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.     

Seasonal changes of Antarctic marine bacterioplankton and sea ice bacterial assemblages

The distributions of bacterial populations in sea ice and underlying seawater were investigated on the continental shelf of the “Terre Adélie” area. A reference station was sampled weekly from January 1991 to January 1992. In winter, the survey included a minimum of six sampling layers: surface and bottom ice, brine, seawater from the interface, and at 0.5 and 2 m depth. In seawater, the total bacterial abundance ranged from 0.5 × 10⁵ cells ml⁻¹ in July to 6.0 × 10⁵ cells ml⁻¹ after ice break. Values reaching 2.5 × 10⁶ cells ml⁻¹ were recorded in the overlying ice cover. Mean cell volumes were twice as high in brine as in seawater. The saprophytic bacterial abundance ranged from 5.0 × 10⁴ CFU (colony-forming units) ml⁻¹ in some winter interface samples to less than 1.0 × 10³ CFU ml⁻¹ in most of the summer seawater samples. In sea ice a clear decreasing gradient for most of the studied bacterial parameters from the surface layers towards the bottom layer was found. The ice cover had a discernible impact on underlying seawater, but its influence was restricted to a limited interface layer.     

Anomalies in the enumeration of starved bacteria on culture media containing nalidic acid and tetracycline

Culturable counts of antibiotic resistant, genetically engineeredPseudomonas fluorescens were determined on antibiotic-containing plate count agar during starvation in water. Prior to starvation, colony counts obtained on all media separated into two groups. The mean of the colony counts on plate count agar with or without tetracycline (4.9 × 10⁶ ml⁻¹) was significantly higher than the mean colony counts on plate count agar containing either nalidixic acid or nalidixic acid plus tetraclycline (2.5×10⁶ ml⁻¹). After 20 days of starvation the highest mean colony counts continued to be obtained on plate count agar (7.2 × 10⁶ ml⁻¹) with slightly, but significantly, lower counts obtained on plate count agar containing either nalidixic acid (5.6 × 10⁶ ml⁻¹) or tetraclycline (1.5×10⁶ ml⁻¹). A combination of nalidixic acid and tetracycline in plate count agar, however, dramatically reduced colony counts (8.3 × 10² ml⁻¹) after this starvation period. The addition of catalase to plate count agar containing nalidixic acid and tetracycline negated the effect caused by this combination of antibiotics. When colony counts obtained over the entire 20 day incubation were considered, the addition of MgSO₄ to plate count agar containing nalidixic acid and tetracycline resulted in a significant increase in colony counts. Other combinations of antibiotics, nalidixic acid+carbenicillin, nalidixic acid+kanamycin, streptomycin+tetracycline, streptomycin+carbenicillin, rifampicin+tetracycline, rifampicin+carbenicillin, and rifampicin+kanamycin, did not inhibit colony formation of starved cells. Antibiotic resistant strains ofP. putida andEscherichia coli also displayed sensitivity to the combination of nalidixic acid and tetracycline in plate count agar after starvation.     

Effects of Acetic Acid on the Regrowth of Heterotrophic Bacteria in the Drinking Water Distribution System

Summary Three laboratory-scale water pipe systems were set up to study the effects of adding two levels of acetic acid (10 and 50 μg acetate eq-C l⁻¹) on the bacterial regrowth in water pipes. The results of the water pipe test showed that nearly all carbon in the acetic acid could be readily utilized by bacteria and resulted in an increase in biomass concentration. The maximum heterotrophic plate counts in biofilm were equal to 3.5 × 10⁴, 8.9 × 10⁵ and 2.9 × 10⁷ c.f.u. cm⁻² while the maximum heterotrophic plate counts of free bacteria were equal to 1.2 × 10³, 5.0 × 10³ and 6.8 × 10⁴ c.f.u. ml⁻¹ for the blank and with addition of 10 and 50 μg acetate eq-C l⁻¹. These results showed that addition of acetic acid to drinking water has a positive effect on the assimilable organic carbon content of drinking water and bacterial regrowth in the distribution system. This effect is enhanced with addition of high-level acetic acid. Batch tests were also conducted using water samples collected from a Taiwanese drinking water distribution system. The bacterial regrowth potentials of the blank were equal to 4.3 × 10³, 1.5 × 10⁴, 4.9 × 10⁴ and 7.5 × 10⁴ c.f.u. ml⁻¹ for water samples collected from treatment plant effluent, commercial area, mixed area, and residential area, respectively. These results showed that the biological stability of drinking water is the highest in treatment plant effluent, followed by distributed water of the commercial area, distributed water of the mixed area, and then the distributed water of residential area.     

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.     

Further investigations on the concentration of marine bacteriophages in the water around Helgoland,with reference to the phage-host systems encountered

Between April 3 and September 24, 1991, the concentrations of bacteriophages infecting bacterial strains, isolated in 1990 and during this investigations, were determined in 35 samples of seawater taken at station ‘Kabeltonne’ adjacent to Helgoland. Similar to the findings of 1990, phage concentrations of several hundred plaque forming units (PFU) ml⁻¹ were observed with a number of indicator strains, the maximum concentration being at least 1.5×10³ PFU ml⁻¹. These high concentrations lasted for only a few days, generally decreasing at rates between 0.6 and 0.9 day⁻¹. Phage concentrations of 0 to 2 PFU ml⁻¹ were found to be predominant until the end of June, occasionally attaining 5 PFU ml⁻¹. From July through September, when high phage concentrations were observed with some indicator strains, between 0 and 10 PFU ml⁻¹ were found in the majority of tests. As revealed by a final phage-host cross-reaction test, the greater part of 138 indicator bacteria is genetically related, and almost half of the 200 phage strains tested are propagated only by their original indicator bacterium. The possible importance of mutational events for the maintenance of phage-host systems in nature is discussed.     

Sensitive Determination of Microbial Growth by Nucleic Acid Staining in Aqueous Suspension

The determination of cell numbers or biomass in laboratory cultures or environmental samples is usually based on turbidity measurements, viable counts, biochemical determinations (e.g., protein and lipid measurements), microscopic counting, or recently, flow cytometric analysis. In the present study, we developed a novel procedure for the sensitive quantification of microbial cells in cultures and most-probable-number series. The assay combines fluorescent nucleic acid staining and subsequent fluorescence measurement in suspension. Six different fluorescent dyes (acridine orange, DAPI [4′,6′-diamidino-2-phenylindole], ethidium bromide, PicoGreen, and SYBR green I and II) were evaluated. SYBR green I was found to be the most sensitive dye and allowed the quantification of 50,000 to up to 1.5 × 10⁸ Escherichia coli cells per ml sample. The rapid staining procedure was robust against interference from rRNA, sample fixation by the addition of glutaric dialdehyde, and reducing agents such as sodium dithionite, sodium sulfide, and ferrous sulfide. It worked well with phylogenetically distant bacterial and archaeal strains. Excellent agreement with optical density measurements of cell increases was achieved during growth experiments performed with aerobic and sulfate-reducing bacteria. The assay offers a time-saving, more sensitive alternative to epifluorescence microscopy analysis of most-probable-number dilution series. This method simplifies the quantification of microbial cells in pure cultures as well as enrichments and is particularly suited for low cell densities.     

Contamination of pear concentrate by Alicyclobacillus from recirculating flume water during fruit concentrate production

Alicyclobacillus and viable aerobic counts were monitored at nine different production stages of pear concentrate, with the functioning of either a recirculating or a one-pass flume water system. Significantly (P < 0.05) higher levels of Alicyclobacillus were detected in the final pasteurized product (102–104°C for 90 s) when the recirculating flume water system was operational. An average of 1.19 Log₁₀ c.f.u. ml⁻¹ vegetative cells and 1.35 Log₁₀ c.f.u. ml⁻¹ endospores were recovered, whereas 0 c.f.u. ml⁻¹ vegetative cells and endospores were detected when the one-pass flume water system was operational. Alicyclobacillus levels did not differ significantly (P > 0.05) in condensate water during the functioning of the two flume water systems, with 1.81 Log₁₀ c.f.u. ml⁻¹ vegetative cells and 1.01 Log₁₀ c.f.u. ml⁻¹ endospores (recirculating system) and 0.78 Log₁₀ c.f.u. ml⁻¹ vegetative cells and 0.42 Log₁₀ c.f.u. ml⁻¹ endospores (one-pass system) recovered, respectively. As a result, water treatment protocols should be established if untreated recirculating flume or condensate water is to be used in order to prevent Alicyclobacillus contamination and accumulation in the processing environment.     

Improved hydrogen photoproduction regulated by carbonylcyanide <Emphasis Type="Italic">m</Emphasis>-chlorophenylhrazone from marine green alga <Emphasis Type="Italic">Platymonas subcordiformis</Emphasis> grown in CO<Subscript>2</Subscript>-supplemented air bubble column bioreactor

To develop an integrated process of CO₂-fixation and H₂ photoproduction by marine green microalga Platymonas subcordiformis, the impact of algal cells grown in CO₂-supplemented air bubble column bioreactor was investigated on H₂ photoproduction regulated by carbonylcyanide m-chlorophenylhrazone. Highest cell growth (3.85 × 10⁶ cells ml⁻¹), starch content (0.25 ± 0.08 mg per 10⁶ cells) and hydrogen production (50 ± 3 ml l⁻¹) were achieved at 3% CO₂-supplemented culture, which are respectively 1.4, 2.1, 1.5-fold of the air-supplemented culture. Improved H₂ production correlated well with the increase in starch accumulation. In this process, the algal cells have been recycled for stable H₂ production of 40–50 ml l⁻¹ over five cycles.     

Antarctic sea ice viral dynamics over an annual cycle

Viral abundance, burst sizes, lytic production and temperate phage were investigated in land-fast ice at two sites in Prydz Bay Antarctica (68°S, 77°E) between April and November 2008. Both ice cores and brine were collected. There was no seasonal pattern in viral or bacterial numbers. Across the two sites virus abundances ranged between 0.5 × 10⁵ and 5.1 × 10⁵ viruses ml⁻¹ in melted ice cores and 0.6 × 10⁵–3.5 × 10⁵ viruses ml⁻¹ in brine, and bacterial abundances between 2.7 × 10⁴ and 17.3 × 10⁴ cells ml⁻¹ in melted ice cores and 3.9 × 10⁴–32.5 × 10⁴ cells ml⁻¹ in brine. Virus to bacterium ratios (VBR) showed a clear seasonal pattern in ice cores with lowest values in winter (range 1.2–20.8), while VBRs in brine were lower (0.2–4.9). Lytic viral production range from undetectable to 2.0 × 10⁴ viruses ml⁻¹ h⁻¹ in ice cores with maximum rates in September and November. In brine maximum, lytic viral production occurred in November (1.18 × 10⁴ viruses ml⁻¹ h⁻¹). Low burst sizes were typical (3.94–4.03 viruses per bacterium in ice cores and 3.16–4.0 viruses per bacterium in brine) with unusually high levels of visibly infected cells—range 40–50%. This long-term investigation revealed that viral activity was apparent within the sea ice throughout its annual cycle. The findings are discussed within the context of limited data available on viruses in sea ice.     

Protozoan Bacterivory in the Ice and the Water Column of a Cold Temperate Lagoon

Abstract Bacterial abundance and bacterivorous protist abundance and activity were examined in ice-brine and water column communities of a cold temperate Japanese lagoon (Saroma-Ko Lagoon, Hokkaido, 44°N, 144°E), during the late winter phase of ice community development (February–March 1992). Bacterial abundance averaged 6 and 1 × 10⁵ cells ml⁻¹ in the ice-brine and plankton samples, respectively, and generally decreased during the sampling period. Bacterivorous protists, identified based on direct observation of short-term (<1 h) ingested fluorescently labeled bacteria (FLB) in their food vacuoles, were largely dominated by flagellates, mainly cryothecomonad-type and chrysomonad-like cells and small dinoflagellates of the genus Gymnodinium. Bacterivorous ciliates included mainly the prostomatid Urotricha sp., the scuticociliates Uronema and Cyclidium, the choreotrichs Lohmaniella oviformis and Strobilidium, and the hypotrich Euplotes sp. Protist abundance averaged 4 × 10³ and 8.1 cells ml⁻¹ in the ice-brine and 0.3 × 10³ and 1.2 cells ml⁻¹ in the plankton, for flagellates and ciliates, respectively. In contrast to bacteria, the abundance of protists generally increased throughout the sampling period, indicating predator–prey interactions. Protistan bacterivory, measured from the rate of FLB disappearance over 24 h, averaged 36% (ice) and 24% (plankton) of bacterial standing stock and exhibited the same seasonal pattern as for protist abundance. The calculated specific clearance (range, 2–67 nl protozoa⁻¹ h⁻¹) and ingestion (<1–26 particles protozoa⁻¹ h⁻¹) rates were likely to be minimal estimates and grazing impact may have been higher on occasion. Indications for the dependence of ``bacterivorous protists' on nonbacterial food items were also provided. Although alternative sources of bacterial loss are likely to be of importance, this study provides evidence for the potential of protozoan assemblages as bacterial grazers in both sea ice-brine biota and water column at the southern limit of sea ice in the northern hemisphere. Received: 30 July 1998; Accepted: 18 November 1998     

Biodegradation of nonylphenol in a continuous bioreactor at low temperatures and effects on the microbial population

A packed-bed bioreactor inoculated with a mixed culture obtained from a contaminated site was used to continuously treat a saturated solution of nonylphenol. The reactor was operated at feeding rates of 13–112 ml h⁻¹ and temperatures of 5.5, 10, and 15°C. Optimal bioreactor performance was achieved at 10°C and at a feeding rate of 84 ml h⁻¹ (with a removal rate of 43 mg l⁻¹ day⁻¹ of nonylphenol). No endocrine activity was observed in the effluent of the bioreactor at any of the temperatures tested, and the only metabolic products found were branched carboxylic acids and alkanes (lacking an aromatic ring). The study of the microbial populations in the biofilm at the three temperatures tested using fluorescence in situ hybridization showed that all the bacterial species that could be identified belonged to the phylum Proteobacteria. The most abundant class identified at all three temperatures was β-Proteobacteria. The proportions of bacteria that bound to the specific probes among the total population, identified with the bacterial probe EUB338MIX, were 60, 43, and 24% at 15, 10, and 5.5°C, respectively.     

Use of lysozyme for treatment of bacterial contamination in <Emphasis Type="Italic">in vitro</Emphasis> shoot cultures of fruit plants

Summary Use of lysozyme was tested for treatment of bacterial contaminations in in vitro shoot cultures of quince (Cydonia oblonga) ‘BA 29’ and the hybrid (Prunus persica × P. amygdalus) rootstock ‘GF 677’. Shoots which had been contaminated for about 1 yr by Bacillus circulans and Sphingomonas paucimobilis were treated in liquid culture, at pH 4.5, with 9–36 mg ml⁻¹ egg white lysozyme (EWL), and compared to each other and to untreated cultures for their growth, proliferation, and number of bacterial colony-forming units in the tissues. EWL did not negatively affect shoot growth up to 18 mg ml⁻¹; furthermore, the proliferation rates of EWL-treated shoots were sometimes higher than those of controls. In contrast, the concentration of 36 mg ml⁻¹ had some deleterious effect on the regrowth capacity and shoot production of ‘GF 677’ at the first subculture to solid medium after EWL, treatments. EWL had a simple bacteriostatic effect against Sphingomonas paucimobilis; in contrast, it was effective at 18 mg ml⁻¹ in eliminating Bacillus circulans in both ‘BA 29’ and ‘GF 677’ cultures, after optimal treatment duration.     

Effects of soil amendment with different carbon sources and other factors on the bioremediation of an aged PAH-contaminated soil

Carbon supplementation, soil moisture and soil aeration are believed to enhance in situ bioremediation of PAH-contaminated soils by stimulating the growth of indigenous microorganisms. However, the effects of added carbon and nitrogen together with soil moisture and soil aeration on the dissipation of PAHs and on associated microbial counts have yet to be fully assessed. In this study the effects on bioremediation of carbon source, carbon-to-nitrogen ratio, soil moisture and aeration on an aged PAH-contaminated agricultural soil were studied in microcosms over a 90-day period. Additions of starch, glucose and sodium succinate increased soil bacterial and fungal counts and accelerated the dissipation of phenanthrene and benzo(a)pyrene in soil. Decreases in phenanthrene and benzo(a)pyrene concentrations were effective in soil supplemented with glucose and sodium succinate (both 0.2 g C kg⁻¹ dry soil) and starch (1.0 g C kg⁻¹ dry soil). The bioremediation effect at a C/N ratio of 10:1 was significantly higher (P < 0.05) than at a C/N of either 25:1 or 40:1. Soil microbial counts and PAH dissipation were lower in the submerged soil but soil aeration increased bacterial and fungal counts, enhanced indigenous microbial metabolic activities, and accelerated the natural degradation of phenanthrene and benzo(a)pyrene. The results suggest that optimizing carbon source, C/N ratio, soil moisture and aeration conditions may be a feasible remediation strategy in certain PAH contaminated soils with large active microbial populations.     

Ostracodology in time and space: looking back on fifteen International Symposia on Ostracoda,and the times in between

Nuclease protection assay (NPA) probes were designed to target the rRNA of Chaetoceros curvisetus and Skeletonema costatum, and quantitative sandwich hybridization integrated with nuclease protection assay (NPA-SH) was developed to detect C. curvisetus and S. costatum in culture and field samples in Jiaozhou Bay, China. The specificity and validity of the NPA-SH technique were tested with cultured pure strains, mixed strains and field samples, and by comparison with that of microscopy observation. The linear detection range for C. curvisetus was 4.2 × 10⁴ to 1.2 × 10⁶ cells with a detection limit of 42 cells ml⁻¹. The linear range for S. costatum was 6.0 × 10⁴ to 1.0 × 10⁶ cells with a detection limit of 60 cells ml⁻¹. The NPA-SH in this study provides a convenient tool for rapid assessment of HAB species in marine environments. Handling editor: D. Hamilton     

A flow cytometric protocol for titering recombinant adenoviral vectors containing the green fluorescent protein

As the use of adenoviral vectors in gene therapy protocols increases, there is a corresponding need for rapid, accurate, and reproducible titer methods. Multiple methods currently exist for determining titers of recombinant adenoviral vector, including optical absorbence, electron microscopy, fluorescent focus assay, and the “gold standard” plaque assay. This paper introduces a novel flow cytometric method for direct titer determination that relies on the expression of the green fluorescent protein (GFP), a tracking marker incorporated into several adenoviral vectors. This approach was compared to the plaque assay using 10⁻⁴-to 10⁻⁶-fold dilutions of a cesium-chloride-purified, GFP expressing adenovirus (AdEasy+GFP+GAL). The two approaches yielded similar titers: 3.25±1.85×10⁹ PFU/mL versus 3.46±0.76×10⁹ green fluorescent units/(gfu/mL). The flow cytometric method is complete within 24 h in contrast to the 7×10 days required by the plaque assay. These results indicate that the GFU/mL is an alternative functional titer method for fluorescent-tagged adenoviral vectors.     

Quantitative Determination of H2-Utilizing Acetogenic and Sulfate-Reducing Bacteria and Methanogenic Archaea from Digestive Tract of Different Mammals

Total number of bacteria, cellulolytic bacteria, and H₂-utilizing microbial populations (methanogenic archaea, acetogenic and sulfate-reducing bacteria) were enumerated in fresh rumen samples from sheep, cattle, buffaloes, deer, llamas, and caecal samples from horses. Methanogens and sulfate reducers were found in all samples, whereas acetogens were not detected in some samples of each animal. Archaea methanogens were the largest H₂-utilizing populations in all animals, and a correlation was observed between the numbers of methanogens and those of cellulolytic microorganisms. Higher counts of acetogens were found in horses and llamas (1 × 10⁴ and 4 × 10⁴ cells ml⁻¹ respectively).     

Recognition of Individual Genes in Diverse Microorganisms by Cycling Primed In Situ Amplification

Cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) was developed to recognize individual genes in a single bacterial cell. In CPRINS, the amplicon was long single-stranded DNA and thus retained within the permeabilized microbial cells. FISH with a multiply labeled fluorescent probe set enabled significant reduction in nonspecific background while maintaining high fluorescence signals of target bacteria. The ampicillin resistance gene in Escherichia coli, chloramphenicol acetyltransferase gene in different gram-negative strains, and RNA polymerase sigma factor (rpoD) gene in Aeromonas spp. could be detected under identical permeabilization conditions. After concentration of environmental freshwater samples onto polycarbonate filters and subsequent coating of filters in gelatin, no decrease in bacterial cell numbers was observed with extensive permeabilization. The detection rates of bacterioplankton in river and pond water samples by CPRINS-FISH with a universal 16S rRNA gene primer and probe set ranged from 65 to 76% of total cell counts (mean, 71%). The concentrations of cells detected by CPRINS-FISH targeting of the rpoD genes of Aeromonas sobria and A. hydrophila in the water samples varied between 2.1 × 10³ and 9.0 × 10³ cells ml⁻¹ and between undetectable and 5.1 × 10² cells ml⁻¹, respectively. These results demonstrate that CPRINS-FISH provides a high sensitivity for microscopic detection of bacteria carrying a specific gene in natural aquatic samples.     

Detection and Quantification with 16S rRNA Probes of Planktonic Methylotrophic Bacteria in a Floodplain Lake

Abstract Two approaches employing 16S rRNA oligonucleotide probes, in situ hybridization combined with ³³P-autoradiography and ³²P-labeled slot-blot hybridizations on nitrocellulose filters, were used to enumerate methylotrophic bacteria in the water column of Ryans 1 Billabong, a small floodplain lake in northeastern Victoria, Australia. Methylotrophic bacterioplankton numbered 0.6–1.2 × 10⁹ cells liter⁻¹ in the winter of 1994, and 0.8–5.5 × 10⁹ cells liter⁻¹ in the summer of 1994–95. This was equivalent to 10–46% of total bacterioplankton cell counts, determined via epifluorescence microscopy. Methylotrophic bacteria were not detected in the water column of the nearby Kiewa River, and a set of laboratory controls indicated that the high abundance of methylotrophs in the billabong samples was not a methodological artifact. There was no change, with water depth, in total bacterioplankton or methylotroph abundance in winter, a result consistent with the water column being well mixed at this time of year (dissolved O₂ concentrations 5–7 mg liter⁻¹; dissolved methane concentrations <60 μg liter⁻¹, or <5% methane saturation, at all depths). In summer the billabong became diurnally stratified (dissolved O₂ concentrations <2 mg liter⁻¹ at water depths of >45 cm; dissolved methane concentrations <100 μg liter⁻¹ at the surface, but >500 μg liter⁻¹ near the sediments) and there was a correspondingly marked increase in the abundance of total bacterioplankton and methylotrophs with depth. In situ hybridizations and slot-blot hybridizations both indicated that type II methylotrophs (detected with a probe specific for the 9-α subgroup of Proteobacteria) were markedly less abundant than were type I and X methylotrophs (detected with a probe specific for the 10-γ subgroup of Proteobacteria). Received: 12 March 1996; Accepted: 2 October 1996     

Natural populations of bacteria in Lake Kinneret: Observations with scanning electron and epifluorescence microscopy

The bacterioplankton assemblage in Lake Kinneret, Israel, sampled on 6 occasions representative of different seasonal conditions was studied with scanning electron microscopy (SEM) and epifluorescence microscopy after acridine-orange staining. In near-surface (1–3 m) samples taken in October 1981 and March 1983, several unusual types of budding, appendaged, and filamentous cells were found. During lake stratification, typical large anaerobic forms (including photosynthetic green sulphur bacteria) were observed in samples from the metalimnion and deep (40 m) hypolimnion. Epifluorescence counts indicated that bacteria in the water column ranged from 0.55 to 2.67 × 10⁶ cells ml^–1.