Technique for Enumeration of Heterotrophic and Phototrophic Nanoplankton, Using Epifluorescence Microscopy, and Comparison with Other Procedures

A new method is described that uses the fluorochrome primulin and epifluorescence microscopy for the enumeration of heterotrophic and phototrophic nanoplankton (2 to 20 μm). Phototrophic microorganisms are distinguished from heterotrophs by the red autofluorescence of chlorophyll a. Separate filter sets are used which allow visualization of the primulin-stained nanoplankton without masking chlorophyll a fluorescence, thus allowing easy recognition of phototrophic cells. Comparison with existing epifluorescence techniques for counting heterotrophic and phototrophic nanoplankton shows that primulin provides more accurate counts of these populations than the fluorescein isothiocyanate or proflavine techniques. Accuracy is comparable to that with the acridine orange technique, but this method requires only one filter preparation for the enumeration of both phototrophic and heterotrophic populations.     

Flow Cytometric Analysis of Marine Bacteria with Hoechst 33342

We investigated the accuracy and precision of flow cytometric (FCM) estimates of bacterial abundances using 4′, 6-diamidino-2-phenylindole (DAPI) and Hoechst 33342 (HO342, a bisbenzamide derivative) on paraformaldehyde-fixed seawater samples collected from two stations near Oahu, Hawaii. The accuracy of FCM estimates was assessed against direct counts by using epifluorescence microscopy. DAPI and HO342 differ in two aspects of their chemistry that make HO342 better suited for staining marine heterotrophic bacteria for FCM analysis. These differences are most important in studies of open-ocean ecosystems that require dual-beam FCM analysis to clearly separate heterotrophic bacterial populations from populations of photosynthetic Prochlorococcus spp. Bacterial populations were easier to distinguish from background fluorescence when stained with HO342 than when stained with DAPI, because HO342 has a higher relative fluorescence quantum yield. A substantially higher coefficient of variation of blue fluorescence, which was probably due to fluorescent complexes formed by DAPI with double-stranded RNA, was observed for DAPI-stained populations. FCM estimates averaged 2.0 and 12% higher than corresponding epifluorescence microscopy direct counts for HO342 and DAPI-stained samples, respectively. A paired-sample t test between FCM estimates and direct counts found no significant difference for HO342-stained samples but a significant difference for DAPI-stained samples. Coefficients of variation of replicate FCM abundance estimates ranged from 0.63 to 2.9% (average, 1.5%) for natural bacterial concentrations of 6 × 10⁵ to 15 × 10⁵ cells ml^-1.     

Impact of UV-B radiation (290–320 nm) upon estuarine bacteria

Summary Effects of ultraviolet radiation on the development of metabolism of estuarine bacterial populations in laboratory microecosystems were studied. When compared with bacterial populations developing under an ultraviolet-deficient condition, the heterotrophic populations from microecosystems exposed to an ultraviolet-supplemented sprectrum displayed an overall decrease in total numbers, an increase in the proportion of pigmented cells, a decrease in the number of cellulolytic microorganisms and an increase in heterotrophic respiration. Ultraviolet radiation in the 290–320 nm waveband was the apparent stressful environmental parameter.     

Assessing Biomass and Production of Bacteria in Eutrophic Lake Mendota, Wisconsin

Estimates were made of the biomass and production of heterotrophic bacteria in the epilimnion of Lake Mendota, Wis. Cell counts were done with epifluorescence microscopy and varied from 3 × 10⁵ bacteria per ml in winter to 3 × 10⁶ bacteria per ml in summer. Cell volumes were measured in scanning electron micrographs. The average cell volume was 0.159 μm³. Annual variations and depth distribution were studied. Production was estimated from the frequency of dividing cells and from dark radioactive sulfate uptake. Annual productivity and daily average productivity were very close with both methods: 107 to 205 g of C per m² per year for sulfate and 89 to 117 g of C per m² per year for frequency of dividing cells. Zooplankton feeding removed 2 to 10% of the bacterial net production annually. When compared with biomass changes and losses due to zooplankton feeding, production values were very high. Therefore, it was suggested that other loss factors have to be more important than zooplankton feeding in controlling the bacterial population. Bacterial heterotrophic production was about 50% of gross primary production.     

Springtime microprotozoan abundance and biomass in the southeastern Bering Sea and Shelik of Strait, Alaska

We surveyed springtime biomass and abundance of the >20 µmmicroprotozoa in surface waters of the SE Bering Sea and Shelikof Strait, Alaska. This study was part of the Fisheries OceanographyCoordinated Investigations (FOCI) program examining processeswhich affect the recruitment variability of walleye pollock(Theragra chalcogramma). Microprotozoa are a potential preyresource for larval pollock which has not been previously examined.In both areas, the >20 µm microprotozoa were predominantlydinoflagellates and ciliates. At the time of sampling (May 1990in Shelikof Strait and April 1992 in the SE Bering Sea), thespring diatom bloom was under way in Shelik of Strait, but notin the SE Bering Sea. Heterotrophic dinoflagellates dominatedthe microprotozoan assemblage in Shelik of Strait, but not inthe SE Bering Sea. In the SE Bering Sea. total microprotozoanabundances ranged from 300 to 6233 organisms 1^–1 and biomassfrom 0.58 to 9.73 µg C 1^–1. In Shelik of Strait,abundance and biomass were higher, ranging from 850 to 14 960organisms 1^–1 and from 1.29 to 70.73 µg C 1^–1,respectively. These biomass levels are comparable to those reportedfrom other coastal and oceanic regions. Microprotozoan biomasslevels were sufficient to support the estimated metabolic needsof first-feeding larval walleye pollock. It remains to be shownwhether larval pollock use this resource.     

Proportion of prokaryotes enumerated as viruses by epifluorescence microscopy

It is well known that there are prokaryotes small in size (e.g. ultra-microprokaryotes) that pass through a 0.2-μm filter. As bacterial and viral abundances are determined by epifluorescence microscopy and the differentiation between them is based on particle size, some bacteria can be erroneously enumerated as viruses, namely in marine waters where bacteria are small. However, there is no information on the proportion of prokaryotes that could be misidentified as viruses by epifluorescence microscopy. In this work, we assessed, in water samples collected in the estuarine system Ria de Aveiro (Portugal), the proportion of prokaryotes that could be counted as viruses by the current widespread epifluorescence microscopy and, for the first time, by fluorescence in situ hybridization (FISH). The total number of particles was determined on membranes of 0.2 and 0.02 μm after staining with 4′,6-diamidino-2-phenylindole (DAPI), and the number of prokaryotes (Bacteria and Archaea) was determined by FISH for both pore size membranes. The results show that, in the marine zone of the estuarine system, 28 % of particles enumerated as virus-like particles were prokaryotes, but, in the brackish water zone, only 13 % of the particles counted as viruses were actually prokaryotic cells. Epifluorescence microscopy overestimates viral abundance, and also the ratio viruses:prokaryotes, and this error must be taken into consideration because it can vary significantly within a system. In fact, in the marine zone of an estuarine system, the overestimation of viral abundance can be twice as high as in the brackish water zone.     

Tracing pollen nuclei in the ovary and ovule of Gasteria verrucosa (Mill.) H. Duval after pollination with DAPI-stained pollen

Summary The addition of DAPI particles to the stigma exudate of Gasteria results in the labelling of the pollen nuclei. By means of epifluorescence microscopy and clearing of the ovule, the labelled nuclei of the sperm cells and, subsequently, the zygotic nucleus can be observed. The method was used in a cross-pollination with low seed setting to examine different types of penetration and transport of the pollen tube nucleus and sperm cell nuclei. More than one pollen tube can penetrate, but generally only one set of sperm cell nuclei is accepted.     

Bivariate cytofluorimetric analysis of DNA and nuclear protein content in plant tissue

Summary Techniques of static biparametric cytofluorimetry were developed to measure DNA and protein fluorescence simultaneously in the same nucleus stained with 4,6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate (FITC) fluorochromes. With these cytofluorimetric procedures, we analysed DNA and nuclear protein content in root apices during the first 72 h of pea seed germination. This method allows a more reliable, rapid and less expensive measurement of DNA and proteins than cytophotometry. Nuclear protein content can be considered as a second parameter to define subcompartments of cell cycle phases; it offers the possibility of studying the progression of plant cells through cell cycle and its control in greater detail.Abbreviations DAPI 4,6-diamidino-2-phenylindole - FITC fluorescein isothiocyanate - PI propidium iodide - Tris Tris(hydroxymethyl) aminomethane     

Detection of toxic phytoplankton species by immunochemical particle analysis based on flow cytometry

Particulate suspended matter in oceanic, coastal, and estuarine regions can be specifically marked immunochemically with a fluorescent probe using antisera recognizing antigens present on their surface. Of the particulate matter, phytoplankton is a major component. Toxic species that may form harmful blooms can be a direct threat to aquaculturing tourism, sea-life and man. In order to detect such species in natural fixed phytoplankton populations, immunochemical tagging has been combined with flow cytometric evaluation Microalgal cells can be labeled with a fluorescent probe (fluorescein isothiocyanate, FITC, is recommended). Labeled cells are counted using a flow cytometer. This method has proved to be applicable in a monitoring programme in the North Sea.     

Autotrophic and heterotrophic nanoplankton in the diet of the estuarine copepods Eurytemora affinis and Acartia bifilosa

The ingestion of autotrophic and heterotrophic nanoplanktonby two estuarine copepods, Eurytenora affinis and Acarith bifilosa,was measured in various environmental conditions using the incubationmethod and epifluorescence microscopy. Egg production of thespecies was also deter mined in order to estimate their carbonrequirements. Assuming a gross efficiency of egg productionof 0.3, nanoplanktonic carbon ingested always met the carbonrequirements suggesting that, most of the time, other carbonsources could be unnecessaly. Nanoplankton ingestion by A.bifilosa(from 128 to 1693 cells ind.^–1 h^–1) was dominatedby autotrophic forms (60–97%) and was seriously affectedby high (>100 mg l^–1 suspended particulate matter (SPM)concentrations. Nanoplankton ingestion by E.affinis (from 300to 1049 cells ind^–1 h^–1) was relatively stable incomparison, but this latter species seemed to switch its grazingpressure from autotrophic to heterotrophic forms when SPM concentrationsincreased. Thus, two copepod species, living in the same estuary,presented two different feeding behaviours, probably to maximizeenergy input per unit of energy expenditure. Such differencescould contribute to the spatial and seasonal segregation ofthese species which is usually observed.      

A method for analysing phosphatase activity in aquatic bacteria at the single cell level using flow cytometry

It has been demonstrated that ELF97-phosphate (ELF-P) is a useful tool to detect and quantify phosphatase activity of phytoplankton populations at a single cell level. Recently, it has been successfully applied to marine heterotrophic bacteria in culture samples, the cells exhibiting phosphatase activity being detected using epifluorescence microscopy. Here, we describe a new protocol that enables the detection of ELF alcohol (ELFA), the product of ELF-P hydrolysis, allowing the detection of phosphatase positive bacteria, using flow cytometry. Bacteria from natural samples must be disaggregated and, in oligotrophic waters, concentrated before they can be analyzed by flow cytometry. The best efficiency for disaggregating/separating bacterial cell clumps was obtained by incubating the sample for 30 min with Tween 80 (10 mg l(-1), final concentration). A centrifugation step (20,000 g; 30 min) was required in order to recover all the cells in the pellet (only 7+/-2% of the cells were recovered from the supernatant). The cells and the ELFA precipitates were resistant to these treatments. ELFA-labelled samples were stored in liquid nitrogen for up to four months before counting without any significant loss in total or ELFA-labelled bacterial cell abundance or in the ELFA fluorescence intensity. We describe a new flow cytometry protocol for detecting and discriminating the signals from both ELFA and different counterstains (4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)) necessary to distinguish between ELFA-labelled and non ELFA-labelled heterotrophic bacteria. The method has been successfully applied in both freshwater and marine samples. This method promises to improve our understanding of the physiological response of heterotrophic bacteria to P limitation.     

Spatial distribution of coastal antarctic seawater bacteria: Relationship with avifauna

Summary Previous antarctic studies have pointed out the ecological importance of ornithogenic soils. However, few data exist to determine the impact of such guanoenriched soils on surrounding seawater microbial populations. In order to evaluate the influence of birds, the relationships of spatial distribution of seawater bacterial microflora to penguin repartition were studied during the antarctic summer 1986 in Terre Adelie land area and in January 1984 in the subantarctic Kerguelen Archipelago. With bacterial estimates as high as 1.7×10⁸ cells ml^-1 for total counts and 2.3×10⁷ CFU ml^-1 for aerobic heterotrophic populations, ornithogenic soil analyses confirmed previous results from similar sites. In seawater a clear decreasing gradient from the shore towards the open sea was found. All bacterial parameters studied (epifluorescence direct counts, frequency of dividing cells estimation and viable counts) were correlated significantly with penguin populations. Complementary numerical taxonomy confirmed the major role played by the bird manuring in such antarctic ecosystems.     

Visualization of vacuoplasts in isolated vacuole preparations from mesophyll protoplasts of periwinkle [Catharanthus roseus (L.) G. Don]

A procedure was developed for the rapid detection of vacuoplasts in vacuole preparations isolated from mesophyll protoplasts of Catharanthus roseus (L.) G. Don (periwinkle). The procedure relies on the staining of surface carbohydrates on the plasma membrane surrounding vacuoplasts with fluorescein-labeled lectins. When isolated under conditions of constant osmotic strength, approximately 15–20% of the vacuoles isolated showed surface labeling with FITC-agglutinin from Abrus precatorius. Isolation of vacuoles after an initial osmotic shock showed much lower (<5%) surface labeling. This lower level of surface labeling correlated well with a lower level of other non-vacuolar marker enzyme activities. A thin layer of cytoplasm was visible in a small number of these stained structures, indicating that they were vacuoplasts.Abbreviations FITC fluorescein isothiocyanate     

CHROOCOCCOID CYANOBACTERIA IN LAKE ONTARIO: VERTICAL AND SEASONAL DISTRIBUTIONS DURING 19821

Chroococcoid cyanobacteria (0.7–1.3 μm in diameter) were discovered to be a significant component of the Lake Ontario plankton. Using epifluorescence microscopy, the densities of these microorganisms were found to vary by four orders of magnitude with a single large peak in abundance (6.5 × 10⁵ cells mL^?1) corresponding to the time of maximum water temperature. The morphology and abundance of these cyanobacteria were similar to those previously found in oceanic systems. They constituted 10% of the bacterial numbers in the epilimnion during this period, approximately 40% of the biomass of prokaryotes less than 2.0 μm, and 30% of the biomass of all microorganisms less than 20 μm in size. Size fractionation studies indicated that they were responsible for approximately 38% of the total primary production during times of peak abundance, and were important in phosphorus uptake. Cyanobacteria observed in the food vacuoles of heterotrophic microflagellates and in the guts of rotifers suggest that the latter organisms may be important consumers of this prokaryote population.     

Dynamics and trophic roles of heterotrophic protists in the plankton of a freshwater tidal estuary

Freshwater tidal estuaries comprise the most upstream reaches of estuaries and are often characterised by the presence of dense bacterial and algal populations which provide a large food source for bacterivorous and algivorous protists. In 1996, the protistan community in the freshwater tidal reaches of the Schelde estuary was monitored to evaluate whether these high food levels are reflected in a similarly high heterotrophic protistan biomass. Protistan distribution patterns were compared to those of metazoan zooplankton to evaluate the possible role of top-down regulation of protists by metazoans. Apart from the algivorous sarcodine Asterocaelum, which reached high densities in summer, heterotrophic protistan biomass was dominated by ciliates and, second in importance, heterotrophic nanoflagellates (HNAN). HNAN abundance was low (annual average 2490 cells ml^–1) and did not display large seasonal variation. It is hypothesised that HNAN were top-down controlled by oligotrich ciliates throughout the year and by rotifers in summer. Ciliate abundance was generally relatively high (annual average 65 cells ml^–1) and peaked in winter (maximum 450 cells ml^–1). The decline of ciliate populations in summer was ascribed to grazing by rotifers, which developed dense populations in that season. In winter, ciliate populations were probably regulated `internally' by carnivorous ciliates (haptorids and Suctoria). Our observations suggest that, in this type of productive ecosystems, the microbial food web is mainly top-down controlled rather than regulated by food availability.     

THE EFFECT OF LABELING INTENSITY,ESTIMATED BY REAL-TIME CONFOCAL LASER SCANNING MICROSCOPY,ON FLOW CYTOMETRIC APPEARANCE AND IDENTIFICATION OF IMMUNOCHEMICALLY LABELED MARINE DINOFLAGELLATES1

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.     

Simplified method for DNA and protein staining of human hematopoietic cell samples

A rapid reproducible method yielding high resolution analysis of DNA and protein in human hematopoietic cell samples has been developed by modification of the propidium iodide and fluorescein isothiocyanate procedure. Cell staining involves sequential addition of each reagent (RNase, fluorescein isothiocyanate and propidium iodide) to ethanol-fixed cells and requires no centrifugation steps. Stained cells are analyzed in the reagent solutions. Analysis of bone marrow samples from multiple myeloma patients showed mixed normal and aneuploid populations with a major portion of the aneuploid cells having a significantly higher protein content. This approach permitted differential cell cycle analysis of normal and the aneuploid populations.     

Biochemical,morphological and flow cytometric evaluation of the effects of hexachlorobenzene on rat liver

This study investigated the ability of HCB (0.1% in the diet for 15 days) to cause early changes in the cellular ploidy of rat liver. Treatment caused marked hepatomegaly, increase of microsomal proteins and cytochrome P-450 content and reduction of hepatocyte microviscosity. Microscopic examination showed that the hepatocytes were enlarged, with hyaline cytoplasm and vacuoles. The size distribution of the isolated hepatocytes showed a larger percentage of bigger cells. Flow-cytometric DNA/protein analysis was performed on whole (fixed) cells and on nuclei. From the combined results of both analyses it was possible to exclude significant changes in the percentages of diploid, mononucleated tetraploid, binucleated tetraploid and octoploid hepatocytes. The DNA and protein content of each subpopulation remained unchanged. Our results suggest that HCB does not cause early diploidization of liver cells and that hepatomegaly and cytochrome P-450 induction seem not to be correlated with effects on total DNA and total protein contents.Abbreviations HCB hexachlorobenzene - PI propidium iodide - FITC fluorescein isothiocyanate - DN diploid nuclei - SN 2N-4N nuclei in S-phase - TN tetraploid nuclei - DC diploid cells - SDC 2N-4N diploid cells in S-phase - TC tetraploid cells - STC 4N-8N tetraploid cells in S-phase - OC octoploid cells - MDC mononucleated diploid cells - SMDC mononucleated diploid cells in S-phase - BOC binucleated octoploid cells - SBTC binucleated tetraploid cells in S-phase - BTC binucleated tetraploid cells - MTC mononucleated tetraploid cells.     

Abundance and productivity of heterotrophic nanoplankton in Georgia coastal waters

The population abundances and rates of biomass production ofheterotrophic nanoplankton (HNAN) in Georgia coastal waterswere evaluated by epifluorescence microscopy. HNAN populations(mostly non-pigmented microflagellates <10 µm in diameter)ranged from 0.3 x 10³ cells ml^–1 in shelf waters 15 kmoffshore to 6.3 x 10³ cells ml^–1 in waters 0.25 km fromthe coast. There was a strong correlation (r = 0.83) betweenHNAN and free bacterioplankton population abundances, but noapparent relation (r = 0.38) between HNAN and phototrophic nanopLankton(PNAN) abundances. HNAN biomass production in estuarine andnearshore shelf waters, as estimated from increases in HNANpopulations during laboratory incubations of natural water samples,ranged from 0.10 to 0.79 mg C m^–3 h^–3, with populationgeneration times of 9.7 to 26.5 h. There was a significant linearrelation (r = 0.95) between HNAN biomass and HNAN productivity.We calculated that HNAN may graze at least 30% to 50% of dailybacterioplankton production in Georgia coastal waters.