Using light scatter signal to estimate bacterial biovolume by flow cytometry.

BACKGROUND: In the past decade, flow cytometry has become a useful and precise alternative to microscopic bacterial cell counts in aquatic samples. However, little evidence of its usefulness for the evaluation of bacterial biovolumes has emerged in from the literature. METHODS: The light scattering and cell volume of starved bacterial strains and natural bacterial communities from the Black Sea were measured by flow cytometry and epifluorescence microscopy, respectively, in order to establish a relationship between light scattering and cell volume. RESULTS: With the arc-lamp flow cytometer, forward angle light scatter (FALS) was related to cell size in both the starved strains and natural communities, although regression parameters differed. We tested the predictive capacity of the FALS verous cell size relationship in a bacterial community from the North Sea. That analysis showed that a reliable bacterial biovolume prediction of a natural bacterial community can be obtained from FALS using a model generated from natural bacterial community data. CONCLUSIONS: Bacterial biovolume is likely to be related to FALS measurements. It is possible to establish a generally applicable model derived from natural bacterial assemblages for flow cytometric estimation of bacterial biovolumes by light scatter.     

Culturability and coexistence of colony-forming and single-cell marine bacterioplankton

Culturability and coexistence of bacterioplankton exhibiting different life strategies were investigated in the Baltic Sea and Skagerrak Sea. Bacterial numbers were estimated using a dilution-to-extinction culturing assay (DCA) and calculated as the most probable number, based on six different methods to detect bacterial growth in the DCA. Irrespective of the method used to detect growth, the fraction of multiplying cells never exceeded 10%, using the total count of 4',6'-diamidino-2-phenylindole (DAPI)-stainable cells as a reference. Furthermore, the data also showed that non-colony-forming bacteria made up the majority of the viable cells, confirming molecular results showing dominance of non-colony-forming bacteria in clone libraries. The results obtained are in agreement with previous observations, indicating that bacterial assemblages in seawater are dominated by small, active subpopulations coexisting with a large group of inactive cells. The ratio of colony-forming to non-colony-forming bacteria was approximately 10 to 20 times higher in the brackish Baltic Sea than in the Skagerrak Sea. These two sea areas differ in (for example) their levels of bacterial production, dissolved organic carbon, and salinity. We suggest that the relative importance of colony-forming versus non-colony-forming bacterioplankton may be linked to environmental characteristics.     

Determination of DNA content of aquatic bacteria by flow cytometry

The distribution of DNA among bacterioplankton and bacterial isolates was determined by flow cytometry of DAPI (4',6'-diamidino-2-phenylindole)-stained organisms. Conditions were optimized to minimize error from nonspecific staining, AT bias, DNA packing, changes in ionic strength, and differences in cell permeability. The sensitivity was sufficient to characterize the small 1- to 2-Mb-genome organisms in freshwater and seawater, as well as low-DNA cells ("dims"). The dims could be formed from laboratory cultivars; their apparent DNA content was 0.1 Mb and similar to that of many particles in seawater. Preservation with formaldehyde stabilized samples until analysis. Further permeabilization with Triton X-100 facilitated the penetration of stain into stain-resistant lithotrophs. The amount of DNA per cell determined by flow cytometry agreed with mean values obtained from spectrophotometric analyses of cultures. Correction for the DNA AT bias of the stain was made for bacterial isolates with known G+C contents. The number of chromosome copies per cell was determined with pure cultures, which allowed growth rate analyses based on cell cycle theory. The chromosome ratio was empirically related to the rate of growth, and the rate of growth was related to nutrient concentration through specific affinity theory to obtain a probe for nutrient kinetics. The chromosome size of a Marinobacter arcticus isolate was determined to be 3.0 Mb by this method. In a typical seawater sample the distribution of bacterial DNA revealed two major populations based on DNA content that were not necessarily similar to populations determined by using other stains or protocols. A mean value of 2.5 fg of DNA cell(-1) was obtained for a typical seawater sample, and 90% of the population contained more than 1.1 fg of DNA cell(-1).     

Relationships among Bacterial Cell Size, Productivity, and Genetic Diversity in Aquatic Environments using Cell Sorting and Flow Cytometry

Abstract The study of relationships between cell size and productivity is of key importance in microbial ecology to understand which members of natural aquatic communities are responsible for the overall activity and/or productivity. Flow sorting of microorganisms from different environmental samples was used to analyze the activity of bacterial cells depending on their biovolume. Bacterial cells from five different natural samples taken along the Mediterranean coast including fresh- and seawaters were incubated with tritiated leucine, then stained with SYTO 13 and sorted by flow cytometry according to their average side-angle-scattered (SSC) light. In all samples, a bell-shaped relationship was found between cell biovolume and activity, whereas activity of a given cell-size class varied between samples. In contrast, an inverse relationship was found between biovolumes and abundances. These results suggest that medium-sized cells with highest growth rates are probably submitted to intense grazing. For one sample, bacteria within five different size classes were sorted and the genetic diversity of cells within each sorted size class and that of the whole community were analyzed by the denaturing gradient gel electrophoresis (DGGE) method. The genetic diversity, as determined at the community level was highly represented into the pool of small cells, whereas only few species were present into larger cell subpopulations. The results suggest that only a few genotypes may be dominant within the largest and most productive cells. Furthermore, cell size polymorphism as well as heterogeneous cellular activities were found within some species. Received: January 2000; Accepted: April 2000; Online Publication: 28 August 2000     

Evaluation of flow cytometry as a method for quantification of circulating blood cell populations in salmonid fish

Flow cytometry was used to estimate the proportions of different blood cell types in brown and rainbow trout. On the basis of forward light scatter and 90° side scatter three populations were differentiated. The relative abundance of these cells correlated with that of erythrocytc (r²= 0.994), lymphocyte plus thrombocyte(r²= 0.676) and neutrophil populations (r²= 0.571) enumerated by direct microscopy. By density gradient separation of cells, cell sorting and acridine orange staining it was confirmed that these cell types could be assigned to the populations detected. Changes in blood cell populations were monitored by flow cytometry in a group of experimental fish placed under confinement stress. Flow cytometry proved to be a rapid and reliable method for monitoring cell population dynamics in fish blood.     

Flow cytometric analysis of a marine LAS-degrading consortia

The specific nucleic acid fluorochrome SYTO-13 was used in flow cytometric analysis to assess changes in the density and heterogeneity of marine bacterial populations which biodegrade linear alkylbenzene sulphonate (LAS). Seawater samples with LAS and incubated in the laboratory (20 degrees C, 100 rpm, 30 days) were used to monitor LAS-degrading consortia. Flow cytometric studies and culture methods were used to characterize the LAS degrading bacterioplankton consortia. Fluorescence and scatter signals enabled us to define three regions (R1, R2 and R3) in the dual parameter cytograms. The distribution of the bacterial counts in these regions allowed us to monitor the formation and evolution of the consortia.     

Identification and characterization of two subpopulations of Encephalitozoon intestinalis

Microsporidia are obligate intracellular protozoa that have been shown to be pathogenic to most living creatures. The development of in vitro cell culture propagation methods has provided researchers with large numbers of spores and facilitated the study of these organisms. Here, we describe heterogeneity within cell culture-propagated Encephalitozoon intestinalis suspensions. Flow cytometer histograms depicting the log side scatter and forward-angle light scatter of spores from nine suspensions produced over 12 months consistently showed two populations differing in size. The suspensions were composed primarily of the smaller-spore subpopulation (76.4% +/- 5.1%). The presence of two subpopulations was confirmed by microscopic examination and image analysis (P < 0.001). Small subpopulation spores were noninfectious in rabbit kidney (RK13) cell culture infectivity assays, while the large spores were infectious when inocula included > or = 25 spores. The small spores stained brilliantly with fluorescein isothiocyanate-conjugated monoclonal antibody against Encephalitozoon genus spore wall antigen, while the large spores stained poorly. There was no difference in staining intensities using commercial (MicroSporFA) and experimental polyclonal antibodies. Vital-dye (DAPI [4',6'-diamidino-2-phenylindole], propidium iodide, or SYTOX Green) staining showed the spores of the small subpopulation to be permeable to all vital dyes tested, while spores of the large subpopulation were not permeable in the absence of ethanol pretreatment. PCR using primers directed to the 16S rRNA or beta-tubulin genes and subsequent sequence analysis confirmed both subpopulations as E. intestinalis. Our data suggest that existing cell culture propagation methods produce two types of spores differing in infectivity, and the presence of these noninfective spores in purified spore suspensions should be considered when designing disinfection and drug treatment studies.     

Size-selective grazing on bacteria by natural assemblages of estuarine flagellates and ciliates

The small average cell size of in situ bacterioplankton, relative to cultured cells, has been suggested to be at least partly a result of selection of larger-sized cells by bacterivorous protozoa. In this study, we determined the relative rates of uptake of fluorescence-labeled bacteria (FLB), of various cell sizes and cell types, by natural assemblages of flagellates and ciliates in estuarine water. Calculated clearance rates of bacterivorous flagellates had a highly significant, positive relationship with size of FLB, over a range of average biovolume of FLB of 0.03 to 0.08 microns3. Bacterial cell type or cell shape per se did not appear to affect flagellate clearance rates. The dominant size classes of flagellates which ingested all types of FLB were 3- to 4-microns cells. Ciliates also showed a general preference for larger-sized bacteria. However, ciliates ingested a gram-positive enteric bacterium and a marine bacterial isolate at higher rates than they did a similarly sized, gram-negative enteric bacterium or natural bacterioplankton, respectively. From the results of an experiment designed to test whether the addition of a preferentially grazed bacterial strain stimulated clearance rates of natural bacterioplankton FLB by the ciliates, we hypothesized that measured differences in rates of FLB uptake were due instead to differences in effective retention of bacteria by the ciliates. In general, clearance rates for different FLB varied by a factor of 2 to 4. Selective grazing by protozoa of larger bacterioplankton cells, which are generally the cells actively growing or dividing, may in part explain the small average cell size, low frequency of dividing cells, and low growth rates generally observed for assemblages of suspended bacteria.     

Soil bacterial DNA and biovolume profiles measured by flow-cytometry

Abstract Flow-cytometry was used to measure cell volumes and DNA contents of single cells in cultures of soil bacteria during exponential growth and starvation conditions. DNA was measured after staining with mitramycin/ethidium bromide. The measurement of DNA was calibrated with rifampicin-treated cells of E. coli containing even numbers of genomes per cell. Cell volumes were assessed by scatter light measurements. Constant DNA to biovolume relations over a range of cell sizes were found for each of the bacteria at exponential growth, and DNA contents per cell varied over a range equivalent to 1–4 genomes per cell. At generation times of 1.0–1.5 h, two genomes were registered as a mean. After starvation of washed cells in a salt solution (24 hrs), a fraction of the cells in each culture had DNA contents equivalent to 1 genome, but significant fractions retained DNA contents equivalent to 2–4 genomes. Attempts to create cells with even numbers of genomes per cell by treatment with rifampicin was successful on an Acinetobacter sp. In contrast, the response to rifampicin was less clear for Pseudomonas fluorescens and P. chlororaphis , and unclear for the gram positive bacteria isolated from soil. The mean decrease in biovolume upon starvation was 4.1 times (range 1.3–8.1 times) and larger than the mean decrease in DNA content of 1.8 (range 1.3–2.7 times). Cell volume determinations by measurements of scatter light was compared with volume determinations by fluorescence microscopy. The amounts of scatter light per volumes was variable, not only did we find large differences between bacterial types, but also between starving and exponentially growing cells of the same isolate. In order to use light scatter as a measure of biovolume, internal standards has to be chosen of comparable size and surface properties as to soil bacteria.     

太湖夏季浮游细菌群落多样性的空间格局

为了研究太湖夏季浮游细菌群落多样性与水体营养盐的关系,在太湖全湖范围内开展了一次大规模浮游细菌采样调查,分析了太湖不同湖区浮游细菌丰度和多样性组成。研究发现,浮游细菌丰度在不同湖区中存在明显的空间差异,从北部和西部湖区沿湖流向东南方向至湖心和南部沿岸再到东部湖区呈下降趋势,这与太湖水体营养水平从高到低变化趋势一致。浮游细菌丰度与营养盐浓度回归分析结果显示,总磷（TP）与细菌丰度存在较好的正相关（R2=0.6392,n=29,P0.01）,而总氮（TN）与细菌丰度无显著相关（R2=0.0663,n=29,P0.05）。因此,磷是太湖夏季浮游细菌生长的限制因子。不同湖区营养盐与浮游细菌群落多样性也具有显著的正相关,随着营养水平的升高,浮游细菌多样性增加。此外,细菌群落的组成在不同湖区间亦具有明显的空间异质性,与不同湖区营养水平空间变化一致。研究结果将有助于人们更好地理解淡水湖泊中微生物循环和生态系统功能。       

Coupling Bacterial Activity Measurements with Cell Sorting by Flow Cytometry

Abstract A new procedure to investigate the relationship between bacterial cell size and activity at the cellular level has been developed; it is based on the coupling of radioactive labeling of bacterial cells and cell sorting by flow cytometry after SYTO 13 staining. Before sorting, bacterial cells were incubated in the presence of tritiated leucine using a procedure similar to that used for measuring bacterial production by leucine incorporation and then stained with SYTO 13. Subpopulations of bacterial cells were sorted according to their average right-angle light scatter (RALS) and fluorescence. Average RALS was shown to be significantly related to the average biovolume. Experiments were performed on samples collected at different times in a Mediterranean seawater mesocosm enriched with nitrogen and phosphorus. At four sampling times, bacteria were sorted in two subpopulations (cells smaller and larger than 0.25 μm³). The results indicate that, at each sampling time, the growth rate of larger cells was higher than that of smaller cells. In order to confirm this tendency, cell sorting was performed on six subpopulations differing in average biovolume during the mesocosm follow-up. A clear increase of the bacterial growth rates was observed with increasing cell size for the conditions met in this enriched mesocosm. Received: 21 January 1999; Accepted: 12 April 1999     

Effect of Protistan Grazing on the Frequency of Dividing Cells in Bacterioplankton Assemblages

Grazing by phagotrophic flagellates and ciliates is a major source of mortality for bacterioplankton in both marine and freshwater systems. Recent studies have demonstrated a positive relationship between clearance rate and prey size for bacterivorous protists. We tested the idea that, by selectively grazing the larger (more actively growing or dividing) cells in a bacterial assemblage, protists control bacterial standing stock abundances by directly cropping bacterial production. Samples of estuarine water were passed through 0.8-μm-pore-size filters (bacteria only) or 20-μm-mesh screens (bacteria and bacterivorous protists) and placed in dialysis tubing suspended in 7 liters of unfiltered water. Changes in total bacterial biovolume per milliliter (bacterial biomass), frequency of dividing cells (FDC), and average per cell biovolume were followed over a period of 24 h. In three experiments, the FDC increased more rapidly and attained higher values in water passed through 0.8-μm-pore-size filters (average, 5.1 to 8.9%; maximum, 15.5%) compared with FDC values in water passed through 20-μm-mesh screens (average, 2.7 to 5.3%; maximum, 6.7%). Increases in bacterial biomass per milliliter lagged behind increases in FDC by about 4 to 6 h. Grazed bacterial assemblages were characterized by lower total biomasses and smaller average cell sizes compared with those of cells in nongrazed assemblages. We conclude that bacterivorous protists control bacterial standing stock abundances partly by preferentially removing dividing cells. Selective grazing of the more actively growing cells may also explain, in part, the ability of slow-growing cells to persist in bacterioplankton assemblages.     

Size-selective feeding by Cyclidium sp. on bacterioplankton and various sizes of cultured bacteria

Abstract Size-selective grazing by Cyclidium sp., isolated as a dominant ciliate bacterivore from the Římov Reservoir (South Bohemia), was examined using fluorescent labelled bacteria (FLB) produced from natural bacterioplankton or pure bacterial cultures. Sizes of ingested bacteria in food vacuoles were measured directly. Three experimental arrangements were used: (1) Ciliates were grown on the pure culture of Alcaligenes xylosoxidans and fed with various proportions of ‘large’ and ‘small’ FLB (mean biovolume, 0.377 and 0.202 μm³, respectively) prepared from the same bacterial species. Results clearly showed significant selection of larger bacteria. (2) Ciliates were grown on natural bacterioplankton from the reservoir and subsequently fed on FLB prepared from the reservoir bacterioplankton (mean biovolume, 0.065 μm³). Independent of either prey or predator abundance, larger FLB (> 0.100 μ m³, and especially those > 0.200 μ m³) were ingested with much higher frequency than their occurrence i the natural assemblage. (3) Ciliates were grown on the reservoir baterioplankton and fed by FLB prepared from the culture of Pseudomonas sp. In contrast with previous results, no size selection of the ciliate was found when FLB were different from the bacterial food used to grow the ciliate. Ecological impacts of size-selective bacterivory are suggested.     

Cell cycle analysis in flow cytometry: Use of BrdU labelling and side scatter for the detection of the different cell cycle phases

Cell cycle analysis in flow cytometry is based on the incorporation of labelled precursors in DNA. The use of BrdU versus SSC, in which side scatter substitutes PI fluorescence, has proved to be useful also for the distinction between G2 and Mitotic cells. Mitoses often produce an SSC decrease due to the morphological changes that happen in the nucleus during this phase of cell cycle. Moreover, DNA accessibility to PI varies during mitosis, as well. However, most of these variations, detectable by flow cytometry appear to be basically dependent on the cell line used.     

Analysis of bacteria contaminating ultrapure water in industrial systems

Bacterial populations inhabiting ultrapure water (UPW) systems were investigated. The analyzed UPW systems included pilot scale, bench scale, and full size UPW plants employed in the semiconductor and other industries. Bacteria present in the polishing loop of the UPW systems were enumerated by both plate counts and epifluorescence microscopy. Assessment of bacterial presence in UPW by epifluorescence microscopy (cyanotolyl tetrazolium chloride [CTC] and DAPI [4',6'-diamidino-2-phenylindole] staining) showed significantly higher numbers (10 to 100 times more bacterial cells were detected) than that determined by plate counts. A considerable proportion of the bacteria present in UPW (50 to 90%) were cells that did not give a positive signal with CTC stain. Bacteria isolated from the UPW systems were mostly gram negative, and several groups seem to be indigenous for all of the UPW production systems studied. These included Ralstonia pickettii, Bradyrhizobium sp., Pseudomonas saccharophilia, and Stenotrophomonas strains. These bacteria constituted a significant part of the total number of isolated strains (>or=20%). Two sets of primers specific to R. pickettii and Bradyrhizobium sp. were designed and successfully used for the detection of the corresponding bacteria in the concentrated UPW samples. Unexpectedly, nifH gene sequences were found in Bradyrhizobium sp. and some P. saccharophilia strains isolated from UPW. The widespread use of nitrogen gas in UPW plants may be associated with the presence of nitrogen-fixing genes in these bacteria.     

Population dynamics of bacterioplankton in an oligotrophic lake

The population ecology of bacterioplankton was studied overa 3 year period in Mirror Lake, an oligotrophic lake in thenortheastern USA. Bacterial population density, biomass, andrates of biomass production in the epilimnion and hypolimnionwere examined for their relationship with several environmentalparameters. Bacterioplankton density fluctuated between 0.5and 7 x l0 bacteria ml^–1, with highest values in the anoxichypolimnion. At all depths there was a trend towards a higherdensity of bacteria from spring to midsummer, followed by adecline in late summer to early autumn. Cocci tended to dominatebacterial cell shapes from winter to midsummer, after whichrod-shaped cells became most abundant. Rod-shaped cells contributedthe most to bacterioplankton biomass at all depths and timesof year. The mean annual biovolume of all bacterioplankton was0.12 µ cell^–1. The mean annual areal bacterioplanktonbiomass was 11–12 mmol C m^–2. The percentage ofbacterial to phytoplankton biomass per volume in summertimewas 27% in the epilimnion and 11% in the hypolimnion. Averageannual and summertime bacterial production estimated using the[3H]thymidine method was similar to previous estimates of bacterialproduction measured in Mirror Lake using other methods. Theaverage ratio of bacterial to net phytoplankton production pervolume was 0.34 in the epilimnion, and between 0.65 and 1 1.depending on depth, in the hypolimnion during summer. Of severalvariables considered in regression analyses, only temperatureexplained >50% of the variance in bacterial production inboth the hypolimnion and epilimnion. Above 14°C, however,bacterial production and growth rate in the epilimnion werenot clearly related to temperature. During the period of midsummerhypolimnetic anoxia, despite colder temperatures in the hypolimnion,bacterial production was up to 10 times greater than in theepilimnion.     

Community composition of marine bacterioplankton determined by 16S rRNA gene clone libraries and fluorescence in situ hybridization

We determined the compositions of bacterioplankton communities in surface waters of coastal California using clone libraries of 16S rRNA genes and fluorescence in situ hybridization (FISH) in order to compare the community structures inferred from these two culture-independent approaches. The compositions of two clone libraries were quite similar to those of clone libraries of marine bacterioplankton examined by previous studies. Clones from gamma-proteobacteria comprised ca. 28% of the libraries, while approximately 55% of the clones came from alpha-proteobacteria, which dominated the clone libraries. The Cytophaga-Flavobacter group and three others each comprised 10% or fewer of the clone libraries. The community composition determined by FISH differed substantially from the composition implied by the clone libraries. The Cytophaga-Flavobacter group dominated 8 of the 11 communities assayed by FISH, including the two communities assayed using clone libraries. On average only 10% of DAPI (4', 6'-diamidino-2-phenylindole)-stained bacteria were detected by FISH with a probe for alpha-proteobacteria, but 30% of DAPI-stained bacteria appeared to be in the Cytophaga-Flavobacter group as determined by FISH. alpha-Proteobacteria were greatly overrepresented in clone libraries compared to their relative abundance determined by FISH, while the Cytophaga-Flavobacter group was underrepresented in clone libraries. Our data show that the Cytophaga-Flavobacter group can be a numerically dominant component of coastal marine bacterioplankton communities.     

Detection, enumeration, and sizing of planktonic bacteria by image-analyzed epifluorescence microscopy.

Epifluorescence microscopy is now being widely used to characterize planktonic procaryote populations. The tedium and subjectivity of visual enumeration and sizing have been largely alleviated by our use of an image analysis system consisting of a modified Artek 810 image analyzer and an Olympus BHT-F epifluorescence microscope. This system digitizes the video image of autofluorescing or fluorochrome-stained cells in a microscope field. The digitized image can then be stored, edited, and analyzed for total count or individual cell size and shape parameters. Results can be printed as raw data, statistical summaries, or histograms. By using a stain concentration of 5 micrograms of 4'6-diamidino-2-phenylindole per ml of sample and the optimal sensitivity level and mode, counts by image analysis of natural bacterial populations from a variety of habitats were found to be statistically equal to standard visual counts. Although the time required to prepare slides, focus, and change fields is the same for visual and image analysis methods, the time and effort required for counting is eliminated since image analysis is instantaneous. The system has been satisfactorily tested at sea. Histograms of cell silhouette areas indicate that rapid and accurate estimates of bacterial biovolume and biomass will be possible with this system.     

Oligotrophic bacterioplankton with a novel single-cell life strategy

A large fraction of the marine bacterioplankton community is unable to form colonies on agar surfaces, which so far no experimental evidence can explain. Here we describe a previously undescribed growth behavior of three non-colony-forming oligotrophic bacterioplankton, including a SAR11 cluster representative, the world's most abundant organism. We found that these bacteria exhibit a behavior that promotes growth and dispersal instead of colony formation. Although these bacteria do not form colonies on agar, it was possible to monitor growth on the surface of seawater agar slides containing a fluorescent stain, 4',6'-diamidino-2-phenylindole (DAPI). Agar slides were prepared by pouring a solution containing 0.7% agar and 0.5 micro g of DAPI per ml in seawater onto glass slides. Prompt dispersal of newly divided cells explained the inability to form colonies since immobilized cells (cells immersed in agar) formed microcolonies. The behavior observed suggests a life strategy intended to optimize access of individual cells to substrates. Thus, the inability to form colonies or biofilms appears to be part of a K-selected population strategy in which oligotrophic bacteria explore dissolved organic matter in seawater as single cells.     

In situ classification and image cytometry of pelagic bacteria from a high mountain lake (gossenkollesee, austria)

We describe a procedure to measure the cell sizes of pelagic bacteria after determinative hybridization with rRNA-targeted fluorescently labeled oligonucleotide probes. Our approach is based on established image analysis techniques modified for objects simultaneously stained with two fluorescent dyes. It allows the estimation of biomass and cell size distribution and the morphological characterization of different bacterial taxa in plankton samples. The protocol was tested in a study of the bacterioplankton community of a high mountain lake during and after the ice break period. Cells that hybridized with a probe for the domain Bacteria accounted for 70% of the bacterial abundance (range, 49 to 83%) as determined by 4(prm1),6(prm1)-diamidino-2-phenylindole staining (K. G. Porter and Y. S. Feig, Limnol. Oceanogr. 25:943-948, 1980), but for >85% of the total biomass (range, 78 to 99%). The size distribution for members of the beta subclass of the Proteobacteria shifted toward larger cells and clearly distinguished this group from the total bacterial assemblage. In the surface water layer beneath the winter cover, bacteria belonging to the beta 1 subgroup constituted about one-half of the beta subclass abundance. The mean cell volume of the beta 1 subgroup bacteria was significantly less than that of the beta subclass proteobacteria, and the beta 1 subgroup accounted for less than 30% of the total beta subclass biovolume. Two weeks later, the biovolume of the beta Proteobacteria had decreased to the level of the beta 1 subgroup, and both the biovolume size distributions and cell morphologies of the beta Proteobacteria and the beta 1 subgroup were very similar. We could thus quantify the disappearance of large, morphologically distinct beta subclass proteobacteria which were not members of the beta 1 subgroup during the ice break period. Our results demonstrate that changes in biovolumes and cell size distributions of different bacterial taxa, and eventually of individual populations, reveal hitherto unknown processes within aquatic bacterial assemblages and may open new perspectives for the study of microbial food webs.