Buma AGJ, Vanhannen EJ, Roza L, Veldhuis MJW, Gieskes WWC (1995) Monitoring Ultraviolet-B-Induced DNA-Damage in Individual Diatom Cells by Immunofluorescent Thymine Dimer Detection. Journal of Phycology 31 :314-321
We developed a method to investigate the effect of ultraviolet-B radiation (UVBR) on the formation of thymine dimers in microalgal DIVA that can be used for both laboratory and in situ research. Antibody labeling of dimers was followed by a secondary antibody (fluorescein isothiocyanate) staining to allow visualization of DNA damage with flow cytometry or fluorescence microscopy. Thymine dimer-specific fluorescence in nuclear DNA of the marine diatom Cyclotella sp. was linearly related to the UVBR dose. Simultaneous measurements of cellular DNA content showed that the vulnerability of G2 cells to DNA damage did not differ significantly from the vulnerability of G1 cells. The formation and removal of thymine dimers in Cyclotella sp. cells was monitored for 3 consecutive days at two realistic UVBR irradiance bevels. Thymine dimers were removed within 24 h when exposed to a saturating photosynthetically active radiation intensity following the UVBR treatment. This new method allows the study of UVBR-induced DNA damage on a cell-to-cell basis. It is also feasible for field studies because cells remain intact and can be recognized readily after antibody treatment.
Cid A, Fidalgo P, Herrero C, Abalde J (1995) Flow cytometry determination of acute physiological changes in a marine diatom stressed by copper. Microbiologia 11 :455-460
Flow cytometry (FCM) was used to determine changes in cellular volume, transmembrane potential, mitochondrial membrane potential and intracellular pH in the marine microalga Phaeodactylum tricornutum immediately (5 to 10 seconds) after the addition of selected concentrations of copper. An acute increase in the forward scatter signal of this diatom was detected after the addition of 10 mg 1-1 of copper. Stress produced by the copper addition resulted in various physiological alterations that can be easily and quickly detected by FCM : (i) the hyperpolarization of the cell membrane, as a result of an immediate increase in the cytoplasmic membrane potential, (ii) the increase of the mitochondrial membrane potential, being maximum at the higher copper concentration assayed, and (iii) the increase in the intracellular pH with the highest copper concentration assayed (10 mg 1-1).
Cid A, Herrero C, Torres E, Abalde J (1995) Copper Toxicity on the Marine Microalga Phaeodactylum-Tricornutum - Effects on Photosynthesis and Related Parameters. Aquatic Toxicology 31 :165-174
Essential heavy metals, as copper, can be toxic for microalgae at high concentrations. Copper affected growth and other parameters closely related to photosynthesis of the marine diatom Phaeodactylum tricornutum. A copper concentration of 0.10 mg l-1 provoked about 50% growth reduction and 1 mg l-1 inhibited the growth. Copper also interfered with photosynthesis and ATP production. A copper concentration of 0.5 mg l-1 reduced in a 50% the photosynthetic rate. Therefore, growth is more affected by copper than photosynthesis. Results of chlorophyll a fluorescence obtained by flow cytometry showed that copper’s inhibitory effect on PS II activity is located on its oxidizing side. The lower copper concentration assayed provoked a significant decrease in the cellular pool of ATP. Pigment analysis by HPLC showed that copper affected the pigment pattern of P. tricornutum. Important changes were observed for chlorophyll a and its allomer : chlorophyll a proportion decreased while its allomer increased with the copper concentration, being maximum at 1 mg Cu 1-1. The study of the intracellular pH by flow cytometry revealed that P. tricornutum cells exposed to 0.5 and 1 mg Cu 1-1 showed an intracellular pH higher than control cultures cells, explaining the high proportion of the chlorophyll a allomer in these cells.
Corzo A, Vergara JJ, GarciaJimenez MC (1995) Isolation and flow cytometric characterization of protoplasts from marine macroalgae. Journal of Phycology 31 :1018-1026
Protoplasts were isolated from Ulva rigida C. Agardh (Chlorophyta) and two species of Rhodophyta, Gracilariopsis lemaneiformis (Bory) Dawson, Acleto et Folvik and Gracilaria tenuistipitata Chang et Xia var. liui with minor modifications (the inclusion of 0.01% agarase in the set of cell-wall-degrading enzymes for the two red algae). Flow cytometric characteristics of freshly isolated protoplasts were determined on a FACScan flow cytometer (FC). The most useful parameters for characterizing protoplasts from marine algae were forward angle light scatter (FSC), orange fluorescence (FL2) and red fluorescence (FL3). Protoplasts from all the species were easily distinguishable when their FSC, FL2, and FL3 signals were combined in the bivariate Plots FL3 vs. FSC and FL3 vs. FL2. Two alternative techniques to help identify protoplasts from debris in the FC computer screen were developed (for FC without sorting capability). Both techniques were based on the ability of new FCs to record time. The first one was based on the induction of rapid changes of cell volume in response to osmotic stress. Only intact protoplasts responded to changes in the osmotic pressure. The second one was based on the uptake and hydrolysis of fluorescein diacetate by intracellular esterases. Viable protoplasts showed a hyperbolic accumulation of fluorescein with time. Semimaximal fluorescein accumulation was attained in 30.5 +/- 9.5 s. Debris was easily recognized since, contrary to protoplasts, it did not show a time-dependent accumulation of fluorescein.
Cullen JJ, Lewis MR (1995) Biological Precesses and Optical Measurements near the Sea-Surface - Some Issues Relevant to Remote-Sensing. Journal of Geophysical Research-Oceans 100 :13255-13266
The advent of remote sensing, the development of new optical instrumentation, and the associated advances in hydrological optics have transformed oceanography : it is now feasible to describe ocean-scale biogeochemical dynamics from satellite observations, verified and complemented by measurements from optical sensors on profilers, moorings, and drifters. Only near-surface observations are common to both remote sensing and in situ observation, so it is critical to understand processes in the upper euphotic zone. Unfortunately, the biological principles that must be used to interpret optical variability near the sea surface are weaker than we would like, because relatively few experiments and analyses have examined bio-optical relationships under high irradiance characteristic of the upper optical depth. Special consideration of this stratum is justified, because there is good evidence that bio-optical relationships are altered near the surface : (1) the fluorescence yield from chlorophyll declines, leading to bias in the estimation of pigment from fluorometry ; (2) the modeled relationship between solar-stimulated fluorescence and photosynthesis seems to deviate significantly from that presented for the lower euphotic zone ; and (3) carbon-specific and cellular attenuation cross sections of phytoplankton change substantially during exposures to bright light. Even the measurement of primary productivity is problematic near the sea surface, because vertical mixing is not simulated and artifactual inhibition of photosynthesis can result. These problems can be addressed by focusing more sampling effort, experimental simulation, and analytical consideration on the upper optical depth and by shortening timescales for the measurement of marine photosynthesis. Special efforts to study near-surface processes are justified, because new bio-optical algorithms will require quantitative descriptions of the responses of phytoplankton to bright light.
Gladu PK, Patterson GW, Wikfors GH, Smith BC (1995) Sterol, Fatty-Acid, and Pigment Characteristics of Utex-2341, a Marine Eustigmatophyte Identified Previously as Chlorella-Minutissima (Chlorophyceae). Journal of Phycology 31 :774-777
An examination of the sterols of UTEX 2341, a small (ca. 2 mu m), nonmotile unicellular marine alga identified as Chlorella minutissima Fott et Novakova, yielded results inconsistent with any of 35 Chlorella strains analyzed previously. UTEX 2341 contained cholesterol as the principal sterol, with 24-methylenecholesterol, fucosterol, and isofucosterol also present ; these are not dominant sterols in any other Chlorella species. Presence of eicosapentaenoic acid in UTEX 2341 also contrasted with fatty acids of Chlorella strains analyzed previously. Pigment analysis of UTEX 2341 revealed that it contained chlorophyll a, but not chlorophylls b or c ; violaxanthin was the only major xanthophyll pigment. Both lipid and pigment compositions suggest that UTEX 2341 is not a member of the genus Chlorella but, rather, belongs in the Eustigmatophyceae it may be Nannochloropsis sp. Cells with possible extracellular structures were present at an appreciable percentage of the stationary-phase population studied ; centrifuging removed or collapsed these structures. The high cholesterol and polyunsaturated fatty acid contents of UTEX 2341 make it attractive as a potential aquaculture feed, provided it is, or can be made, digestible.
Hemendinger RA, Miller MM, Bloom SE (1995) Selective expression of major histocompatibility complex (MHC) antigens and modulation of T-cell differentiation in chickens with increased MHC-chromosome dosages. Vet Immunol Immunopathol 46 :303-316
Increased dosage of genes belonging to the immunoglobulin superfamily may be responsible for some of the less noticeable but targeted phenotypic disturbances seen in trisomy conditions of humans and animals. We used an avian aneuploidy model to study the specific effects of extra major histocompatibility complex (MHC)-microchromosome dosage on the progression of thymocyte differentiation through a broad period of embryonic and neonatal development. The particular goal in the present investigation was to determine whether a reduction in the number of thymocytes, previously observed in the developing thymus of MHC aneuploids, is accompanied by particular alterations in thymocyte differentiation. We hypothesized that the subpopulation structure and/or developmental pattern for thymocyte differentiation are characteristically perturbed (delayed or modified) by increased MHC-chromosome dosage in cells. The regulation of MHC surface antigen expression in aneuploid thymocytes was also studied to detect dosage-dependent expression for one and possibly more sub-regions (class I, II, IV) of the avian MHC. Surface densities of MHC class I antigens on thymocytes were increased significantly at all ages studied, for example by 15% and 45% in trisomics and tetrasomics, respectively at 22 days post-hatching. The surface density of CT1 antigen, a thymocyte-specific marker, was also increased in a dosage-dependent manner, but only in juveniles. Increases in the proportion of alpha beta 1, TCR+ and CD3+ thymocytes were observed in juveniles, with no alterations in other TCR-expressing thymocytes. No major alterations in CD4 and CD8 thymocyte populations were observed. These results demonstrate a targeted effect of extra MHC-chromosome dosage towards enhanced class I and CT1, and not class II or IV, expression. The increased MHC-microchromosome dosage appears to influence primarily immature thymocytes expressing alpha beta 1 TCR and CD3.
Johnson DW, Pieniazek NJ, Griffin DW, Misener L, Rose JB (1995) Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples. Appl Environ Microbiol 61 :3849-3855
The development of a reliable method of using PCR for detection of Cryptosporidium oocysts in environmental samples with oligonucleotide primers which amplify a portion of the sequence encoding the small (18S) subunit of rRNA producing a 435-bp product was demonstrated. The PCR assay was found to provide highly genus-specific detection of Cryptosporidium spp. after release of nucleic acids from oocysts by a simple freeze-thaw procedure. The assay routinely detected 1 to 10 oocysts in purified oocyst preparations, as shown by direct microscopic counts and by an immunofluorescence assay. The sensitivity of the PCR assay in some seeded environmental water samples was up to 1,000-fold lower. However, this interference was eliminated by either flow cytometry or magnetic-antibody capture. Sensitivity was also improved 10- to 1,000-fold by probing of the PCR product on dot blots with an oligonucleotide probe detected by chemiluminescence. Confirmation of the presence of Cryptosporidium oocysts in water samples from the outbreak in Milwaukee, Wis., was obtained with this technique, and PCR was found to be as sensitive as immunofluorescence for detection of oocysts in wastewater concentrates.
Landry MR, Kirshtein J, Constantinou J (1995) A Refined Dilution Technique for Measuring the Community Grazing Impact of Microzooplankton, with Experimental Tests in the Central Equatorial Pacific. Marine Ecology-Progress Series 120 :53-63
The standard dilution technique can provide unbiased estimates of phytoplankton growth and microzooplankton grazing rates only when certain restrictive assumptions are met. The most important of these assumptions - that grazing impact Varies in direct proportion to the dilution of grazer population density - can be easily violated when clearance rate of individual grazers and/or growth response of the grazer population vary significantly with food concentration over the course of the incubation. We have developed a modified protocol which now allows the dilution technique to be applied unambiguously, even when its original assumptions may be violated. The new protocol uses flow-cytometry measured disappearance of fluorescently labeled tracer cells (FLB or FLA) as an internal measure of ’relative grazing activity’ in each dilution treatment. Coefficients of phytoplankton growth and mortality rates are determined from Model II regression analyses of ’net growth’ versus ’relative grazing’, rather than the usual Model I regressions of ’net growth’ versus ’dilution factor’. Tests of this hybrid experimental design in the central equatorial Pacific during an EQPAC cruise in August 1992 gave results essentially identical to the standard dilution interpretation.
Lipschultz F (1995) Nitrogen-Specific Uptake Rates of Marine-Phytoplankton Isolated from Natural-Populations of Particles by Flow-Cytometry. Marine Ecology-Progress Series 123 :245-258
Phytoplankton were isolated from natural, heterogeneous populations of particles in Boothbay Harbor, Maine, USA using flow cytometry with cell sorting to measure true rates of nitrogen-specific uptake of N-15 without the bias due to detritus, bacteria or other heterotrophs that affects traditional filtration techniques. After incubation under Light or dark conditions with (NH4+)-N-15 Or (NO3-)-N-15, particles were either directly filtered or were sorted to isolate phytoplankton and then filtered for isotopic analysis. Nitrogen-specific (mu(N) time(-1)) and absolute uptake rates (mass volume(-1) time(-1) of ammonium were greater for small (3-10 mu M) phytoplankton than for larger (10-53 mu m) phytoplankton, whereas the opposite was observed for uptake of nitrate. Accounting for pronounced diel variations in uptake from both nitrogen sources, algal growth rates were calculated to be 0.38 d(-1) on ammonium and 0.05 d(-1) on nitrate. The absolute rate of NH4+ uptake by all particles as assayed by filtration was approximately balanced by the summed rates of the 2 sorted fractions and the small (<3 mu m) particles that were removed prior to sorting : phytoplankton represented only 50% of the total rate. In contrast, phytoplankton uptake of nitrate represented only about one-third of the total uptake, and the remaining fractions contributed only 10%, leaving about 60% unaccounted for. Future applications of flow cytometry with stable isotopes could be used to investigate diel variations in mu(N) and mu(C) (carbon-specific uptake rates) and provide an estimate of phytoplankton growth rates.
Malinskyrushansky N, Berman T, Dubinsky Z (1995) Seasonal Dynamics of Picophytoplankton in Lake Kinneret, Israel. Freshwater Biology 34 :241-254
1. Picophytoplankton (picocyanobacteria and picoeukaryotes) communities in Lake Kinneret were studied from 1988 to 1992. No prochlorophytes were observed in the lake. 2. Picocyanobacteria were a prominent and ubiquitous component of the phytoplankton, being present at all depths throughout the year, with concentrations ranging from 2 X 10-8 x 10(5) cells ml(-1). Low cell numbers in winter and spring were followed at the end of the annual dinoflagellate bloom by maximal abundances in summer-autumn in the epilimnion. High cell numbers (> 10(4) cells ml(-1)) were sometimes also found in the anaerobic hypolimnion. Net growth rates for picocyanobacteria ranged from 0.29 to 0.60 divisions day(-1). 3. Picoeukaryotes were a very minor constituent of the picoplankton, mostly present in winter and spring, and sometimes at the end of autumn, with concentrations ranging from 44 to 5700 cells ml(-1). Higher cell numbers tended to occur in the near surface water layers. In August-September, picoeukaryotes were found only in the hypolimnion. In December, the occurrence of picoeukaryotes in the deep water layers probably resulted from advection with cold water currents from the Jordan river. Net growth rates for picoeukaryotes ranged from 0.26 to 0.43 divisions day(-1). 4. Overall, the contribution of picophytoplankton to the phytoplankton standing crop in Lake Kinneret was limited ; picocyanobacteria and picoeukaryotes accounted for no more than 7.0 and 0.1% of total algal biomass (semiannual average), respectively. 5. Picophytoplankton cell numbers in pelagic waters were usually similar to those in shallower lake stations. 6. Picocyanobacteria appear to be an autochthonous population, whereas picoeukaryotes are probably brought annually by the Jordan River and do not maintain themselves in the lake.
Orellana MV, Perry MJ (1995) Optimization of an Immunofluorescent Assay of the Internal Enzyme Ribulose-1,5-Bisphosphate Carboxylase (Rubisco) in Single Phytoplankton Cells. Journal of Phycology 31 :785-794
Immunochemical probes are widely used to identify different species and to quantify and understand the role that different antigens play within cells. We optimized a single-cell immunofluorescent assay for the carbon fixation enzyme ribulose-1,5-bisphosphate carboxylase (Rubisco) in order to quantify the enzyme by flow cytometry in phytoplankton cells. The criteria for optimization of the immunofluorescent assay for Rubisco in single cells included maximization of Rubisco immunogenicity, minimization of Rubisco diffusion out of the cells, minimization of cell breakage, and maximization of the cell labeling. Several fixatives (cross-linkers and denaturing) and permeabilizing agents were tested on 26 species of phytoplankton. The only fixative/permeabilizing agent that fulfilled the criteria established for the assay was 96% ethanol. Phytoplankton cells collected from the field needed further treatment with a strong oxidant to permeabilize ethanol-fixed cells and thus allow the antibody probe to access the Rubisco antigen. This study should have a general applicability to the study of other soluble photosynthetic antigens in single phytoplankton cells.
Porter J, Diaper J, Edwards C, Pickup R (1995) Direct Measurements of Natural Planktonic Bacterial Community Viability by Flow-Cytometry. Applied and Environmental Microbiology 61 :2783-2786
A range of fluorescent viability dyes were used in conjunction with flow cytometry to rapidly enumerate viable bacteria from freshwater environments. Optimal labelling was achieved by using carboxyfluorescein diacetate or chemchrome B with a detergent-mediated permeabilization step. The viable bacterial count under optimal conditions was 7% in oligotrophic lake water and 75% in polluted river water.
Porter J, Edwards C, Pickup RW (1995) Rapid Assessment of Physiological Status in Escherichia-Coli Using Fluorescent-Probes. Journal of Applied Bacteriology 79 :399-408
Rapid and direct viability assessment of Escherichia coli in filtered, sterile lake water was possible using multiparameter flow cytometry. Fluorescent dyes were used as probes for different cellular functions (membrane potential, membrane integrity and intracellular enzyme activity), which were correlated with the ability of the cells to respond to nutrient addition while in a stressed state, Measurement of several criteria circumvented limitations imposed by other methods, and provided extensive evidence for the validity of the methods for monitoring cell viability during adoption of a viable-but-non-culturable state in starved E. coli. Macromolecular staining was concomitantly used to monitor changes in cellular protein, RNA and DNA as additional indicators of physiological status during starvation/stress.
Sieracki ME, Haugen EM, Cucci TL (1995) Overestimation of Heterotrophic Bacteria in the Sargasso-Sea - Direct Evidence by Flow and Imaging Cytometry. Deep-Sea Research Part I-Oceanographic Research Papers 42 :1399-&
Accurate measurements of bacterial biomass in the ocean are needed for modeling marine microbial food webs and global biogeochemical cycling. We present direct evidence that previous estimates of heterotrophic bacteria biomass in the oligotrophic ocean are confounded by the presence of the abundant photosynthetic procaryote, Prochlorococcus. The chlorophyll autofluorescence of these photosynthetic bacterial cells is very faint and fades rapidly under epifluorescence microscopy. Detection and enumeration of these cells thus far has almost exclusively been by flow cytometry. Using a cooled, charge-coupled device (CCD) camera we were able to image these cells for direct biovolume measurements. A double-exposed image of DAPI-stained Prochlorococcus cells shows that they are indistinguishable from heterotrophic bacteria in standard slide preparations. At two Sargasso Sea stations Prochlorococcus could cause an overestimation of surface (top 150 m) integrated heterotrophic bacterial biovolume (biomass) of 18 and 22% determined by standard microscope methods. At the subsurface chlorophyll maximum Prochlorococcus was 33 and 43% of the heterotrophic bacterial biovolume (biomass) at these stations. Prochlorococcus cell size increased from 0.05 mu m(3) in the surface mixed layer to about 0.2 mu m(3) below 100 m, confirming previous interpretations of flow cytometric light scatter measurements. Shifting biomass from the heterotrophic bacteria pool to the primary producer compartment has significant implications for ecosystem structure and trophic transfer in marine food webs.
Simon N, Lebot N, Marie D, Partensky F, Vaulot D (1995) Fluorescent in-Situ Hybridization with Ribosomal-Rna-Targeted Oligonucleotide Probes to Identify Small Phytoplankton by Flow-Cytometry. Applied and Environmental Microbiology 61 :2506-2513
Because of their tiny size (0.2 to 2 mu m), oceanic picophytoplanktonic cells (either cultured strains or natural communities) are difficult to identify, and some basic questions concerning their taxonomy, physiology, and ecology are still largely unanswered. The present study was designed to test the suitability of in situ hybridization with rRNA fluorescent probes detected by flow cytometry for the identification of small photosynthetic eukaryotes. Oligonucleotide probes targeted against regions of the 18S rRNAs of Chlorophyta lineage (CHLO probe) and of non-Chlorophyta (NCHLO probe) algal species were designed. The CHLO and NCHLO probes, which differed by a single nucleotide, allowed discrimination of chlorophyte from nonchlorophyte cultured strains. The sensitivity of each probe was dependent upon the size of the cells and upon their growth stage. The mean fluorescence was 8 to 80 times higher for specifically labeled than for nonspecifically labeled cells in exponential growth phase, but it decreased sharply in stationary phase. Such taxon-specific probes should increase the applicability of flow cytometry for the rapid identification of cultured pico- and nanoplanktonic strains, especially those that lack taxonomically useful morphological features.
Suzuki K, Handa N, Kiyosawa H, Ishizaka J (1995) Distribution of the Prochlorophyte Prochlorococcus in the Central Pacific-Ocean as Measured by Hplc. Limnology and Oceanography 40 :983-989
The abundance of the prochlorophyte Prochlorococcus along a 56 degrees-long meridional (175 degrees E) transect in the central Pacific was measured by determining divinyl chlorophyll a (divinyl chl a) during August and September 1993. The aims of this study were to investigate the large-scale spatial distribution patterns of Prochlorococcus, to evaluate its contribution to total phytoplankton biomass, and to identify the environmental boundaries of its distribution in the central Pacific Ocean. Prochlorococcus was detected at depths below the euphotic zone (<250 m) at stations south of 40 degrees N by the presence of divinyl Chl a. For most of the subtropical and tropical central Pacific, Prochlorococcus accounted for >50% of the total Chl a (divinyl Chl a + Chl a) above the 1% isolume and generally occurred between 15-30 degrees C and 0-10 mu M nitrate plus nitrite.
Waite AM, Olson RJ, Dan HG, Passow U (1995) Sugar-containing compounds on the cell surfaces of marine diatoms measured using concanavalin A and flow cytometry. Journal of Phycology 31 :925-933
Some diatom exudates may remain attached to the exterior cell surface, potentially altering cell stickiness and affecting important aspects of the diatom’s ecology such as aggregation rates and grazing rates. We measured the accumulation of cell-surface sugar-containing compounds by labeling cultured marine diatoms with fluorescent-tagged sugar-binding lectins and measuring the fluorescence associated with each cell using flow cytometry. The binding of FITC-labeled concanavalin A (FITC-ConA), a lectin that binds to glucose and mannose residues, varied 5-fold among different diatom species in exponential growth (on a per-cell basis) and 2-3-fold within a given species in different physiological states. Although transparent exopolymer particles followed a simple accumulation curve over time in batch culture, FITC-ConA . cell(-1) did not follow the same pattern, suggesting that surface sugar accumulation is not driven simply by the accumulation of such particles in the medium. For Thalassiosira pseudonana (Hustedt) Hasle and Heimdal (3H clone), the amount of sugar-containing compounds on the cell surface increased transiently as growth rate slowed in early stationary phase under both N and Si limitation. For Chaetoceros neograciie (Schuett) van Landingham, FITC-ConA . cell(-1) had a strong inverse relationship with growth rate across several Si-limited batch culture experiments. Both results suggest some biological mediation of cell-surface sugar-containing compounds. Our study reveals the great potential for using lectin binding to investigate cell-surface sugars on diatoms. Lectins allow us to investigate noninvasively the role of cell-surface sugar-containing compounds in modifying cell stickiness and aggregation, as well as the partitioning of exuded phytoplankton carbon. We suggest that cell-surface sugar accumulation may be related to diatom stickiness, based on a correlation between our FITC-ConA measurements and stickiness estimates in the literature on several of the species we studied.
Wallner G, Erhart R, Amann R (1995) Flow Cytometric Analysis of Activated-Sludge with Ribosomal-Rna-Targeted Probes. Applied and Environmental Microbiology 61 :1859-1866
Samples from a wastewater treatment plant were hybridized with fluorescein labeled oligonucleotide probes specific for members of the domains Bacteria and Eucarya ; the alpha, beta, and gamma subclasses of the class Proteobacteria ; or the genus Acinetobacter. Subsequently, they were counterstained with the DNA-specific dye Hoechst 33342 and analyzed by flow cytometry. By quantifying forward angle light scatter and Hoechst- and probe-conferred fluorescence as measures for cell size, DNA content, and rRNA content, respectively, not only relative abundances but also assessments of general metabolic activity for each of these groups were obtained. Hybridizations with a positive control probe binding to all bacteria showed that in the activated-sludge samples examined, 70 to 80% of the Hoechst-stained cells could unambiguously be identified by this method. The majority of the detected cells (approximately 40%) were beta-subclass Proteobacteria. Flow cytometric and microscopic counts were in general agreement. Discrepancies were found in particular for those populations that occurred predominantly in flocs (alpha subclass of the Proteobacteria) or chains (Acinetobacter spp.). Although the dispersal of aggregates needs to be improved, how cytometry combined with rRNA-based in situ probing appears to be a powerful tool for the rapid and highly automated analysis of the microbial communities in activated sludge.