1994

mardi 21 avril 2009
par   G. Grégori

Berreur-Bonnenfant J, Ammar M, Dubreuil A, Kiefer H, Diogene G, Metezeau P, Puiseux-Dao S (1994) Modulation of fibroblast response to maitotoxin along the cell division cycle. Cell Biol Toxicol 10 :423-427

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Maitotoxin (MTX) induces an increase of [Ca2+]i and of phosphoinositide breakdown in various cell types. The [Ca2+]i increase followed with fluorescent probes on cell suspensions has been described as slow and lasting, in contrast to the "signal" induced by calcium ionophores such as ionomycin. MTX effects have been studied on two fibroblastic cell lines, BHK21 C13 and FR 3T3, synchronized by serum deprivation treatment performed in an isoleucine-free medium for BHK21 C13 cells. In BHK21 C13 cells, flow cytometry analysis showed that two stages, G1/S and G2/M, were particularly susceptible to MTX treatment. Scanning laser cytometry demonstrated that calcium response of FR 3T3 fibroblasts followed with Indo-1 varied during the cell division cycle. The [Ca2+]i increase was almost always vertical, but its delay after MTX addition lasted from zero (S and G2/M transition) to 10-20 min (G1) or more (G2). No [Ca2+]i change could be detected during mitosis. The [Ca2+]i response at the S phase was biphasic. These observations suggest that (1) the lasting response described in the literature represents a global cell population effect, and (2) cells are more sensitive to MTX at specific stages of the cell division cycle, which could correspond to periods when calcium signals have been detected in different cell types.


Boddy L, Morris CW, Wilkins MF, Tarran GA, Burkill PH (1994) Neural network analysis of flow cytometric data for 40 marine phytoplankton species. Cytometry 15 :283-293

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Flow cytometry data (time of flight, horizontal and vertical forward light scatter, 90 degrees light scatter, and "red" and "orange" integral fluorescence) were collected for laboratory cultures of 40 species of marine phytoplankton, from the following taxonomic classes, the Dinophyceae, Bacillariophyceae, Prymnesiophyceae, Cryptophyceae, and other flagellates. Single-hidden-layer "back-propagation" neural networks were trained to discriminate between species by recognising patterns in their flow cytometric signatures, and network performance was assessed using an independent test data set. Two approaches were adopted employing : (1) a hierarchy of small networks, the first identifying to which major taxonomic group a cell belonged, and then a network for that taxonomic group identified to species, and (2) a single large network. Discriminating some of the major taxonomic groups was successful but others less so. With networks for specific groups, cryptophyte species were all identified reliably (probability of correct classification always being > 0.75) ; in the other groups half of the species were identified reliably. With the large network, dinoflagellates, cryptomonads, and flagellates were identified almost as well as by networks specific for these groups. The application of neural computing techniques to identification of such a large number of species represents a significant advance from earlier studies, although further development is required.


Boddy L, Morris CW, Wilkins MF, Tarran GA, Burkill PH (1994) Neural-Network Analysis of Flow Cytometric Data for 40 Marine-Phytoplankton Species. Cytometry 15 :283-293

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Flow cytometry data (time of flight, horizontal and vertical forward light scatter, 90-degrees light scatter, and ’’red’’ and ’’orange’’ integral fluorescence) were collected for laboratory cultures of 40 species of marine phytoplankton, from the following taxonomic classes, the Dinophyceae, Bacillariophyceae, Prymnesiophyceae, Cryptophyceae, and other flagellates. Single-hidden-layer ’’back-propagation’’ neural networks were trained to discriminate between species by recognising patterns in their flow cytometric signatures, and network performance was assessed using an independent test data set. Two approaches were adopted employing : (1) a hierarchy of small networks, the first identifying to which major taxonomic group a cell belonged, and then a network for that taxonomic group identified to species, and (2) a single large network. Discriminating some of the major taxonomic groups was successful but others less so. With networks for specific groups, cryptophyte species were all identified reliably (probability of correct classification always being > 0.75) ; in the other groups half of the species were identified reliably. With the large network, dinoflagellates, cryptomonads, and flagellates were identified almost as well as by networks specific for these groups. The application of neural computing techniques to identification of such a large number of species represents a significant advance from earlier studies, although further development is required. (C) 1994 Wiley-Liss, Inc.


Brzezinski MA, Conley DJ (1994) Silicon Deposition during the Cell-Cycle of Thalassiosira-Weissflogii (Bacillariophyceae) Determined Using Dual Rhodamine-123 and Propidium Iodide Staining. Journal of Phycology 30 :45-55

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The relatively non-toxic dye, rhodamine 123 (R123), was incorporated into the frustule of Thalassiosira weissflogii Grun. clone ACTIN in direct proportion to biogenic silica (BSi). R123 was used together with the DNA stain propidium iodide to track and quantify Si deposition during the cell cycle of T. weissflogii using flow cytometry. Silicon deposition was not continuous through the cell cycle. Deposition of the valves occurred during M phase. The hypocingulum was largely deposited during G1 with some suggestion of minor girdle band deposition during G2. Silicon deposition did not occur during S phase. Assuming that a complete frustule consists of an epivalve, epicingulum, hypocingulum, and hypovalve, then 40% of cellular BSi was contained within the cingulum of T. weissflogii with 60% present in the valves. These percentages correspond to 0.38 pmol Si in the two cingula and 0.57 pmol Si in the valves. Temporal differences in the timing of silicic acid uptake and deposition during the cell cycle of T. weissflogii suggested that deposition of both the new valves and the cingulum is supported by an internal pool of dissolved Si acquired during G2.


Campbell L, Nolla HA, Vaulot D (1994) The Importance of Prochlorococcus to Community Structure in the Central North Pacific-Ocean. Limnology and Oceanography 39 :954-961

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Assessments of plankton community structure in the oligotrophic oceans based solely on microscopy may overstate the importance of heterotrophic bacterial biomass. Using flow cytometry to distinguish heterotrophic bacteria from the photosynthetic procaryotes Prochlorococcus spp., we found that Prochlorococcus contributed 31% of total bacterial counts in the upper 100 m at station ALOHA (22-degrees 45’N, 158-degrees-W). In terms of carbon, procaryotic biomass was the largest component (greater-than-or-equal-to 80%) of the microbial community, but almost half of this was photosynthetic biomass contributed by Prochlorococcus. Overall, the total 200-m integrated photosynthetic biomass exceeded heterotrophic bacterial biomass (55 vs. 45%). We suggest that the relative proportion of photosynthetic to heterotrophic bacterial biomass varies among oligotrophic regions of the ocean and that dominance by heterotrophic bacteria is not typical.


Cariou V, Casotti R, Birrien JL, Vaulot D (1994) The Initiation of Phaeocystis Colonies. Journal of Plankton Research 16 :457-470

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This study was designed to elucidate the sequence of events that leads to the formation of new colonies of Phaeocystis sp. (strain PCC 540) starting from single cells released from mature colonies. Colonies were first isolated by filtration onto a 10 mum mesh. Colonial cells were then liberated by shaking and inoculated into individual culture wells containing medium with a PO42-concentration of approximately 1 muM. Cell size and shape were determined daily by image analysis. while chlorophyll and DNA distributions were estimated by flow cytometry. Released cells were non-flagellated and mostly located in the G1 phase of the cell cycle. They developed flagella and up to 90% became motile within 24 h. Swarmers lost motility rapidly, became elongated, began to cycle again, excreted a mucilaginous compound and divided leading to new colonies within a few days. During this reproducible process, no change of ploidy could be observed. Colonies initially adhered to the bottom of culture wells. Frequent mixing drastically reduced the fraction of colonies produced and their volume. High initial pO42- concentrations (5 muM) delayed colony appearance. whereas low concentrations (0.3 muM) prevented colony formation. The two main conclusions of this study are : (i) under favorable conditions (approximately 1 muM PO42-, no mixing), a large percentage of released colonial cells give back colonies after going through a flagellated stage ; (ii) sexuality does not appear to be involved in this process.


Cleveland JS, Perry MJ (1994) A Model for Partitioning Particulate Absorption into Phytoplanktonic and Detrital Components. Deep-Sea Research Part I-Oceanographic Research Papers 41 :197-221

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A model for partitioning total particulate absorption, measured on glass fiber filters, into phytoplanktonic and detrital components is developed. The model reconstructs absorption spectra for living phytoplankton using total particulate absorption at the red absorption maxima for chlorophylls a and b, concentrations of chlorophyll a and pheopigment, and mean normalized absorption spectra for laboratory-grown algal cultures. The model was developed in stages for two types of phytoplankton assemblages. Section A of the model applies to waters dominated by eukaryotic algae and is based on absorption spectra for chromophytic (phytoplankton containing chlorophyll c) and chlorophytic (containing chlorophyll b) species. Section B of the model. allowing more variability in spectral shape, was developed for algal communities with more diverse pigmentation. All spectra are processed through Section A. with an internal evaluation determining whether processing continues through Section B. Detrital spectra, generated as the difference between total particulate and modelled phytoplanktonic spectra, included pheopigment absorption and had high blue absorption. Blind tests on samples of known composition predicted absorption within 8-10% at 436 nm and 1-13% when averaged from 400 to 700 nm, which is within the expected accuracy of the glass fiber filter method. No true measure of phytoplankton absorption in field samples is available for testing the model, but results from methanol-extractions were used for comparison despite inclusion of pheopigment absorption as ’’phytoplankton’’. For samples collected from coastal waters of Washington State, the Sargasso Sea and coastal waters of Norway, modelled absorption (averaged over 400-700 nm) ranged from 25% lower to 0.5% higher than the methanol-extraction results ; pheopigment absorption inappropriately included in the phytoplankton component accounts for the higher phytoplanktonic absorption estimated by the methanol technique. Successful application of the model to samples from widespread locations, of different absorption magnitudes, and with varied spectral shapes indicate that the model can be used in diverse environments.


Gala WR, Giesy JP (1994) Flow Cytometric Determination of the Photoinduced Toxicity of Anthracene to the Green-Alga Selenastrum-Capricornutum. Environmental Toxicology and Chemistry 13 :831-840

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Certain PAHs are photosensitizers and in the presence of solar radiation can cause toxicity to aquatic plants and animals. The photoinduced toxicity of anthracene to the green alga Selenastrum capricornutum was assessed by the use of flow cytometry to measure cell size, cellular chlorophyll concentration, and cell viability. Anthracene was slightly toxic in the absence of UV-A radiation. The detection of the direct toxicity of anthracene in this study at a concentration of 19 mug/L anthracene resulted from the use of sensitive flow cytometric measures. There was a significant interaction between anthracene and UV-A radiation, which, in combination, caused significant toxic effects on Selenastrum capricornutum. The most sensitive flow cytometric measure of toxicity was the stress index (SI), which was predictive of longer term effects on cell growth. The 28-h EC50 and EC10 for the SI for Selenastrum capricornutum were 16.1 and 8.3 mug/L anthracene, respectively, at 125 muW/cm2 UV-A. All combinations of anthracene and UV-A that inhibited algal growth also caused a significantly greater number of nonviable cells. The flow cytometric methods used in this study proved to be sensitive, predictive measures of the direct and photoinduced toxicity of anthracene and UV-A radiation to Selenastrum capricornutum.


Galbreath PF, Stjean W, Anderson V, Thorgaard GH (1994) Fresh-Water Performance of All-Female Diploid and Triploid Atlantic Salmon. Aquaculture 128 :41-49

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Comparisons of freshwater survival and growth were made between diploid and triploid all-female Atlantic salmon (Salmo salar). All-female fish were produced using sperm from sex-reversed gynogenetic males and triploidy was induced by heat shock. At 4 months post-initiation of feeding the fry were placed in separate replicated rearing tanks for a 16-week early growth trial. Initial and final average weights were 1.3 and 11.0 g/fish, respectively, for diploids, and 1.1 and 11.9 g/fish, respectively, for triploids. Growth rate of triploids was significantly higher than that of diploids (P = 0.028). Following the early growth trial the triploids and diploids were pooled in a single large tank and raised until completion of smoltification at 17 months post-initiation of feeding. Average weight at this time was significantly greater for diploids (94 g/fish) than for triploids (76 g/fish) (P < 0.00002).


Galbreath PF, Thorgaard GH (1994) Viability and Fresh-Water Performance of Atlantic Salmon (Salmo-Salar) X Brown Trout (Salmo-Trutta) Triploid Hybrids. Canadian Journal of Fisheries and Aquatic Sciences 51 :16-24

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Survival to initiation of feeding and early freshwater growth were measured for both diploid (2N) and triploid (3N) Atlantic salmon (Salmo salar) and 2N and 3N hybrids between female Atlantic salmon and male brown trout (Salmo trutta) to evaluate the triploid hybrid’s potential for commercial culture. Crosses were made in 1990 and 1991 and triploidy was induced by heat shock. Average survival to initiation of feeding generally did not differ among the 2N Atlantic salmon and hybrids within years, nor between the 3N crosses within years. Average survival to initiation of feeding of the 3N Atlantic salmon and hybrids relative to the corresponding 2N crosses was reduced by 2 and 9%, respectively, in 1990, and by 55 and 62%, respectively, in 1991. Five freshwater growth trials were conducted to compare the different crosses. Results indicated no consistent differences in relative growth rates, although the hybrids demonstrated greater variability in growth at age 0+.


Glazer AN (1994) Phycobiliproteins - a Family of Valuable, Widely Used Fluorophores. Journal of Applied Phycology 6 :105-112

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Phycobiliproteins are brilliantly colored, highly fluorescent components of the photosynthetic light-harvesting antenna complexes of cyanobacteria (blue-green algae), red algae and cryptomonads. These proteins carry covalently attached linear tetrapyrrole pigments related structurally to biliverdin. Phycobiliproteins, purified from certain organisms, are isolated as either trimers, (alphabeta)3, of approximately M(r) 110-120 x 10(3) (e.g., allophycocyanins), or hexamers, (alphabeta)6gamma, of about M(r) 250 x 10(3) (certain phycoerythrins). Three phycobiliproteins R-phycoerythrin, B-phycoerythrin, and allophycocyanin serve as valuable fluorescent tags with numerous applications in flow cytometry, fluorescence activated cell sorting, histochemistry and, to a limited degree, in immunoassay and detection of reactive oxygen species. These applications exploit the unique physical and spectroscopic properties of phycobiliproteins.


Heldal M, Norland S, Bratbak G, Riemann B (1994) Determination of Bacterial-Cell Number and Cell-Volume by Means of Flow-Cytometry, Transmission Electron-Microscopy, and Epifluorescence Microscopy. Journal of Microbiological Methods 20 :255-263

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Comparative measurements of bacterial total counts and volumes by flow cytometry (FCM), transmission electron microscopy (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1.10(6)-6.10(6) cells ml(-1), 1.10(6)-3.10(6) cells ml(-l), and 5.10(5) cells ml(-1) respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12-0.15 mu m(3) at the start of the experiment to 0.39-0.53 mu m(3) at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 mu m(3) were not detected by flow cytometry and these counts were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was larger than 0.3 mu m(3), all three methods were in agreement both with respect to counts and volume estimates.


Kaprelyants AS, Mukamolova GV, Kell DB (1994) Estimation of Dormant Micrococcus-Luteus Cells by Penicillin Lysis and by Resuscitation in Cell-Free Spent Culture-Medium at High Dilution. Fems Microbiology Letters 115 :347-352

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Micrococcus luteus starved for 2-7 months in spent medium following growth to stationary phase in batch culture exhibited a culturability (as estimated by direct plating on nutrient agar plates) of < 0.001%. However, following a lag, some 70% of the cells could be lysed upon inoculation into and cultivation in fresh lactate minimal medium containing penicillin, showing the capability of a significant portion of the cells at least to enlarge (and thus potentially to resuscitate). When the viable cell count was estimated using the most probable number method, by incubation of high dilutions of starved cells in liquid growth media, the number of culturable or resuscitable cells was very low, and little different from the viable cell count as assessed by plating on solid ; media. However, the apparent viability of these populations evidenced with the most probable number method was 1000-100000-fold greater when samples were diluted into liquid media containing supernatants taken from the stationary phase of batch cultures of the organism, suggesting that viable cells can produce a factor which stimulates the resuscitation of dormant cells. Both approaches show, under conditions in which the growth of a limited number of viable cells during resuscitation is excluded, that a significant portion of the apparently non-viable cell population in an extended stationary phase is dormant, and not dead.


Keller MD, Shapiro LP, Haugen EM, Cucci TL, Sherr EB, Sherr BF (1994) Phagotrophy of Fluorescently Labeled Bacteria by an Oceanic Phytoplankter. Microbial Ecology 28 :39-52

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Using fluorescently-labeled bacteria and detection by flow cytometry and epifluorescence microscopy, we demonstrate inducible mixotrophy in a marine photosynthetic flagellate, Ochromonas sp. (class Chrysophyceae). Phagotrophic uptake of bacteria increases under conditions of low or limiting light and nutrients, but deceases in periods of prolonged darkness ; sustained phagotrophy may require light. In addition, this alga appears to discriminate between and preferentially ingest different types of bacteria. Although this clone is primarily photosynthetic, phagotrophy contributes to its nutrition, especially when light or nutrients limit photosynthesis.


Lebaron P, Joux F (1994) Flow cytometric analysis of the cellular DNA content of Salmonella typhimurium and Alteromonas haloplanktis during starvation and recovery in seawater. Appl Environ Microbiol 60 :4345-4350

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Flow cytometry was used to investigate the heterogeneity of the DNA content of Salmonella typhimurium and Alteromonas haloplanktis cells that were starved and allowed to recover in seawater. Hoechst 33342 (bisbenzimide) was used as a DNA-specific dye to discriminate between DNA subpopulations. The DNA contents of both strains were heterogeneous during starvation. S. typhimurium cells contained one or two genomes, and A. haloplanktis cells contained up to six genomes. S. typhimurium genomes were fully replicated at the onset of starvation. Each replication cycle was completed in the early stage of starvation for A. haloplanktis by stopping cells in the partition step of the cell cycle prior to division. Multigenomic marine cells can undergo rapid cell division without DNA synthesis upon recovery, resulting in large fluctuations in the DNA contents of individual cells. In contrast, the heterogeneity of the DNA distribution of S. typhimurium cells was preserved during recovery. The fluctuations in the DNA fluorescence of this strain seem to be due to topological changes in DNA. Flow cytometry may provide a new approach to understanding dynamic and physiological changes in bacteria by detecting cellular heterogeneity in response to different growth conditions.


Lebaron P, Joux F (1994) Flow Cytometric Analysis of the Cellular DNA Content of Salmonella-Typhimurium and Alteromonas-Haloplanktis during Starvation and Recovery in Seawater. Applied and Environmental Microbiology 60 :4345-4350

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Flow cytometry was used to investigate the heterogeneity of the DNA content of Salmonella typhimurium and Alteromonas haloplanktis cells that were starved and allowed to recover in seawater. Hoechst 33342 (bisbenzimide) was used as a DNA-specific dye to discriminate between DNA subpopulations. The DNA contents of both strains were heterogeneous during starvation. S. typhimurium cells contained one or two genomes, and A. haloplanktis cells contained up to six. genomes. S. typhimurium genomes were fully replicated at the onset of starvation. Each replication cycle was completed in the early stage of starvation for A. haloplanktis by stopping cells in the partition step of the cell cycle prior to division. Multigenomic marine cells can undergo rapid cell division without DNA synthesis upon recovery, resulting in large fluctuations in the DNA contents of individual cells. In contrast, the heterogeneity of the DNA distribution of S. typhimurium cells was preserved during recovery. The fluctuations in the DNA fluorescence of this strain seem to be due to topological changes in DNA. Flow cytometry may provide a new approach to understanding dynamic and physiological changes in bacteria by detecting cellular heterogeneity in response to different growth conditions.


Lebaron P, Troussellier M, Got P (1994) Accuracy of Epifluorescence Microscopy Counts for Direct Estimates of Bacterial Numbers. Journal of Microbiological Methods 19 :89-94

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The aim of this study was to determine the extent and source of inaccuracies for total counts of microorganisms by filtration followed by epifluorescent microscopy. The extent of these inaccuracies were determined by comparing the microscopic method with viable plate counts while their possible sources were investigated using flow cytometry and fluorescent microbeads. One factor responsible for these inaccuracies was found to be the microscope conversion factor which did not take into account the total filter surface area on which the bacteria were impacted. Approximately a two-fold underestimation was observed when the filtration area rather than the effective area over which the cells are distributed were compared. Thus it is recommended that the latter be accurately measured when calculating the conversion factor in order to reduce the inaccuracy of direct microscopic counts.


Mcmurter HJG, Pick FR (1994) Fluorescence Characteristics of a Natural Assemblage of Fresh-Water Picocyanobacteria. Journal of Plankton Research 16 :911-925

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The fluorescence characteristics of a freshwater assemblage of picocyanobacteria were determined in a mesotrophic lake using microspectrofluorometry. In Jack’s Lake, Ontario, 72-98% of the assemblage was comprised of cells with a single excitation peak for chlorophyll (Chl) a (emission at 680 nm) at 565 (+/-3) nm, the excitation spectra for chlorophyll (Chl) a composition of downwelling irradiance. The assemblage was, therefore, dominated by a single phycobiliprotein pigment type, similar to type 2 phycoerythrin (PE) marine Synechococcus strains. The shape of excitation spectra did not change significantly with depth down to 0.6% of incident irradiance or between sampling dates, although the relative intensity of the PL excitation peak was generally greater for populations below the thermocline compared to surface populations during summer stratification. Two separate populations of PE-containing picocyanobacteria, distinguished based on their morphology and plane of division, could be further separated based on their emission spectra (using blue excitation) : a Synechocystis type cell (PE-Sys) consistently had a more pronounced peak at 665 nm from allophycocyanin compared to a Synechocystis (PE-Syn) type cell. In addition, the ratio of the PE to Chl a peak emissions was higher in PE-Sys and increased significantly with depth below the thermocline. While nitrogen was limiting in the lake in summer, experimental additions of nitrogen did not significantly affect this ratio in surface water populations, but increased the ratio in PE-Syn populations at the base of the photic zone. For surface assemblages of picocyanobacteria, high irradiance may be more important in regulating fluorescence characteristics than nitrogen stress.


Monfort P, Baleux B (1994) Effects of Environmental-Factors Present in the St-Lawrence Estuary (Quebec, Canada) on Experimental Survival of Salmonella-Salamae as Determined by Flow-Cytometry. Canadian Journal of Microbiology 40 :712-719

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Survival of Salmonella salamae in the St. Lawrence Estuary was studied experimentally during an oceanographic cruise using in situ exposure diffusion chambers. The abundance distribution (colony-forming units) of culturable S. salamae on media was compared with the distribution of cells enumerated by flow cytometry. Flow cytometry was also used to characterize the size distribution and DNA content of cells exposed to various environmental factors. Solar radiation, starvation, and a gradual increase in salinity led to an abrupt loss of the ability of S. salamae cells to form cultures and to a gradual reduction in the cell size and DNA content. Conversely, starvation combined with a gradual increase in salinity in the absence of sunlight led to st gradual loss of the cells’ ability to form cultures and an abrupt reduction in cell size and DNA content (i.e., a rapid increase in cell damage). Mortality (i.e., a decrease in total cell count) of S. salamae placed in darkness began at a lower salinity (11.4 parts per thousand) than did the mortality of cells exposed to sunlight (23.1 parts per thousand). Therefore, the S. salamae cells exposed to sunlight seemed to be more resistant to gradual salinity stress than the cells that were not subjected to sunlight.


Palenik B (1994) Cyanobacterial Community Structure as Seen from Rna-Polymerase Gene Sequence-Analysis. Applied and Environmental Microbiology 60 :3212-3219

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PCR was used to amplify DNA-dependent RNA polymerase gene sequences specifically from the cyanobacterial population in a seawater sample from the Sargasso Sea. Sequencing and analysis of the cloned fragments suggest that the population in the sample consisted of two distinct clusters of Prochlorococcus-like cyanobacteria and four clusters of Synechococcus-like cyanobacteria. The diversity within these clusters was significantly different, however. Clones within each Synechococcus-like cluster were 99 to 100% identical, while each Prochlorococcus-like cluster was only 91% identical at the nucleotide level. One Prochlorococcus-like cluster was significantly more closely related to a Mediterranean Sea (surface) Prochlorococcus isolate than to the other cluster, showing the highly divergent nature of this group even in one sample. The approach described here can be used as a general method for examining cyanobacterial diversity, while an oligotrophic ocean ecosystem such as the Sargasso Sea may be an ideal model for examining diversity in relation to environmental parameters.


Poryvkina L, Babichenko S, Kaitala S, Kuosa H, Shalapjonok A (1994) Spectral Fluorescence Signatures in the Characterization of Phytoplankton Community Composition. Journal of Plankton Research 16 :1315-1327

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Fluorescence spectral signatures from 28 algal cultures are described. The cultures are split into four groups according to their accessory pigments. Phycocyanin and phycoerythrin, character istic pigments of cyanobacteria, form groups I and II. The characteristic pigment found in group III is chlorophyll b (green and prasinophyte algae) and in group IV it is chlorophyll c (diatoms, dinophytes and some other algae). This preliminary catalogue of spectral signatures was used to characterize five natural phytoplankton communities from brackish water environments as a comparison with phytoplankton species found in the samples. Accessory pigments such as phycocyanin and phycoerythrin, characterizing groups I and II, can be used for identification without confusion. Distinguishing between groups III and IV is more complicated, because their accessory pigments do. not have their own fluorescence. These groups can be characterized by increased fluorescence of chlorophyll a induced by energy excited through chlorophyll b and c. The possibilities of applying the spectral fluorescence signatures approach to the characterization of natural phytoplankton communities are discussed.


Reiriz S, Cid A, Torres E, Abalde J, Herrero C (1994) Different responses of the marine diatom Phaeodactylum tricornutum to copper toxicity. Microbiologia 10 :263-272

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Different responses of the marine diatom Phaeodactylum tricornutum (Bohlin) to toxic copper concentrations were investigated. Besides the classical variables applied to toxicity studies in microalgae, such as growth or chlorophyll a content, other variables analyzed by flow cytometry were used. Toxic effects due to copper concentration were observed. Cell density reached in the stationary phase was reduced to 50% in cultures with 20 mg Cu/l, with respect to control cultures without copper. Cell light scatter properties (related to cell volume and intracellular granularity) and chlorophyll a fluorescence of microalgal cells were determined by flow cytometry analysis at the beginning of growth, 1 h after copper exposure, and when cultures reached the stationary phase (72 h). After 1 h of exposure to metal, no differences were observed, but when cultures reached the stationary phase, a gradual increase in the variables analyzed by flow cytometry was observed as the copper concentration increased. The increase in chlorophyll a fluorescence detected by flow cytometry was not correlated with an increase in the cell content of this photopigment, thus indicating an inhibitory effect of copper on photosystem II.


Robins DB, Bedo AW (1994) Quantitative determination of particle concentrations in experimental and marine environmental samples. Cytometry 17 :179-184

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As the application of flow cytometry increases across the environmental sciences, there is a need to expand instrument flexibility. In particular, some of the major applications within aquatic research demand an ability to quantify the particles under investigation, expressing them as numbers per unit of sample volume. Two of the principal areas of flow cytometric investigations are the characterisation of naturally occurring particulate assemblages in the oceans and investigations into the feeding behaviour of "filter feeding" organisms on the mixtures of such particles. In both cases some of the most useful data derived from flow cytometry require that analyses be determined in terms of particle concentrations. Therefore, a simple and practical modification is described, which allows known sample volumes to be analysed on flow cytometers. The method has a high degree of flexibility in sample volume and is cost effective and reliable. It involves introducing a standard chromatography "loop injection valve" into the sample-handling fluidics system. Use of fluorescent beads shows that a range of particle sizes and concentrations can be used with this system. Tests, also using fluorescent beads and an algal culture, show that replicate 100 microliter sample volumes can be analysed and counted with a coefficient of variation of less than 3%.


Robins DB, Bedo AW (1994) Quantitative-Determination of Particle Concentrations in Experimental and Marine Environmental-Samples. Cytometry 17 :179-184

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As the application of flow cytometry increases across the environmental sciences, there is a need to expand instrument flexibility. In particular, some of the major applications within aquatic research demand an ability to quantify the particles under investigation, expressing them as numbers per unit of sample volume. Two of the principal areas of flow cytometric investigations are the characterisation of naturally occurring particulate assemblages in the oceans and investigations into the feeding behaviour of ’’filter feeding’’ organisms on the mixtures of such particles. In both cases some of the most useful data derived from flow cytometry require that analyses be determined in terms of particle concentrations. Therefore, a simple and practical modification is described, which allows known sample volumes to be analysed on flow cytometers. The method has a high degree of flexibility in sample volume and is cost effective and reliable. It involves introducing a standard chromatography ’’loop injection valve’’ into the sample-handling fluidics system. Use of fluorescent beads shows that a range of particle sizes and concentrations can be used with this system. Tests, also using fluorescent beads and an algal culture, show that replicate 100 mu l sample volumes can be analysed and counted with a coefficient of variation of less than 3%. (C) 1994 Wiley-Liss, Inc.


Simon N, Barlow RG, Marie D, Partensky F, Vaulot D (1994) Characterization of Oceanic Photosynthetic Picoeukaryotes by Flow-Cytometry. Journal of Phycology 30 :922-935

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To interpret flow cytometric data that are routinely obtained on natural oceanic communities, 23 strains of photosynthetic picoeukaryotes belonging to four classes (Prasinophyceae, Chlorophyceae, Pelagophyceae, and Prymnesiophyceae) and six pigment types were investigated for their light scattering in the forward and right-angle directions, chlorophyll fluorescence, and DNA content as measured by flow cytometry. Cell size was assessed by Coulter counter, and pigment composition was measured by reverse-phase high-performance liquid chromatography. The size and GC% of the nuclear genome of cultured picoeukaryotes was measured from fluorescence of DNA specific dyes. Using these two parameters, we could discriminate species within pigment groups. DNA staining of preserved natural samples may also prove useful in discriminating cooccurring populations in situ as long as the communities are not tao complex. Using the relationships that we established between size and light-scattering properties of the cells, we estimated eqzcivalent diameters of picoeukaryotes in natural populations to be between 1.3 and 2 mu m. Chlorophyll a content was between 6 and 16 fg.cell(-1) as calculated from relationships that we established between chlorophyll a content and red fluorescence of the cultured strains. With respect to size, chlorophyll a content, and pigment composition, Pelagomonas sp. strains (Pelagophyceae) appeared to be the most representative of the natural communities in subtropical ocean waters. In contrast, green coccoid strains, which often outcompete other strains in culture, might only be minor contributors to these communities.


Unson MD, Holland ND, Faulkner DJ (1994) A Brominated Secondary Metabolite Synthesized by the Cyanobacterial Symbiont of a Marine Sponge and Accumulation of the Crystalline Metabolite in the Sponge Tissue. Marine Biology 119 :1-11

<Go to ISI> ://A1994NK27500001

The dictyoceratid marine sponge Dysidea herbacea (Keller, 1889) is common in shallow waters of the tropical Pacific Ocean. Polybrominated biphenyl ethers such as 2-(2’,4’-dibromophenyl)-4,6-dibromopheno (1) are characteristic secondary metabolites of some specimens of this sponge and may represent as much as 12% of the dry weight. We have found 1 to be deposited as conspicuous crystals throughout the sponge tissue. The dominant prokaryotic endosymbiont in the mesohyl of the sponge is a filamentous cyanobacterium (Oscillatoria spongeliae), although a vacuole-containing, heterotrophic bacterium is also present. The cyanobacteria were separated from the sponge cells and heterotrophic bacteria by flow cytometry. Coupled gas chromatography-mass spectrometry and proton nuclear magnetic-resonance spectroscopy revealed that the major brominated Compound 1 isolated from the intact symbiotic association is found in the cyanobacteria and not in the sponge cells or heterotrophic bacteria. This suggests that the production of the compound is due to the cyanobacterium, and not to the sponge or symbiotic heterotrophic bacteria, as had been suggested earlier.


Vaulot D, Birrien JL, Marie D, Casotti R, Veldhuis MJW, Kraay GW, Chretiennotdinet MJ (1994) Morphology, Ploidy, Pigment Composition, and Genome Size of Cultured Strains of Phaeocystis (Prymnesiophyceae). Journal of Phycology 30 :1022-1035

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We examined cell morphology, ploidy level, cell size, pigment composition, and genome size in 16 cultured strains of Phaeocystis Lagerheim. Two strains originated from the Antarctic, 3 from the tropical Western Atlantic, and 11 from temperate regions (Eastern Atlantic, English Channel, North Sea, and Mediterranean Sea). Thirteen strains made colonies morphologically similar to P. globosa Scherffel, whereas three never formed colonies under any circumstances. Five-rayed star-like structures with filaments were observed in 11 strains. In several strains, two ploidy levels were observed, one (haploid) linked to flagellates and one (diploid) linked to colonies. Cell size did not appear to be a very good criterion for distinguishing strains since size distributions overlapped. Pigment analysis by reversed-phase-high-performance liquid chromatography allowed the strains to be grouped into three clusters that differed from each other mainly by the relative proportions of three carotenoids : fucoxanthin, 19’-hexanoyloxyfucoxanthin, and diadinoxanthin. All strains contained low levels of 19’-butanoyloxyfucoxanthin. Differences in genome size measured by flow cytometry delimited at least five groups. On the basis of bath pigment composition and genome size, six clusters were defined, one corresponding to an Antarctic species (possibly P. antarctica), one to P. globosa, and the rest probably to several yet-undescribed species or subspecies. Two main conclusions emerge from this study. First, the taxonomy of the genus Phaeocystis needs to be clarified through a combination of morphological, biochemical, and molecular studies. Second, sexuality is a prevalent phenomenon in Phaeocystis, but controls of the sexual cycle are most likely strain-dependent,


Vesey G, Narai J, Ashbolt N, Williams K, Veal D (1994) Detection of Specific Microorganisms in Environmental-Samples Using Flow-Cytometry. Methods in Cell Biology, Vol 42 42 :489-522

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Vrieling EG, Peperzak L, Gieskes WWC, Veenhuis M (1994) Detection of the Ichthyotoxic Dinoflagellate Gyrodinium (Cf) Aureolum and Morphologically Related Gymnodinium Species Using Monoclonal-Antibodies - a Specific Immunological Tool. Marine Ecology-Progress Series 103 :165-174

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Sixteen monoclonal antibodies which recognize different cell surface antigens of the ichthyotoxic marine dinoflagellate Gyrodinium cf. aureolum were prepared and characterized for use in identification by both immunofluorescence microscopy and flow cytometry. Based on the labeling results obtained from indirect immunofluorescence assays the monoclonals could be divided into 3 groups : (I) fluorescence of the overall cell surface, (II) fluorescence of the overall cell surface together with the flagella, and (III) fluorescence of granular-like structures randomly distributed along the cell surface and at the edges of the sulcus and cingulum. Cross-reactivity of 6 selected monoclonals with other phytoplankton species revealed that labeling was restricted to G. aureolum/G. cf. aureolum and to the morphologically closely related species Gymnodinium nagasakiense and G. mikimotoi. This suggests a taxonomic proximity of these species. Fixation of samples with paraformaldehyde produced the optimal immunochemical response, resulting in the strongest fluorescence intensities. In flow cytometric analyses, labeled cells of G. cf. aureolum could be distinguished sufficiently from unlabeled controls when monoclonals of Group II and a pooled serum (combined antibodies of Groups I, II, and III) were used. This immunochemical technique will be applied in an early warning system to detect G. aureolum cells at dilute concentrations using flow cytometry or, alternatively, image analysis.


Wilkins MF, Morris CW, Boddy L (1994) A comparison of Radial Basis Function and backpropagation neural networks for identification of marine phytoplankton from multivariate flow cytometry data. Comput Appl Biosci 10 :285-294

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7922685

Two artificial neural network classifiers, the well-known Multi-layer Perception (MLP) (also known as the ’backpropagation network’), and the more recently developed Radial Basis Function (RBF) network, were evaluated and compared for their ability to identify multivariate flow cytometric data from five North Sea plankton groups (Dinoflagellidae, Bacillariophyceae, Prymnesiomonadida, Cryptomonadida, and other flagellates). RBF networks generally performed similarly to MLPs, and slightly better in cases where the data were markedly multimodal ; RBF networks also have much shorter training times. The performance of MLPs was improved greatly by the use of a symmetrical bipolar ’transfer function’ as opposed to the commonly-used asymmetric form. The issues of network optimisation and computational efficiency in use are discussed.


Wilkins MF, Morris CW, Boddy L (1994) A Comparison of Radial Basis Function and Backpropagation Neural Networks for Identification of Marine-Phytoplankton from Multivariate Flow-Cytometry Data. Computer Applications in the Biosciences 10 :285-294

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Two artifical neural network classifiers, the well-known Multi-layer Perceptron (MLP) (also known as the ’backpropagation network’), and the more recently developed Radial Basis Function (RBF) network, were evaluated and compared for their ability to identify multivariate flow cytometric data from Jive North Sea plankton groups (Dinoflagellidae, Bacillariophyceae, Prymnesiomonadida, Cryptomonadida, and other flagellates). RBF networks generally performed similarly to MLPs, and slightly better in cases where the data M !ere markedly multimodal ; RBF networks also have much shorter training times. The performance of MLPs was improved greatly by the use of a symmetrical bipolar ’transfer function’ as opposed to the commonly-used asymmetric form. The issues of network optimisation and computational efficiency in use are discussed.


Yentsch CM, Pomponi SA (1994) Strategies for flow cytometric analysis of marine microalgae and sponge cells. Methods Cell Biol 42 Pt B :523-538

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7533255


Yu FP, Mcfeters GA (1994) Rapid in-Situ Assessment of Physiological Activities in Bacterial Biofilms Using Fluorescent-Probes. Journal of Microbiological Methods 20 :1-10

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Two rapid in situ enumeration methods using fluorescent probes were used to assess the physiological activities of Klebsiella pneumoniae biofilms on stainless steel. Fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and rhodamine 123 (Ph 123), were chosen to perform this study. CTC is a soluble redox indicator which can be reduced by respiring bacteria to fluorescent CTC-formazan crystals. Ph 123 is incorporated into bacteria with respect to cellular proton motive force. The intracellular accumulation of these fluorescent dyes can be determined using epifluorescence microscopy. The results obtained with these two fluorescent probes in situ were compared to the plate count (PC) and in situ direct viable count (DVC) methods. Viable cell densities within biofilms determined by the three in situ methods were comparable and always showed approximately 2-fold higher values than those obtained with the PC method. As an additional advantage, the results were observed after 2 h, which was shorter than the 4 h incubation time required for the DVC method and 24 h for colony formation. The results indicate that staining with CTC and Rh 123 provides rapid information regarding cell numbers and physiological activities of bacteria within biofilms.