Seasonal and spatial variability of virio-, bacterio-, and picophytoplanktonic abundances in three peri-alpine lakes

Flow cytometry (FCM) was used to assess microbial community abundances and patterns in three natural, large and deep peri-alpine hydrosystems, i.e., lakes Annecy (oligotrophic), Bourget, and Geneva (mesotrophic). Picocyanobacteria, small eukaryotic autotrophs, heterotrophic prokaryotes, and viruses were studied in the 0–50 m surface layers to highlight the impact of both physical and chemical parameters as well as possible biotic interactions on the functioning of microbial communities. Some specificities were recorded according to the trophic status of each ecosystem such as the higher number of viruses and heterotrophic bacteria in mesotrophic environments (i.e., Lakes Geneva and Bourget) or the higher abundance of picocyanobacteria in the oligotrophic Lake Annecy. However, both seasonal (temperature) and spatial (depth) variations were comparatively more important than the trophic status in driving the microbial communities’ abundances in these three lakes, as revealed by principal component analysis (PCA). A strong viral termination of the heterotrophic bacterial blooms could be observed in autumn for each lake, in parallel to the mixing of the upper lit layers. As virus to bacteria ratio (VBR) was indeed very high at this period with values varying between 87 and 114, such important relationships between viruses and bacteria were likely. The magnitudes of seasonal variations in VBR, with the highest values ever reported so far, were largely greater than the magnitude of theoretical variations due to the trophic status, suggesting also a strong seasonality in virioplankton production associated to prokaryotic dynamics. FCM analyses allowed discriminating several viral groups. Virus-Like Particles group 1 (VLP1) and group 2 (VLP2) were always observed and significantly correlated to bacteria for the former and chlorophyll a and picocyanobacteria for the latter, suggesting that most of VLP1 and VLP2 could be bacteriophages and cyanophages, respectively. On the basis of these results, new ways of investigation emerge concerning the study of relationships between specific picoplanktonic groups; and overall these results provide new evidence of the necessity to consider further viruses for a better understanding of lake plankton ecology. Handling editor: Luigi Naselli-Flores     

Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy

Comparative measurements of bacterial total counts and volumes of flow cytometry (FCM), transmission electron (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1 · 10⁶−6 cells ml⁻¹, 1 · 10⁶−3 · 10¹⁶ cells ml⁻¹, and 5 · 10⁵ cells ml⁻¹ respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12–0.15 μm³ at the start of the experiment to 0.39–0.53 μm³ at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 μm³ were not detected by flow cytometry and these were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was longer than 0.3 μm³, all three methods were in agreement both with respect to counts and volume estimates.     

Quantitative intercomparison of transmission electron microscopy,flow cytometry,and epifluorescence microscopy for nanometric particle analysis

Nanometric biological particles such as viruses have received increased attention in a wide range of scientific fields. Evaluation of viral contributions to environmental processes and the use of viruses in medical applications such as gene therapy require viruses to be routinely and accurately enumerated. There are a variety of existing techniques for counting viruses, namely, plaque assays, transmission electron microscopy (TEM), epifluorescence microscopy (EFM), and flow cytometry (FCM); each has advantages and disadvantages. While there have been attempts to intercompare some of these techniques to determine the most effective means to count viruses, no previous study used a technique-independent standard for quantitative comparison of collection efficiency, accuracy, and precision. In this work, polystyrene nanospheres were used as standards for the intercomparison of performance characteristics for TEM, EFM, FCM, as well as a custom-built flow cytometer (the Single Nanometric Particle Enumerator, SNaPE). EFM and SNaPE exhibited the highest degree of accuracy and precision, with particle concentrations deviating < or =5% from true and relative errors less than half that of TEM, EFM and SNaPE are also significantly more time and cost efficient than TEM.     

Variations in Abundance,Genome Size,Morphology, and Functional Role of the Virioplankton in Lakes Annecy and Bourget over a 1-Year Period

We sampled the surface waters (2–50 m) of two deep peri-alpine lakes over a 1-year period in order to examine (1) the abundance, vertical distribution, genome size, and morphology structures of the virioplankton; (2) the virus-mediated bacterial mortality; and (3) the specific genome size range of double-stranded DNA (dsDNA) phytoplankton viruses. Virus-like particle (VLP) concentrations varied between 4.16?×?10⁷ (January) and 2.08?×?10⁸ part mL^?1 (May) in Lake Bourget and between 2.7?×?10⁷ (June) and 8.39?×?10⁷ part mL^?1 (November) in Lake Annecy. Our flow cytometry analysis revealed at least three viral groups (referred to as virus-like particles 1, 2, and 3) that exhibited distinctive dynamics suggestive of different host types. Phage-induced bacterial mortality varied between 6.1 % (June) and 33.2 % (October) in Lake Bourget and between 7.4 % (June) and 52.6 % (November) in Lake Annecy, suggesting that viral lysis may be a key cause of mortality of the bacterioplankton. Virioplankton genome size ranged from 27 to 486 kb in Lake Bourget, while it reached 620 kb in Lake Annecy for which larger genome sizes were recorded. Our analysis of pulsed field gel electrophoresis bands using different PCR primers targeting both cyanophages and algal viruses showed that (1) dsDNA viruses infecting phytoplankton may range from 65 to 486 kb, and (2) both cyanophage and algal “diversity” were higher in Lake Annecy. Lakes Annecy and Bourget also differed regarding the proportions of both viral families (with the dominance of myoviruses vs. podoviruses) and infected bacterial morphotypes (short rods vs. elongated rods), in each of these lakes, respectively. Overall, our results reveal that (1) viruses displayed distinct temporal and vertical distribution, dynamics, community structure in terms of genome size and morphology, and viral activity in the two lakes; (2) the Myoviridae seemed to be the main cause of bacterial mortality in both lakes and this group seemed to be related to VLP2; and (3) phytoplankton viruses may have a broader range of genome size than previously thought. This study adds to growing evidence that viruses are diverse and play a significant role in freshwater microbial dynamics and more globally lake functioning. It highlights the importance of further considering this biological compartment for a better understanding of plankton ecology in peri-alpine lakes.     

Estimates of protozoan- and viral-mediated mortality of bacterioplankton in Lake Bourget (France)

1. We performed three, 1‐week in situ experiments in March‐April (expt 1), May (expt 2) and August (expt 3) 2003 in order to assess protozoan and virus‐induced mortality of heterotrophic bacteria in a French lake. Viral and bacterial abundances were obtained using flow cytometry (FCM) while protozoa were counted using epifluorescence microscopy (EFM). 2. A dilution approach, applied to pretreated grazer‐free samples, allowed us to estimate that viral lysis could be responsible for 60% (expt 1), 35% (expt 2) and 52% (expt 3) of daily heterotrophic bacterial mortality. Flagellate (both mixotrophic and heterotrophic) grazing in untreated samples, was responsible for 56% (expt 1), 63% (expt 2) and 18% (expt 3) of daily heterotrophic bacteria removal. 3. These results therefore suggest that both viral lysis and flagellate grazing had a strong impact on bacterial mortality, and this impact varied seasonally. 4. From parallel transmission electron microscopy (TEM) analysis, we found that the burst size (i.e. the number of viruses potentially released per lysed cell) ranged from nine to 25 (expt 1), 10 to 35 (expt 2) and eight to 25 (expt 3). The percentage of infected heterotrophic bacteria was 5.7% (expt 1), 3.4% (expt 2) and 5.7% (expt 3) so that the calculated percentage of bacterial mortality induced by viruses was 6.3% (expt 1), 3.7% (expt 2) and 6.3% (expt 3). 5. It is clear that the dilution‐FCM and TEM methods yielded different estimates of viral impact, although both methods revealed an increased impact of viruses during summer.     

Assessment of Factors Influencing Direct Enumeration of Viruses within Estuarine Sediments

Accurate enumeration of viruses within environmental samples is critical for investigations of the ecological role of viruses and viral infection within microbial communities. This report evaluates differences in viral and bacterial direct counts between estuarine sediment samples which were either immediately processed onboard ship or frozen at −20°C and later processed. Viral and bacterial abundances were recorded at three stations spanning the length of the Chesapeake Bay in April and June 2003 within three sediment fractions: pore water (PW), whole sediment (WS), and sediment after pore water removal (AP). No significant difference in viral abundance was apparent between extracts from fresh or frozen sediments. In contrast, bacterial abundance was significantly lower in the samples subjected to freezing. Both bacterial and viral abundance showed significant differences between sediment fractions (PW, WS, or AP) regardless of the fresh or frozen status. Although pore water viral abundance has been used in the past as a measurement of viral abundance in sediments, this fraction accounted for only ca. 5% of the total sediment viral abundance across all samples. The effect of refrigerated storage of sediment viral extracts was also examined and showed that, within the first 2 h, viral abundance decreased ca. 30% in formalin-fixed extracts and 66% in unfixed extracts. Finally, the reliability of direct viral enumeration via epifluorescence microscopy was tested by using DNase treatment of WS extractions. These tests indicated that a large fraction (>86%) of the small SYBR gold fluorescing particles are likely viruses.     

Assessment of factors influencing direct enumeration of viruses within estuarine sediments

Accurate enumeration of viruses within environmental samples is critical for investigations of the ecological role of viruses and viral infection within microbial communities. This report evaluates differences in viral and bacterial direct counts between estuarine sediment samples which were either immediately processed onboard ship or frozen at -20 degrees C and later processed. Viral and bacterial abundances were recorded at three stations spanning the length of the Chesapeake Bay in April and June 2003 within three sediment fractions: pore water (PW), whole sediment (WS), and sediment after pore water removal (AP). No significant difference in viral abundance was apparent between extracts from fresh or frozen sediments. In contrast, bacterial abundance was significantly lower in the samples subjected to freezing. Both bacterial and viral abundance showed significant differences between sediment fractions (PW, WS, or AP) regardless of the fresh or frozen status. Although pore water viral abundance has been used in the past as a measurement of viral abundance in sediments, this fraction accounted for only ca. 5% of the total sediment viral abundance across all samples. The effect of refrigerated storage of sediment viral extracts was also examined and showed that, within the first 2 h, viral abundance decreased ca. 30% in formalin-fixed extracts and 66% in unfixed extracts. Finally, the reliability of direct viral enumeration via epifluorescence microscopy was tested by using DNase treatment of WS extractions. These tests indicated that a large fraction (>86%) of the small SYBR gold fluorescing particles are likely viruses.     

Counting Viruses and Bacteria in Photosynthetic Microbial Mats

Viral abundances in benthic environments are the highest found in aquatic systems. Photosynthetic microbial mats represent benthic environments with high microbial activity and possibly high viral densities, yet viral abundances have not been examined in such systems. Existing extraction procedures typically used in benthic viral ecology were applied to the complex matrix of microbial mats but were found to inefficiently extract viruses. Here, we present a method for extraction and quantification of viruses from photosynthetic microbial mats using epifluorescence microscopy (EFM) and flow cytometry (FCM). A combination of EDTA addition, probe sonication, and enzyme treatment applied to a glutaraldehyde-fixed sample resulted in a substantially higher viral (5- to 33-fold) extraction efficiency and reduced background noise compared to previously published methods. Using this method, it was found that in general, intertidal photosynthetic microbial mats harbor very high viral abundances (2.8 × 10¹⁰ ± 0.3 × 10¹⁰ g⁻¹) compared with benthic habitats (10⁷ to 10⁹ g⁻¹). This procedure also showed 4.5- and 4-fold-increased efficacies of extraction of viruses and bacteria, respectively, from intertidal sediments, allowing a single method to be used for the microbial mat and underlying sediment.     

Effect of grazers and viruses on bacterial community structure and production in two contrasting trophic lakes

Background

Over the last 30 years, extensive studies have revealed the crucial roles played by microbes in aquatic ecosystems. It has been shown that bacteria, viruses and protozoan grazers are dominant in terms of abundance and biomass. The frequent interactions between these microbiological compartments are responsible for strong trophic links from dissolved organic matter to higher trophic levels, via heterotrophic bacteria, which form the basis for the important biogeochemical roles of microbial food webs in aquatic ecosystems. To gain a better understanding of the interactions between bacteria, viruses and flagellates in lacustrine ecosystems, we investigated the effect of protistan bacterivory on bacterial abundance, production and structure [determined by 16S rRNA PCR-DGGE], and viral abundance and activity of two lakes of contrasting trophic status. Four experiments were conducted in the oligotrophic Lake Annecy and the mesotrophic Lake Bourget over two seasons (early spring vs. summer) using a fractionation approach. In situ dark vs. light incubations were performed to consider the effects of the different treatments in the presence and absence of phototrophic activity.

Results

The presence of grazers (i.e. < 5-μm small eukaryotes) affected viral production positively in all experiments, and the stimulation of viral production (compared to the treatment with no eukaryotic predators) was more variable between lakes than between seasons, with the highest value having been recorded in the mesotrophic lake (+30%). Viral lysis and grazing activities acted additively to sustain high bacterial production in all experiments. Nevertheless, the stimulation of bacterial production was more variable between seasons than between lakes, with the highest values obtained in summer (+33.5% and +37.5% in Lakes Bourget and Annecy, respectively). The presence of both predators (nanoflagellates and viruses) did not seem to have a clear influence upon bacterial community structure according to the four experiments.

Conclusions

Our results highlight the importance of a synergistic effect, i.e. the positive influence of grazers on viral activities in sustaining (directly and indirectly) bacterial production and affecting composition, in both oligotrophic and mesotrophic lakes.     

Interannual dynamics of viriobenthos abundance and morphological diversity in Chesapeake Bay sediments

Despite significant implications of viral activity in sediment ecosystems, there are limited data describing how sediment viral assemblages respond to broader ecosystem changes. To document this, the spatial and temporal dynamics of viral and bacterial abundance (BA) and changes in the morphological distribution of viruses were examined within three salinity regions over 2 years. Viral abundances (VA) ranged from 0.2 to 17 × 10(10) viruses mL(-1) sediment while direct bacterial counts ranged from 3.8 to 37 × 10(8) cells mL(-1) sediment. Peaks and valleys in the abundance of extracted viruses and bacteria from surface sediments occurred simultaneously, with lows in February 2004 and highs in April 2003. Across all samples, viral and BA were positively correlated (P < 0.001). Vertical profiles showed a decrease in viral and BA with depth in sediments. Based on transmission electron microscopy results, viruses with diminutive capsids (20-50 nm) and from the Myoviridae and Podoviridae viral family types were dominant within surface sediments. The most morphologically diverse viral assemblages occurred in autumn samples from the sandy, polyhaline station and spring samples from the mesohaline station. Seasonal changes showed an average 72% decrease in VA from spring to winter. These observations support the view that viriobenthos assemblages are responsive to seasonal environmental changes and that viral processes have significant implications for the biogeochemical processes mediated by bacterial communities within Bay sediments.     

Rapid detection of efflux pumps and their relation with drug resistance in yeast cells

BACKGROUND: Cell drug resistance can be due to the presence of active efflux pumps (AEP). Identification of yeast cells with a resistance phenotype is important either from a clinical, agricultural or biotechnological point of view. Rapid and reliable methods to detect AEP can be therefore very useful. METHODS: Some yeast cells change their staining by calcein-AM, BCECF-AM, rhodamine 123 and DiOC(5), when pretreated with verapamil, CCCP or ATP depletion, or when pretreated with specific antimicrobial agents. This fact may be interpreted as an indication of the presence/absence of AEP. Six yeast species were tested with a flow cytometric method (FCM) and an epifluorescence microscopic method (EFM), and ten other species were evaluated only by EFM. The minimum inhibitory concentration (MIC) of penconazol, benomyl and cycloheximide for Saccharomyces cerevisiae and Kluyveromyces marxianus, were determined by growth inhibition on solid medium and were compared to the staining changes detected by FCM. RESULTS: The FCM and the EFM allowed the detection of AEP in all the yeast species tested. High MIC values for a drug were related with the presence of at least one AEP indicated by the cytometric data. CONCLUSIONS: The FCM revealed to be a robust assay whereas the EFM can be used as a preliminary test. It is possible to identify resistance/sensitivity patterns in yeast cells through cytometric detection methods of different efflux pumping systems.     

Rapid detection of viable yeasts and bacteria in wine by flow cytometry

The potential of using flow cytometry (FCM) in combination with fluorescent dyes for rapidly estimating counts of yeasts and malolactic bacteria in laboratory media and wines was examined. In general, there was a good correlation (regression coefficient, 0.94) between viable counts of yeasts determined by FCM and by standard plate assay. The FCM detection limit of yeasts in YPDE medium and in Pinot noir must was 10(3) cells/ml. The lowest bacterial concentration detected by FCM was 10(4) cells/ml. When yeast and malolactic bacteria populations were simultaneously analysed in wine by FCM without any previous sample treatment, difficulties were encountered in the count of bacterial cells due to their size, which is similar to natural debries present in wine. However, after the optimisation of the sample preparation, the technique appeared promising in determining the presence of such microorganisms in wine with one single measurement. Because it is rapid and easy to use, flow cytometry can be considered a useful method for microbiological quality control in wineries and for the investigation of the growth dynamics of microorganisms in wine.     

Responses of Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus to simulated food processing treatments, determined using fluorescence-activated cell sorting and plate counting

Three common food pathogenic microorganisms were exposed to treatments simulating those used in food processing. Treated cell suspensions were then analyzed for reduction in growth by plate counting. Flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) were carried out on treated cells stained for membrane integrity (Syto 9/propidium iodide) or the presence of membrane potential [DiOC2(3)]. For each microbial species, representative cells from various subpopulations detected by FCM were sorted onto selective and nonselective agar and evaluated for growth and recovery rates. In general, treatments giving rise to the highest reductions in counts also had the greatest effects on cell membrane integrity and membrane potential. Overall, treatments that impacted cell membrane permeability did not necessarily have a comparable effect on membrane potential. In addition, some bacterial species with extensively damaged membranes, as detected by FCM, appeared to be able to replicate and grow after sorting. Growth of sorted cells from various subpopulations was not always reflected in plate counts, and in some cases the staining protocol may have rendered cells unculturable. Optimized FCM protocols generated a greater insight into the extent of the heterogeneous bacterial population responses to food control measures than did plate counts. This study underlined the requirement to use FACS to relate various cytometric profiles generated by various staining protocols with the ability of cells to grow on microbial agar plates. Such information is a prerequisite for more-widespread adoption of FCM as a routine microbiological analytical technique.     

Distribution of viral abundance in the reef environment of Key Largo, Florida.

The distribution of viral and microbial abundance in the Key Largo, Fla., reef environment was measured. Viral abundance was measured by transmission electron microscope direct counts and plaque titer on specific bacterial hosts in water and sediment samples from Florida Bay (Blackwater Sound) and along a transect from Key Largo to the outer edge of the reef tract in Key Largo Sanctuary. Water column viral direct counts were highest in Blackwater Sound of Florida Bay (1.2 x 10(7) viruses per ml), decreased to the shelf break (1.7 x 10(6) viruses per ml), and were inversely correlated with salinity (r = -0.97). Viral direct counts in sediment samples ranged from 1.35 x 10(8) to 5.3 x 10(8)/cm(3) of sediment and averaged nearly 2 orders of magnitude greater than counts in the water column. Viral direct counts (both sediment and water column measurements) exceeded plaque titers on marine bacterial hosts (Vibrio natriegens and others) by 7 to 8 orders of magnitude. Water column viral abundance did not correlate with bacterial direct counts or chlorophyll a measurements, and sediment viral parameters did not correlate with water column microbial, viral, or salinity data. Coliphage, which are indicators of fecal pollution, were detected in two water column samples and most sediment samples, yet their concentrations were relatively low (<2 to 15/liter for water column samples, and <2 to 108/cm(3) of sediment). Our findings indicate that viruses are abundant in the Key Largo environment, particularly on the Florida Bay side of Key Largo, and that processes governing their distribution in the water column (i.e., salinity and freshwater input) are independent of those governing their distribution in the sediment environment.     

High concentrations of viruses in the sediments of Lac Gilbert, Québec

Viruses were found to be very abundant in the top layer of the sediments of Lac Gilbert, Québec. Viruses were extracted from the sediments using pyrophosphate buffer, and viruses from the diluted extracts were pelleted onto grids and enumerated using transmission electron microscopy. Viral abundance in the sediments ranged from 6.5 × 10⁸ to 1.83 × 10¹⁰ ml^–1, which is 10- to 1,000-fold greater than the number observed in the water column. This increase corresponds well with the 100- to 1,000-fold increase in bacterial abundance in the sediments. Viral abundance differed significantly among the surface sediment samples taken at different bottom depths and among samples taken at different depths of the water column. Viral abundance also varied significantly between the oxic and anoxic zones of the water column and the sediments. The virus-to-bacteria ratio varied greatly among the different sediment sites but not among depths in the water column. Viral abundance in the water column was related to bacterial abundance and chlorophyll concentration, whereas viruses in the sediments were most abundant in sediments with high organic matter content. Elevated viral abundance and their erratic distribution in the sediments suggest that viruses might play an important role in sediment microbial dynamics. Correspondence to: Roxane Maranger     

Accurate estimation of viral abundance by epifluorescence microscopy

Virus enumeration by epifluorescence microscopy (EFM) is routinely done on preserved, refrigerated samples. Concerns about obtaining accurate and reproducible estimates led us to examine procedures for counting viruses by EFM. Our results indicate that aldehyde fixation results in rapid decreases in viral abundance. By 1 h postfixation, the abundance dropped by 16.4% +/- 5.2% (n = 6), and by 4 h, the abundance was 20 to 35% lower. The average loss rates for glutaraldehyde- and formaldehyde-fixed samples over the first 2 h were 0.12 and 0.13 h(-1), respectively. By 16 days, viral abundance had decreased by 72% (standard deviation, 6%; n = 6). Aldehyde fixation of samples followed by storage at 4 degrees C, for even a few hours, resulted in large underestimates of viral abundance. The viral loss rates were not constant, and in glutaraldehyde- and formaldehyde-fixed samples they decreased from 0.13 and 0.17 h(-1) during the first hour to 0.01 h(-1) between 24 and 48 h. Although decay rates changed over time, the abundance was predicted by using separate models to describe decay over the first 8 h and decay beyond 8 h. Accurate estimates of abundance were easily made with unfixed samples stained with Yo-Pro-1, SYBR Green I, or SYBR Gold, and slides could be stored at -20 degrees C for at least 2 weeks or, for Yo-Pro-1, at least 1 year. If essential, samples can be fixed and flash frozen in liquid nitrogen upon collection and stored at -86 degrees C. Determinations performed with fixed samples result in large underestimates of abundance unless slides are made immediately or samples are flash frozen. If protocols outlined in this paper are followed, EFM yields accurate estimates of viral abundance.     

Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR gold

A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.     

Application of Digital Image Analysis and Flow Cytometry To Enumerate Marine Viruses Stained with SYBR Gold

A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.     

Distribution of virus-like Particles in an Oligotrophic Marine Environment (Alboran Sea,Western Mediterranean)

Viruses are abundant in a variety of aquatic environments, often exceeding bacterial abundance by one order of magnitude. In the present study, the spatial distribution of viruses in offshore waters of the Alboran Sea (Western Mediterranean) have been studied to determine the relationships between viruses and host communities in this oligotrophic marine environment. Viral abundance was determined using two methods: (i) epifluorescence light microscopy using the dsDNA binding fluorochrome DAPI, and (ii) direct counts by transmission electron microscopy (TEM). The results obtained were significantly different; the highest viral counts were obtained by mean of TEM analyses. In all the samples tested the number of viruses was exceeded by the bacterial concentrations, with a ratio between viral and bacterial titers varying between 1.4 and 20. VLP (virus-like particle) counts were not significantly correlated (p > 0.001) with chlorophyll a concentration or the abundance of cyanobacteria. However, there was a positive and significant correlation with bacterial abundance (p < 0.001). The analysis of size and morphology of viral particles by TEM and the correlation obtained between the numbers of VLP and bacteria suggest that the majority of the viral particles in the Alboran Sea are bacteriophages. None of the indirect evidence suggested that eukaryotic algae or cyanobacteria were important host organisms in these waters.