1997

mardi 21 avril 2009
par   G. Grégori

Barcina I, Lebaron P, VivesRego J (1997) Survival of allochthonous bacteria in aquatic systems : A biological approach. Fems Microbiology Ecology 23 :1-9

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The survival of allochthonous bacteria in aquatic systems is affected by biotic and abiotic environmental factors. Grazing by protozoa is one of the main biological processes that control allochthonous bacterial density. Its extent depends on the concentration of bacteria and the digestion capacity of the grazer. The physiological state of bacteria is affected by multiple physicochemical stresses, to which they respond by entering a dormant, viable but non-culturable state. Starved bacteria show a tendency to shrink, and a generally enhanced resistance to heat, oxidative and osmotic shock is observed. Nutrient scarcity, temperature, osmotic stress and visible light seem to be the abiotic factors that most negatively influence survival. The negative effect of light upon the culturability of enterobacteria in aquatic systems has long been recognized. In relation to the influence of plasmids on bacterial survival, heterogeneous and contradictory results have been reported. Some authors reported that plasmid-bearing strains can survive as well as their wild-type counterparts or even better, whereas in other reports the effects of various plasmids on the survival of their hosts were very variable. Plasmid transfer could be affected by the physiological status of donors and recipients during survival. Flow cytometry is a recent approach with great potential, especially for assessing the heterogeneity of cell size, metabolic state and molecular content in the population.


Blanchot J, Andre JM, Navarette C, Neveux J (1997) Picophytoplankton dynamics in the equatorial Pacific : diel cycling from flow-cytometer observations. Comptes Rendus De L Academie Des Sciences Serie Iii-Sciences De La Vie-Life Sciences 320 :925-931

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The French JGOFS cruise FLUPAC was conducted in October-November 1994 in the western and central equatorial Pacific Ocean. During a 7-day time series at 150 degrees W, in addition to conventional sampling (four times per day from discrete depths between 0 and 150 m), a high frequency (hourly) experiment was performed by continuously pumping water at 5 m below the surface over 24 h. Flow cytometric measurements allowed us to recognise and to follow separately the two major components of equatorial picophytoplankton, the Prochlorococcus and the picoeukaryotes. The hourly surface experiment confirmed the synchrony of Prochlorococcus cell division and showed that picoeukaryotes exhibited a similar behaviour. The main consequence is that the maximum potential growth rate of picophytoplankton is one doubling per day for both cell groups. The vertical profiles indicated that the diel cycling extends throughout the surface layer for both algal groups. The cells were observed to divide daily from late afternoon to the middle of the night, and then to disappear, probably as a result of grazing. In the surface layer variations of abundance allowed us to estimate a growth rate of about 0.6 day(-1). Mean cell light scattering <(FS)over bar> as measured by the cytometer indicated a decrease in cell size concurrent with cell division and an increase during the photosynthetic growth phase.


Campbell L, Liu HB, Nolla HA, Vaulot D (1997) Annual variability of phytoplankton and bacteria in the subtropical North Pacific Ocean at Station ALOHA during the 1991-1994 ENSO event. Deep-Sea Research Part I-Oceanographic Research Papers 44 :167-&

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Time-series data on community structure in the upper 200 m at Station ALOHA in the subtropical North Pacific were collected at approximately monthly intervals from December 1990 through to March 1994 during an extended El Nino-Southern Oscillation (ENSO) event ; Samples were analyzed by flow cytometry to enumerate Prochlorococcus, Synechococcus, picoeucaryotes, 3-20 mu m algae, and heterotrophic bacteria, as well as to quantify cellular chlorophyll fluorescence for the autotrophic components. A significant seasonal cycle was evident in cellular chlorophyll fluorescence for each of the autotrophic components, with maxima occurring each winter as a consequence of photoacclimation. Abundance of each picophytoplankton component exhibited temporal variability on both seasonal and interannual scales. Although the magnitude gf the seasonal cycles in the abundance was relatively small, the cycles appeared to be out of Phase. Typically, abundance maxima of Synechococcus occurred in winter, of picoeucaryotes in spring, and of Prochlorococcus during summer/fall. The different timing in these cycles may explain why the presence of a seasonal pattern in total phytoplankton biomass has been difficult to establish. Abundance of the larger 3-20 mu m algae varied over two orders of magnitude during the time series, with no obvious seasonal pattern. The 3-20 mu m algae were a small percentage of the total estimated carbon biomass (similar to 8%). Heterotrophic bacteria were the most numerous of the picoplankton, and the seasonal pattern in their 200-m integrated abundance paralleled Prochlorococcus over the time series. Together, the procaryotes contributed 60-90% of the total estimated microbial carbon. Significant interannual variation in the total 200-m integrated microbial carbon estimates may be related to the effects of the extended ENSO event, which began in 1991. (C) 1997 Elsevier Science Ltd.


Cid A, Torres E, Herrero C, Abalde JE (1997) Disorders provoked by copper in the marine diatom Phaeodactylum tricornutum in short-time exposure assays. Cahiers De Biologie Marine 38 :201-206

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Copper toxicity on the marine diatom Phaeodactylum tricornutum was assessed after 24 hours of exposure, using different parameters : growth, C-14-bicarbonate assimilation, H-3-thymidine incorporation, photosynthetic pigment content, and others assayed by flow cytometry, such as cellular volume, chlorophyll a autofluorescence and cell viability. Different degrees of sensitivity were observed for the different analysis. Cell viability assayed by flow cytometry was the less sensitive parameter.


delGiorgio PA, Prairie YT, Bird DF (1997) Coupling between rates of bacterial production and the abundance of metabolically active bacteria in lakes, enumerated using CTC reduction and flow cytometry. Microbial Ecology 34 :144-154

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In natural bacterioplankton assemblages, only a fraction of the total cell count is active, and, therefore, rates of bacterial production should be more strongly correlated to the number of active cells than to the total number of bacteria. However, this hypothesis has seldom been tested. Herein we explore the relationship between rates of bacterial production (measured as leucine uptake) and the number of active bacteria in 14 lakes in southern Quebec. Active bacteria are defined as those cells capable of reducing the tetrazolium salt CTC to its fluorescent formazan ; these cells were enumerated using now cytometry. Bacterial production varied two orders of magnitude in the lakes studied, as did the number of active bacteria, whereas the total number of bacteria varied by only sixfold. The number and proportion of active bacteria were similar among lake strata, but rates of bacterial production were highest in the epilimnion and lo west in the hypolimnion. As expected, bacterial production was better correlated to the number of active cells, and bacterial growth rates calculated for active cells ranged from 0.7 to 1.8 day(-1), on average threefold higher than those calculated on the basis of total bacterial abundance. Growth rates scaled to active cells were, on average, similar among lake strata and did not show any pattern along a gradient of increasing chlorophyll concentration, so there was no systematic change of bacterial growth rates with lake productivity. In contrast, growth rates scaled to the entire bacterial assemblage were positively correlated to chlorophyll, were tenfold more variable among lakes than growth rates of active cells, and showed larger differences among lake strata. Scaling bacterial production to either the total number or the number of active cells thus results in very different patterns in bacterial growth rates among aquatic systems.


Faber MJ, Smith LMJ, Boermans HJ, Stephenson GR, Thompson DG, Solomon KR (1997) Cryopreservation of fluorescent marker-labeled algae (Selenastrum capricornutum) for toxicity testing using flow cytometry. Environmental Toxicology and Chemistry 16 :1059-1067

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A rapid, two-stain (fluorescein diacetate and ethidium homodimer-l) Row cytometric assay to evaluate viability and cytotoxicity of the alga Selenastrum capricornutum in preserved samples is described. For storage, stained cells were fixed in glutaraldehyde, flash frozen in liquid nitrogen, and stored frozen (-20 degrees C) for assessment at a later date. Weekly analysis of frozen samples showed that fluorescence was stable for 7 weeks. A mixture of 50% healthy and 50% heat-killed cells of S. capricornutum showed 36.1% healthy cells, 13.2% compromised cells, 12% nonstained cells, and 38.7% dead cells. This technique was tested using sodium dodecyl sulfate (SDS) and phenol as toxicants. Threshold concentrations for toxicant impact were in the range of 1 to 10 mg/L for SDS and 10 to 100 mg/L for phenol. Estimates of mortality showed 96-h median lethal concentration (LC50) values of 2,340 mg/L and 6,970 mg/L for SDS and phenol, respectively. This two-stain flow cytometric procedure has proven to be a reliable, sensitive assay for determining viability of S. capricornutum in preserved samples. The sensitivity of this dual-color assay and the ability to store samples for later analysis are significant improvements over current techniques.


Fronicke L, MullerNavia J, Romanakis K, Scherthan H (1997) Chromosomal homeologies between human, harbor seal (Phoca vitulina) and the putative ancestral carnivore karyotype revealed by Zoo-FISH. Chromosoma 106 :108-113

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We report on the construction of the first comparative Zoo-FISH map of a marine mammal. Zoo-FISH with DNA probes from a human chromosome-specific library to metaphase spreads of the harbor seal (Phoca vitulina) disclosed 31 conserved syntenic segments covering the complete autosomal complement and the X chromosome. Comparison with Zoo-FISH maps of other species reveals that the harbor seal shares a high degree of karyotypic homeology with the human complement and an even higher degree with the conordinal cat complement. These findings suggest that pinniped, felid and human karyotypes have maintained conserved complements. Based on data of Zoo-FISH and comparative cytogenetics, a Zoo-FISH map of the ancestral carnivore karyotype (Z-CAR) is proposed. Flow cytometry revealed that the DNA value of the harbor seal genome is 79% that of the human genome.


Fronicke L, Muller-Navia J, Romanakis K, Scherthan H (1997) Chromosomal homeologies between human, harbor seal (Phoca vitulina) and the putative ancestral carnivore karyotype revealed by Zoo-FISH. Chromosoma 106 :108-113

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9215560

We report on the construction of the first comparative Zoo-FISH map of a marine mammal. Zoo-FISH with DNA probes from a human chromosome-specific library to metaphase spreads of the harbor seal (Phoca vitulina) disclosed 31 conserved syntenic segments covering the complete autosomal complement and the X chromosome. Comparison with Zoo-FISH maps of other species reveals that the harbor seal shares a high degree of karyotypic homeology with the human complement and an even higher degree with the conordinal cat complement. These findings suggest that pinniped, felid and human karyotypes have maintained conserved complements. Based on data of Zoo-FISH and comparative cytogenetics, a Zoo-FISH map of the ancestral carnivore karyotype (Z-CAR) is proposed. Flow cytometry revealed that the DNA value of the harbor seal genome is 79% that of the human genome.


Guindulain T, Comas J, VivesRego J (1997) Use of nucleic acid dyes SYTO-13, TOTO-1, and YOYO-1 in the study of Escherichia coli and marine prokaryotic populations by flow cytometry. Applied and Environmental Microbiology 63 :4608-4611

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Three nucleic acid dyes (SYTO-13, TOTO-1, and YOYO-1) were tested on cultures of Escherichia coli and marine prokaryote populations. These dyes stain the RNA and DNA in E. coli but only respond to DNA in marine populations, according to the histograms obtained after DNase and RNase treatments.


Guindulain T, Comas J, Vives-Rego J (1997) Use of nucleic acid dyes SYTO-13, TOTO-1, and YOYO-1 in the study of Escherichia coli and marine prokaryotic populations by flow cytometry. Appl Environ Microbiol 63 :4608-4611

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Three nucleic acid dyes (SYTO-13, TOTO-1, and YOYO-1) were tested on cultures of Escherichia coli and marine prokaryote populations. These dyes stain the RNA and DNA in E. coli but only respond to DNA in marine populations, according to the histograms obtained after DNase and RNase treatments.


Jansson JK, Prosser JI (1997) Quantification of the presence and activity of specific microorganisms in nature. Molecular Biotechnology 7 :103-120

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Traditional techniques for assessment of microbial numbers and activity generally lack the specificity required for risk assessment following environmental release of genetically engineered microbial inocula. Immunological and molecular-based techniques, such as DNA probing and genetic tagging, were initially used to determine the presence or absence of microorganisms in environmental samples. Increasingly they are being developed for quantification of populations of specific organisms, either indigenous or introduced, in the environment. In addition, they are being used to quantify the activity of particular organisms or groups of organisms, greatly extending the range of techniques available to the microbial ecologist. This article reviews the use of traditional techniques for the quantification of microbial population size and activity and the application of molecular techniques, including DNA probing, genetic marking, use of fluorescent probes, and quantitative PCR, in combination with advanced cell detection techniques such as confocal laser scanning microscopy and flow cytometry.


Jones JG (1997) The microbiological quality of water : the nature of the problem. Journal of Water Services Research and Technology-Aqua 46 :346-352

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Improvements in methods for the detection and enumeration of microbes in water, particularly the application of techniques of molecular biology, have highlighted shortcomings in the ’standard methods’ for assessing water quality. Higher expectations from the consumer and increased publicity associated with pollution incidents can lead to an uncoupling of the cycle which links methodological development with standard-setting and legislation.
The new methodology has also highlighed problems within the water cycle, related to the introduction, growth and metabolism of microbes. A greater understanding of the true diversity of the microbial community and the ability to transmit genetic information within aquatic systems ensures that the subject of this symposium and volume provides an ideal forum to discuss the problems encountered by both researcher and practitioner.


Joux F, Lebaron P, Troussellier M (1997) Changes in Cellular States of the Marine Bacterium Deleya aquamarina under Starvation Conditions. Appl Environ Microbiol 63 :2686-2694

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In this study, we have used different fluorescent dyes and techniques to characterize the heterogeneity and changes of the physiological states encountered by the marine bacterium Deleya aquamarina during a 92-day starvation survival experiment at 20 and 5(deg)C. Changes of physiological states were investigated on a single-cell basis by flow cytometry and epifluorescence microscopy in conjunction with fluorescent dyes specific for various cellular functions and constituents. Heterogeneities within populations with regard to functions (respiration, substrate responsiveness, enzymatic activity, and cytoplasmic membrane permeability), constituent (DNA), and cell volume (light scatter) were compared to the evolution of viable plate counts (CFU). At 20(deg)C, CFU changes were divided into three stages corresponding to stability up to day 13 followed by a rapid drop between days 13 and 42 and then by stabilization at a level of 10 to 20% during the remaining survival period. Most of the cellular fractions showing a metabolic activity were close to the evolution of the culturable cells, suggesting the absence of viable but nonculturable cells. On the other hand, cells with selective cytoplasmic membrane permeability but without any metabolic activity were observed, and this stage was followed by DNA alteration occurring at different rates after the loss of membrane cytoplasmic permeability. We observed a greater maintenance of culturability, physiological functions, DNA, and cellular volume at the lower temperature. These results have different ecological implications from both methodological and conceptual viewpoints.


Joux F, Lebaron P, Troussellier M (1997) Succession of cellular states in a Salmonella typhimurium population during starvation in artificial seawater microcosms. Fems Microbiology Ecology 22 :65-76

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The succession of the physiological states of Salmonella typhimurium cells under starvation-survival conditions in artificial seawater was investigated by now cytometry and epifluorescence microscopy using direct examination of different cell functions. Measurements of substrate responsiveness (DVC method), real and potential respiration (redox dye 5-cyano-2,3-ditolyl tetrazolium chloride, CTC), membrane permeability (BacLight(TM) kit), as well as DNA content (Hoechst 33342 staining) reveal an important heterogeneity within the population, and suggest a progressive physiological cell alteration throughout the starvation process. The transition steps between culturable and prelytic cells are described.


Lantoine F, Neveux J (1997) Spatial and seasonal variations in abundance and spectral characteristics of phycoerythrins in the tropical northeastern Atlantic Ocean. Deep-Sea Research Part I-Oceanographic Research Papers 44 :223-246

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A simple and rapid spectrofluorimetric method to quantify phycoerythrins (PEs) from the area below the fluorescence excitation spectra is proposed. In contrast to other methods which used measurements at fixed wavelengths, this method takes into account the spectral diversity of these pigments. It was applied in the tropical northeastern Atlantic during the EUMELI program at three stations which were characterized by different trophic status (eutrophic, mesotrophic and oligotrophic). Sampling was made at various periods of the year. The results showed the constant presence of phycoerythrin nearly always associated with cyanobacteria (Synechococcus). At the oligotrophic site, high phycourobilin (PUB) populations and a great stability in the abundance and spectral characteristics of PE were observed. The eutrophic and mesotrophic sites showed higher PE abundance and spectral variability, and were essentially dominated by high phycoerythrobilin (FEB) populations. A general increase with depth in the PUB/PEB ratio at the eutrophic and mesotrophic sites was observed. This increase could be regular or relatively sharp through physical discontinuity (thermocline or halocline). The presence of Cryptophyceae PE was occasionally noted. The PE diversity was most probably related to the presence of different populations, but some variations could be also influenced by photoacclimation processes. Further progress in the spectrofluorimetric analysis of PE could be achieved by using three-dimensional spectra. (C) 1997 Elsevier Science Ltd.


Lefebvre A, Guelorget O, Perthuisot JP, Courties C, Millet B (1997) Hydrobiological organization of a bahira type paralic basin : Kalloni bay (Lesbos, Greece). Oceanologica Acta 20 :757-768

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The deep, elongated paralic basin of Kalloni bay has been studied through comparisons between several computed hydrodynamical situations and data collected in similar situations from its water body. These include salinities and phytoplanktonic biomasses. Additionally, sedimentological data, which integrate the environmental variations on the long term, have been collected in order to help in understanding the average biogeological functioning of the system. Depending upon its direction and speed, wind forcing induces different salinity and phytoplanktonic patterns. These generally display rather weak gradients from the entrance and the axial deepest channel towards the farthest northeastern reaches of the basin. Moreover, the ranges of salinities and phytoplanktonic biomasses display a very low degree of restriction from the sea (confinement), while the global phytoplanktonic composition, analysed by flow cytometry, shows the prominent marine influence over the basin.
The average hydrodynamical conditions are dominated by the axial influx of marine water on the bottom, and by the surface-reflux of basinal waters along both side banks.
Such a prevailing configuration is also displayed by sedimentological features. Kalloni bay appears as one of the less confined systems in the Mediterranean region, due to its shape and bathymetry, the absence of a sill between the basin and the sea, and active exchanges with the sea under the influence of wind activity.


LopezAmoros R, Castel S, ComasRiu J, VivesRego J (1997) Assessment of E-coli and Salmonella viability and starvation by confocal laser microscopy and flow cytometry using rhodamine 123, DiBAC4(3), propidium iodide, and CTC. Cytometry 29 :298-305

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Assessment of tell viability using methods which do not require cell culture is essential in the field of aquatic microbiology, since many bacteria known to be present in aquatic environments cannot be grown in culture. The study of bacterial biofilms, which previously needed an epifluorescent microscope, has recently been enhanced by the use of flow cytometry and confocal scanning laser microscopy (CSLM), A method based on the combination of several membrane potential related dyes, a membrane integrity dye and a redox probe was used to measure cell viability by flow cytometry and confocal laser microscopy, Rhodamine-propidium iodide (PI) double staining was used to discriminate viable from nonviable cells in CSLM observations. Membrane depolarization during E. coli and Salmonella starvation measured by DiBAC4(3) incorporation (flow cytometry and CSLM) was found to be in concordance with respiratory activity as detected by a tetrazolium salt (CTC) reduction. (C) 1997 Wiley-Liss, Inc.


Lopez-Amoros R, Castel S, Comas-Riu J, Vives-Rego J (1997) Assessment of E. coli and Salmonella viability and starvation by confocal laser microscopy and flow cytometry using rhodamine 123, DiBAC4(3), propidium iodide, and CTC. Cytometry 29 :298-305

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9415412

Assessment of cell viability using methods which do not require cell culture is essential in the field of aquatic microbiology, since many bacteria known to be present in aquatic environments cannot be grown in culture. The study of bacterial biofilms, which previously needed an epifluorescent microscope, has recently been enhanced by the use of flow cytometry and confocal scanning laser microscopy (CSLM). A method based on the combination of several membrane potential related dyes, a membrane integrity dye and a redox probe was used to measure cell viability by flow cytometry and confocal laser microscopy. Rhodamine-propidium iodide (PI) double staining was used to discriminate viable from nonviable cells in CSLM observations. Membrane depolarization during E. coli and Salmonella starvation measured by DiBAC4(3) incorporation (flow cytometry and CSLM) was found to be in concordance with respiratory activity as detected by a tetrazolium salt (CTC) reduction.


Marie D, Partensky F, Jacquet S, Vaulot D (1997) Enumeration and Cell Cycle Analysis of Natural Populations of Marine Picoplankton by Flow Cytometry Using the Nucleic Acid Stain SYBR Green I. Appl Environ Microbiol 63 :186-193

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The novel dye SYBR Green I binds specifically to nucleic acids and can be excited by blue light (488-nm wavelength). Cell concentrations of prokaryotes measured in marine samples with this dye on a low-cost compact flow cytometer are comparable to those obtained with the UV-excited stain Hoechst 33342 (bis-benzimide) on an expensive flow cytometer with a water-cooled laser. In contrast to TOTO-1 and TO-PRO-1, SYBR Green I has the advantage of clearly discriminating both heterotrophic bacteria and autotrophic Prochlorococcus cells, even in oligotrophic waters. As with TOTO-1 and TO-PRO-1, two groups of heterotrophic bacteria (B-I and B-II-like types) can be distinguished. Moreover, the resolution of DNA distribution obtained with SYBR Green I is similar to that obtained with Hoechst 33342 and permits the analysis of the cell cycle of photosynthetic prokaryotes over the whole water column.


Nakayasu C, Yoshitomi T, Oyamatsu T, Okamoto N, Ikeda Y (1997) Separation of carp (Cyprinus carpio L.) thrombocytes by using a monoclonal antibody, and their aggregation by collagen. Vet Immunol Immunopathol 57 :337-346

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A monoclonal antibody against carp peripheral blood leucocytes was produced, and its reactivity analysed by flow cytometry and electron microscopy. The antibody reacted positively with 10-35% of the cells in a fraction of lymphocytes and thrombocytes that was separated by flow cytometry. Electron microscopy using immunogold labelling showed that this antibody reacted strongly with thrombocytes, but not with other leucocytes. By using a magnetic separator, leucocytes that were positive and negative for this antibody were separated. The positive cells were uniform, spindle-type cells that aggregated in the presence of collagen, while the negative cells did not aggregate. Light and electron microscopy showed that many positive cells changed to a spherical form after the addition of collagen and then 40-60% of these cells aggregated.


Pichard SL, Campbell L, Carder K, Kang JB, Patch J, Tabita FR, Paul JH (1997) Analysis of ribulose bisphosphate carboxylase gene expression in natural phytoplankton communities by group-specific gene probing. Marine Ecology-Progress Series 149 :239-253

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To understand the composition and photosynthetic carbon fixing activities of natural phytoplankton communities, we employed group-specific ribulose bisphosphate carboxylase (RubisCO) large subunit gene probes (rbcL) to examine RubisCO gene expression. The rbcL genes from Synechococcus PCC6301 (cyano) and from Cylindrotheca sp. (chrome) were used as probes at select stations to examine levels of rbcL mRNA in specific size fractions (>5 mu m, 1-5 mu m, <1 mu m) in surface waters of the mouth of Tampa Bay (estuarine), West Florida Shelf (coastal), and from the offshore Gulf of Mexico. Using DNA purified from algal isolates, we demonstrated that the cyano probe was specific for the chlorophyte/cyanobacterial RubisCO evolutionary lineage and the chrome probe was specific for the chromophyte evolutionary Lineage (diatoms, prymnesiophytes, and other non-green microalgae). For coastal/estuarine environments, both cyano and chrome rbcL mRNA was predominately confined to the >5 mu m size fraction, whereas in offshore oligotrophic environments, the cyano mRNA was associated with smaller cells (<1 mu m). Similarly, C-14 carbon fixation rates and chi a were predominately associated with the >5 mu m fraction in coastal/estuarine environments, while in offshore environments, a greater percentage was present in the <1 mu m fraction. In profiles through the euphotic zone, cyano rbcL mRNA exhibited maximal values at depths above 65 m at all stations where the waters were dominated by Synechococcus and Prochlorococcus. In contrast, chrome rbcL mRNA increased with depth from undetectable levels in surface waters to its highest levels al or below the subsurface chlorophyll maximum (SCM, 67 m or deeper). Carbon fixation rates were generally elevated in both surface waters and around the SCM. The SCM was dominated by chromophytic picoeucaryotes, as detected by HPLC pigment analysis and flow cytometry. Such analyses are consistent with the rbcL gene probe patterns of euphotic zones of offshore oligotrophic environments. This study demonstrates the utility of group-specific gene probes for examining the expression of carbon fixing genes in phytoplankton and is a first approach to understanding the active phytoplankton community structure and its relationship to the fixation of inorganic carbon in marine environments.


Pile AJ, Patterson MR, Savarese M, Chernykh VI, Fialkov VA (1997) Trophic effects of sponge feeding within Lake Baikal’s littoral zone .2. Sponge abundance, diet, feeding efficiency, and carbon flux. Limnology and Oceanography 42 :178-184

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Endemic freshwater demosponges in the littoral zone of Lake Baikal, Russia, dominate the benthic biomass, covering 44% of the benthos. We measured in situ sponge abundance and,orating and calculated sponge-mediated Fluxes of picoplankton (plankton <2 mu m) for two common species, Baikalospongia intermedia and Baikalospongia bacillifera. By means of dual-beam how cytometry, we found retention efficiencies ranging from 58 to 99% for four types of picoplankton : heterotrophic bacteria, Synechococcus-type cyanobacteria, autotrophic picoplankton with one chloroplast, and autotrophic picoplankton with two chloroplasts. By using a general model for organism-mediated fluxes, we conservatively estimate that through active suspension feeding, sponges are a sink for 1.97 g C d(-1) m(-1), mostly from procaryotic cell types. Furthermore, grazing by these extensive sponge communities can create a layer of picoplankton-depleted water overlying the benthic community in this unique lake.


Porter J, Deere D, Hardman M, Edwards C, Pickup R (1997) Go with the flow - use of flow cytometry in environmental microbiology. Fems Microbiology Ecology 24 :93-101

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Microbiological activity in the natural world is of key importance in the integrated functioning of ecosystems, yet we remain largely ignorant of the role and relevance of the vast majority of microorganisms. This ignorance is largely due to widely acknowledged, but unresolved problems in methodologies. Application of flow cytometry to such studies has already revolutionised our understanding of marine photosynthetic planktonic microorganisms, revealed new levels of complexity in the behaviour of bacterial populations and produced a reliable screening protocol for eukaryotic water-borne pathogens. Advances in fluorescent probe technology now offer realistic approaches for direct cell identification, viability assessment and responses to environmental changes using basic, single light-source flow cytometers. Here we review current applications of flow cytometry in environmental microbiology and present a case for the adoption of the technique as a necessary and routine research instrument.


Potter D, Lajeunesse TC, Saunders GW, Anderson RA (1997) Convergent evolution masks extensive biodiversity among marine coccoid picoplankton. Biodiversity and Conservation 6 :99-107

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Coccoid picoplankters are minute unicellular algae that, when viewed with a light microscope, appear as little ’balls’. They comprise an important component of open-ocean phytoplankton, and, except for colour differences (i.e. red, green, brown), many eukaryotic picoplankters are morphologically similar. To evaluate the biological diversity of the ’little brown ball’ subpopulation of ’little balls’, we randomly selected nine undescribed algal strains and compared the nucleotide sequences of their nuclear 18S ribosomal RNA genes. The results indicate that ’little brown balls’ have evolved independently in three distinct eukaryotic lineages (heterokont algae, haptophyte algae, and green algae), and at least four taxonomic classes, and that, even within the four classes, considerable genetic diversity exists. These findings suggest that a tiny coccoid morphology confers some adaptive advantage in the open ocean, that repeated convergent evolution has occurred, and that molecular data may be necessary for taxonomic distinction of closely related coccoid picoplankters.


Reckermann M, Veldhuis MJW (1997) Trophic interactions between picophytoplankton and micro- and nanozooplankton in the western Arabian Sea during the NE monsoon 1993. Aquatic Microbial Ecology 12 :263-273

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The grazing pressure of micro- and nanozooplankton on phytoplankton was estimated in serial dilution experiments in the northwestern Arabian Sea and its adjacent areas (the Somali Current, the Somali Basin, the Gulf of Aden and the southern Red Sea) during the NE monsoon 1992-1993. Microzooplankton grazing rates (g) on total phytoplankton (analyzed as chi a) were generally exceeded by phytoplankton growth rates (g = 0.2 to 1.19 d(-1), mean 0.48 d(-1) ; mu = 0.52 to 1.12 d(-1), mean 0.72 d(-1)), resulting in an average daily consumption of 38 % of the phytoplankton standing stock and 67 % of the primary production. Microzooplankton grazing on 4 picophytoplankton groups (Prochlorococcus spp., Synechococcus spp., and 2 picoeukaryotes) analyzed by flow cytometry showed growth (mu = 0.27 to 0.92 d(-1), mean 0.68 d(-1)) and grazing mortality rates (g = 0.26 to 0.73 d(-1), mean 0.67 d(-1)) well in balance, with an average of 49 % of the standing stock and 102% of the primary production grazed per day. Picophytoplankton growth and grazing mortality rates increased dramatically when grazers >10 mu m were removed. These results suggest a control of the small grazers by larger ones (trophic cascade) and a close coupling between picoautotrophic prey and small grazers. The trophic cascade within the microbial food web of the nanoplankton encompasses 3 trophic levels : picoplankton - small HNF - larger flagellates and ciliates.


Rice J, Sleigh MA, Burkill PH, Tarran GA, O’Connor C D, Zubkov MV (1997) Flow Cytometric Analysis of Characteristics of Hybridization of Species-Specific Fluorescent Oligonucleotide Probes to rRNA of Marine Nanoflagellates. Appl Environ Microbiol 63 :938-944

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Identification problems restrict quantitative ecological research on specific nanoflagellates. Identification by specific oligonucleotide probes permits use of flow cytometry for enumeration and measurement of size of nanoflagellates in statistically meaningful samples. Flow cytometry also permits measurement of intensity of probe binding by cells. Five fluorescent probes targeted to different regions of the small subunit rRNA of the common marine flagellate Paraphysomonas vestita all hybridized with cells of this flagellate. Cells fixed with trichloroacetic acid gave detectable signals at a probe concentration of 15 aM and specific fluorescence increased almost linearly to 1.5 fM, but at higher concentrations nonspecific binding increased sharply. Three flagellates, P. vestita, Paraphysomonas imperforata, and Pteridomonas danica, all bound a general eukaryotic probe approximately in proportion to their cell size, but the specific P. vestita probe gave 14 times more fluorescence with P. vestita than with either of the other flagellates. Cell fluorescence increased during the early growth of a batch culture and decreased toward the stationary phase ; cell size changed in a comparable manner. Cell fluorescence intensity may allow inferences about growth rate, but whether fluorescence (assumed to reflect ribosome number) merely correlates with cell biomass or changes in a more complex manner remains unresolved.


Rice J, Sleigh MA, Burkill PH, Tarran GA, OConnor CD, Zubkov MV (1997) Flow cytometric analysis of characteristics of hybridization of species-specific fluorescent oligonucleotide probes to rRNA of marine nanoflagellates. Applied and Environmental Microbiology 63 :938-944

<Go to ISI> ://A1997WL14400019

Identification problems restrict quantitative ecological research on specific nanoflagellates. Identification by specific oligonucleotide probes permits use of flow cytometry for enumeration and measurement of size of nanoflagellates in statistically meaningful samples, Flow cytometry also permits measurement of intensity of probe binding by cells, Five fluorescent probes targeted to different regions of the small subunit rRNA of the common marine flagellate Paraphysomonas vestita all hybridized with cells of this flagellate, Cells fixed with trichloroacetic acid gave detectable signals at a probe concentration of 15 aM and specific fluorescence increased almost linearly to 1.5 fM, but at higher concentrations nonspecific binding increased sharply. Three flagellates, P. vestita, Paraphysomonas imperforata, and Pteridomonas danica, all bound a general eukaryotic probe approximately in proportion to their cell size, but the specific P. vestita probe gave 14 times more fluorescence with P. vestita than with either of the other flagellates. Cell fluorescence increased during the early growth of a batch culture and decreased toward the stationary phase ; cell size changed in a comparable manner, Cell fluorescence intensity may allow inferences about growth rate, but whether fluorescence (assumed to reflect ribosome number) merely correlates with cell biomass or changes in a more complex manner remains unresolved.


Schonhuber W, Fuchs B, Juretschko S, Amann R (1997) Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification. Appl Environ Microbiol 63 :3268-3273

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9251215

The substrate fluorescein-tyramide was combined with oligonucleotide probes directly labeled with horseradish peroxidase to improve the sensitivity of in situ hybridization of whole fixed bacterial cells. Flow cytometry and quantitative microscopy of cells hybridized by this technique showed 10- to 20-fold signal amplifications relative to fluorescein-monolabeled probes. The application of the new technique to the detection of natural bacterial communities resulted in very bright signals ; however, the number of detected cells was significantly lower than that detected with fluorescently monolabeled, rRNA-targeted oligonucleotide probes.


Sgorbati S, Barbesti S, Neri MG, Davegna C, Citterio S (1997) Rapid flow cytometric immunodetection of bacteria to monitor aquatic environments. Eur J Histochem 41 Suppl 2 :187-188

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9859842


Shalapenok LS, Shalapenok A (1997) Heterogeneous pigment composition of phycoerythrin-containing picocyanobacteria Synechococcus spp in the Black Sea. Microbiology 66 :80-84

<Go to ISI> ://A1997WG63900016

Natural picoplankton communities in the Black Sea involve phycoerythrin-containing cyanobacteria of two spectral types, which differ in the position of phycoerythrin (PE) fluorescence maximum. Type 1 exhibits a maximum at 558-569 nm, while type 2, at 570-579 nm. The abundance and distribution of various spectral groups of Synechococcus spp, in the Black Sea differ from those in the ocean, although, in both cases, ’’shorter-wavelength’’ forms dominate in deep waters. Namely, cyanobacteria containing PEs of the WH8103 type (with a superhigh content of phycourobilin) dominate in the open ocean, while those of type 1 (the relative content of phycourobilin is unknown) are prevalent in the open Black Sea. At the same time, dominant cyanobacteria living in the coastal waters are of the WH7803 type (the ocean) and type 2 (the Black Sea). The intensity of cell fluorescence changes with depth.


Simon N, Brenner J, Edvardsen B, Medlin LK (1997) The identification of Chrysochromulina and Prymnesium species (Haptophyta, Prymnesiophyceae) using fluorescent or chemiluminescent oligonucleotide probes : a means for improving studies on toxic algae. European Journal of Phycology 32 :393-401

<Go to ISI> ://000071195200009

Chrysochromulina and Prymnesium are important bloom-forming organisms in marine and brackish waters, respectively. Both genera include toxic species, which are primarily implicated in fish kills. Previous analyses of small subunit (SSU) rDNA sequences from Chrysochromulina and Prymnesium spp. indicate that Chrysochromulina is paraphyletic. C. polylepis, which produced a spectacular, harmful bloom in 1988, is more closely related to toxic Prymnesium species than to most other Chrysochromulina species based on rDNA sequence comparisons. Signatures were identified in the SSU rRNA gene specific for a clade that comprised primarily toxic taxa (C. polylepis, P. parvum, P. patelliferum and P. calathiferum) and that recognized C. polylepis alone. Oligonucleotide probes complementary to these regions were designed, and their specificity tested using dot-blot hybridization on PCR products of the SSU rRNA gene from 28 strains of Chrysochromulina and Prymnesium. Whole-cell hybridizations were performed with FLUOS- as well as Cy3-labelled probes on cultured species from both genera, and were detected with both epifluorescence microscopy and flow cytometry. The probes afforded easy identification of clonal isolates of C. polylepis and a cluster of closely related species including C. polylepis and Prymnesium spp. The feasibility of using these probes for species identification and studies of population dynamics in the field is discussed.


Turon X, Galera J, Uriz MJ (1997) Clearance rates and aquiferous systems in two sponges with contrasting life-history strategies. Journal of Experimental Zoology 278 :22-36

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The clearance rates and microarchitecture of the aquiferous systems of two sympatric sponge species, Crambe crambe (Schmidt) and Dysidea avara (Schmidt), are compared. We performed a filtration experiment with fluorescent latex microspheres with diameters ranging from 0.2 to 4 mu m. Microsphere concentration in the water was measured by flow cytometry, and the particles ingested were detected within the sponge cells through confocal microscopy. The two species studied showed contrasting life-history strategies, reflected by different structural organizations of the aquiferous system, which in turn correlate with clearance rates measured in the filtration experiment. The species with higher growth and regeneration rates also showed the highest clearance rates at all particle sizes assayed and displayed larger ostia, a thicker choanosome layer, and larger flagellate chambers and choanocytes. The particle size most efficiently retained by both species was 1 mu m, and maximal clearance rates were obtained in all cases after 15 min of incubation. The sites of particle capture were the choanocytes, which retained small particles (0.2, 0.5, and 1 mu m) and, in the case of D. avara, also 4 mu m particles. The pinacocytes captured the largest particles assayed (6 mu m) and, in the case of C. crambe, also retained particles of the smaller sizes. It is concluded that there is an adaptive interspecific variation in structure and efficiency of the filtering systems in sponges which correlates with diverse biological strategies. The clearance rates obtained, coupled with the abundance of sponge populations in the community studied, point to a significant grazing impact of sponges on the picoplankton of the area. (C) 1997 Wiley-Liss, Inc.


Veldhuis MJW, Cucci TL, Sieracki ME (1997) Cellular DNA content of marine phytoplankton using two new fluorochromes : Taxonomic and ecological implications. Journal of Phycology 33 :527-541

<Go to ISI> ://A1997XH98800023

Two new fluorochromes, PicoGreen(R) and SYTOX Green(TM) stain (Molecular Probes, inc.), are useful with flow cytometry for quantitative detection of cellular DNA in a variety of marine phytoplankton. The basic instrument configuration of modern low-power flow cytometers (15 mW, 488 nm excitation) is sensitive enough to detect the DNA signal in nearly all of the 121 strains (from 12 taxonomic classes) examined. The major advantages of these dyes over others are 1) suitability for direct use in seawater, 2) green fluorescence emission of the DNA-dye complex (wavelength 525 +/- 15 nm) showing no overlap with the autofluorescence of the plankton pigments in the red band, 3) high fluorescence yield of the DNA-dye complex with an increase in fluorescence >100-fold compared to the unstained cell, and 4) dyes can be used to quantify double-stranded DNA. The high sensitivity allowed the quantification of the DNA of the smallest known phytoplankter (Prochlorococcus) as well as bacteria found in some of the algal cultures. Of the 12 taxonomic classes tested, only the 3 Nannochloropsis spp. (Eustigmatophyceae) stained poorly, and a few members of the Chlorophyceae and Pelagophyceae showed poor staining occasionally. In general, maximal fluorescence was achieved within 15 min after addition of the dye. Although the PicoGreen dye stained some living phytoplankton species, presentation is recommended for quantitation. SYTOX Green did not stain live cells. The combination of the dyes, therefore, allows the discrimination between live and dead cells in some algal groups (Prochlorococcus, diatoms, prasinophytes, and pelagophytes). Paraformaldehyde was preferred over glutaraldehyde for fixation to avoid (induced) green autofluorescence. Total DNA values measured in 90 algal species (ca. 121 strains) vaned by a factor of 20,000. The lowest values were found in Prochlorococcus and the highest in a large dinoflagellate (Prorocentrum micans). DNA content appears to be a scaleable cell component covarying with the carbon and nitrogen contents of the phytoplankton cells. This covariation allows the total DNA content to be used as an accurate, independent estimate of total cell carbon biomass in unicellular pelagic phytoplankton.


Wilson WH, Mann NH (1997) Lysogenic and lytic viral production in marine microbial communities. Aquatic Microbial Ecology 13 :95-100

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It is now well established that viruses are an abundant component of marine ecosystems and they are being increasingly recognised and accepted as important contributors to element cycling within the microbial loop. However, some of the key questions regarding the ecological significance of viruses in the marine environment still remain largely unanswered. Thus, particular interest is currently focused on the extent to which lyric production or lysogeny predominates and the nature of factors in the marine environment, particularly nutrient availability and multiplicity of infection (MOI), which might influence the lysis/lysogeny ’decision’. The present evidence is still insufficient to unambiguously assess the relative ecological significance of lysogeny versus lysis and progress in this area will rely on the development and application of new techniques. This review attempts to collect recent information relating to this central question, focusing particularly on those viruses which infect the bacterioplankton and nano- and picophytoplankton.


Yamaguchi N, Nasu M (1997) Flow cytometric analysis of bacterial respiratory and enzymatic activity in the natural aquatic environment. Journal of Applied Microbiology 83 :43-52

<Go to ISI> ://A1997XL41700007

The respiratory and enzymatic activity of bacteria in polluted and unpolluted river water was investigated by flow cytometry. Double staining with 6CFDA (6-carboxy fluorescein diacetate) and PI (propidium iodide) was used to examine bacterial esterase activity. CTC (5-cyano-2,3-ditolyl tetrazolium chloride) was employed as the indicator of bacterial respiration. The ratios of colony-forming units to total bacterial number were less than 2%, except highly polluted sites. The number of respiring bacteria determined by flow cytometry amounted to 10-15% of the total bacterial number at both unpolluted and polluted sampling stations, while it was 30% at the highly polluted station. Almost 50% of total bacteria demonstrated esterase activity in the unpolluted areas, whereas 70-90% of total bacteria retained this enzymatic activity in the polluted areas. Thus, many of the non-culturable bacteria in the natural aquatic environment have enzymatic activity regardless of the pollution level, and 6CFDA was more superior in the sensitivity for the evaluation of physiological activity of bacteria in the natural environment. The ratio of bacteria with esterase activity increased in direct proportion to the pollution level of river water and suggested that this ratio would be a useful indicator in evaluating the pollution levels in an aquatic environment.