mardi 21 avril 2009
par   G. Grégori

Bamdad M, Brousseau P, Denizeau F (1999) Identification of a multidrug resistance-like system in Tetrahymena pyriformis : evidence for a new detoxication mechanism in freshwater ciliates. FEBS Lett 456 :389-393


The freshwater ciliate Tetrahymena pyriformis is an ubiquitous organism that is present in all aquatic ecosystems. This protozoan showed a clear resistance against some polycyclic aromatic hydrocarbons which can be attributed to an efflux pump probably of the multidrug resistance (MDR) type. Immunocytochemical detection showed a positive stain of ciliate cells using the monoclonal antibodies 4E3, raised against P-glycoprotein (P-gp). The kinetics of P-gp expression were studied for control cultures and cultures treated with 15 microM benzo(a)pyrene. Western blot analysis using the Ab1, anti-P-gp polyclonal antibodies indicates the presence of two bands of 66 and 96 kDa of which the intensity increased with time in benzo(a)pyrene-treated ciliates. Uptake experiments with target compounds for the MDR pump, namely adriamycin, rhodamine 123 and two polycyclic aromatic hydrocarbons, benzo(a)pyrene and 7,12-dimethylbenzanthracene, were carried out by flow cytometry, in the presence or absence of cyclosporin (an inhibitor of the multidrug resistant pump). The data indicate that the accumulation of these compounds by ciliate cells is significantly enhanced in the presence of cyclosporin. This suggests that Tetrahymena is provided with a P-gp-like system that is functionally active in a way similar to that of the mammalian P-gp.

Bihari N, Batel R, Zahn RK (1999) Flow cytometry in marine environmental research. Periodicum Biologorum 101 :151-155

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Background and purpose : Exposure of marine organisms to environmental contaminants is a global problem. In order to establish effective pollution, control measurement for water conservation, besides emission and contaminant levels, their effect on hereditary material (DNA) must be taken into consideration. Better understanding of responses of the organism to these environmental impacts could be obtained by flow cytometry analysis of cellular DNA content.
Materials and methods : The DNA content of hemocytes of the mussel Mytilus galloprovincialis transferred from uncontaminated mariculture area to the area contaminated with industrial waste waters ("mussel watch") was investigated. The measurement of DNA content using flow cytometry is based on the ability of certain fluorescent dyes (DAPI) to bind stoichiometrically to DNA under appropriate staining conditions. By comparing the intensity of each mussel hemocyte to the intensity of each hemocyte containing normal diploid amounts of DNA, the relative quantity of DNA in the hemocytes of interest is determined.
Results and conclusions : For the first time ii was shown that haemocyte DNA content in the mussels from the vicinity of Rovinj, Istrian coast, Northern Adriatic, Croatia, was influenced by industrial waste wafers. We can conclude that G(1)-arrest and G(2)-delay appear to represent an active response to DNA damage in mussel hemocytes caused by environmental contamination. Thus, applications of flow cytometry in marine environmental research give us information about health status of organisms as well as about environmental conditions themselves.

Bouchard N, Pelletier E, Fournier M (1999) Effects of butyltin compounds on phagocytic activity of hemocytes from three marine bivalves. Environmental Toxicology and Chemistry 18 :519-522

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Effects of tributyltin (TBT), dibutyltin (DBT), and monobutyltin (MBT) on the in vitro phagocytic activity of hemocytes from three marine bivalve species, Mytilus edulis, Mya arenaria, and Mactromeris polynyma, were determined using flow cytometry. Phagocytosis was reduced with increasing doses of TBT and DBT The toxicity of butyltins on hemocytes decreased in the order DBT > TBT > MET, and comparison of the relative sensitivity of the three species showed that blue mussels (M. edulis) were more tolerant of butyltin compounds than both clam species. Toxicity mechanisms of butyltins are discussed and compared to those of other metals.

Brussaard CPD, Thyrhaug R, Marie D, Bratbak G (1999) Flow cytometric analyses of viral infection in two marine phytoplankton species, Micromonas pusilla (Prasinophyceae) and Phaeocystis pouchetii (Prymnesiophyceae). Journal of Phycology 35 :941-948

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Cell characteristics of two axenic marine phytoplankton species, Micromonas pusilla (Butscher) Manton et Parke and Phaeocystis pouchetii (Hariot) Lagerheim, were followed during viral infection using flow cytometry, Distinct differences between noninfected and infected cultures were detected in the forward scatter intensities for both algal species. Changes in side scatter signals on viral infection were found only for P, pouchetii. Chlorophyll red fluorescence intensity per cell decreased gradually over time in the infected cultures, DNA analyses were performed using the nucleic acid-specific fluorescent dye SYBR Green I. Shortly after infection the fraction of algal cells with more than one genome equivalent increased for both species because of the replication of viral DNA in the infected cells. Over time, a population of algal cells with low red autofluorescence and low DNA fluorescence developed, likely representing algal cells just prior to viral lysis, The present study provides insight into basic virus-algal host cell interactions. It shows that flow cytometry can be a useful tool to discriminate between virus infected and noninfected phytoplankton cells.

Catala P, Parthuisot N, Bernard L, Baudart J, Lemarchand K, Lebaron P (1999) Effectiveness of CSE to counterstain particles and dead bacterial cells with permeabilised membranes : application to viability assessment in waters. Fems Microbiology Letters 178 :219-226

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The CSE dye (Chemunex, Maisons-Alfort, France) was combined with an activity marker to improve bacterial activity assessment in natural waters. Its effectiveness to counterstain dead cells with permeabilised membranes was investigated on live and dead cells of a variety of strains from collections or isolated from the natural environment. Cells were killed by hear treatment. For all strains tested, the fluorescent dye showed an intense staining of killed cells having permeabilised membranes while no significant signal was detected when applied to live cells. Furthermore, the CSE dye had no toxicity on viable cells. Then, CSE was combined with the ChemChrome V6 dye (Chemunex) to assess the activity of bacterial cells in different waters. Both fluorescences were analysed simultaneously by solid-phase cytometry. The active cell counts were sometimes lower when both dyes were combined suggesting that CSE was able to counterstain cells having a residual esterase activity and compromised membranes. These cells were subtracted from the active cell counts determined with ChemChrome V6. In most samples, active cell counts were congruent with those determined by the direct viable count method. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Cavender-Bares KK, Mann EL, Chisholm SW, Ondrusek ME, Bidigare RR (1999) Differential response of equatorial Pacific phytoplankton to iron fertilization. Limnology and Oceanography 44 :237-246

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Recent unenclosed iron-fertilization experiments in the equatorial Pacific Ocean have shown that phytoplankton biomass can be increased substantially by the addition of iron. Analyses of size-fractionated chlorophyll indicate that much of the increase during the most recent fertilization experiment, IronEx II, occurred in the >10-mu m size fraction. We used flow cytometry, combined with taxon-specific pigment measurements by high-performance liquid chromatography (HPLC), to analyze the responses of five different groups of phytoplankton : Prochlorococcus, Synechococcus, ultraplankton, nanoplankton, and pennate diatoms. These results are unique in the suite of measurements from the IronEx studies in that they simultaneously examine individual cell properties, which are grazer independent, and population dynamics, which reflect the net result of growth and grazing. Our results show that the overall increase of chlorophyll a (Chl a) in the patch was due in part to increases in chlorophyll content per cell and in part to increases in cell numbers of specific groups. Cellular fluorescence was stimulated by iron addition in all five groups to a qualitatively similar degree and was correlated with taxon-specific changes in cellular pigments. In terms of net cell growth, however, these groups responded very differently. The groups that dominated the community before the addition of iron increased at most twofold in cell number ; Prochlorococcus actually decreased. In contrast, the initially rare pennate diatoms increased 15-fold in number by the peak of the iron-induced bloom. Within 1 week, this differential response led to a dramatic change in the phytoplankton community structure, from one dominated by picoplankton to one dominated by large diatoms. It is not known whether this shift would be sustained over extended periods of fertilization, a response that would ultimately change the structure of the food web.

Cembella AD, Lewis NI, Quilliam MA (1999) Spirolide composition of micro-extracted pooled cells isolated from natural plankton assemblages and from cultures of the dinoflagellate Alexandrium ostenfeldii. Nat Toxins 7 :197-206


A novel micro-extraction technique was applied to the extraction of biologically active macrocyclic imines known as spirolides from pooled individual cells isolated from spirolide-rich plankton material. For comparison, this method was also applied to pooled individual cells isolated from a unialgal culture of the marine dinoflagellate Alexandrium ostenfeldii (Paulsen) Balech & Tangen, a species known to produce spirolides. Both athecate cells and motile forms of gonyaulacoid dinoflagellates derived from size-fractionated plankton material from Nova Scotia, Canada were sorted and pooled by the glass micropipette isolation technique and by flow cytometry. The development of a highly sensitive analytical method for spirolides (detection limit 2 ng ml(-1) for spirolide B) using liquid chromatography-mass spectrometry (LC-MS) and application to micro-extracted samples allowed the accurate determination of spirolide composition in as few as 50 cells. Total spirolide concentrations (fmol cell(-1)) calculated from pooled micropipette isolated cells were very consistent with those based upon bulk- or micro-extractions of A. ostenfeldii cells from unialgal batch cultures in exponential growth phase. The results of the pooled cell selection from field material from two sites in Nova Scotia confirmed the association of spirolides with vegetative cells of A. ostenfeldii and related athecate forms. Combining these techniques represents a highly sensitive method for the analysis of marine toxins within complex plankton matrices, even when the toxigenic species is in low abundance, by enrichment of the target organism.

Choi JW, Sherr BF, Sherr EB (1999) Dead or alive ? A large fraction of ETS-inactive marine bacterioplankton cells, as assessed by reduction of CTC, can become ETS-active with incubation and substrate addition. Aquatic Microbial Ecology 18 :105-115

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The majority of bacteria suspended in seawater do not appear to be metabolically active or in good physiological condition as assessed by various methods. We tested the idea that a large fraction of ’inactive’ bacterial cells can become ’active’ with respect to detectable cell-specific electron transport system (ETS) activity, determined by the ability of cells to reduce the fluorogenic tetrazolium salt, CTC, when incubated for periods of time with or without additional substrate. Aliquots of 1.0 mu m filtered seawater were amended with mixed antibiotics to inhibit DNA synthesis and thus cell division, and incubated at in situ (12.8 and 16.4 degrees C) temperature or at 20 degrees C. Additions included : phosphate (0.83 mM.P, 5.3 mgP l(-1)), ammonium (1.67 mM N, 23.4 mgN l(-1)), and organic carbon as glucose, mixed amino acids or yeast extract (8.33 mM C, 100 mgC l(-1)). At 20 degrees C, the addition of mixed amino acids and yeast extract resulted in a large increase of % ETS-active cells (CTC-positive [CTC+] cells), from 1.9-2.4% at 0 h to 55-87% CTC+ cells by 21 to 28 h. At in situ temperature, the increase in % CTC+ cells was less, and the glucose addition caused the greatest increase in % CTC+ cells. Under conditions of increased temperature and high concentration of organic substrate, a large proportion of the apparently ’inactive’ bacteria can become highly ETS-active within a day, suggesting that these cells are in fact alive, and are capable of attaining significant metabolic activity. The different response patterns of the bacterial assemblages at 20 degrees C compared to those at 12.8 and 16.4 degrees C suggests that temperature can be an important factor in bacterioplankton response to increase in substrate concentration.

Christaki U, Jacquet S, Dolan JR, Vaulot D, Rassoulzadegan F (1999) Growth and grazing on Prochlorococcus and Synechococcus by two marine ciliates. Limnology and Oceanography 44 :52-61

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The two most abundant marine autotrophic prokaryotes, Prochlorococcus and Synechococcus, often have different distributions in the ocean. For example, Synechococcus is restricted to the first 100 m, whereas Prochlorococcus extends much deeper in oligotrophic waters. This is in part explained by differences in adaptation to nutrient and light regimes. However, they could also be subjected to different predation rates. To explore this hypothesis, we compared the consumption of these two picoplankters by an algivorous ciliate, Strombidium sulcatum, and a bactivorous ciliate. Uronema sp. For both ciliate species, removal rates were higher by a factor of 3 to 10, for Synechococcus compared to Prochlorococcus when prey items were presented alone or together. The growth of the two ciliates fed Synechococcus and/or Prochlorococcus also differed. S. sulcatum grew well on both prey items, whether alone or together, whereas Uronema sp. grew slowly when fed Synechococcus and very poorly when fed Prochlorococcus either alone or with Synechococcus. Our results suggest that Prochlorococcus may be less subject to ciliate predation than Synechococcus.

Claustre H, Morel A, Babin M, Cailliau C, Marie D, Marty JC, Tailliez D, Vaulot D (1999) Variability in particle attenuation and chlorophyll fluorescence in the tropical Pacific : Scales, patterns, and biogeochemical implications. Journal of Geophysical Research-Oceans 104 :3401-3422

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The variability in particle attenuation (c(p)) and in chlorophyll in situ fluorescence (F-is) was examined in November 1994 along 150 degrees W in the Pacific Ocean. Two main sources of variation in c(p) and F-is profiles are identified by analyzing data from a 16 degrees S-1 degrees N transect, and from two 5 day stations (5 degrees S and 16 degrees S). The first source reflects changes in the trophic status resulting from prevailing hydrodynamical regimes at large scales. By using flow cytometric data and some assumptions about the size distribution of the different biological stocks, a decomposition of c(p) into its vegetal (c(veg)) and nonvegetal (c(nveg)) components is attempted. Within the euphotic layer, c(veg) accounts for 43% of the total c(p) Signal at the equator and for only 20% in the South Pacific gyre. The nonvegetal component is then subdivided into heterotrophic organisms and detritus contributions. The detrital material is an important contributor with 43% of c(p) at 5 degrees S and 55% at 16 degrees S. A further decomposition of F-is and c(veg) into the three dominant phytoplanktonic groups (Prochlorococcus, Synechococcus, and picoeucaryotes) confirms that picoeucaryotes are important contributors of the vegetal biomass, especially within and below the deep chlorophyll maximum (DCM) (>50% of the algal stock) at 16 degrees S. The second, and essentially local, source of variation is related to specific rhythms in biological and physiological processes. The prominent signals detected during the time series occur at the daily scale : besides the pronounced fluorescence depression at noon in upper layers, particle attenuation in all the layers examined and fluorescence in the DCM display conspicuous daily oscillations. They result from the balance between daytime accumulation and night removal of particles, of algal cells in particular. Finally, the estimation of cp-based growth rates points out the surprisingly rapid turnover time of the whole particulate matter stock in oligotrophic waters (16 degrees S), not only in the euphotic zone (0.63 d(-1)) but also within the dimly lit layers of the DCM (0.36 d(-1)). The corresponding growth rate at 5 degrees S, within a quasi-mesotrophic regime, is 0.47 d(-1) within the euphotic zone.

Collier JL, Campbell L (1999) Flow cytometry in molecular aquatic ecology. Hydrobiologia 401 :33-53

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In working towards understanding ecosystems that are often dominated by microorganisms, aquatic ecologists have historically relied on measuring bulk, community-level properties and synecological processes. However, developing a mechanistic and predictive explanation for the factors structuring aquatic ecosystems will require understanding the roles that individual microorganisms play in these higher-order phenomena. The application of molecular biological techniques to examine nucleic acids extracted in bulk from microbial communities can provide information about the taxonomic structure of microbial communities and the physiological ecology of particular types of organisms at various levels of specificity. Yet, even if accomplished at the ’species’ level, these data still represent bulk parameters because they can reveal only an average value for the organisms and community of interest. A more detailed view may be gained by investigations performed at a single-cell level. Flow cytometry allows the measurement of one cell at a time, at a rate of thousands of cells per second. When combined with fluorescent stains, including nucleic acid and antibody-based molecular probes, flow cytometry permits rapid analysis of cell-specific information for particular types of microbes within complex microbial assemblages. Thus, the autecology of microbial populations and structure of microbial communities can be examined from the vantage point of the individual cells comprising them. By bringing the level of analysis closer to the relevant scale of the organisms being investigated, the combination of molecular tools and flow cytometry will bring powerful new insights into the autecology of aquatic microorganisms.

Corzo A, Jimenez-Gomez F, Gordillo FJL, Garcia-Ruiz R, Niell FX (1999) Synechococcus and Prochlorococcus-like populations detected by flow cytometry in a eutrophic reservoir in summer. Journal of Plankton Research 21 :1575-1581

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Particles with characteristics similar to marine free-living prochlorophytes have been detected by flow cytometry during summer in a eutrophic reservoir in the south of Spain. The Prochlorococcus-like particles showed a vertical distribution similar to Synechococcus. Both populations displayed a subsuperficial maximum at 5 m.

Dubelaar GB, Gerritzen PL, Beeker AE, Jonker RR, Tangen K (1999) Design and first results of CytoBuoy : a wireless flow cytometer for in situ analysis of marine and fresh waters. Cytometry 37 :247-254


BACKGROUND:The high costs of microscopical determination and counting of phytoplankton often limit sampling frequencies below an acceptable level for the monitoring of dynamic ecosystems. Although having a limited discrimination power, flow cytometry allows the analysis of large numbers of samples to a level that is sufficient for many basic monitoring jobs. For this purpose, flow cytometers should not be restricted to research laboratories. We report here on the development of an in situ flow cytometer for autonomous operation inside a small moored buoy or on other platforms. METHODS AND RESULTS : Operational specifications served a wide range of applications in the aquatic field. Specific conditions had to be met with respect to the operation platform and autonomy. A small, battery-operated flow cytometer resulted, requiring no external sheath fluid supply. Because it was designed to operate in a buoy, we call it CytoBuoy. Sampling, analysis, and radio transmission of the data proceed automatically at user-defined intervals. A powerful feature is the acquisition and radio transmission of full detector pulse shapes of each particle. This provides valuable morphological information for particles larger than the 5-microm laser focus. CONCLUSIONS:CytoBuoy allows on-line in situ particle analysis, estimation of phytoplankton biomass, and discrimination between different phytoplankton groups. This will increase the applicability of flow cytometry in the field of environmental monitoring.

Dubelaar GBJ, Gerritzen PL, Beeker AER, Jonker RR, Tangen K (1999) Design and first results of CytoBuoy : A wireless flow cytometer for in situ analysis of marine and fresh waters. Cytometry 37 :247-254

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Background : The high costs of microscopical determination and counting of phytoplankton often limit sampling frequencies below an acceptable level for the monitoring of dynamic ecosystems. Although having a limited discrimination power, flow cytometry allows the analysis of large numbers of samples to a level that is sufficient for many basic monitoring jobs. For this purpose, flow cytometers should not be restricted to research laboratories. We report here on the development of an in situ flow cytometer for autonomous operation inside a small moored buoy or on other platforms.
Methods and Results : Operational specifications served a wide range of applications in the aquatic field. Specific conditions had to be met with respect to the operation platform and autonomy. A small, battery-operated flow cytometer resulted, requiring no external sheath fluid supply. Because it was designed to operate in a buoy, we call it CytoBuoy. Sampling, analysis, and radio transmission of the data proceed automatically at user-defined intervals. A powerful feature is the acquisition and radio transmission of full detector pulse shapes of each particle. This provides valuable morphological information for particles larger than the 5-mu m laser focus.
Conclusions : CytoBuoy allows on-line in situ particle analysis, estimation of phytoplankton biomass, and discrimination between different phytoplankton groups. This will increase the applicability of flow cytometry in the field of environmental monitoring. Cytometry 37:247-254, 1999. (C) 1999 Wiley-Liss, Inc.

Gasol JM, Moran XAG (1999) Effects of filtration on bacterial activity and picoplankton community structure as assessed by flow cytometry. Aquatic Microbial Ecology 16 :251-264

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We used flow cytometry of autofluorescent and Syto13-stained marine bacteria and the uptake of tritiated leucine to assess the effects of different filter types on picoplankton abundance, community structure and bacterial activity in the filtrate. Coastal and oceanic samples from the NW and SW Mediterranean and the Atlantic coast of Galicia were size-fractionated using polycarbonate (PC), mixed cellulose esters (CE), aluminum oxide (IM) and glass fiber (GF) filters of 0.2 to 1.2 mu m nominal pore size from different brands. Flow cytometry of Syto13-stained marine bacteria and autofluorescent photosynthetic prokaryotes was used to analyze picoplankton abundance, size structure and community composition before and after filtration. We combined this capability with the detection of the changes in cell-specific heterotrophic activity in the filtrates. We found that the CE filters retained picoplankton better than the PC filters. The PC filters did not discriminate prokaryotes according to size as much as the GF and the CE filters did. In our hands the IM filters were no better than the CE filters. Bacterial activity in the filtrates increased in the PC and in the CE filtrates and this stimulation of bacterial activity was more important in the less productive environments. We conclude that care must be taken when PC filters are used for generating bacteria-free water, and that the use of CE 0.22 mu m filters is the best way of creating picoplankton-free water. However, the picoplankters that will go through the filters may encounter increased nutrient levels.

Gasol JM, Zweifel UL, Peters F, Fuhrman JA, Hagstrom A (1999) Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria. Appl Environ Microbiol 65 :4475-4483


Total bacterial abundances estimated with different epifluorescence microscopy methods (4’,6-diamidino-2-phenylindole [DAPI], SYBR Green, and Live/Dead) and with flow cytometry (Syto13) showed good correspondence throughout two microcosm experiments with coastal Mediterranean water. In the Syto13-stained samples we could differentiate bacteria with apparent high DNA (HDNA) content and bacteria with apparent low DNA (LDNA) content. HDNA bacteria, "live" bacteria (determined as such with the Molecular Probes Live/Dead BacLight bacterial viability kit), and nucleoid-containing bacteria (NuCC) comprised similar fractions of the total bacterial community. Similarly, LDNA bacteria and "dead" bacteria (determined with the kit) comprised a similar fraction of the total bacterial community in one of the experiments. The rates of change of each type of bacteria during the microcosm experiments were also positively correlated between methods. In various experiments where predator pressure on bacteria had been reduced, we detected growth of the HDNA bacteria without concomitant growth of the LDNA bacteria, such that the percentage contribution of HDNA bacteria to total bacterial numbers (%HDNA) increased. This indicates that the HDNA bacteria are the dynamic members of the bacterial assemblage. Given how quickly and easily the numbers of HDNA and LDNA bacteria can be obtained, and given the similarity to the numbers of "live" cells and NuCC, the %HDNA is suggested as a reference value for the percentage of actively growing bacteria in marine planktonic environments.

Gisselson LA, Graneli E, Carlsson P (1999) Using cell cycle analysis to estimate in situ growth rate of the dinoflagellate Dinophysis acuminata : drawbacks of the DNA quantification method. Marine Ecology-Progress Series 184 :55-62

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In an attempt to use cell cycle analysis to estimate in situ gross growth rate of the dinoflagellate Dinophysis acuminata, epifluorescence microscopy in combination with an image analysis system was used to measure the relative DNA content of DAPI stained D. acuminata nuclei. To be able to estimate growth rate with this method, the time it takes for a cell to synthesise a second DNA copy and complete mitosis (the duration of the terminal event in the cell cycle) must be known or measurable. The duration of the terminal event is normally estimated graphically from diurnal variations in the phase fraction curves. No diurnal variation in the phase fractions was detected in this study, and consequently no reliable estimate of the duration of the terminal event could be obtained. The main drawback was the difficulty in delineating the S phase in DNA histograms based on only a few hundred cells. No dear S phase maximum could be obtained during our 48 h study. The presence of double-nucleated cells and a constantly high percentage (23 to 43%) of cells with double genomes (G2 + M phase cells) suggests, under the assumption that no cells can arrest in the G2 or M phase, that the population was actively dividing, but not clearly in phase with a diurnal cycle. Chang & Carpenter (1991) previously estimated the duration of the terminal event (the duration of the S + G2 + M phases) in this species to be 11 to 13 h. A 12 h duration of the terminal event in this study would yield specific growth rates of 0.69 to 0.75 d(-1). We conclude that the number of cells that can be measured using epifluorescence microscopy (a few hundred per sample) is too low to allow detection of a low degree of synchronisation, especially with regard to the S phase. Estimations of in situ growth rate of poorly synchronised populations of phytoplankton using the cell cycle technique will require DNA measurements on several thousand cells per sample, e.g. using flow cytometry or automated image cytometry.

Groben R, Colijn F, Medlin LK (1999) Meeting report : aquatic flow cytometry : achievements and prospects, Research- and Technology Centre Westcoast (FTZ), Busum, Germany, October 15-16, 1998. Protist 150 :7-10


Guillou L, Moon-Van Der Staay SY, Claustre H, Partensky F, Vaulot D (1999) Diversity and abundance of bolidophyceae (Heterokonta) in two oceanic regions. Applied and Environmental Microbiology 65 :4528-4536

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The diversity and abundance of the Bolidophyceae (Heterokonta), a newly described picoplanktonic algal class which is a sister group to the diatoms, was assessed in the equatorial Pacific Ocean and in the Mediterranean Sea by culture isolation, molecular biology techniques, and pigment analyses. Eight strains of Bolidophyceae were isolated in culture from different mesotrophic and oligotrophic areas. The corresponding small subunit (SSU) rRNA gene sequences allowed us to design two probes specific for the Bolidophyceae. These probes have been used in natural samples (i) to selectively amplify and detect Bolidophyceae sequences and (ii) to quantify the relative abundance of Bolidophyceae within the picoeukaryote community. Sequences available to date indicate that the class Bolidophyceae comprises at least three different clades, two corresponding to the previously described species Bolidomonas pacifica and Bolidomonas mediterranea and the third one corresponding to a subspecies of B. pacifica. Amplification of the SSU rRNA gene from natural samples with universal primers and hybridization using a Bolidomonas-specific probe followed by a eukaryote-specific probe allowed us to estimate the contribution of the Bolidophyceae to the eukaryotic DNA in both Pacific and Mediterranean waters to be lower than 1%. Similarly, high-performance liquid chromatography analyses of fucoxanthin, the major carotenoid present in Bolidophyceae, indicated that less than 4% of the total chlorophyll a in the picoplanktonic fraction in the equatorial Pacific was due to Bolidophyceae. Consequently, although strains of Bolidophyceae have been isolated from samples collected at several stations, this new class seems to have been a minor component of the natural picoeukaryotic populations in the ecosystems investigated, at least during the periods sampled.

Hjort K, Bernander R (1999) Changes in cell size and DNA content in Sulfolobus cultures during dilution and temperature shift experiments. Journal of Bacteriology 181 :5669-5675

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Stationary-phase cultures of different hyperthermophilic species of the archaeal genus Sulfolobus were diluted into fresh growth medium and analyzed by flow cytometry and phase-fluorescence microscopy, After dilution, cellular growth started rapidly but no nucleoid partition, cell division, or chromosome replication took place until the cells had been increasing in size for several hours, Initiation of chromosome replication required that the cells first go through partition and cell division, revealing a strong interdependence between these key cell cycle events. The time points at which nucleoid partition, division, and replication occurred after the dilution were used to estimate the relative lengths of the cell cycle periods. When exponentially growing cultures were diluted into fresh growth medium, there was an unexpected transient inhibition of growth and cell division, showing that the cultures did not maintain balanced growth. Furthermore, when cultures growing at 79 degrees C were shifted to room temperature or to ice-water baths, the cells were found to "freeze" in mid-growth. After a shift back to 79 degrees C, growth, replication, and division rapidly resumed and the mode and kinetics of the resumption differed depending upon the nature and length of the shifts. Dilution of stationary-phase cultures provides a simple protocol for the generation of partially synchronized populations that may be used to study cell cycle-specific events.

Husby MP, McBee K (1999) Nuclear DNA content variation and double-strand DNA breakage in white-footed mice (Peromyscus leucopus) collected from abandoned strip mines, Oklahoma, USA. Environmental Toxicology and Chemistry 18 :926-931

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White-footed mice (Peromyscus leucopus) were collected during the spring, summer, and fall from four metal-polluted abandoned mines and three reference sites in eastern Oklahoma. Two mines in east-central (Okmulgee County) Oklahoma, USA, were matched with a nearby reference site, as were two mines in northeastern Oklahoma (Craig County). A third, remote reference sire was located outside the Oklahoma coal belt in north-central Oklahoma (Payne County). Intercellular DNA content variation, measured as a coefficient of variation (CV) of nuclear DNA content among 20,000 cells per animal, was determined for splenocytes of individuals by Bow cytometry. Double-strand breakage in liver DNA from the same animals was compared using agarose gel electrophoresis. Mice trapped from mines were expected to have more intercellular DNA content variation and more DNA breakage when compared with mice from the matched and remote reference sites. With one exception, mice from mine sites did not show any significant increase in nuclear DNA content variation during any season. Electrophoresis revealed that mice from both Okmulgee County mines had significantly more DNA breakage compared with the intracounty reference site in the spring. In the summer, mice from one Craig County mine had significantly higher levels of DNA breakage compared with the remote reference.

Jochem FJ, Meyerdierks D (1999) Cytometric measurement of the DNA cell cycle in the presence of chlorophyll autofluorescence in marine eukaryotic phytoplankton by the blue-light excited dye YOYO-1. Marine Ecology-Progress Series 185 :301-307

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The novel blue-light excited fluorescent DNA dye YOYO-1(R) was tested on cultures of 8 eukaryotic phytoplankton species from the classes of Prymnesiophyceae, Bacillariophyceae, Coscinodiscophyceae, Prasinophyceae and Dinophyceae, and the coccoid cyanobacterium Synechococcus sp. In all tested species, YOYO staining of formalin fixed cells allowed the differentiaton of cell cycle phases of G1, S, and G2+M in the presence of chlorophyll autofluorescence in flow cytometric analyses. Diel cell cycle patterns of Emiliania huxleyi and Skeletonema costatum grown under a light:dark cycle and under continuous light could be established. Under the light/dark cycle, DNA replication occurred during the night and cell division in the early morning in both species. However, S. costatum spent a longer fraction of their cell cycle in S and G2+M phases than E. huxleyi did. Changes in cellular chlorophyll content and cell size could be observed in relation to cell cycle phasing. Cell division phasing was nearly lost under continuous light. Growth rates calculated from DNA cell cycle analyses agreed well with those established from cell counts.

Johnson Z, Landry ML, Bidigare RR, Brown SL, Campbell L, Gunderson J, Marra J, Trees C (1999) Energetics and growth kinetics of a deep Prochlorococcus spp. population in the Arabian Sea. Deep-Sea Research Part Ii-Topical Studies in Oceanography 46 :1719-1743

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During the US JGOFS process studies in the Arabian Sea (1995), secondary fluorescence maxima (SFM) were observed frequently at the oxic-anoxic interface at the extreme base of the euphotic zone. These secondary peaks were most prominent during the early NE monsoon in the central oligotrophic portion of the Arabian Sea, although they were spatially and temporally variable. Based on high performance liquid chromatography (HPLC) and flow cytometry analyses, SFM were determined to be populated almost exclusively by the marine cyanobacterium Prochlorococcus spp. While SFM were about half the magnitude of primary fluorescence peaks, chlorophyll a biomass was typically an order of magnitude less than at the primary maxima (although total chlorophyll (a + b) differed only by a factor of two). Photosynthesis versus irradiance response curves revealed an efficient population adapted to extremely low light (similar to 0.02-0.05% surface irradiance) largely through increased light absorption capabilities. A theoretical spectral irradiance absorption efficiency model based on available spectral irradiance, individual cell properties, and bulk particulate spectral absorption also supports a well-adapted low-light population. Deck-incubated C-14 uptake as well as dilution growth experiments revealed instantaneous growth rates on the order of mu = 0.01 d(-1). However, additional in situ observations suggest SFM populations may be more dynamic than the growth rates estimates from shipboard bottle incubations predict. We advance four hypotheses for the regulation of SFM populations including : (1) reduced loss rates, (2) discontinuous environmental conditions. (3) enhanced sub-oxic growth, and (4) physical mechanisms. (C) 1999 Elsevier Science Ltd. All rights reserved.

Joux F, Jeffrey WH, Lebaron P, Mitchell DL (1999) Marine bacterial isolates display diverse responses to UV-B radiation. Appl Environ Microbiol 65 :3820-3827


The molecular and biological consequences of UV-B radiation were investigated by studying five species of marine bacteria and one enteric bacterium. Laboratory cultures were exposed to an artificial UV-B source and subjected to various post-UV irradiation treatments. Significant differences in survival subsequent to UV-B radiation were observed among the isolates, as measured by culturable counts. UV-B-induced DNA photodamage was investigated by using a highly specific radioimmunoassay to measure cyclobutane pyrimidine dimers (CPDs). The CPDs determined following UV-B exposure were comparable for all of the organisms except Sphingomonas sp. strain RB2256, a facultatively oligotrophic ultramicrobacterium. This organism exhibited little DNA damage and a high level of UV-B resistance. Physiological conditioning by growth phase and starvation did not change the UV-B sensitivity of marine bacteria. The rates of photoreactivation following exposure to UV-B were investigated by using different light sources (UV-A and cool white light). The rates of photoreactivation were greatest during UV-A exposure, although diverse responses were observed. The differences in sensitivity to UV-B radiation between strains were reduced after photoreactivation. The survival and CPD data obtained for Vibrio natriegens when we used two UV-B exposure periods interrupted by a repair period (photoreactivation plus dark repair) suggested that photoadaptation could occur. Our results revealed that there are wide variations in marine bacteria in their responses to UV radiation and subsequent repair strategies, suggesting that UV-B radiation may affect the microbial community structure in surface water.

Kfoury JR, Jr., Nakayasu C, Rodrigues Souza JC, Okamoto N (1999) Characterization of a monoclonal antibody specific to rainbow trout thrombocytes. J Exp Zool 284 :309-316


A monoclonal antibody (MAb) specific for rainbow trout thrombocytes was produced and its reactivity was demonstrated by flow cytometry and immuno-electron microscopy. Flow cytometry analysis showed that this MAb (TTL-7D11) reacted positively with about 30% of the peripheral blood leucocytes (PBL) and about 1%, 2%, and 11% of the pronephros, mesonephros, and spleen cells, respectively. Electron microscopy using immunogold labeling demonstrated that this MAb reacted strongly with thrombocytes, where gold beads could be seen attached only to the membrane and canalicular system of these cells. Positive and negative leucocytes for this MAb were obtained by magnetic cell separation. In the positive fraction, 96% of the cells were thrombocytes, while in the negative fraction no more than 3% were, which clearly showed a high purity of the positive fraction. Aggregation studies showed that about 75% of the positive fraction cells aggregated after being mixed with U-46619 thromboxane-mimetic, whereas in the negative fraction only 10% of the cells did so. Thus, utilizing the TTL-7D11 we have succeeded in isolating a pure thrombocyte population, and this would facilitate further studies, particularly on their characteristics and function(s).

Knowles JF (1999) Long-term irradiation of a marine fish, the plaice Pleuronectes platessa : an assessment of the effects on size and composition of the testes and of possible genotoxic changes in peripheral erythrocytes. Int J Radiat Biol 75 :773-782


PURPOSE : Previous investigation showed the very significant effects of chronic gamma-radiation on plaice testes at mean absorbed dose rates as low as 1.3 mGy h(-1) given over a period of 168 days (accumulated dose 4.7 Gy). The present paper examines the effects on the testes of exposure to even lower dose rates of gamma-radiation given over periods of 73 days or 197 days. In addition, the use of micronucleus counts and flow-cytometric measurement of nuclear DNA content in samples of peripheral blood for monitoring genotoxic effects has been assessed. MATERIALS AND METHODS : In Experiment 1, adult male plaice were exposed at mean absorbed dose rates of 0.25, 0.5 or 1.2 mGy h(-1) for 73 days (mean accumulated doses of 0.43, 0.85 and 2.03 Gy, respectively) and in Experiment 2 to 0.24, 0.5 or 1.0 mGy h(-1) for 197 days (mean accumulated doses of 1.07, 2.24 and 4.57 Gy, respectively). At termination the testes were removed, weighed and sections were prepared and examined histometrically. In addition, in Experiment 2, blood samples were taken during exposure and at termination. Blood smears were scored for micronuclei and samples processed and examined for nuclear DNA content by flow cytometry. RESULTS : Significant reductions in testis weight were seen in all radiation groups after 197 days of exposure, which were predominantly due to decreased amounts of sperm. In plaice killed after 73 days (at an earlier stage of spermatogenesis), there were no significant differences in weight compared with controls but amounts of spermatogonia were significantly reduced in irradiated fish. CONCLUSIONS : Exposure to dose rates as low as 0.24 mGy h(-1) of gamma-radiation given over a period of 197 days significantly reduced the weights of plaice testes, this being consequent on reductions in the amounts of sperm. Although there was some evidence of radiation affecting the numbers of spermatogonia it was not possible to determine the primary target for radiation damage which eventually caused the sperm reductions. Along with the related work described by Greenwood and Knowles (1996) this is the first investigation of a marine fish and it indicates that plaice testes are probably more radiosensitive than those previously described in tropical fish and of a similar radiosensitivity to mammalian testes. Although significant effects were observed after the lowest dose rate used of 0.24 mGy h(-1), this is still a factor of about 400 times greater than the estimated absorbed dose rate to plaice testes in the north-east Irish Sea off Sellafield at the present time. Micronucleus counts and flow-cytometric analysis of blood DNA both failed to show any evidence of genotoxic damage.

Kuipers BR, Witte HJ (1999) Grazing impact of microzooplankton on different size classes of algae in the North Sea in early spring and mid-summer. Marine Ecology-Progress Series 180 :93-104

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The impact of microzooplankton community grazing on different size classes of algae was investigated at 11 stations between Degger Bank and the Shetlands in early spring (March-April) and in summer (July-August). This work is part of a larger study designed to test the hypothesis that size-differential grazing of phytoplankton populations plays a crucial role in regulating food web structure. Dilution experiments, in which loss rates of the algae due to microzooplankton grazing can be estimated from the relation between growth rate of the prey and dilution, failed in many cases due to high variance. The present paper analyses the problem and puts forward a solution which involved pooling data from comparable stations into 1 average grazing estimate per algal size class. In early spring, estimates of grazing from measurements of chlorophyll a (chl a) were obtained only for algae >5 mu m, and average grazing rate was 0.23 d(-1) at the deeper stations. At the shallower more southern stations where a phytoplankton bloom was in progress the average grazing rate on algae >5 mu m was 0.5 d(-1). Flow cytometry was more successful in the <5 mu m algal fraction, yielding grazing rates of 0.25 d(-1) for the southern and 0.31 d(-1) for the northern stations. In summer, microzooplankton grazing incubations yielded significant results only when flow cytometry was used, and only when results were pooled for different areas. Grazing rates ranged from 0.07 d(-1) in the 1-2 mu m algal size class to 0.74 d(-1) for algae of 3-4 mu m and were 0.25 d(-1) for the <5 mu m cluster as a whole.

Lebaron P, Servais P, Troussellier M, Courties C, Vives-Rego J, Muyzer G, Bernard L, Guindulain T, Schafer H, Stackebrandt E (1999) Changes in bacterial community structure in seawater mesocosms differing in their nutrient status. Aquatic Microbial Ecology 19 :255-267

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Quantitative and qualitative changes in bacterial communities from the Mediterranean Sea were analysed under eutrophication conditions simulated in batch mesocosms (addition of inorganic nutrients or phytoplanktonic lysate). A wide variety of methods including traditional microbial ecology techniques, molecular biology and flow cytometry were combined to determine abundances, production, cell size, activity, culturability, and genetic and taxonomic diversity. In all mesocosms, the increase in biomass was rapidly controlled by protozoan grazing. Morphological and physiological changes 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. Grazing eliminates preferentially active and medium-sized cells within communities regulating bacterial productivity. Small and large cells were produced as a consequence of grazing pressure, and the large active cells contributed to the remaining productivity after grazing. Although grazing had an effect on the genetic diversity of bacterial communities by eliminating some populations, other species were preserved. It seems that some species such as Alteromonas macleodii may have developed defence strategies to escape predation. We hypothesize that such species may escape grazing by producing small and/or large cells during their growing phase.

Lee Y, Huang TS, Yan ML, Huang LR, Chen CH, Lu FJ (1999) Peroxisome proliferation, adipocyte determination and differentiation of C3H10T1/2 fibroblast cells induced by humic acid : Induction of PPAR in diverse cells. Journal of Cellular Physiology 179 :218-225

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Humic acid, a high-molecular-weight polyphenolic compound, exists abundantly in soil, natural water, and various terrestrial and aquatic environments. Humic acid causes peroxisome proliferation in mouse liver and induces the expression of peroxisome proliferator activated receptor (PPAR) in BNL CL.2 cells. Both cytotoxicity and flow cytometry show that humic acid inhibits the growth of C3H10T1/2 cells at G(1) phase. C3H10T1/2 fibroblast cells express PPAR gamma and the adipocyte P2 (aP2) genes which convert into adipocytes after being treated with humic acid. Our findings may provide a unique model for studying the molecular control of determination and differentiation of mesodermal cell lineages, (C) 1999 Wiley-Liss, Inc.

Lee Y, Huang TS, Yang ML, Huang LR, Chen CH, Lu FJ (1999) Peroxisome proliferation, adipocyte determination and differentiation of C3H10T1/2 fibroblast cells induced by humic acid : induction of PPAR in diverse cells. J Cell Physiol 179 :218-225


Humic acid, a high-molecular-weight polyphenolic compound, exists abundantly in soil, natural water, and various terrestrial and aquatic environments. Humic acid causes peroxisome proliferation in mouse liver and induces the expression of peroxisome proliferator activated receptor (PPAR) in BNL CL.2 cells. Both cytotoxicity and flow cytometry show that humic acid inhibits the growth of C3H10T1/2 cells at G1 phase. C3H10T1/2 fibroblast cells express PPARgamma and the adipocyte P2 (aP2) genes which convert into adipocytes after being treated with humic acid. Our findings may provide a unique model for studying the molecular control of determination and differentiation of mesodermal cell lineages.

Marie D, Brussaard CPD, Thyrhaug R, Bratbak G, Vaulot D (1999) Enumeration of marine viruses in culture and natural samples by flow cytometry. Appl Environ Microbiol 65 :45-52


Flow cytometry (FCM) was successfully used to enumerate viruses in seawater after staining with the nucleic acid-specific dye SYBR Green-I. The technique was first optimized by using the Phaeocystis lytic virus PpV-01. Then it was used to analyze natural samples from different oceanic locations. Virus samples were fixed with 0.5% glutaraldehyde and deep frozen for delayed analysis. The samples were then diluted in Tris-EDTA buffer and analyzed in the presence of SYBR Green-I. A duplicate sample was heated at 80 degreesC in the presence of detergent before analysis. Virus counts obtained by FCM were highly correlated to, although slightly higher than, those obtained by epifluorescence microscopy or by transmission electron microscopy (r = 0.937, n = 14, and r = 0.96, n = 8, respectively). Analysis of a depth profile from the Mediterranean Sea revealed that the abundance of viruses displayed the same vertical trend as that of planktonic cells. FCM permits us to distinguish between at least two and sometimes three virus populations in natural samples. Because of its speed and accuracy, FCM should prove very useful for studies of virus infection in cultures and should allow us to better understand the structure and dynamics of virus populations in natural waters.

Minor EC, Eglinton TI (1999) Molecular-level variations in particulate organic matter subclasses along the Mid-Atlantic Bight. Marine Chemistry 67 :103-122

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A significant portion of oceanic primary productivity occurs on continental margins. As the fate of this primary production has not been clearly determined, continental margins have been the focus of increased research over the past two decades. In the present study, molecular-level characteristics of particulate organic matter (POM) subclasses from the Mid-Atlantic Eight (MAB) were explored in an attempt to further our understanding of organic matter cycling in productive continental shelf and slope waters. Small-particle POM (2 —> 53 mu m), large-particle POM (> 53 mu m), "phytoplankton" (2 —> 53 mu m) and "detritus" (2 —> 53 mu m) were isolated from surface waters collected from different regimes (mid-shelf, shelf-edge, and slope) along the MAB in March 1996. Broad-band molecular-level variations within these subclasses were explored using direct temperature-resolved mass spectrometry (DT-MS) and multivariate analysis techniques. Correlations between molecular-level compositional differences and external environmental and biological variables such as salinity, temperature, and chlorophyll a concentrations were also examined. Both large-particle and small-particle POM exhibited molecular-level variations related to regime. Large-particle POM from the continental shelf exhibited chemical characteristics consistent with a greater zooplankton biomass component, as indicated by an enhanced chitin, protein, and cholesterol signature. In contrast, large-particle POM from slope waters appeared to contain a greater phytodetrital component, as indicated by enrichment in molecular-level characteristics similar to those of phytoplankton. A higher relative proportion of phytoplankton biomass was present in small-particle POM from the shelf-edge than from the mid-shelf or the slope. Ln addition, there was also an alongshelf (roughly north-south) trend in the composition of 2 —> 53 mu m POM, with samples farther south exhibiting enrichment in polysaccharides, fatty acids, chlorophyll, and diglycerides (the latter may result from triglycerides, phospholipids, or diglycerides within the samples). The molecular-level composition of "phytoplankton" also appeared to vary as a function of location, while the composition of "detritus" illustrated no easily interpretable variations with either location or external variables. (C) 1999 Elsevier Science B.V. All rights reserved.

Monger BC, Landry MR, Brown SL (1999) Feeding selection of heterotrophic marine nanoflagellates based on the surface hydrophobicity of their picoplankton prey. Limnology and Oceanography 44 :1917-1927

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Theory suggests that variation in the attractive solvation force associated with cell-surface hydrophobicity can significantly affect contact rates among small cells in aqueous environments and consequently may influence rates and selective impacts of marine nanoflagellate grazers feeding on picoplankton assemblages. To investigate this hypothesis, we assayed the natural range in hydrophobic characteristics of subtropical picoplankton from the oligotrophic subtropical Pacific (Station Aloha, 22 degrees 45’N, 158 degrees W) and mesotrophic Kaneohe Bay, Hawaii, using hydrophobic interaction chromatography (HIC) in conjunction with analytical flow cytometry. Variability in a relative index of cell-surface hydrophobicity (HIC index) for heterotrophic bacteria, Prochlorococcus and Synechococcus, exhibited some consistent spatial patterns. The HIC index for Prochlorococcus at Station Aloha varied about threefold, being consistently more hydrophobic in the upper 80 m of the water column and dropping abruptly below this depth. Heterotrophic bacteria were more hydrophobic near the surface and decreased slightly, but steadily, with increasing depth. The hydrophobicity of heterotrophic bacteria steadily increased along a Kaneohe Bay transect extending from oligotrophic to mesotrophic conditions. In experiments involving nanoflagellates grazing on laboratory cultures of Prochlorococcus, cell cultures exhibiting the highest HIC indices were grazed upon at the highest rates. An additional experiment involving mixtures of Prochlorococcus cells exhibiting high and low hydrophobicities showed that the average hydrophobicity of the uningested prey mixture was driven progressively toward lower hydrophobicity as the more hydrophobic cells were selectively removed through time. If these laboratory grazing results hold in nature, the rate at which picoplankton cells are cleared from suspension by nanoflagellates could vary by as much as twofold due solely to natural variation in cell surface hydrophobicity.

Mostajir B, Sime-Ngando T, Demers S, Belzile C, Roy S, Gosselin M, Chanut JP, de Mora S, Fauchot J, Vidussi F, Levasseur M (1999) Ecological implications of changes in cell size and photosynthetic capacity of marine Prymnesiophyceae induced by ultraviolet-B radiation. Marine Ecology-Progress Series 187 :89-100

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A natural planktonic assemblage of organisms (<240 mu m) was studied in mesocosm experiments for 7 d under varying conditions of ultraviolet-B radiation (WB : 280 to 320 nm) : UVB excluded, natural radiation and WE enhanced at 2 different levels. Specifically. the changes in Prymnesiophyceae abundance and Light scatter properties attributed to cell size (CS) were investigated by flow cytometry during the first 72 h with 4 h temporal resolution and were thereafter examined during the 4 following days with twice daily sampling. In addition, the specific rate of photosynthesis (p(cell)) of phytoplankton <5 mu m, ciliate abundance (predator 15 to 35 mu m) and dynamics of nutrients were monitored. Prymnesiophyceae ranged in size between 2.7 and 4 mu m and dominated the phytoplanktonic community <5 mu m (more than 94%). Prymnesiophyceae exhibited marked diel variability with synchronized cell division : CS increased during the day and diminished at nighttime, indicating cell division. Shortterm UVB exposures during the first 3 d of the experiment did not affect CS, probably due to vertical water mixing in the mesocosms moderating harmful UVB exposure. In contrast, long-term UVB treatments (3 to 7 d) induced progressive CS increases in Prymnesiophyceae as a function of increasing UVB doses. The successive inhibition of p(cell) of phytoplankton <5 mu m was also observed as a function of increasing WE doses. The results suggest that enhanced UVB provokes the retardation of cell division and inhibition of p(cell) which causes Prymnesiophyceae CS enlargement. CS enlargement and probably a change in the food quality of Prymnesiophyceae could result in food limitation for the ciliate population. although ciliates seem to be directly affected by WE enhancement. This study highlights the ecological implications of CS changes and photosynthetic capacity of phytoplankton, with respect to predator-prey interactions in response to WE enhancement.

Neveux J, Lantoine F, Vaulot D, Marie D, Blanchot J (1999) Phycoerythrins in the southern tropical and equatorial Pacific Ocean : Evidence for new cyanobacterial types. Journal of Geophysical Research-Oceans 104 :3311-3321

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Quantitative and qualitative investigations of phycoerythrins (PE) were achieved in the central southern tropical and equatorial Pacific during the Flux dans l’ouest du Pacifique equatorial (FLUPAC : September-October 1994) and Oligotrophie en Pacifique (OLIPAC : November 1994) cruises. We observed mainly high-phycourobilin (PUB) PE related to small Synechococcus spp. (0.6-1.4 mu m) This PE was characterized by fluorescence excitation peaks at 496 and 550 nm in 50% glycerol. Highest concentrations (0.1-0.2 mu g L-1) were recorded either in the mixed layer (nutrient-enriched area) or at depth in the vicinity of the 0.1 mu M NO3- isopleth (oligotrophic waters). Maximum abundance of Synechcoccus did not exceed 31 x 10(3) cell mL(-1). No subpopulations of Synechococcus were evidenced by flow cytometry. Nevertheless, in a few samples, two new PE spectral types were observed. The first type was a high-PUB PE with two fluorescence excitation peaks at 494 and 564 nm. It appears to be attributable to nonmotile round cells, 2-3 mu m in size and easily detected by flow cytometry, likely cyanobacteria. They were only observed in very oligotrophic waters south of 15 degrees 30’S along 150 degrees W in the top 60 m. In this area, nitrate, nitrite, and ammonia were undetectable above 120 m, while phosphate was always recorded. This suggests that these larger cyanobacteria may fix dinitrogen (N-2) to supply their growth and therefore possibly play a significant role in oceanic new production. The second type, a high-phycoerythrobilin (PEB) PE was observed in three samples obtained at or near the equator. It displayed two fluorescence excitation peaks at 496 and 536 nm. The organisms that contained this PE type were not identified. These data suggest that PE is more diverse in oceanic waters than was previously assumed.

O’Dowd AM, Ellis AE, Secombes CJ (1999) Binding of soluble immune complexes to fractionated Atlantic salmon (Salmo salar L.) leucocytes. Vet Immunol Immunopathol 68 :149-157


Binding of a fluorescent-labelled soluble immune complex to different types of Atlantic salmon leucocytes was investigated using flow cytometry. Peripheral blood leucocytes (PBL) were separated into sIg+ and sIg enriched populations by magnetic activated cell sorting, blood neutrophils were identified by electronic gating, and kidney macrophages were selected by plastic-adherence. About 60% of both sIg+ and sIg- enriched PBL, 44% of neutrophils and 34% of macrophages bound the soluble immune complexes.

Okochi M, Taguchi T, Tsuboi M, Nakamura N, Matsunaga T (1999) Fluorometric observation of viable and dead adhering diatoms using TO-PRO-1 iodide and its application to the estimation of electrochemical treatment. Applied Microbiology and Biotechnology 51 :364-369

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A rapid method for the direct measurement of viable and dead adhering diatoms was developed using a fluorescent dye, TO-PRO-1 iodide. By staining the marine diatom, Nitzchia closterium, with TO-PRO-1 iodide, viable and dead cells were identified as red and yellow cells respectively, under an epifluorescence microscope employing blue excitation. Only dead cells were stained with TO-PRO-1 iodide. Viable cells were observed as red because of autofluorescence arising from intracellular chlorophyll, whereas dead cells were observed as yellow because of the fluorescence of TO-PRO-1 iodide. The percentage of TO-PRO-l-iodide-stained was correlated with the percentage of dead cells in N. closterium cells exposed to heat (60 degrees C, 15 min). Other microalgae containing intracellular chlorophyll could be also distinguished as viable or dead cells by this fluorometric staining method. This method was applied for the assessment of N. closterium cells killed by the electrochemical treatment and used to monitor biofouling populations and their viability directly on the electrode surface. When 1.0 V was applied against a saturated calomel electrode, 99% of the cells attached to graphite electrode were killed in 1 h.

Olson RJ, Sosik HM, Chekalyuk AM (1999) Photosynthetic characteristics of marine phytoplankton from pump-during-probe fluorometry of individual cells at sea. Cytometry 37 :1-13


BACKGROUND : Active fluorescence techniques are becoming commonly used to monitor the state of the photosynthetic apparatus in natural populations of phytoplankton, but at present these are bulk water measurements that average all the fluorescent material in each sample. Here we describe two instruments that combine individual-cell "pump-during-probe" (PDP) measurements of chlorophyll (Chl) fluorescence induction, on the time scale of 30 to 100 micros, with flow cytometric or visual characterization of each cell. METHODS : In the PDP flow cytometer, we measure Chl fluorescence yield as a function of time during a 150 micros excitation flash provided by an argon ion laser ; each particle is subsequently classified as in a conventional flow cytometer. In the PDP microfluorometer, individual cells in a sample chamber are visually identified, and fluorescence excitation is provided by a blue light-emitting diode that can be configured to provide a saturating flash and also a subsequent series of short flashlets. This sequence allows both saturation and relaxation kinetics to be monitored. RESULTS : Phytoplankton from natural samples and on-deck iron-enrichment incubation experiments in the Southern Ocean were examined with each PDP instrument, providing estimates of the potential quantum yield of photochemistry and the functional absorption cross section for photosystem 2, for either individuals (for cells larger than a few micrometers) or populations (for smaller cells). CONCLUSIONS : Results from initial field applications indicate that single-cell PDP measurements can be a powerful tool for investigating the nutritional state of phytoplankton cells and the regulation of phytoplankton growth in the sea.

Pan Y, Cembella AD, Quilliam MA (1999) Cell cycle and toxin production in the benthic dinoflagellate Prorocentrum lima. Marine Biology 134 :541-549

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Profiles of diarrhetic shellfish poisoning (DSP) toxins produced throughout the growth cycle and the cell cycle of the toxigenic marine dinoflagellate Prorocentrum lima were studied in triplicate unialgal batch cultures. Cells were pre-conditioned at 18 +/- 1 degrees C, under a photon flux density (PFD) of 90 +/- 5 mu mol m(-2) s(-1) on a 14 h light:10 h dark photoperiod. In exponential growth phase, cultures were synchronized in darkness for 17 d. After dark synchronization, cultures were transferred back to the original photoperiod regime. Cells were harvested for DSP toxin analysis by LC-MS (liquid chromatography with mass spectrometry), and double-stranded (nuclear) DNA was quantified by flow cytometry. The cell populations became asynchronous within approximately 3 d after transition from darkness to the 14 h light:10 h dark photoperiod. This may be due to the prolonged division cycle (5 to 7 d) that is not tightly phased by the photoperiod. Unlike other planktonic Prorocentrum spp., cytokinesis in P. lima occurred early in the dark and ceased by "midnight". Cellular levels of the four principal DSP toxins, okadaic acid (OA), OA C8-diol-ester (OA-D8), dinophysistoxin-1 (DTX1) and dinophysistoxin-4 (DTX4), ranged from 0.37 to 6.6, 0.02 to 1.5, 0.04 to 2.6, and 1.8 to 7.8 fmol cell(-1), respectively. No toxin production was evident during the extended period of dark synchronization nor during the initial period when NH4 was consumed as the major nitrogen source. Soon after the cells were returned to the 14 h light:10 h dark cycle and they began to take up NO3, cellular levels of all four toxins gradually increased. This increase in DSP toxins usually occurred in the light, marked by a rise in DTX4 levels that preceded an increase in the cellular concentration of OA and DTX1 (delayed by 3 to 6 h), Thus, DTX4 synthesis is initiated in the G1 phase of the cell cycle and persists into S phase ("morning" of the photoperiod), whereas OA and DTX1 production occurs later during S and G2 phases ("afternoon"). No toxin production was measured during cytokinesis, which happened early in the dark. The evidence indicates that toxin synthesis is restricted to the light period and is coupled to cell cycle events.

Putland JN, Rivkin RB (1999) Influence of storage mode and duration on the microscopic enumeration of Synechococcus from a cold coastal ocean environment. Aquatic Microbial Ecology 17 :191-199

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Photosynthetic picoplankton of the genus Synechococcus can represent a substantial proportion of planktonic community biomass and production in many oceanic provinces. These cells are typically enumerated by visualizing the autofluorescence of phycoerythrin using epifluorescence microscopy. Detailed studies of bacterioplankton and preliminary studies with photosynthetic pico- and nanoplankton suggest that the number of cells which can be visualized changes with mode and duration of sample storage. Inaccurate estimates of Synechococcus abundance may bias the interpretation of the distribution and turnover of microbial stocks. We carried out a comprehensive, long-term (similar to 0.9 yr) time-course study to determine if storage mode and duration influence microscopic estimates of Synechococcus abundance. Seawater samples preserved with gluteraldehyde were either stored at 4 degrees C until counting (i.e. RS-refrigerated in suspension) or slides were prepared and stored at -20 degrees C until counting (i.e. FF-filtered and frozen). Over time, both methods converged on an apparent cell loss (ACL ; i.e, loss of epifluorescence-detectable cells) of ca 45%. Significant (p less than or equal to 0.05) ACL occurred within 1 mo for the RS method, whereas cell numbers were unchanged for the first 2 mo for the FF method. Apparent cell loss was hyperbolic for both storage modes and the rate constants for decay were similar. Our results are consistent with the suggestion in the Literature that ACL may have been due to persistence of intracellular autolytic enzymes in the preserved cells. We also examined the influence of the excitation wavebands on the estimation of Synechococcus abundances. About twice as many Synechococcus were observed using green (490 to 545 nm) compared to blue (450 to 490 nm) excitation epifluorescence microscopy and this increase was significant (p < 0.001). Based on our results, we recommend that samples for the enumeration of Synechococcus be immediately preserved, filtered, frozen, and counted using green excitation within 2 mo.

Raychoudhury SS, Blake CA, Millette CF (1999) Toxic effects of octylphenol on cultured rat spermatogenic cells and Sertoli cells. Toxicol Appl Pharmacol 157 :192-202


Alkylphenols, including the estrogenic 4-tert-octylphenol (OP), are environmental pollutants. Because administration of OP to adult male rats impairs spermatogenesis and OP has been shown to be toxic to aquatic animals and to mammalian splenocytes in vitro, we studied whether OP exerts direct toxic effects on cultured spermatogenic cells and Sertoli cells isolated from male rats. Cell viability was assessed with a Live/Dead Eukolight viability/cytotoxicity kit. Culture of mixed spermatogenic cells from adult rats with 10(-8) M OP or Sertoli cells from 19- to 21-day-old rats with 10(-12) M OP, but not with 0.08% EtOH (vehicle) or 10(-6) M 17beta-estradiol (E2) or dexamethasone (DEX), significantly decreased the percentage of viable cells after 24 h of treatment. None of the treatments significantly altered total cell number. Flow cytometric analyses of spermatogenic cells revealed that exposure to 10(-4) or 10(-6) M OP yielded abnormal relative ploidy classes. Four hours of treatment with 10(-6) M OP, but not with 10(-6) M E2 or DEX, caused significant chromatin condensation in Sertoli cells as observed with acridine orange staining. The decreased percentage of viable cells after 24 h of exposure to 10(-6) M OP remained when Sertoli cells were cultured in Ca2+-free medium. Sertoli cells contained nuclei of reduced size and labeled 3’-OH DNA ends as detected by microscopic analyses when the cells had been incubated for 24 h with 10(-6) M OP but not with vehicle or 10(-6) M E2 or DEX. The results demonstrate that OP, but not E2 or DEX, is directly toxic to cultured rat spermatogenic cells and Sertoli cells and suggest that this toxic effect in Sertoli cells is exerted through Ca2+-independent apoptosis.

Reynolds DT, Fricker CR (1999) Application of laser scanning for the rapid and automated detection of bacteria in water samples. Journal of Applied Microbiology 86 :785-795

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It is widely accepted that the heterotrophic plate count method may not support the growth of all viable bacteria which may be present within a water sample and that alternative procedures using ’viability markers’ may yield additional information. In this study, ChemChrome B (CB), which is converted to a fluorescent product by esterase activity, was used to stain viable bacteria (captured by membrane filtration) from potable water samples. The labelled bacteria from each sample were subsequently enumerated using a novel laser scanning instrument (ChemScan). Analysis of 107 potable water samples using this procedure demonstrated the presence of a significantly greater number of bacteria than were detected by culture (x-test, P < 0.05). The mean number of bacteria isolated by culture on R(2)A agar incubated at 22 degrees C for 7 d was only 25.2% of the total number of viable bacteria detected using the CB/ChemScan viability assay. Further analysis of 81 water samples using a 5-cyano-2,3,4-tolyl-tetrazolium chloride (CTC) viability assay also demonstrated the presence of many viable bacteria which were not capable of growth under the culture conditions employed in this study. However, the results indicate that ChemChrome B has the ability to stain a significantly greater number of heterotrophs than CTC (x-test, P < 0.05). In contrast, six potable waters were identified in which the CTC viability assay resulted in counts greater than those obtained using CB. The ChemScan instrument was successfully used for rapid and accurate enumeration of labelled micro-organisms, allowing information on the total viable microbial load of a water sample to be determined within 1 h. Furthermore, the ChemScan system has the potential for use in detecting specific organisms labelled with fluorescently-labelled antibodies or nucleic acid probes.

Satoh M, Karaki E, Kakehashi M, Okazaki E, Gotoh T, Oyama Y (1999) Heavy-metal induced changes in nonproteinaceous thiol levels and heavy-metal binding peptide in Tetraselmis tetrathele (Prasinophyceae). Journal of Phycology 35 :989-994

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The effects of mercury and cadmium on the intracellular level of nonproteinaceous thiols in a unicellular preen alga Tetraselmis tetrathele (West) Butcher (Prasinophyceae) were investigated by using a fluorescent dye, 5-chloromethylfluorescein (5CMF), as a probe for nonproteinaceous thiols, The 5CMF fluorescence was observed in cytoplasm, and the intensity of the fluorescence was decreased by exposure of the cells to HgCl2, Analysis of the fluorescent intensity of 5CMF by flow cytometry made it possible to distinguish cells in three states during the dying process caused by HgCl2 : a normal state, a thiol-depleted state, and a dead state. Depletion of nonproteinaceous thiols began within 30 min, and they were completely depleted at 2 h, Most cells died after 24 h of exposure to more than 3.0 mu M HgCl2, whereas exposure up to 1.0 mM CdCl2 did not cause depletion of nonproteinaceous thiols or cell death within 48 h.
HPLC analyses revealed that glutathione was a major nonprotein thiol in T, tetrathele and that it was oxidized by exposing the cells to HgCl2. Phytochelatins, which play a great role in the tolerance to heavy metals of higher plants and many algae, could not be found in T. tetrathele. However, a tripeptide, Arg-Arg-Glu, was found to be abundant, and it showed ability to bind Hg2+, suggesting that it functions to scavenge heavy metals as well as thiol molecules.

Servais P, Courties C, Lebaron P, Troussellier M (1999) Coupling bacterial activity measurements with cell sorting by flow cytometry. Microbial Ecology 38 :180-189

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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 mu m(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 mel in this enriched mesocosm.

Sieracki ME, Cucci TL, Nicinski J (1999) Flow cytometric analysis of 5-cyano-2,3-ditolyl tetrazolium chloride activity of marine bacterioplankton in dilution cultures. Appl Environ Microbiol 65 :2409-2417


The respiratory activity of marine bacteria is an important indication of the ecological functioning of these organisms in marine ecosystems. The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is reduced intracellularly in respiring cells to an insoluble, fluorescent precipitate. This product is detectable and quantifiable by flow cytometry in individual cells. We describe here an evaluation of flow cytometry for measuring CTC activity in natural assemblages of marine bacteria growing in dilution cultures. We found that more CTC-positive cells are detected by flow cytometry than by visual epifluorescence microscopy. Samples can be stored refrigerated or frozen in liquid nitrogen for at least 4 weeks without a significant loss of total cells, CTC-positive cells, or CTC fluorescence. Cytometry still may not detect all active cells, however, since the dimmest fluorescing cells are not clearly separated from background noise. Reduction of CTC is very fast in most active cells, and the number of active cells reaches 80% of the maximum number within 2 to 10 min. The proportion of active cells is correlated with the growth rate, while the amount of fluorescence per cell varies inversely with the growth rate. The CTC reduction kinetics in assemblages bubbled with nitrogen and in assemblages bubbled with air to vary the oxygen availability were the same, suggesting that CTC can effectively compete with oxygen for reducing power. A nonbubbled control, however, contained more CTC-positive cells, and the amount of fluorescence per cell was greater. Activity may have been reduced by bubble-induced turbulence. Addition of an artificial reducing agent, sodium dithionite, after CTC incubation and fixation resulted in a greater number of positive cells but did not "activate" a majority of the cells. This indicated that some of the negative cells actually transported CTC across their cell membranes but did not reduce it to a detectable level. Automated analysis by flow cytometry allows workers to study single-cell variability in marine bacterioplankton activity and changes in activity on a small temporal or spatial scale.

Stramski D (1999) Refractive index of planktonic cells as a measure of cellular carbon and chlorophyll a content. Deep-Sea Research Part I-Oceanographic Research Papers 46 :335-351

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Current methods for determining carbon content in individual planktonic cells from particle volume alone may involve large errors, and no routine technique exists for determining chlorophyll a content in individual phytoplankters. In this study the concept of using the refractive index of cells as a measure of intracellular concentrations of carbon and chlorophyll a is discussed. Specifically, the real part of the refractive index n (at light wavelengths where absorption is negligible or very small) is shown to correlate well with the intracellular concentration of carbon, C-i. The imaginary part of the refractive index n’ (in the red band of chlorophyll a) correlates well with the intracellular chlorophyll concentration, Chl(i). These relationships were found to be nearly identical for two species, a cyanobacterium Synechococcus and a diatom Thalassiosira a pseudonana, over a two-fold range in C-i and Chl(i), This range was associated with interspecies differences and intraspecies variations in the cell properties over a day-night cycle. These observations and the underlying theoretical considerations suggest that the relationships C-i vs. n and Chl(i) vs, n’ may be robust and hold for a variety of planktonic species regardless of interspecies and intraspecies variability in cellular carbon content, Chi a content, and cell size. In addition, these relationships may be applicable to single-particle analysis of natural water samples, which promises a unique capability to acquire information about the distribution of carbon and chlorophyll a among individual cells, different size classes, and taxonomic groups of planktonic microorganisms in the ocean. Further research with various planktonic species is needed to examine the generality of the relationships C-i vs, n and Chl(i) vs, n’ before the approach can be implemented in field studies. (C) 1999 Elsevier Science Ltd. All rights reserved.

Suzuki MT (1999) Effect of protistan bacterivory on coastal bacterioplankton diversity. Aquatic Microbial Ecology 20 :261-272

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Four protist exclusion experiments were conducted to test the hypothesis that marine bacterivorous protists selectively feed on different bacterioplankton genotypes and affect the taxonomic diversity of coastal marine bacterioplankton communities. In these experiments, the changes in bacterial community composition of seawater samples from which protists were removed by filtration were followed and compared to those of untreated control water samples. Bacterioplankton community structure was inferred from the relative abundance of bacterial small subunit rRNA genes (SSU rDNAs) by a recently developed technique (length heterogeneity analysis by PCR (LH-PCR ; Suzuki et al. 1998). The results of Me experiments show that the community structure did not dramatically change up to a 24 h incubation period in any of the treatments. However there were significant differences in filtered water samples and controls between 24 and 48 h of incubation. In the absence of bacterivores some SSU rDNAs that were rare in the original water samples dominated the bacterioplankton SSU rDNA pool after 48 h of incubation. Protists appeared to be capable of controlling bacterioplankton taxonomic diversity under these manipulated conditions. The results also agree with the hypothesis that aquatic bacterioplankton communities are composed of small cells that escape predation, and are in a state of low physiological turnover.

Toledo G, Palenik B, Brahamsha B (1999) Swimming marine Synechococcus strains with widely different photosynthetic pigment ratios form a monophyletic group. Applied and Environmental Microbiology 65 :5247-5251

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Unicellular marine cyanobacteria are ubiquitous in both coastal and oligotrophic regimes. The contribution of these organisms to primary production and nutrient cycling is substantial on a global scale. Natural populations of marine Synechococcus strains include multiple genetic lineages, but the link, if any, between unique phenotypic traits and specific genetic groups is still not understood. We studied the genetic diversity (as determined by the DNA-dependent RNA polymerase rpoC1 gene sequence) of a set of marine Synechococcus isolates that are able to swim, Our results show that these isolates form a monophyletic group. This finding represents the first example of correspondence between a physiological trait and a phylogenetic group in marine Synechococcus. In contrast, the phycourobilin (PUB)/phycoerythrobilin (PEB) pigment ratios of members of the motile clade varied considerably. An isolate obtained from the California Current (strain CC9703) displayed a pigment signature identical to that of nonmotile strain WH7803, which is considered a model for low-PUB/PEB-ratio strains, whereas several motile strains had higher PUB/PEB ratios than strain WH8103, which is considered a model for high-PUB/PEB-ratio strains. These findings indicate that the PUB/FEB pigment ratio is not a useful characteristic for defining phylogenetic groups of marine Synechococcus strains.

Troussellier M, Courties C, Lebaron P, Servais P (1999) Flow cytometric discrimination of bacterial populations in seawater based on SYTO 13 staining of nucleic acids. Fems Microbiology Ecology 29 :319-330

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Flow cytometric (FCM) counts of bacteria stained with SYTO 13, a cyanine dye, were highly correlated with DAPI epifluorescence microscopic counts in coastal seawater samples. Fluorescence intensity of stained cells appeared to depend on nucleic acid content and on the polarization of cell membranes. Right angle light scarier values of bacterial populations were clearly related to cell size. By FCM analysis of SYTO 13-stained samples from a batch mesocosm of Mediterranean seawater, several bacterial clusters having different apparent sizes and physiological status were discriminated showing that FCM analysis allows key cell categories to be followed for understanding bacterial community dynamics. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Van Dolah FM, Leighfield TA (1999) Diel phasing of the cell-cycle in the Florida red tide dinoflagellate, Gymnodinium breve. Journal of Phycology 35 :1404-1411

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The diel cycle is a key regulator of the cell-cycle in many dinoflagellates, but the mechanisms by which the diel cycle entrains the cell-cycle remain poorly understood. In this study, we describe diel phasing of the cell-cycle in the Florida red tide dinoflagellate Gymnodinium breve Davis, determine the diel cue which serves to entrain the cell-cycle, and provide evidence for the presence of cyclin-dependent kinase (CDK), a cell-cycle regulator which may be responsive to this cue. Four laboratory isolates from the West Coast of Florida were compared. When gown on a 16:8 h LD cycle, all isolates displayed phased cell division, with the S-phase beginning 6-8 h into the light phase, and mitosis following 12-14 h later, as determined by flow cytometry, A naturally occurring bloom of G. breve, studied over one diel cycle, displayed diel cell-cycle phasing similar to that in the laboratory cultures, with the S-phase beginning during daylight and the peak of mitosis occurring approximately 4 h after sunset. In the laboratory cultures, the dark/light "dawn" transition was found to provide the diel cue which serves to entrain the G, breve cell-cycle, whereas the light/ dark "dusk" transition did not appear to be involved. Evidence for the presence of CDK in G. breve was obtained using two approaches : (1) identification of a 34-kDa protein, immunoreactive to an antibody against a conserved amino acid sequence (alpha-PSTAIR) unique to the CDK protein family and (2) inhibition of the cell-cycle by olomoucine, a selective CDK inhibitor. Together, these results provide the basis from which one can begin addressing mechanisms by which the diel cycle regulates the cell-cycle in G. breve.

Vaulot D, Marie D (1999) Diel variability of photosynthetic picoplankton in the equatorial Pacific. Journal of Geophysical Research-Oceans 104 :3297-3310

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The diel variability in cell abundance, light scatter, and pigment fluorescence of three autotrophic picoplankton groups (Prochlorococcus, Synechococcus, picoeukaryotes) measured by flow cytometry was investigated in surface waters of the equatorial Pacific Ocean (5 degrees S, 150 degrees W) during 5 days with about 1 hour temporal resolution. The diel variability of vertical profiles was examined at the same station on days 2 and 4. Prochlorococcus division rate was also estimated from cell cycle measurements. A more limited data set was obtained at a station located in very oligotrophic waters (16 degrees S, 150 degrees W). All three picoplankton populations exhibited very marked diel variability. Cell division was highly synchronized but not phased identically for all three populations : Synechococcus divided first, followed 2 hours ;later by Prochlorococcus and 7 hours later by picoeukaryotes. Cells grew in size only once the sun had risen, but growth did continue in the dark for a short period. Growth processes occurred in parallel at the top and the bottom of the mixed layer, inducing uniform profiles for cell abundance and scatter. For chlorophyll fluorescence, in contrast, prokaryotes displayed opposite patterns during the light period between surface (decrease due to very strong quenching) and depth (increase). This created steep vertical gradients during the day that vanished at night because of convective mixing. In the top 25 m, strong light intensities (including UV radiation) had very pronounced detrimental effects on prokaryotes, especially on Prochlorococcus, inducing fluorescence quenching, slowed down growth, and retardation of DNA synthesis.

Vazquez-Dominguez E, Peters F, Gasol JM, Vaque D (1999) Measuring the grazing losses of picoplankton : methodological improvements in the use of fluorescently labeled tracers combined with flow cytometry. Aquatic Microbial Ecology 20 :119-128

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Fluorescently labeled tracers (FLT) are often used to estimate the loss rates of picoplankton to grazers. These tracers are commonly enumerated by epifluorescence microscopy, although flow cytometry is a viable alternative in the detection of FITC (fluorescein-5-isothiocyanate)- or DTAF (5-([4,6-dichlorotriazin-2-yl)amino]-fluorescein)-stained bacterial tracers. However, the bacterivory measured with FLT has hardly been applied to routine monitoring of oceanic waters, partly because of the time-consuming preparation of the tracers and other problems associated with the long-term incubations needed to generate detectable rates of tracer change. In addition, these long-term incubations make samples especially sensitive to the unwanted addition of nutrients carried over with the tracers. Here we present some experiments designed to ease the estimation of grazing rates on bacteria with this technique. Two bacterial strains and 2 fluorescent dyes were tested : Escherichia coli minicells (0.065 mu m(3)) and Pseudomonas diminuta (0.064 mu m(3)), stained with DTAF or with FITC. In addition, instead of the common use of pyrophosphate buffer during the staining protocol, the use of carbonate-bicarbonate buffer and cells scraped directly from solid media is suggested to avoid the problems associated with phosphorus enrichment of the sample that at times can occur in oligotrophic water samples. The FITC- or DTAF-stained tracers can be observed with either epifluorescence microscopy or flow cytometry. However, FITC- or DTAF-stained P, diminuta were more easily resolved with the flow cytometer than stained minicells. Flow cytometric detection of P. diminuta tracers, prepared in bicarbonate-buffer and stained with FITC, is a fast protocol for the estimation of the grazing loss rates of bacteria in oceanic environments.

Vives-Rego J, Guindulain T, Vazquez-Dominguez E, Gasol JM, Lopez-Amoros R, Vaque D, Comas J (1999) Assessment of the effects of nutrients and pollutants on coastal bacterioplankton by flow cytometry and SYTO-13 staining. Microbios 98 :71-85

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The specific nucleic acid fluorochrome SYTO-13 was used to assess changes in density and heterogeneity of marine bacterial populations exposed to nutrients and common pollutants. Bacterial counts were determined by flow cytometry calibrated using epifluorescence microscopy and image analysis. The results obtained by both methods were significantly correlated. Seawater samples from a coastal area of Barcelona were incubated for 30 days, after the addition of several pollutants (Hg and surfactants) and organic nutrients. The evolution of bacterioplankton abundance over time was similar in most cases. It occurred in three phases : (1) a progressive increase in bacterial density ; (2) a subsequent decrease, and then (3) a fluctuating stationary phase. Variations in fluorescence and scatter signals showed changes in the populations of bacteria observed during the evolution of bacterial abundance. Bacterioplankton growth or inhibition obtained after exposure to nutrients or toxic compounds can be rapidly and easily evaluated by this technique.

Wilkins MF, Boddy L, Morris CW, Jonker RR (1999) Identification of phytoplankton from flow cytometry data by using radial basis function neural networks. Appl Environ Microbiol 65 :4404-4410


We describe here the application of a type of artificial neural network, the Gaussian radial basis function (RBF) network, in the identification of a large number of phytoplankton strains from their 11-dimensional flow cytometric characteristics measured by the European Optical Plankton Analyser instrument. The effect of network parameters on optimization is examined. Optimized RBF networks recognized 34 species of marine and freshwater phytoplankton with 91. 5% success overall. The relative importance of each measured parameter in discriminating these data and the behavior of RBF networks in response to data from "novel" species (species not present in the training data) were analyzed.

Wood AM, Lipsen M, Coble P (1999) Fluorescence-based characterization of phycoerythrin-containing cyanobacterial communities in the Arabian Sea during the Northeast and early Southwest Monsoon (1994-1995). Deep-Sea Research Part Ii-Topical Studies in Oceanography 46 :1769-1790

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Scanning fluorescence spectroscopy was used to investigate the spatial and temporal variability in the fluorescence signature of phycoerythrin-containing organisms in the Arabian Sea during the early Northeast and early Southwest Monsoon (1994-1995), Phycoerythrin (PE) emission spectra were relatively invariant among all the samples collected on either cruise ; the relatively symmetrical PE emission peaks showed maxima at wavelengths ranging from 563-572 nm. PE excitation spectra always showed either a strong shoulder or a peak at wavelengths absorbed maximally by phycourobilin (PUB) chromophores as well as a peak at wavelengths absorbed maximally by phycoerythrobilin (PEB) chromophores. Thus, the Arabian Sea appears to be different from the Black Sea or Gulf of Maine in that PUB-lacking forms of PE rarely, if ever, dominate the PE signal. Fluorescence excitation signatures differed in the relative excitation of PE emission by wavelengths absorbed by PUB (similar to 495 nm, Ex(PUB)) and by wavelengths absorbed by PEB (similar to 550 nm, Ex(PEB)) ; these were distinguished by having either very low (similar to 0.6), very high (similar to 1.8), or intermediate Ex(PUB):Ex(PEB) ratios. The distribution of samples with different PE fluorescence signatures was investigated extensively during the early Southwest Monsoon, and communities characterized by the low Ex(PUB):Ex(PEB) ratios were closely associated with cooler (24-27 degrees C), fresher (35.7-36.25 psu) water influenced by coastal upwelling. In general, "ambient" surface water of the Arabian Sea during the early Southwest Monsoon was of intermediate temperature (27-29 degrees C) and salinity (36.15-36.4 psu) and showed intermediate or high values for Ex(PUB):Ex(PEB). This suggests that the PE fluorescence signature can be used to follow the fate of upwelling-influenced water masses and the populations they transport. (C) 1999 Elsevier Science Ltd. All rights reserved.

Wright AC, Powell JL, Tanner MK, Ensor LA, Karpas AB, Morris JG, Jr., Sztein MB (1999) Differential expression of Vibrio vulnificus capsular polysaccharide. Infect Immun 67 :2250-2257


Vibrio vulnificus is a human pathogen whose virulence has been associated with the expression of capsular polysaccharide (CPS). Multiple CPS types have been described ; however, virulence does not appear to correlate with a particular CPS composition. Reversible-phase variation for opaque and translucent colony morphologies is characterized by changes in CPS expression, as suggested by electron microscopy of cells stained nonspecifically with ruthenium red. Isolates with opaque colony morphologies are virulent and appear to be more thickly encapsulated than naturally occurring translucent-phase variants, which have reduced, patchy, or absent CPS. Previously, we have shown that the virulence of translucent-phase variants was intermediate between opaque-phase variants and acapsular transposon mutants, suggesting a correlation between virulence and the amount of CPS expressed. In the present study, CPS expression of phase variants and genetically defined mutants of V. vulnificus M06-24/O was examined by using a CPS-specific monoclonal antibody with an enzyme-linked immunosorbent assay, flow cytometry, and immunoelectron microscopy. Semiquantitative analyses of CPS expression correlated well among these assays, confirming that the translucent-phase variant was intermediate in CPS expression and retained type I CPS-specific epitopes. Cell surface expression of CPS varied with the growth phase, increasing during logarithmic growth and declining in stationary culture. Significantly greater CPS expression (P = 0.026) was observed for cells grown at 30 degrees C than for those at 37 degrees C. These studies confirm that phase variation and virulence in V. vulnificus correlate with the amount of CPS expressed and demonstrate the fluidity of bacterial polysaccharide expression in response to environmental conditions.

Zubkov MV, Fuchs BM, Eilers H, Burkill PH, Amann R (1999) Determination of total protein content of bacterial cells by SYPRO staining and flow cytometry. Appl Environ Microbiol 65 :3251-3257


An assay has been developed for measuring protein biomass of marine planktonic bacteria by flow cytometry. The method was calibrated by using five species of Bacteria (an Arcobacter sp., a Cytophaga sp., an Oceanospirillum sp., a Pseudoalteromonas sp., and a Vibrio sp.) recently isolated from seawater samples and grown in culture at different temperatures. The intensity of SYPRO-protein fluorescence of these bacteria strongly correlated with their total protein content, measured by the bicinchoninic acid method to be in the range of 60 to 330 fg of protein cell-1 (r2 = 0.93, n = 34). According to the calibration, the mean biomass of planktonic bacteria from the North Sea in August 1998 was 24 fg of protein cell-1.