1993

mardi 21 avril 2009
par   G. Grégori

Andreoli C, Scarabel LR, Tolomio C (1993) The Distribution of Photoautotrophic Picoplancton in the Terra-Nova (Ross Sea, Antarctica) during Austral Summer 1989-1990. Archiv Fur Hydrobiologie :123-132

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In this paper results obtained from a study carried out during the austral summer 1989-90 on photoautotrophic picoplankton in the Antarctic Ross Sea are reported. The observations with the flow cytometry on water samples collected at different depth and at several stations have revealed the presence of the picoplankton. The registred quantities have been generally low (< 100 cells . ml-1, sometimes up to 500 cells . ml-1) and almost completely consisting of cyanobacteria. The water samples have also been used to determine the concentrations of the main nutrients (nitrite, nitrate, ammoniacal nitrogen and orthophosphate) as well as of chlorophyll a.


Bedo AW, Acuna JL, Robins D, Harris RP (1993) Grazing in the Micron and the Submicron Particle-Size Range - the Case of Oikopleura-Dioica (Appendicularia). Bulletin of Marine Science 53 :2-14

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We used procaryotic and eucaryotic phytoplankton (1.21 and 4.5 mum = Equivalent Spherical Diameter) and fluorescent carboxylated beads (0.2 and 0.75 mum ESD) to obtain direct measurements of grazing by Oikopleura dioica in the micron (0.914-6.025 mum ESD) and submicron particle size range (0.2-0.75 mum ESD). Flow cytometry, used quantitatively, detected processes in the sub-micron range that could not be differentiated by other direct methods, such as low aperture techniques for electronic particle counting. We conclude from our observations that (1) Based on the decrease of particle concentration in the food suspension, filtration by the food concentration filter is a non selective process down to 0.2 mum. (2) Collection by the pharyngeal filter, and consequently ingestion, of the very fine 0.2 mum beads is less efficient than that of the larger 0.75 mum microspheres and apparently diminishes with their concentration in the food suspension. (3) The fraction of 0.2 mum beads resuspended after sieving by the pharyngeal filter is accumulated in the filter house (possibly by adhering on the internal walls of the house due to physico-chemical interactions of their surfaces, though the properties of the beads are probably different from naturally occurring colloids), rather than being expelled as fecal pellets through the water sphincter exit. These observations suggest that, as producers of biogenic particles, O. dioica influence the fate of the fine colloidal material (<0.2 mum) in a different way than that of the slightly larger material (e.g., 0.75 mum), the latter being exported preferentially with fecal pellets while the finer particles would essentially contribute to the enrichment of the filter-house community microcosm.


Buma AGJ, Noordeloos AAM, Larsen J (1993) Strategies and Kinetics of Photoacclimation in 3 Antarctic Nanophytoflagellates. Journal of Phycology 29 :407-417

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Three Antarctic nanophytoflagellates (two cryptophyte species and a Pyramimonas sp.) were compared for their capacity to photoacclimate and for their kinetic responses in changing photic environments. Division rate, cell size, cellular fluorescence, and chlorophyll a content were measured during steady and transient states Of semi-continuous cultures maintained at 1.0-degrees-C. Of all parameters tested, cell size was most affected by irradiance. Acclimation kinetics were modelled using a first-order equation. Rates of change in cell size following shifts in irradiance were comparable with rates of change in chemical composition reported for temperate algae. Response rates of cellular in vivo red and orange fluorescence were lower. In many cases, however, responses could not be described by the first-order kinetic model. Division rates remained high for approximately 3 days following a shift down in irradiance, after which new division rates were established. The nanoflagellates studied here appear to respond to small irradiance perturbations at low rates. However, they may, fail to adapt to large and abrupt changes in photon flux density (PFD). When shade-adapted (25 mumol . m-2 . s-1) cells were exposed to high PFD (400 mumol . m-2. s-1)for 1-3 days, cells were incapable of readapting division rate and pigment content to the initial irradiance condition (25 mumol . m-2 . s-1)for about 1 month following the shift-down step. The ecological role of the kinetics of photoacclimation in nanophytoflagellate growth performance in Antarctic ecosystems is discussed.


Button DK, Schut F, Quang P, Martin R, Robertson BR (1993) Viability and Isolation of Marine Bacteria by Dilution Culture : Theory, Procedures, and Initial Results. Appl Environ Microbiol 59 :881-891

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16348896

Dilution culture, a method for growing the typical small bacteria from natural aquatic assemblages, has been developed. Each of 11 experimental trials of the technique was successful. Populations are measured, diluted to a small and known number of cells, inoculated into unamended sterilized seawater, and examined three times for the presence of 10 or more cells per ml over a 9-week interval. Mean viability for assemblage members is obtained from the frequency of growth, and many of the cultures produced are pure. Statistical formulations for determining viability and the frequency of pure culture production are derived. Formulations for associated errors are derived as well. Computer simulations of experiments agreed with computed values within the expected error, which verified the formulations. These led to strategies for optimizing viability determinations and pure culture production. Viabilities were usually between 2 and 60% and decreased with >5 mg of amino acids per liter as carbon. In view of difficulties in growing marine oligobacteria, these high values are noteworthy. Significant differences in population characteristics during growth, observed by high-resolution flow cytometry, suggested substantial population diversity. Growth of total populations as well as of cytometry-resolved subpopulations sometimes were truncated at levels of near 10 cells per ml, showing that viable cells could escape detection. Viability is therefore defined as the ability to grow to that population ; true viabilities could be even higher. Doubling times, based on whole populations as well as individual subpopulations, were in the 1-day to 1-week range. Data were examined for changes in viability with dilution suggesting cell-cell interactions, but none could be confirmed. The frequency of pure culture production can be adjusted by inoculum size if the viability is known. These apparently pure cultures produced retained the size and apparent DNA-content characteristic of the bulk of the organisms in the parent seawater. Three cultures are now available, two of which have been carried for 3 years. The method is thus seen as a useful step for improving our understanding of typical aquatic organisms.


Button DK, Schut F, Quang P, Martin R, Robertson BR (1993) Viability and Isolation of Marine-Bacteria by Dilution Culture - Theory, Procedures, and Initial Results. Applied and Environmental Microbiology 59 :881-891

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Dilution culture, a method for growing the typical small bacteria from natural aquatic assemblages, has been developed. Each of 11 experimental trials of the technique was successful. Populations are measured, diluted to a small and known number of cells, inoculated into unamended sterilized seawater, and examined three times for the presence of 10(4) or more cells per ml over a 9-week interval. Mean viability for assemblage members is obtained from the frequency of growth, and many of the cultures produced are pure. Statistical formulations for determining viability and the frequency of pure culture production are derived. Formulations for associated errors are derived as well. Computer simulations of experiments agreed with computed values within the expected error, which verified the formulations. These led to strategies for optimizing viability determinations and pure culture production. Viabilities were usually between 2 and 60% and decreased with >5 mg of amino acids per liter as carbon. In view of difficulties in growing marine oligobacteria, these high values are noteworthy. Significant differences in population characteristics during growth, observed by high-resolution flow cytometry, suggested substantial population diversity. Growth of total populations as well as of cytometry-resolved subpopulations sometimes were truncated at levels of near 10(4) cells per ml, showing that viable cells could escape detection. Viability is therefore defined as the ability to grow to that population ; true viabilities could be even higher. Doubling times, based on whole populations as well as individual subpopulations, were in the 1-day to 1-week range. Data were examined for changes in viability with dilution suggesting cell-cell interactions, but none could be confirmed. The frequency of pure culture production can be adjusted by inoculum size if the viability is known. These apparently pure cultures produced retained the size and apparent DNA-content characteristic of the bulk of the organisms in the parent seawater. Three cultures are now available, two of which have been carried for 3 years. The method is thus seen as a useful step for improving our understanding of typical aquatic organisms.


Campbell L, Vaulot D (1993) Photosynthetic Picoplankton Community Structure in the Subtropical North Pacific-Ocean near Hawaii (Station Aloha). Deep-Sea Research Part I-Oceanographic Research Papers 40 :2043-2060

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The structure of the picoplankton community in the subtropical Pacific was examined on four depth profiles, one from each season, sampled at the Hawaii Ocean Time-series station ALOHA (22 degrees 45’N, 158 degrees W). Three cell populations were discriminated by flow cytometry : Prochlorococcus prochlorophytes, Synechococcus cyanobacteria, and picoeukaryotes. Prochlorococcus were the most abundant component (maximum ca 2 x 10(5) cells ml(-1)). Unlike previous reports, their concentration was almost constant down to roughly 100 m, with a slight maximum at the surface or near the chlorophyll maximum. Cellular chlorophyll fluorescence increased 50-fold between surface and deep populations. One distinguishing feature of the community off Hawaii was the co-occurrence near the chlorophyll maximum of at least two distinct Prochlorococcus populations with different chlorophyll and DNA contents. Throughout the year, Synechococcus abundance was two orders of magnitude lower and there was no seasonal alternation between Prochlorococcus and Synechococcus, as observed in the northern Sargasso Sea. Synechococcus populations did not extend below 120 m and were dominated by high phycourobilin cell types. Picoeukaryote abundance was quite similar to that of Synechococcus, but these cells extended deeper in the water column. Their chlorophyll fluorescence exhibited much less depth variation than Prochlorococcus or Synechococcus. Seasonal variability was small (<2- to 3-fold) for all three components of the picoplankton, not only for cell abundance but also for cellular parameters such as light scatter or pigment fluorescence. Synechococcus populations exhibited the largest seasonal changes (e.g. abundance maximum and chlorophyll fluorescence varied 3-fold). Picoplankton community structure in the Pacific Ocean appears to be distinct from previous reports for other areas. In comparing station ALOHA to the Atlantic Ocean (especially the Sargasso Sea) and the Mediterranean Sea, depth-integrated abundances of Prochlorococcus were higher, that of Synechococcus were lower, and that of picoeukaryotes were similar. We believe this structure, dominated by Prochlorococcus, may be typical for subtropical open-ocean regions.


Cowles TJ, Desiderio RA, Neuer S (1993) Insitu Characterization of Phytoplankton from Vertical Profiles of Fluorescence Emission-Spectra. Marine Biology 115 :217-222

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Vertical profiling of the upper ocean with a laser/fiber optic fluorometer enabled the determination of fluorescence emission spectra of photosynthetic pigments over small vertical scales. Simultaneous acquisition of phycoerythrin (PE) and chlorophyll (chl) emission spectra allowed in situ differentiation between PE-containing cells (cryptomonads and cyanobacteria) and other chl-containing autotrophs. Further, fluorescence spectral peak shifts associated with different species of PE-containing cells resulted in even finer scale in situ taxonomic differentiation. We found that the phycoerythrin fluorescence emission maxima shifted from 578 nm near the surface, to 585 mum at the base of the shallow thermocline (30% light level), and to 590 nm below the thermocline at the base of the euphotic zone (1% light level). These shifts in peak emission coincided with a taxonomic change in the PE-containing cells (as determined from analysis of discrete bottle samples) from a greater proportion of Synechococcus spp. in the upper water column to a greater proportion of cryptomonads at the base of the euphotic zone. These results indicate that the composition of the phytoplankton assemblage may be assessed in situ without sample collection.


Fouchet P, Jayat C, Hechard Y, Ratinaud MH, Frelat G (1993) Recent advances of flow cytometry in fundamental and applied microbiology. Biol Cell 78 :95-109

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8220231

This review focuses on the recent applications of flow cytometry (FCM) in microbiological research (1987-mid 1992). It tries to give a scope of the important breakthroughs which occurred in this field during this period. The technical difficulties of microorganism analysis by flow cytometry is briefly appraised. The significance and the limits of the different microbial cell parameters attainable by flow analyses are systematically evaluated : light scatter for cell size and structure, fluorescence measurements for quantification of cellular components, microbial antigen detection and cell physiological activity estimation. Emphasis is given on the new technological advances which appeared in the last two years. The second part of the review is devoted to the analysis of the usefulness of flow cytometric approach in the different fields of microbiology : fundamental studies in microbial physiology, differentiation, microbial ecology and aquatic sciences, medical microbiology, parasitology, microbial pharmacology and biotechnology.


Fouchet P, Jayat C, Hechard Y, Ratinaud MH, Frelat G (1993) Recent Advances of Flow-Cytometry in Fundamental and Applied Microbiology. Biology of the Cell 78 :95-109

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This review focuses on the recent applications of flow cytometry (FCM) in microbiological research (1987-mid 1992). It tries to give a scope of the important breakthroughs which occurred in this field during this period. The technical difficulties of microorganism analysis by flow cytometry is briefly appraised. The significance and the limits of the different microbial cell parameters attainable by flow analyses are systematically evaluated : light scatter for cell size and structure, fluorescence measurements for quantification of cellular components, microbial antigen detection and cell physiological activity estimation. Emphasis is given on the new technological advances which appeared in the last two years. The second part of the review is devoted to the analysis of the usefulness of flow cytometric approach in the different fields of microbiology : fundamental studies in microbial physiology, differentiation, microbial ecology and aquatic sciences, medical microbiology, parasitology, microbial pharmacology and biotechnology.


Goericke R, Repeta DJ (1993) Chlorophyll-a and Chlorophyll-B and Divinyl Chlorophyll-a and Chlorophyll-B in the Open Subtropical North-Atlantic Ocean. Marine Ecology-Progress Series 101 :307-313

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Divinyl chlorophyll a (Chl a2) and divinyl chlorophyll b (chl b2) are chemotaxonomic tracers for the marine photooxytrophic procaryote Prochlorococcus marinus. Here we report the complete separation of chlorophyll a (chl a1) and chl a2 on a reverse-phase high-pressure liquid chromatography system that also achieves good separation of most other chemotaxonomically important pigments. Chlorophyll b (chl b1) and chl b2 are partially resolved, and their relative abundances are estimated with an on-line spectrophotometric method. Using these methods, we determined that chl a2 and chl b2 contributed up to 40 % to total chl a (the sum of chl a1 and a2) and up to 95 % to total chl b, respectively, in samples from the subtropical North Atlantic. The results suggest that Prochlorococcus represented a significant fraction of the total phytoplanktonic biomass. A comparison of chl b/a ratios observed in the field and chl b/a ratios measured in cultures of P marinus suggests the presence of 2 strains of this organism in the subtropical North Atlantic. The spectroscopic differences between chl a1 and chl a2 would have led to small underestimates of total chl a in these samples had these been analyzed by spectrophotometric methods. However, the standard fluorometric method would have underestimated total chl a on the average by 8 % with maximum values of 20 %.


Goericke R, Welschmeyer NA (1993) The Marine Prochlorophyte Prochlorococcus Contributes Significantly to Phytoplankton Biomass and Primary Production in the Sargasso Sea. Deep-Sea Research Part I-Oceanographic Research Papers 40 :2283-2294

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The newly-discovered prochlorophyte Prochlorococcus marinus is often numerically dominant in the euphotic zone of the tropical and subtropical ocean ; however, its contribution to phytoplankton biomass and primary production is largely unknown. Using its unique pigment divinyl-chlorophyll a (Chl a(2)) as a chemosystematic marker, we show that Prochlorococcus is present at a station in the Sargasso Sea throughout most of the year. Whereas it is only found at depth during the early summer, it can be found throughout the euphotic zone during the rest of the year. Averaged over the year Prochlorocaccus pigment-biomass constitutes about 30% of the total. Its growth rate, estimated from the incorporation of C-14 into Chl a(2) ranged from values of 0.3 day(-1) in the surface layer to values less than 0.1 day(-1) at the bottom of the euphotic zone. Averaged over the seasons, approximately 25% of the total productivity was due to Prochlorococcus. Prochlorococcus clearly is an important component of the ecosystem in the Sargasso Sea, and perhaps the world ocean.


Hu LB, Lucio B, Schat KA (1993) Depletion of CD4+ and CD8+ T lymphocyte subpopulations by CIA-1, a chicken infectious anemia virus. Avian Dis 37 :492-500

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The suppressive effect of chicken infectious anemia virus (CIAV) on T-lymphocyte subpopulation was evaluated in vivo by flow cytometry and dual immunostaining on frozen sections. Between 14 and 21 days postinoculation (PI), the percentage of CD4-, CD8-, and CT1-positive (CD4+, CD8+, and CT1+) cells was significantly lower in chickens infected at 1 day of age with CIA-1 strain of CIAV than in controls. The mean percentage of CD4+ cells in the thymus was only 43%, whereas in the controls it was 77%. The mean percentages of CD8+ cells in the thymus in infected and control chickens was 54% and 90%, respectively, and of CT1+ cells, 44% and 92%, respectively. At 28 days PI, the percentage of CD4+, CD8+, and CT1+ cells was similar in infected and control chickens. Also at 14 and 21 days PI, immunoperoxidase staining demonstrated fewer CD4+, CD8+, and CT1+ cells in the thymus of infected chickens than in controls. In frozen sections of thymus stained with CIA-1 antibodies and CD4, CD8, or CT1, few of the cells positive for CIAV antigen seen in the outer zone of the cortex carried CD4, CD8, or CT1 molecules. These results suggest that CIA-1 infection either destroys cells expressing CD4, CD8, or CT1 molecules on their surface or interferes with the expression of these molecules.


Legall Y, Brown S, Marie D, Mejjad M, Kloareg B (1993) Quantification of Nuclear-DNA and G-C Content in Marine Macroalgae by Flow-Cytometry of Isolated-Nuclei. Protoplasma 173 :123-132

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The amounts of nuclear DNA in ten species of seaweeds belonging to the Rhodophyceae, Phaeophyceae, and Chlorophyceae were determined by flow cytometric analysis of nuclei isolated from protoplasts. Genome size was determined from the fluorescence of the nuclei stained with ethidium bromide. The size of the nuclear genome ranged from 0.13 pg per cell in the 1 C population of Ulva rigida to 3.40 pg per cell in the 2 C population of Sphacelaria sp. GC % analysis was based on staining with either Hoechst 33342 or mithramycin A, two fluorochromes specific for the bases A-T and G-C, respectively. Two models were used for the estimation of the proportion of guanine plus cytosine in the nuclear genome. The first one was based on the linear relationships mithramycin A fluorescence/G/C content and ethidium bromide fluorescence/total DNA content. The second model, based on the curvilinear relationships Hoechst 33342 fluorescence/A-T content and mithramycin A fluorescence/G-C content, resulted in comparatively more homogenous and consistent data and appears more accurate. Comparison with previous reports from other methods for the physical investigation of nuclear genomes shows that flow cytometry of nuclei isolated from protoplasts is an accurate, convenient and robust technique to assay for genome sizes and base pair composition in marine macroalgae.


Lepesteur M, Martin JM, Fleury A (1993) A Comparative-Study of Different Preservation Methods for Phytoplankton Cell Analysis by Flow-Cytometry. Marine Ecology-Progress Series 93 :55-63

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Ten methods to preserve phytoplankton populations for flow cytometric analyses were tested. These methods were differentiated by the rate of freezing and thawing, and the use or non-use of cryoprotectants (DMSO and/or glycerol) and chemical fixation. After freezing, the samples were stored in liquid nitrogen. These methods were tested on 3 freshwater and marine algal species. Different intensity parameters and 2 properties were considered. Firstly the number of cells lost, which was more significant with rapid freezing and chemical fixation, and less significant with the addition of cryoprotectants. Secondly, the preservation of both light scattering and fluorescence, which was better with slow freezing than with cryoprotectants. Slow freezing followed by chemical fixation appeared to be the best protocol studied and even if glycerol addition without chemical fixation seemed to be overall the best method, implying the use of cryoprotectant, all these techniques had to be tested on a case by case basis, particularly when phycocyanin and chlorophyll fluorescence were studied.


Lim EL, Amaral LA, Caron DA, Delong EF (1993) Application of Ribosomal-Rna-Based Probes for Observing Marine Nanoplanktonic Protists. Applied and Environmental Microbiology 59 :1647-1655

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The use of small-subunit rRNA-based oligonucleotides as probes for detecting marine nanoplanktonic protists was examined with a ciliate (an Uronema sp.), a flagellate (a Cafeteria sp.), and mixed assemblages of protists from enrichment cultures and natural seawater samples. Flow cytometry and epifluorescence microscopy analyses demonstrated that hybridizations employing fluorescein-labeled, eukaryote-specific probes intensely stained logarithmically growing protists, whereas these same protist strains in late stationary growth were barely detectable. The fluorescence intensity due to probe binding was significantly enhanced by the use of probes end labeled with biotin, which were detected by fluorescein-labeled avidin. The degree of signal amplification ranged from two- to fivefold for cultured protists in both logarithmic and stationary growth phases. Mixed assemblages of heterotrophic protists from enrichment cultures were also intensely labeled by rRNA-targeted oligonucleotide probes by the biotin-avidin detection system. Protists in late stationary growth phase and natural assemblages of protists that were otherwise undetectable when hybridized with fluorescein-labeled probes were easily visualized by this approach. In the latter samples, hybridization with multiple, biotin-labeled probes was necessary for detection of naturally occurring marine protists by epifluorescence microscopy. The signal amplification obtained with the biotin-avidin system should increase the utility of rRNA-targeted probes for identifying protists and facilitate characterization of the population structure and distribution of protists in aquatic environments.


Lim EL, Amaral LA, Caron DA, DeLong EF (1993) Application of rRNA-based probes for observing marine nanoplanktonic protists. Appl Environ Microbiol 59 :1647-1655

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8517756

The use of small-subunit rRNA-based oligonucleotides as probes for detecting marine nanoplanktonic protists was examined with a ciliate (an Uronema sp.), a flagellate (a Cafeteria sp.), and mixed assemblages of protists from enrichment cultures and natural seawater samples. Flow cytometry and epifluorescence microscopy analyses demonstrated that hybridizations employing fluorescein-labeled, eukaryote-specific probes intensely stained logarithmically growing protists, whereas these same protist strains in late stationary growth were barely detectable. The fluorescence intensity due to probe binding was significantly enhanced by the use of probes end labeled with biotin, which were detected by fluorescein-labeled avidin. The degree of signal amplification ranged from two- to fivefold for cultured protists in both logarithmic and stationary growth phases. Mixed assemblages of heterotrophic protists from enrichment cultures were also intensely labeled by rRNA-targeted oligonucleotide probes by the biotin-avidin detection system. Protists in late stationary growth phase and natural assemblages of protists that were otherwise undetectable when hybridized with fluorescein-labeled probes were easily visualized by this approach. In the latter samples, hybridization with multiple, biotin-labeled probes was necessary for detection of naturally occurring marine protists by epifluorescence microscopy. The signal amplification obtained with the biotin-avidin system should increase the utility of rRNA-targeted probes for identifying protists and facilitate characterization of the population structure and distribution of protists in aquatic environments.


Marian T, Krasznai Z, Balkay L, Balazs M, Emri M, Bene L, Tron L (1993) Hypoosmotic Shock Induces an Osmolality-Dependent Permeabilization and Structural-Changes in the Membrane of Carp Sperm. Journal of Histochemistry & Cytochemistry 41 :291-297

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We carried out spectrofluorimetric and flow cytometric measurements to investigate the effect of hypo-osmotic shock on cell membranes of common carp sperm. The time course of the permeability of the sperm cell membrane, as monitored by DNA-related propidium iodide (PI) fluorescence, was followed for 30 min after dilution of semen in hypoosmotic environments of different ionic strengths. Spectrofluorimetric measurements indicated a continuous increase in the total PI emission intensity of a sperm suspension. Cell-by-cell flow cytometric measurements suggested that the permeability changes were of the all-or-none type. The permeabilized fraction of cells in the individual samples was time and osmolality dependent. The number and percentage of cells in which DNA was stained by PI increased gradually over time and reached a steady-state plateau value after 5-15 min. This equilibrium fraction of cells with a PI-permeable cytoplasmic membrane displayed an inverse relationship with the osmolality of the diluent, having a near 100% value for fresh water and distilled water. Dilution of sperm in hypo-osmotic medium brought about a fast decrease in the forward light-scattering signal on a short time scale compared to the pre-steady-state time of the permeabilization. With the addition of extracellular Ca2+ (1.8 mM), restoration of the light scattering signal was observed. Permeabilization of the membrane and restoration of light scattering were not coincident in time. We propose a two-dimensional reorganization of the lipid structure as the underlying mechanism of the latter process.


Millie DF, Paerl HW, Hurley JP (1993) Microalgal Pigment Assessments Using High-Performance Liquid-Chromatography - a Synopsis of Organismal and Ecological Applications. Canadian Journal of Fisheries and Aquatic Sciences 50 :2513-2527

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Past and current efforts at identifying microalgal phylogenetic groups rely largely on microscopic evaluation, which requires a high level of taxonomic skill, may take considerable time, can be variable among personnel, and does not allow characterization of the physiological status of the taxa. High-performance liquid chromatography (HPLC) has proven effective in rapidly separating and distinguishing chlorophylls, chlorophyll-degradation products, and carotenoids within monotypic and mixed algal samples. When coupled with absorbance and/or fluorescence spectroscopy, HPLC can accurately characterize phylogenetic groups and changes in community composition and yield information concerning microalgal physiological status, production, trophic interaction, and paleolimnology/paleooceanography. The recent widespread occurrence of toxic and noxious phytoplankton blooms has necessitated the use of remote imagery of pigment and reflectance ’’signatures’’ for monitoring and predicting bloom distribution. Because HPLC allows the processing of large numbers of samples from numerous locations relatively quickly, it is ideally suited for large-scale ’’ground truthing’’ of remotely sensed imagery. Coupled with rapidly evolving computer-based remote sensing technologies, HPLC-based pigment analyses may provide accurate assessments of aquatic biogeochemical flux, primary production, trophic state, water quality, and changes therein un local, regional, and global scales.


Moreiraturcq P, Martin JM, Fleury A (1993) Chemical and Biological Characterization of Particles by Flow-Cytometry in the Krka Estuary, Croatia. Marine Chemistry 43 :115-126

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Flow cytometry was used to characterize suspended particles in the Krka estuary (Croatia). This technique allows a rapid characterization and quantification of individual particles ; it is based on the simultaneous measurement of the light scatter and natural or induced fluorescence. The relative size of the particles was determined by light scatter signals and by a calibration with fluorescent beads.
The determination of organic particles was carried out by staining with fluorescein isothiocyanate (FITC). The autofluorescence of photosynthetic pigments (chlorophyll and phycoerythrin) present in the phytoplankton population was measured. The carbohydrates (galactose and glucose-mannose) were stained by lectins conjugated to FITC. The abundance of organic coatings or mineral particles and detritus was obtained from the difference between the total number of particles stained by specific carbohydrate fluorochromes and living cells.
Results show that the Krka estuary is characterized by the occurrence of very small particles. About 80% of suspended particles are organic, most of these particles being living cells. The only exception is the Guduca River, a small tributary,


Morel A, Ahn YH, Partensky F, Vaulot D, Claustre H (1993) Prochlorococcus and Synechococcus - a Comparative-Study of Their Optical-Properties in Relation to Their Size and Pigmentation. Journal of Marine Research 51 :617-649

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Three unialgal strains of Prochlorococcus and four of Synechococcus were grown in batch culture at low irradiances. The spectral values of light absorption, scattering and backscattering of intact cells in suspension were determined, together with cell counts, size distribution and pigment composition (via HPLC). The spectral efficienCY factors Q(a), Q(b), Q(bb) for light absorption, scattering and backscattering respectively, were derived, as well as the corresponding chlorophyll-specific coefficients a*, b* and b(b)*. The pigment used when normalizing is ’’true’’ chlorophyll a for Synechococcus, and divinyl-chlorophyll a for Prochlorococcus. In correspondence with small sizes (0.6 mum, on average) Prochlorococcus exhibits Q(b) values below those of Synechococcus (size about 0.9 mum, on average). In contrast, Q(a) is higher for Prochlorococcus than for Synechococcus, in response to high internal divinyl-chlorophyll content. In the blue part of the spectrum the probability for photons of being absorbed by a Prochlorococcus cell exceeds that of being scattered. Such a combination has never been found before for other algal cells, consistently more efficient as scatterers than as absorbers. The magnitude of the three efficiency Q-factors, as well as their spectral variations, can be understood and reconstructed in the frame of the Mie theory. The impact of these small organisms, dominant in oligotrophic environment, upon the optical properties of such waters are discussed on the basis of their chlorophyll-specific optical coefficients. Their absorption capabilities (per unit of chlorophyll) are not far from being maximum, to the extent that the package effect is rather reduced. With respect to scattering, Prochlorococcus cells have a minute signature compared to that of Synechococcus. This point is illustrated using vertical profiles of fluorescence, attenuation coefficient, cell number, Chl a and divinyl-Chl a concentrations, as observed in an oligotrophic tropical situation. Even if the backscattering-to-scattering ratio is, as theoretically expected, higher for Prochlorococcus than for all other algae (including Synechococcus), their light backscattering capacity definitely remains negligible.


Morgan JAW, Rhodes G, Pickup RW (1993) Survival of Nonculturable Aeromonas-Salmonicida in Lake Water. Applied and Environmental Microbiology 59 :874-880

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The survival of Aeromonas salmonicida subsp. salmonicida was investigated in sterile and untreated lake water. In sterile lake water (filtered and autoclaved), it was found that cells of A. salmonicida entered a nonculturable but viable condition. Viability was determined by flow cytometry with the dye rhodamine 123, which is taken up and maintained within cells with a membrane potential. For survival studies in untreated lake water, A. salmonicida was marked with the xylE gene by using the plasmid pLV1013. Marked cells were detected by growth on tryptone soy agar and tryptone soy agar supplemented with kanamycin. Cells were also detected by polymerase chain reaction DNA amplification of the xylE gene and a chromosomal DNA fragment specific for A. salmonicida (pLV1013). The results indicated that A. salmonicida entered a nonculturable condition in untreated lake water over a 21-day study. The viability of nonculturable cells could not be determined in mixed samples ; however, the presence of nonculturable cells containing both chromosomal and plasmid DNA was confirmed.


Partensky F, Hoepffner N, Li WKW, Ulloa O, Vaulot D (1993) Photoacclimation of Prochlorococcus Sp (Prochlorophyta) Strains Isolated from the North-Atlantic and the Mediterranean-Sea. Plant Physiology 101 :285-296

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Two Atlantic (SARG and NATL1) strains and one Mediterranean (MED) strain of Prochlorococcus sp., a recently discovered marine, free-living prochlorophyte, were grown over a range of ’’white’’ irradiances (I(g)) and under low blue light to examine their photoacclimation capacity. All three strains contained divinyl (DV) chlorophylls (Chl) a and b, both distinguishable from :normal’ Chls by their red-shifted blue absorption maximum, a Chl c-like pigment at low concentration, zeaxanthin, and a-carotene. The presence of two phaeophytin b peaks in acidified extracts from both Atlantic strains grown at high I(g) suggests that these strains also had a normal Chl b-like pigment. In these strains, the total Chl b to DV-Chl a molar ratio decreased from about 1 at 7.5 mumol quanta m-2 s-1 to 0.4 to 0.5 at 133 mumol quanta m-2 S-1. In contrast, the MED strain always had a low DV-Chl b to DV-Chl a molar ratio, ranging between 0.13 at low I(g) and 0.08 at high 1, The discrepancies between the Atlantic and MED strains could result from differences either in the number of light-harvesting complexes (LHC) II per photosystem II or in the Chl b-binding capacity of the apoproteins constituting LHC II. Photosynthesis was saturated at approximately 5 fg C(fg Chl)-1 h-1 or 6 fg C cell-1 h-1, and growth was saturated at approximately 0.45 d-1 for both MED and SARG strains at 18-degrees-C, but saturating irradiances differed between strains. Atlantic strains exhibited increased light-saturated rates and quantum yield for carbon fixation under blue light.


Press CM, Hein WR, Landsverk T (1993) Ontogeny of leucocyte populations in the spleen of fetal lambs with emphasis on the early prominence of B cells. Immunology 80 :598-604

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7508421

The presence and distribution of B cells and other early leucocyte populations are described in the spleen of fetal lambs from 40 to 134 days of gestation (length of gestation 150 days). Computer-assisted morphometric analysis and flow cytometry were used to quantify the early predominance of B cells in mid-gestation. B cells appeared at about 48 days and increased in number to occupy over 20% of the spleen area at 77 days. All spleens were collected on their respective livers and at no stage did the livers contain more than a few IgM-positive (+) cells, which were usually close to blood vessels. Two-colour flow cytometry demonstrated that only 1-2% of IgM+ cells expressed CD5 at 81 days. Beyond 77 days, with the expanding presence of T cells, the percentage of area occupied by IgM+ cells declined to stabilize at about 7% during late gestation. The conventional organization of the splenic white pulp was observed from 90 days along with 5’ nucleotidase-positive primary follicles. Double staining technique using immunohistochemical methods demonstrated that IgM+ cells were proliferating in the spleen from as early as 51 days and that clusters of proliferating IgM+ cells were prominent between 60 and 77 days. The results of the present study suggest that during the ontogeny of fetal lambs the spleen is a site of B-cell development or expansion before colonization of the ileal Peyer’s patch and the subsequent generation of the preimmune antibody repertoire.


Schut F, de Vries EJ, Gottschal JC, Robertson BR, Harder W, Prins RA, Button DK (1993) Isolation of Typical Marine Bacteria by Dilution Culture : Growth, Maintenance, and Characteristics of Isolates under Laboratory Conditions. Appl Environ Microbiol 59 :2150-2160

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16348992

Marine bacteria in Resurrection Bay near Seward, Alaska, and in the central North Sea off the Dutch coast were cultured in filtered autoclaved seawater following dilution to extinction. The populations present before dilution varied from 0.11 x 10 to 1.07 x 10 cells per liter. The mean cell volume varied between 0.042 and 0.074 mum, and the mean apparent DNA content of the cells ranged from 2.5 to 4.7 fg of DNA per cell. All three parameters were determined by high-resolution flow cytometry. All 37 strains that were obtained from very high dilutions of Resurrection Bay and North Sea samples represented facultatively oligotrophic bacteria. However, 15 of these isolates were eventually obtained from dilution cultures that could initially be cultured only on very low-nutrient media and that could initially not form visible colonies on any of the agar media tested, indicating that these cultures contained obligately oligotrophic bacteria. It was concluded that the cells in these 15 dilution cultures had adapted to growth under laboratory conditions after several months of nutrient deprivation prior to isolation. From the North Sea experiment, it was concluded that the contribution of facultative oligotrophs and eutrophs to the total population was less than 1% and that while more than half of the population behaved as obligately oligotrophic bacteria upon first cultivation in the dilution culture media, around 50% could not be cultured at all. During one of the Resurrection Bay experiments, 53% of the dilution cultures obtained from samples diluted more than 2.5 x 10 times consisted of such obligate oligotrophs. These cultures invariably harbored a small rod-shaped bacterium with a mean cell volume of 0.05 to 0.06 mum and an apparent DNA content of 1 to 1.5 fg per cell. This cell type had the dimensions of ultramicrobacteria. Isolates of these ultramicrobacterial cultures that were eventually obtained on relatively high-nutrient agar plates were, with respect to cell volume and apparent DNA content, identical to the cells in the initially obligately oligotrophic bacterial dilution culture. Determination of kinetic parameters from one of these small rod-shaped strains revealed a high specific affinity for the uptake of mixed amino acids (a degrees (A), 1,860 liters/g of cells per h), but not for glucose or alanine as the sole source of carbon and energy (a degrees (A), +/- 200 liters/g of cells per h). The ultramicrobial strains obtained are potentially a very important part of picoplankton biomass in the areas investigated.


Schut F, Devries EJ, Gottschal JC, Robertson BR, Harder W, Prins RA, Button DK (1993) Isolation of Typical Marine-Bacteria by Dilution Culture - Growth, Maintenance, and Characteristics of Isolates under Laboratory Conditions. Applied and Environmental Microbiology 59 :2150-2160

<Go to ISI> ://A1993LL31000024

Marine bacteria in Resurrection Bay near Seward, Alaska, and in the central North Sea off the Dutch coast were cultured in filtered autoclaved seawater following dilution to extinction. The populations present before dilution varied from 0.11 x 10(9) to 1.07 x 10(9) cells per liter. The mean cell volume varied between 0.042 and 0.074 mum3, and the mean apparent DNA content of the cells ranged from 2.5 to 4.7 fg of DNA per cell. All three parameters were determined by high-resolution flow cytometry. All 37 strains that were obtained from very high dilutions of Resurrection Bay and North Sea samples represented facultatively oligotrophic bacteria. However, 15 of these isolates were eventually obtained from dilution cultures that could initially be cultured only on very low-nutrient media and that could initially not form visible colonies on any of the agar media tested, indicating that these cultures contained obligately oligotrophic bacteria. It was concluded that the cells in these 15 dilution cultures had adapted to growth under laboratory conditions after several months of nutrient deprivation prior to isolation. From the North Sea experiment, it was concluded that the contribution of facultative oligotrophs and eutrophs to the total population was less than 1% and that while more than half of the population behaved as obligately oligotrophic bacteria upon first cultivation in the dilution culture media, around 50% could not be cultured at all. During one of the Resurrection Bay experiments, 53% of the dilution cultures obtained from samples diluted more than 2.5 x 10(5) times consisted of such obligate oligotrophs. These cultures invariably harbored a small rod-shaped bacterium with a mean cell volume of 0.05 to 0.06 mum3 and an apparent DNA content of 1 to 1.5 fg per cell. This cell type had the dimensions of ultramicrobacteria. Isolates of these ultramicrobacterial cultures that were eventually obtained on relatively high-nutrient agar plates were, with respect to cell volume and apparent DNA content, identical to the cells in the initially obligately oligotrophic bacterial dilution culture. Determination of kinetic parameters from one of these small rod-shaped strains revealed a high specific affinity for the uptake of mixed amino acids (a-degrees(A) 1,860 liters/g of cells per h), but not for glucose or alanine as the sole source of carbon and energy (a-degrees(A) +/- 200 liters/g of cells per h). The ultramicrobial strains obtained are potentially a very important part of picoplankton biomass in the areas investigated.


Spinrad RW, Brown J (1993) Effects of Asphericity on Single-Particle Polarized-Light Scattering. Applied Optics 32 :6151-6158

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Polarized light scattering from individual particles has been analyzed to determine the effects of particle shape. Flow cytometric techniques were used on samples of spherical microspheres and naturally occurring marine algae. An analog of the depolarization ratio was obtained by using crossed polarizers in the source and detector of the flow cytometer. Results suggest that differences between the polarized light scattering of spheres and aspherical particles are not discernible unless the scattered intensities are normalized to the forward scattering, which is roughly equivalent to particulate cross section. This research indicates that polarized light scattering, when normalized to particle size, may provide an indication of the extent of asphericity of hydrosols.


Troussellier M, Courties C, Vaquer A (1993) Recent applications of flow cytometry in aquatic microbial ecology. Biol Cell 78 :111-121

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8220221

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


Troussellier M, Courties C, Vaquer A (1993) Recent Applications of Flow-Cytometry in Aquatic Microbial Ecology. Biology of the Cell 78 :111-121

<Go to ISI> ://A1993MA01500013

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


Unson MD, Faulkner DJ (1993) Cyanobacterial Symbiont Biosynthesis of Chlorinated Metabolites from Dysidea-Herbacea (Porifera). Experientia 49 :349-353

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The tropical marine sponge Dysidea herbacea contains large numbers of a symbiotic filamentous cyanobacterium identified on the basis of a detailed ultrastructural study as Oscillatoria spongeliae. We report the flow-cytometric separation of the symbiont from the sponge cells, and demonstrate by chemical analyses that a unique group of polychlorinated compounds isolated from the whole sponge tissue is limited to the cyanobacterial filaments, whereas the accompanying sesquiterpenoids are found only in the sponge cells. This is the first demonstration that secondary metabolites ascribed to a sponge are localized in prokaryotic symbiont cells.


Veldhuis MJW, Kraay GW (1993) Cell Abundance and Fluorescence of Picoplankton in Relation to Growth Irradiance and Nitrogen Availability in the Red-Sea. Netherlands Journal of Sea Research 31 :135-145

<Go to ISI> ://A1993LW78200003

The vertical distribution and cellular fluorescence characteristics (chlorophyll and phycoerythrin, PE) of picoplankton (cyanobacteria and prochlorophytes) in the southern Red Sea were investigated in relation to physico-chemical properties of the water column. At all stations two subpopulations of Synechococcus sp. (type A and B) co-occurred, with maximal numbers up to 75000.CM-3 . Type B, with dim fluorescence signals, dominated the surface waters whereas type A, with bright fluorescence signals, dominated at greater depth. The divinyl a and b containing Prochlorococcus sp. peaked below the cyanobacteria at the deep chlorophyll maximum (DCM) with maximal cell numbers of 276000.cm.3.
Due to thermal stratification the cellular fluorescence (chlorophyll and phycoerythrin) increased with decreasing growth (PAR) irradiance, in an S-shaped manner, but magnitude and slope for the three picoplankters differed.The Synechococcus sp. type B had only a moderate increase in chlorophyll and phycoerythrin fluorescence signals with depth (3.4 and 6.6 fold, respectively), with values saturating at 3% (I(d)) of the surface irradiance. The deeper-water type A not only possessed much higher values for cellular fluorescence than the B type, but the increase with decreasing light level was also much higher (for chlorophyll by a factor of 11 and PE increased by a factor of 23). In addition, maximal values for these fluorescence signals occurred at an isolume of 1 to 0.5%. These differences in concentrations and responses of the pigment content to the prevailing light climate explain the variation in abundance of both types over the water column.
Although the prochlorophytes dominated almost the entire euphotic zone, their adaptation to low light levels was even better than in the two types of cyanobacteria. With depth their increase in chlorophyll fluorescence was similar to that observed in the cyanobacteria (with an increase from surface to bottom of the euphotic zone by a factor of 25). The maximum fluorescence signal was not reached until an I(d) of 0.01%. The great ability of the prochlorophytes to adapt their pigmentation to changing light and nitrogen conditions suggests that they can maintain growth under a wide range of environmental conditions.


Veldhuis MJW, Kraay GW, Gieskes WWC (1993) Growth and Fluorescence Characteristics of Ultraplankton on a North South Transect in the Eastern North-Atlantic. Deep-Sea Research Part Ii-Topical Studies in Oceanography 40 :609-626

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In the summer of 1989 vertical profiles of chlorophyll a were taken in the North Atlantic. Stations were located along a transect following longitude 20-degrees-W, between 60 and 33-degrees-N. Maximum chlorophyll a levels were located near the surface in the north (2 mugl-1) but became gradually deeper towards the south (0.3 mug l-1 at a depth of 80-110m). Primary production varied only by a factor of 2.3 (226-533 mg C m-2 day-1), whereas the assimilation ratio showed far less variation (15.46 +/- 4.61 mg C mg Chl-1 day-1, n = 14). The northern part of the transect showed no thermal stratification, and nutrients were plentiful. Phytoplankton was distributed homogeneously, with a nearly constant signature of the cellular fluorescence characteristics as monitored by flow-cytometry. The dominant taxa were small prymnesiophytes (Emiliania huxleyi) and chroococcoid phycoerythrin-containing cyanobacteria. Cells were healthy, resulting in net growth rates (growth rate minus grazing) of up to 0.75 divisions per day. Of the total primary production, 68% was actually converted into new plant carbon. Midway along the transect (47-degrees-N) there was a sharp thermocline at a depth of 35 m. All nutrients (nitrogen, phosphate and silicate) were depleted in the upper water layer. Cyanobacteria dominated the phytoplankton community in numbers varying from 12,000 to 39,000 cells ml-1. Stabilization of the water column was apparently in progress, as could be derived from the gradual increase in mean cellular red fluorescence of the cyanobacteria with depth. Furthermore, net growth rates of the algae were relatively low, as was the proportion of primary production resulting in an actual increase of plant carbon (42%). At the southernmost station (33-degrees-N), the situation was typical of the oligotrophic subtropics : a sharp thermocline at 35 m, the 0.1% surface incident irradiance level at 150 m, and a nitracline at a depth of 100-120m. The most prominent (pico)plankters present were prochlorophytes, predominantly at the deep chlorophyll maximum (80-100 m, up to 95,000 cells ml-1). Cyanobacteria co-occurred but only in small numbers (maximum of 5500 ml-1), mainly in surface waters. The vertical (physical) stability of the water column was pronounced, and the mean cellular red fluorescence signal of cells found at the bottom of the euphotic zone was consequently higher than in surface waters. Most remarkable were the net growth rates of the various species suggesting a highly dynamic phytoplankton community. Extreme growth rates included positive as well as negative values, which were not always consistent with the greatest abundance of certain species. Despite these rapid changes, the actual increase in plant carbon was only 14% of the primary production.


Vrieling EG, Draaijer A, Vanzeijl WJM, Peperzak L, Gieskes WWC, Veenhuis M (1993) The Effect of Labeling Intensity, Estimated by Real-Time Confocal Laser Scanning Microscopy, on Flow Cytometric Appearance and Identification of Immunochemically Labeled Marine Dinoflagellates. Journal of Phycology 29 :180-188

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Two different fluorescein isothiocyanate (FITC) conjugates were used to analyze the effect of labeling intensity on the flow cytometric appearance of marine dinoflagellates labeled with antibodies that specifically recognized the outer cell wall. Location of the labeling was revealed by epifluorescence and real-time confocal laser scanning microscopy using an anti-rabbit IgG/FITC-conjugated secondary antiserum. Flow cytometric measurements showed that cells of Prorocentrum species labeled this way could not always be distinguished from unlabeled cells. The labeling intensity increased several times when a biotinylated anti-rabbit IgG secondary antiserum was used in combination with a streptavidin/FITC conjugate. Flow cytometry indicated that the labeling intensity had increased 50%, which resulted in an improved separation of clusters of labeled and unlabeled cells.


Whiteley AS, Burkill PH, Sleigh MA (1993) Rapid method for cell cycle analysis in a predatory marine dinoflagellate. Cytometry 14 :909-915

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7507024

Oxyrrhis marina (Dujardin) is a predatory marine dinoflagellate that feeds phagocytically on live phytoplanktonic "prey" cells from the surrounding environment. A rapid method was developed to separate the cell cycle characteristics of these predators from their prey cells in order to study the cell cycle dynamics of this organism. Nuclei from Oxyrrhis were isolated in low salt buffer (PBS) using detergent and mechanical agitation and the DNA stained with Hoechst 33258 in a one step procedure. The method permitted the isolation of nuclei from the Oxyrrhis cells with > 95% efficiency. Discrimination between prey cell nuclei and those of Oxyrrhis was achieved during flow cytometric analysis which yielded routinely G1 CVs of 3-6% for exponentially growing cell populations and 2-3% for stationary phase cells. The method was used to demonstrate the changes in cell cycle dynamics during the exponential and stationary phases of growth. Results indicated that in contrast to most mammalian and phytoplankton cell types Oxyrrhis spent the major portion (ca. 50%) of its cell cycle in G2 + M when actively dividing. Analysis of stationary phase populations also suggests that specific cell cycle control (or restriction) points were present in both G1 and G2 in this species.