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The influence of dissolved inorganic nitrogen (DIN) enrichments on cell-normalized carbon uptake rate, chlorophyll a content, and apparent cell size of a picoeukaryote (<1 microm) ( Ostreococcus tauri, the smallest eukaryotic cell) from a natural summer phytoplanktonic assemblage (<200 microm) in a northern Mediterranean Lagoon (Thau Lagoon) was studied in 20-L enclosures in June 1995. The natural planktonic community was incubated in situ for 24 h with initial ammonium and nitrate enrichments and compared to a control without enrichment. O. tauri cell-normalized productivity was estimated from the combination of flow cytometric (FCM) enumeration and 2-h (radioactive) carbonate incorporation measured on post-incubation size fractions (<1microm). No difference between the effects of the two DIN sources of enrichment on the studied biological parameters was measured during this experiment. Growth of natural O. tauri was perturbed by the low DIN availability in the control with drastic changes in cell productivity, chlorophyll content, and cell cycle (from the variations in apparent cell size) as compared to the DIN sufficiency conditions. On the other hand, a very high specific growth rate for natural O. tauri, up to 8 day(-1) under DIN enrichments, has been estimated from production and abundance data obtained during this experiment. This supports values measured in culture and suggests that the yearly high contribution of picophytoplankton to the total primary production in Thau Lagoon is likely to be due to their high growth rate rather than the previously suggested lack of grazing pressure.

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We compared the characteristics of ingestion of Prochlorococcus and Synechococcus by the marine heterotrophic nanoflagellate Pseudobodo sp. and by a mixed nanoflagellate culture (around 3 microm in size) obtained from an open sea oligotrophic area. Maximum ingestion rate on Synechococcus (2.7 Syn flagellate(-1) h(-1)) was reached at concentrations of 5 x 10(5) Syn mL(-1) and decreased between 6 x 10(5) and 1.5 x 10(6) Syn mL(-1). In order to validate laboratory data, one set of data on Synechococcus grazing was obtained during a field study in the oligotrophic northeastern Mediterranean Sea. Ingestion rates by heterotrophic nanoflagellates were related to Synechococcus abundance in the water, and the feeding rate showed a clear diel rhythm with consumption being highest during the night, declining during the day hours, and being lowest at dusk. Ingestion rates on Prochlorococcus increased linearly for the whole range of prey density used (i.e., from 1 x 10(3) to 3 x 10(6) Proc mL(-1)), with maximum ingestion of 6.7 Proc flagellate(-1) h(-1). However, for prey concentrations in the range of 10(3)-10(5), which are usually encountered in aquatic systems, ingestion rates were significantly less than on Synechococcus. In our experiments, both Prochlorococcus and Synechococcus proved to be poor food items for support of nanoflagellate growth.

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BACKGROUND: The present review is based on the identification of four major environmental crises that have been approached from a biological oceanographic viewpoint. These crises are the release of contaminants in near shore marine waters, the collapse of marine resources that were renewable until recently, the loss of biodiversity, and global climate change METHODS: The review examines the contribution of cytometry-based biological oceanography to the resolution of the four environmental crises. Using a database of 302 papers, flow cytometric (FCM) studies in biological oceanography over the 1989--1999 decade are examined. Future biological oceanographic applications of FCM are discussed. RESULTS: Most of the published FCM oceanographic studies focus on phytoplankton and bacterioplankton. Analysis of our 1989-1999 database shows the predominance of studies dedicated to phytoplankton (77%), followed by heterotrophic bacteria (21%). The latter progressively increased over the last decade, together with the improved understanding of the biogeochemical and trophic roles of marine bacteria. Most studies on these two microorganisms were conducted in vitro until 1996, after which the trend reversed in favor of in situ research. The most investigated areas were those with major international sampling efforts, related to the changing climate. Concerning environmental topics, 62% of papers on phytoplankton and bacterioplankton focused on the structure of microbial communities and fluxes (e.g., production, grazing); this provides the basis for biological oceanographic studies on resources and climate change. CONCLUSIONS: Future progress in the biological oceanographic use of FCM will likely fall into two categories, i.e., applications where FCM will be combined with the development of other methods and those where FCM will be the main analytical tool. It is expected that FCM and other cytometric approaches will improve the ability of biological oceanography to address the major environmental challenges that are confronting human societies.

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BACKGROUND: In the past decade, flow cytometry has become a useful and precise alternative to microscopic bacterial cell counts in aquatic samples. However, little evidence of its usefulness for the evaluation of bacterial biovolumes has emerged in from the literature. METHODS: The light scattering and cell volume of starved bacterial strains and natural bacterial communities from the Black Sea were measured by flow cytometry and epifluorescence microscopy, respectively, in order to establish a relationship between light scattering and cell volume. RESULTS: With the arc-lamp flow cytometer, forward angle light scatter (FALS) was related to cell size in both the starved strains and natural communities, although regression parameters differed. We tested the predictive capacity of the FALS verous cell size relationship in a bacterial community from the North Sea. That analysis showed that a reliable bacterial biovolume prediction of a natural bacterial community can be obtained from FALS using a model generated from natural bacterial community data. CONCLUSIONS: Bacterial biovolume is likely to be related to FALS measurements. It is possible to establish a generally applicable model derived from natural bacterial assemblages for flow cytometric estimation of bacterial biovolumes by light scatter.

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The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.

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The 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining method is commonly and increasingly used to detect and to enumerate actively respiring cells (CTC+ cells) in aquatic systems. However, this method remains controversial since some authors promote this technique while others pointed out several drawbacks of the method. Using flow cytometry (FCM), we showed that CTC staining kinetics vary greatly from one sample to another. Therefore, there is no universal staining protocol that can be applied to aquatic bacterial communities. Furthermore, using (3)H-leucine incorporation, it was shown that the CTC dye has a rapid toxic effect on bacterial cells by inhibiting protein synthesis, a key physiological function. The coupling of radioactive labelling with cell sorting by FCM suggested that CTC+ cells contribute to less than 60% of the whole bacterial activity determined at the community level. From these results, it is clearly demonstrated that the CTC method is not valid to detect active bacteria, i.e. cells responsible for bacterial production.

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BACKGROUND: Discrimination among viable, active, and inactive cells in aquatic ecosystems is of great importance to understand which species participate in microbial processes. In this study, a new approach combining flow cytometry (FCM), cell sorting, and molecular analyses was developed to compare the diversity of viable cells determined by different methods with the diversity of total cells and active cells. METHODS: Total bacteria were determined by SYBR-II staining. Viable bacteria were determined in water samples from different sites by plate count techniques and by the direct viable count (DVC) method. Substrate-responsive cells (i.e., DVC(+) cells) were distinguished from nonresponsive cells (i.e., DVC(-) cells) by FCM and sorted. The genetic diversity of the sorted cell fraction was compared with the diversity of the total microbial community and with that of the culturable cell fraction by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. The same approach was applied to a seawater sample enriched with nutrients. In this case, actively respiring cells (CTC+) were also enumerated by FCM, sorted, and analyzed by DGGE. RESULTS: The diversity of viable cells varied depending on the methods (traditional culture or DVC) used for viability assessment. Some phylotypes detected in the fraction of viable cells were not detectable at the community level (from total DNA). Similar results were found for actively respiring cells. Inversely, some phylotypes found at the community level were not found in viable and active cell-sorted fractions. It suggests that diversity determined at the community level includes nonactive and nonviable cells. CONCLUSION: This new approach allows investigation of the genetic diversity of viable and active cells in aquatic ecosystems. The diversity determined from sorted cells provides relevant ecological information and uncultured organisms can also be detected. New investigations in the field of microbial ecology such as the identification of species able to maintain cellular activity under environmental changes or in the presence of toxic compounds are now possible.

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A mesocosm experiment was performed to study the influence of nutrients on activity and diversity of bacterial assemblages from the Mediterranean Sea. Changes in the diversity of the predominant bacterial populations were monitored by DGGE fingerprinting of PCR products derived from 16S rRNA encoding genes. Fluctuations in the diversity of the most active populations was inferred by performing the DGGE fingerprinting on the basis of the cellular rRNA after reverse transcription and PCR amplification. DNA-derived DGGE patterns obtained from duplicate control and nutrient-enriched mesocosms showed differences in the development of the bacterial communities between control and nutrient-enriched experimental mesocosms. Multidimensional scaling analysis of the DNA-derived DGGE fingerprints indicated that duplicate treatments were reproducible. DNA- and RNA-derived DGGE fingerprints of bacterial assemblages changed over time, showing that the composition of the bacterial assemblages, as well as the most active bacterial populations changed during different phases of the incubation. Sequences of predominant DGGE bands in RNA-derived patterns were similar to 16S rRNA gene sequences of members of the alpha-, gamma- and delta-Proteobacteria and of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB). Bands corresponding to Ruegeria-like bacteria and members of the CFB became especially dominant during the course of incubation, suggesting that these populations were important contributors to bacterial production and activity in the post-grazing phase of the experiment.

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Quantitative and qualitative changes in bacterial communities from the Mediterranean Sea were compared in duplicate batch mesocosms with or without addition of inorganic nutrients. Methods including traditional microbial ecology techniques, molecular biology and flow cytometry were combined to determine abundances, production, cell size, activity, culturability and taxonomic diversity of bacterial cells. Addition of nutrients and confinement resulted in an increase of bacterial densities which were rapidly controlled by protozoan grazing. Changes in bacterial activity and morphology were observed during the growth phase of bacteria and under grazing pressure. The proportion of medium-size and culturable cells increased during the growth phase. These cells were preferentially consumed by grazers resulting in a strong limitation of bacterial production. As a consequence of the grazing pressure, large cells were produced and contributed to the remaining bacterial productivity after grazing. Grazing had an effect on the taxonomic composition of bacterial communities by preferentially eliminating gamma-Proteobacteria, alpha-Proteobacteria were preserved. It seems that some species from the genera Ruegeria and Cytophaga may have developed defence strategies to escape predation.

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The study of relationships between cell size and productivity is of key importance in microbial ecology to understand which members of natural aquatic communities are responsible for the overall activity and/or productivity. Flow sorting of microorganisms from different environmental samples was used to analyze the activity of bacterial cells depending on their biovolume. Bacterial cells from five different natural samples taken along the Mediterranean coast including fresh- and seawaters were incubated with tritiated leucine, then stained with SYTO 13 and sorted by flow cytometry according to their average side-angle-scattered (SSC) light. In all samples, a bell-shaped relationship was found between cell biovolume and activity, whereas activity of a given cell-size class varied between samples. In contrast, an inverse relationship was found between biovolumes and abundances. These results suggest that medium-sized cells with highest growth rates are probably submitted to intense grazing. For one sample, bacteria within five different size classes were sorted and the genetic diversity of cells within each sorted size class and that of the whole community were analyzed by the denaturing gradient gel electrophoresis (DGGE) method. The genetic diversity, as determined at the community level was highly represented into the pool of small cells, whereas only few species were present into larger cell subpopulations. The results suggest that only a few genotypes may be dominant within the largest and most productive cells. Furthermore, cell size polymorphism as well as heterogeneous cellular activities were found within some species.

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> Abstract A new procedure to investigate the relationship between bacterial cell size and activity at the cellular level has been developed; it is based on the coupling of radioactive labeling of bacterial cells and cell sorting by flow cytometry after SYTO 13 staining. Before sorting, bacterial cells were incubated in the presence of tritiated leucine using a procedure similar to that used for measuring bacterial production by leucine incorporation and then stained with SYTO 13. Subpopulations of bacterial cells were sorted according to their average right-angle light scatter (RALS) and fluorescence. Average RALS was shown to be significantly related to the average biovolume. Experiments were performed on samples collected at different times in a Mediterranean seawater mesocosm enriched with nitrogen and phosphorus. At four sampling times, bacteria were sorted in two subpopulations (cells smaller and larger than 0.25 µm(3)). The results indicate that, at each sampling time, the growth rate of larger cells was higher than that of smaller cells. In order to confirm this tendency, cell sorting was performed on six subpopulations differing in average biovolume during the mesocosm follow-up. A clear increase of the bacterial growth rates was observed with increasing cell size for the conditions met in this enriched mesocosm.http://link.springer-ny.com/link/service/journals/00248/bibs/38n2p180.html

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Microorganisms (unicellular algae, bacteria) constitute fundamental compartments of aquatic ecosystems because of their high concentrations and activities. The evaluation and understanding of their behavior and role raise different problems for which traditional methodologies are often inadequate, whether they refer to global or classical microscopic analyses. Flow cytometry (FCM) has been recently used to study microorganisms in aquatic environments. Although this technology is still applied on a limited scale in our field, a large number of works has been done showing that FCM seems to be a promising tool for aquatic microbial ecology. This paper summarizes, from the literature produced during the last decade and with original data obtained in our laboratory, the main questions related to the cell identification, the evaluation of cell viability, biomasses and productions and the measurements of bacterial and phytoplanktonic activities. The representatives of sampling and observation scales is also discussed within the framework of the FCM measurements.

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A simple method was developed to preserve marine phytoplankton populations so that delayed flow cytometric analyses could be performed. The method consisted of immediate fixation with 1% glutaraldehyde (final concentration) followed by storage in liquid nitrogen. The method was tested on individual algal species and on natural samples from both coastal and pelagic waters. In most cases, it caused little cell loss and preserved well both forward angle light scatter and chlorophyll fluorescence, but phycoerythrin fluorescence sometimes was significantly increased. The technique performed best for the small-sized picoplankton (below 2 microns) such as Synechococcus cyanobacteria or the newly discovered oceanic prochlorophytes. For larger-sized cells it had to be applied on a case by case basis as some fragile species, particularly dinoflagellates and cryptophytes, were poorly preserved.

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