and calcafluor white proved much more useful.

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.

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Flow cytometry was used to study the lag, exponential, stationary and death phases of non-fixed cultures of Escherichia coli. Fluctuations in the forward angle scatter signal (FALS) were compared with cell size as measured by scanning electron microscopy at low temperature and image analysis. A correlation between FALS and cell size was not observed, although a correlation (r = -0.8) was obtained between FALS and the age of the culture for the first eleven days of incubation. Marked increases in FALS were observed during the lag phase, which were attributed both to changes in size and changes in structure or chemical composition. The distribution of FALS for all culture phases was asymmetric, and was associated with the cell size distribution.

Flow cytometry was used to study starvation of Escherichia coli in artificial sea water. Flow cytometric narrow‐angle light scatter was compared and assessed in relation to the cell sizes obtained by scanning electron microscopy at low temperature, and by image analysis. A correlation between narrow‐angle light scatter and cell size was not observed, although an acceptable correlation (γ= ‐0.845) between narrow‐angle light scatter and the starvation period was observed. On the other hand, the distribution of narrow‐angle light scatter at any given moment of culture is asymmetric and may be associated with the cell size distribution at the specific moment of starvation.

Viable, total and metabolically active bacteria were determined during linear alkylbenzene sulfonate degradation in coastal seawater. Viable bacteria were estimated by plate counts on marine agar media while the total and metabolically active bacteria were determined with the nucleic acid stain SYTO‐13 and the tetrazolium salt 5‐cyano‐2,3‐ditolyl tetrazolium chloride, respectively, in double stain procedures analyzed by flow cytometry. The double stain SYTO‐13/5‐cyano‐2,3‐ditolyl tetrazolium chloride is a rapid and simple method that discriminates bacterioplankton populations according to nucleic acid content and formazan formation. Linear alkylbenzene sulfonate degradation was monitored by high‐performance liquid chromatography analysis. Bacterioplankton degraded linear alkylbenzene sulfonate by growing to communities with a high percentage of viable and metabolically active bacteria. The bacteria produced were rapidly grazed by protozoa; however, the grazing took place mostly on metabolically active cells, which were larger than the rest of the population.

Viable and total bacteria were determined during linear alkylbenzene sulfonate (LAS) degradation in coastal seawater. Viable bacteria were determined by plate counts on marine agar media, while total bacteria were determined by flow cytometry after SYTO-13 staining. LAS degradation was monitored by high-performance liquid chromatography analysis. Seawater samples with LAS at 5 mg/L, incubated in the laboratory (20°C, 100 rpm, 30 d), showed in most cases a similar evolution in bacterioplankton abundance over time, characterized by three phases: (i) a progressive increase in bacterial density; (ii) a later decrease; and (iii) a fluctuating stationary phase. Bacterioplankton degraded the LAS by growing to populations with a high percentage of viable bacteria. The bacteria were rapidly grazed by protozoa, preventing anomalous high bacterial growth and ensuring the later channeling of LAS carbon to upper trophic levels.Paper no. S1154 in JSD 3, 303-308 (July 2000). KEY WORDS:Biodegradation, flow cytometry, LAS, marine bacteria, sea water, SYTO-13.Linear alkylbenzene sulfonate (LAS) is an anionic, biodegradable compound and one of the most widely used surfactants (1). Although LAS biodegradation has been frequently reported (2-5), studies on LAS biodegradation in the marine environment are relatively scarce (6-11). Coastal biodegradation data are of relevance, since littoral water is the final recipient of domestic and industrial wastewater. An understanding of the microbial and ecological basis of LAS degradation is important because of the volume and diversity of its usage and disposal. However, a deep understanding of LAS degradation processes in Nature is difficult, because enclosed experiments do not fully represent the marine reality, and pure bacterial cultures capable of LAS mineralization are lacking. This last situation suggests that LAS is predominantly biodegraded by a consortia of bacteria, as the reports of several groups affirm (12-15).The assessment of the impact of surfactants on coastal bacterioplankton is crucial because naturally occurring bacteria are the main (if not the only) agents responsible for the degradation and recycling of xenobiotics in Nature. However, studies of the effects of surfactants on naturally-occurring bacteria are scarce (16-20), mostly due to the lack of adequate techniques to study the changes in aquatic bacterial populations and their activities. A limiting step when studying LAS-degrading marine consortia is that most (>90%) marine bacteria are not cultivable in standard media (21,22), and so when a LAS-degrading consortium is used to isolate its individuals, some basic strains may be missed. It has already been reported that bacterial biomass produced when marine microorganism growth takes place at the expense of LAS is channeled to higher trophic levels by protozoa grazing (23,24). Protozoa, one of the most sensitive bioindicators of ecotoxicity in Nature, are more sensitive than bacteria, algae, invertebrates, and fish to the toxic effects of pollutants. Recent technol...