Enumeration and biomass estimation of planktonic bacteria and viruses by transmission electron microscopy

Bacteria and virus particles were harvested from water samples by ultracentrifugation directly onto Formvar-coated electron microscopy grids and counted in a transmission electron microscope. With this technique, we have counted and sized bacteria and viruses in marine water samples and during laboratory incubations. By X-ray microanalysis, we could determine the elemental composition and dry-matter content of individual bacteria. The dry weight/volume ratio for the bacteria was 600 fg of dry weight microns-3. The potassium content of the bacteria was normal compared with previous estimates from other bacterial assemblages; thus, this harvesting procedure did not disrupt the bacterial cells. Virus particles were, by an order of magnitude, more abundant than bacteria in marine coastal waters. During the first 5 to 7 days of incubation, the total number of viruses increased exponentially at a rate of 0.4 day-1 and thereafter declined. The high proliferation rate suggests that viral parasitism may affect mortality of bacteria in aquatic environments.     

Proportion of prokaryotes enumerated as viruses by epifluorescence microscopy

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

Abundance and diversity of viruses in six Delaware soils

The importance of viruses in marine microbial ecology has been established over the past decade. Specifically, viruses influence bacterial abundance and community composition through lysis and alter bacterial genetic diversity through transduction and lysogenic conversion. By contrast, the abundance and distribution of viruses in soils are almost completely unknown. This study describes the abundance and diversity of autochthonous viruses in six Delaware soils: two agricultural soils, two coastal plain forest soils, and two piedmont forest soils. Viral abundance was measured using epifluorescence microscopy, while viral diversity was assessed from morphological data obtained through transmission electron microscopy. Extracted soil virus communities were dominated by bacteriophages that demonstrated a wide range of capsid diameters (20 nm to 160 nm) and morphologies, including filamentous forms and phages with elongated capsids. The reciprocal Simpson's index suggests that forest soils harbor more diverse assemblages of viruses, particularly in terms of morphological distribution. Repeated extractions of virus-like particles (VLPs) from soils indicated that the initial round of extraction removes approximately 70% of extractable viruses. Higher VLP abundances were observed in forest soils (1.31 x 10(9) to 4.17 x 10(9) g(-1) dry weight) than in agricultural soils (8.7 x 10(8) to 1.1 x 10(9) g(-1) dry weight). Soil VLP abundance was significantly correlated to moisture content (r = 0.988) but not to soil texture. Land use (agricultural or forested) was significantly correlated to both bacterial (r = 0.885) and viral (r = 0.812) abundances, as were soil organic matter and water content. Thus, land use is a significant factor influencing viral abundance and diversity in soils.     

流式细胞仪检测纳帕海高原湿地浮游病毒和浮游细菌丰度

【背景】浮游病毒是水体微生物群落中重要的组成成分,深入研究浮游病毒的时空分布有助于更好地保护和开发当地的微生物资源。【目的】对采集到的纳帕海高原湿地水样中的浮游病毒和浮游细菌进行计数,揭示纳帕海高原湿地浮游病毒的分布规律。【方法】采用流式细胞仪检测2013年12月和2014年9月纳帕海高原湿地7个水样的浮游病毒与浮游细菌丰度,并对影响浮游病毒丰度的因素,如细菌丰度、叶绿素a含量以及其他环境因子进行了相关性分析。【结果】季节分布上,雨季浮游病毒和浮游细菌丰度高于旱季;水平分布上,原水样品的浮游病毒高于湿地水和淤泥水。旱季水样的浮游病毒丰度受到细菌丰度及叶绿素a浓度的影响较大;雨季水样的浮游病毒丰度受到水体的p H值和温度的影响较大。【结论】纳帕海高原湿地的浮游病毒和浮游细菌是比较活跃的。浮游病毒丰度在不同季节、不同采样点受到细菌丰度和叶绿素a浓度等因素的不同影响。在旱季噬菌体而非噬藻体或浮游植物病毒是纳帕海高原湿地中浮游病毒的优势种群。     

Effects of water fluctuations on microbial mass and activity in soil

When previously dried soil was remoistened, a series of microbial events occurred. The bacterial plate count population increased rapidly, with a doubling time of 4–5 h. The length of fungal hyphae and microscopic counts of bacteria increased more slowly. The microscopically counted bacterial population was estimated to have a doubling time of about 90 h. The respiratory burst occurring after 2–3 days coincided with the maximal growth rate of the bacterial plate count population. From the respiratory data, plate count bacteria were estimated to have a cell mass of 0.4 pg dry weight, whereas the mass of microscopically counted bacteria was only 10% of this. Changes in bacterial DNA content corresponded to changes in the microscopic count, whereas changes in soil catalase activity mainly corresponded to changes in the fungal biomass, which was dominant.It is suggested that bacterial plate counts and microscopic counts represent two distinct populations of bacteria, which for practical purposes may be termed zymogenous and autochthonous, respectively.     

Abundance and Diversity of Viruses in Six Delaware Soils

The importance of viruses in marine microbial ecology has been established over the past decade. Specifically, viruses influence bacterial abundance and community composition through lysis and alter bacterial genetic diversity through transduction and lysogenic conversion. By contrast, the abundance and distribution of viruses in soils are almost completely unknown. This study describes the abundance and diversity of autochthonous viruses in six Delaware soils: two agricultural soils, two coastal plain forest soils, and two piedmont forest soils. Viral abundance was measured using epifluorescence microscopy, while viral diversity was assessed from morphological data obtained through transmission electron microscopy. Extracted soil virus communities were dominated by bacteriophages that demonstrated a wide range of capsid diameters (20 nm to 160 nm) and morphologies, including filamentous forms and phages with elongated capsids. The reciprocal Simpson's index suggests that forest soils harbor more diverse assemblages of viruses, particularly in terms of morphological distribution. Repeated extractions of virus-like particles (VLPs) from soils indicated that the initial round of extraction removes approximately 70% of extractable viruses. Higher VLP abundances were observed in forest soils (1.31 × 10⁹ to 4.17 × 10⁹ g⁻¹ dry weight) than in agricultural soils (8.7 × 10⁸ to 1.1 × 10⁹ g⁻¹ dry weight). Soil VLP abundance was significantly correlated to moisture content (r = 0.988) but not to soil texture. Land use (agricultural or forested) was significantly correlated to both bacterial (r = 0.885) and viral (r = 0.812) abundances, as were soil organic matter and water content. Thus, land use is a significant factor influencing viral abundance and diversity in soils.     

Loss of Estuarine Bacteria by Viral Infection and Predation in Microcosm Conditions

The bacterioplankton density in Ria de Aveiro, a shallow estuarine ecosystem, varied in the broad range of 1.9-10.6 109 cells L-1. The range of values was about 2 times higher in brackish water than in marine water. At high tide bacterial abundance was 2-3 times lower than at low tide. The overall variation in virioplankton was in the range of 2.4-25.0 1010 particles L-1. Brackish water was about 2 times richer in viral particles than the marine water. Near low tide the virioplankton was 2-3 times higher that at high tide. Viral density followed the pattern of bacterial abundance (it explained 40% of virioplankton variation). The viruses to bacterium ratio varied, throughout tidal cycles, by a factor of about 10 establishing the range 4.7-55.6 (average 17.6). This ratio was rather similar in the two estuarine zones. We compared the effects of infection and predation on the control of bacterioplankton size in the two zones of the estuary. The approach to this question was conducted in experimental microcosms, set up in six combinations of plankton variables affecting the presence/absence of predators, virus-to-bacterium ratio (10-fold increase), virus-to-bacterium distance (2.2-fold increase), and bacterial growth rate. The results showed that predation was similar, in a percent basis, in marine (69%) and brackish water (73%). Viral infection was, however, higher in brackish water (59%) than in the marine water (36%). We conclude that the bacterioplankton along the salinity gradient evolves under biological pressures that are in different balance in the marine and brackish water zones. The effect of viral lysis on bacterial communities with enhanced growth (after yeast extract addition) was masked even when the initial ratio was 10-fold greater than in the natural samples. The high density of the virioplankton did not preclude the large and rapid increase in bacterial density. We suggest that the dynamics of the equilibrium between bacteria and viruses in the environment is driven to higher numerical levels during periods of intensive bacterial growth. On the contrary, at low bacterial growth rates the temporarily increased virus-to-bacterium ratio may drive the equilibrium to its lowest levels.     

Virus-bacterium interactions in water and sediment of West African inland aquatic systems

The ecology of virioplankton in tropical aquatic ecosystems is poorly documented, and in particular, there are no references concerning African continental waters in the literature. In this study, we examined virus-bacterium interactions in the pelagic and benthic zones of seven contrasting shallow inland waters in Senegal, including one hypersaline lake. SYBR Gold-stained samples revealed that in the surface layers of the sites, the numbers of viruses were in the same range as the numbers of viruses reported previously for productive temperate systems. Despite high bacterial production rates, the percentages of visibly infected cells (as determined by transmission electron microscopy) were similar to the lowest percentages (range, 0.3 to 1.1%; mean, 0.5%) found previously at pelagic freshwater or marine sites, presumably because of the local environmental and climatic conditions. Since the percentages of lysogenic bacteria were consistently less than 8% for pelagic and benthic samples, lysogeny did not appear to be a dominant strategy for virus propagation at these sites. In the benthic samples, viruses were highly concentrated, but paradoxically, no bacteria were visibly infected. This suggests that sediment provides good conditions for virus preservation but ironically is an unfavorable environment for proliferation. In addition, given the comparable size distributions of viruses in the water and sediment samples, our results support the paradigm that aquatic viruses are ubiquitous and may have moved between the two compartments of the shallow systems examined. Overall, this study provides additional information about the relevance of viruses in tropical areas and indicates that the intensity of virus-bacterium interactions in benthic habitats may lower than the intensity in the adjacent bodies of water.     

Virus-Bacterium Interactions in Water and Sediment of West African Inland Aquatic Systems

The ecology of virioplankton in tropical aquatic ecosystems is poorly documented, and in particular, there are no references concerning African continental waters in the literature. In this study, we examined virus-bacterium interactions in the pelagic and benthic zones of seven contrasting shallow inland waters in Senegal, including one hypersaline lake. SYBR Gold-stained samples revealed that in the surface layers of the sites, the numbers of viruses were in the same range as the numbers of viruses reported previously for productive temperate systems. Despite high bacterial production rates, the percentages of visibly infected cells (as determined by transmission electron microscopy) were similar to the lowest percentages (range, 0.3 to 1.1%; mean, 0.5%) found previously at pelagic freshwater or marine sites, presumably because of the local environmental and climatic conditions. Since the percentages of lysogenic bacteria were consistently less than 8% for pelagic and benthic samples, lysogeny did not appear to be a dominant strategy for virus propagation at these sites. In the benthic samples, viruses were highly concentrated, but paradoxically, no bacteria were visibly infected. This suggests that sediment provides good conditions for virus preservation but ironically is an unfavorable environment for proliferation. In addition, given the comparable size distributions of viruses in the water and sediment samples, our results support the paradigm that aquatic viruses are ubiquitous and may have moved between the two compartments of the shallow systems examined. Overall, this study provides additional information about the relevance of viruses in tropical areas and indicates that the intensity of virus-bacterium interactions in benthic habitats may lower than the intensity in the adjacent bodies of water.     

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.     

土壤含水量驱动土壤病毒和细菌多度及其与土壤异养呼吸关系的变化

土壤微生物是维持陆地生态系统稳定性和功能的重要组成部分。病毒是地球上数量最多的生物实体,也是若干类型生境中微生物数量的重要调节者。因此,了解病毒与微生物的相互作用,对深入认识包括碳循环在内的生态系统过程具有重要意义。在实验室建立土壤微宇宙实验系统,跟踪调查恒定低含水量、恒定高含水量和波动含水量3种水分处理下土壤病毒和细菌多度的变化,以及土壤异养呼吸速率对土壤病毒-细菌相互作用的响应。相较于低水分处理,高水分处理显著增加了病毒多度（P<0.001）和病毒-细菌多度比（P=0.0026）,波动水分处理显著增加了病毒多度（P<0.001）。在高水分处理的土壤微宇宙中,细菌和病毒多度呈现出随时间动荡的信号,即细菌多度表现出增加-降低-增加的趋势,而病毒多度则表现出增加-降低的趋势,且其变化滞后于细菌。土壤异养呼吸速率与土壤含水量（P<0.001）、细菌多度（P=0.0045）和病毒多度（P<0.001）都具有显著的正相关关系。这些结果说明：病毒导致的下行控制可能是细菌多度的重要影响因子,在水分增加情形下,病毒有可能通过加速细菌的更新速率进而加速土壤呼吸。因此,病毒与细菌的相互作用可能是碳循环的重要决定因素。     

Significance of Bacteriophages for Controlling Bacterioplankton Growth in a Mesotrophic Lake

Bacterium-specific viruses have attracted much interest in aquatic microbial ecology because they have been shown to be about 10 times more abundant than planktonic bacteria. So far most of the studies of interactions of planktonic bacteria and viruses have been done in marine environments, and very little is known about these interactions in lakes. Therefore, we studied phage proliferation in Lake Constance, a large mesotrophic lake in Germany. We enumerated bacteria and quantified the fraction of bacteria with mature intracellular phage particles and the number of free viruses by transmission electron microscopy. Between the end of March and early August 1992, peaks of bacterial abundance were followed in 1 to 2 weeks by peaks in the fraction of bacteria containing visible phage particles (0 to 1.7%) and in the number of free viruses (1 x 10(sup7) to 4 x 10(sup7) ml(sup-1)). We estimated that 1 to 17% +/- 12% of all bacteria were phage infected, implying that phage-induced mortality was <34% +/- 24% of total mortality. A direct comparison between phage-induced mortality, the net decrease of bacterial numbers, and bacterial growth rates indicated that phage-induced mortality accounted for <11% of total bacterial mortality during the phytoplankton spring bloom and 18 to 21% following the bloom. Estimated burst sizes ranged from 21 to 121 phages. Phage production rates of 0.5 x 10(sup6) to 2.5 x 10(sup6) ml(sup-1) day(sup-1) accounted for 70 to 380% of the observed net increase rates of free phages, implying high rates of simultaneous phage decay. The cyclic dynamics between bacteria and phages and the varying size structure of the intracellular mature phage particles suggested that phage infection was important in structuring the bacterial host assemblage during the study period.     

Response of Bacterial Metabolic Activity to Riverine Dissolved Organic Carbon and Exogenous Viruses in Estuarine and Coastal Waters: Implications for CO2 Emission

A cross-transplant experiment between estuarine water and seawater was conducted to examine the response of bacterial metabolic activity to riverine dissolved organic carbon (DOC) input under virus-rich and virus-free conditions, as well as to exogenous viruses. Riverine DOC input increased bacterial production significantly, but not bacterial respiration (BR) because of its high lability. The bioavailable riverine DOC influenced bulk bacterial respiration in two contrasting ways; it enhanced the bulk BR by stimulating bacterial growth, but simultaneously reduced the cell-specific BR due to its high lability. As a result, there was little stimulation of the bulk BR by riverine DOC. This might be partly responsible for lower CO₂ degassing fluxes in estuaries receiving high sewage-DOC that is highly labile. Viruses restricted microbial decomposition of riverine DOC dramatically by repressing the growth of metabolically active bacteria. Bacterial carbon demand in the presence of viruses only accounted for 7–12% of that in the absence of viruses. Consequently, a large fraction of riverine DOC was likely transported offshore to the shelf. In addition, marine bacteria and estuarine bacteria responded distinctly to exogenous viruses. Marine viruses were able to infect estuarine bacteria, but not as efficiently as estuarine viruses, while estuarine viruses infected marine bacteria as efficiently as marine viruses. We speculate that the rapid changes in the viral community due to freshwater input destroyed the existing bacteria-virus relationship, which would change the bacterial community composition and affect the bacterial metabolic activity and carbon cycling in this estuary.     

Arsenic resistance and removal by marine and non-marine bacteria

Arsenic resistance and removal was evaluated in nine bacterial strains of marine and non-marine origins. Of the strains tested, Marinomonas communis exhibited the second-highest arsenic resistance with median effective concentration (EC(50)) value of 510 mg As l(-1), and was capable of removing arsenic from culture medium amended with arsenate. Arsenic accumulation in cells amounted to 2290 microg As g(-1) (dry weight) when incubated on medium containing 5 mg As l(-1) of arsenate. More than half of the arsenic removed was related to metabolic activity: 45% of the arsenic was incorporated into the cytosol fraction and 10% was found in the lipid-bound fraction of the membrane, with the remaining arsenic considered to be adsorbed onto the cell surface. Potential arsenic resistance and removal were also examined in six marine and non-marine environmental water samples. Of the total bacterial colony counts, 28-100% of bacteria showed arsenic resistance. Some of the bacterial consortia, especially those from seawater enriched with arsenate, exhibited higher accumulated levels of arsenic than M. communis under the same condition. These results showed that arsenic resistant and/or accumulating bacteria are widespread in the aquatic environment, and that arsenic-accumulating bacteria such as M. communis are potential candidates for bioremediation of arsenic contaminated water.     

Characterizing aquatic bacteria according to population, cell size, and apparent DNA content by flow cytometry

Flow cytometry offers a rapid method for characterizing aquatic populations according to the properties of individual cells. This technology has been extended to aquatic bacteria by using high-intensity UV excitation, condensing the laser beam onto a small area, using blemish-free flow cells, optimizing organism staining protocol, segregating the optical signal produced with high-transmittance optical filters, collecting the signal with sensitive photomultipliers, and expanding the range of data displayed from individual samples with calibrated circuitry. Bacteria could be counted according to event frequency, and populations agreed with direct counts by epifluorescence microscopy. Forward scatter intensity was a linear function of volume for bacterial cells between 1.3 and 0.25 micron 3 as calibrated by Coulter impedance. Plastic spheres down to 0.014 micron 3, 0.3 micron in diameter, were resolved. Aquatic bacteria 0.05 micron 3 in volume were clearly resolved according to DNA content by staining with DAPI. The observed signal was DNA-dependent because DNase treatment eliminated most fluorescence. These procedures are suitable for direct analysis of the bacteria in marine and freshwater samples without interference from algae, sediment, or most DNA-free organic particles. Cytograms indicated one or more clearly resolved subpopulations of bacteria of substantially smaller size and DNA content than the laboratory organisms typically classified.     

Bacteria and viruses in the water column of tropical freshwater reservoirs

In tropical freshwater reservoirs of Sri Lanka, which are linked in an aquatic network, bacterial abundance and production as well as virus abundance, frequency of viral infection and virus production were investigated together with a set of nutrient species (Kjeldahl-N, NO3-N, total P, soluble P, PO4-P). At two characteristic seasons (wet season, dry season), samples were taken from two types of reservoirs (new upland impoundment and ancient, shallow lowland reservoir), each during 4 days at various depths of the entire water columns. Kjeldahl-N and total P were greatly elevated in the wind-mixed water body of the shallow impoundment during the dry season, whereas the deeper reservoir type exhibited no obvious seasonality. In SYBR green trade mark -stained samples, bacterial abundance showed no seasonal pattern in either reservoir type. Bacterial secondary production, however, was significantly elevated in the entire water column of the shallow impoundment under wind-mixed conditions in the dry season. Highest abundance of virus particles and elevated frequency of bacteria containing mature phages were also observed in the shallow reservoir during the dry season indicating favourable conditions for virus propagation. Data from this aquatic network show that most virus parameters, such as abundance or frequency of visibly infected cells, were positively linked to bacterial abundance and production, but also to organic nitrogen or some phosphorus species. We calculated that between 13.2% and 46.1% of the bacterial standing stocks would be subjected to virus-mediated mortality. Estimates of bacteriophage production revealed that from 10 x 10(9) up to 98 x 10(9) phages were produced per litre and day. Bacteria and viruses in the studied tropical freshwater system appear to be linked to various environmental conditions and may affect processes at the ecosystem scale.     

Method for enumeration of 5-cyano-2,3-ditoyl tetrazolium chloride (CTC)-active cells and cell-specific CTC activity of benthic bacteria in riverine, estuarine and coastal sediments

Bacteria are the most abundant and active organisms in marine sediments and are critical for nutrient cycling and as a food source to many benthic and pelagic organisms. Bacteria are found both as free-living cells and as particle-associated cells, which can make investigations of these communities difficult. We found that common procedures for extracting bacteria from sediments leave the bacteria clay particle-associated and the clay particles clump, which reduce the reproducibility of direct counts. We optimized a sonication/surfactant method that produces a homogeneous suspension of bacterial cells against a uniform background of clay particles, which results in reproducible samples for epifluorescence microscopy. We developed a method to estimate CTC-positive cells and cell-specific CTC content in intact cores of surficial sediment communities from riverine, estuarine and coastal sites. Benthic bacterial abundances averaged 4.9x10(8) cells/g dry wt sediments in Apalachicola River, Florida sediments, 4.9-13.8x10(9) cells/g dry wt sediments in a variety of Apalachicola Bay sediments and 3.6x10(8) cells/g dry weight in shallow, anoxic Gulf of Mexico sediments. Percent CTC-positive cells ranged from low values of 9-10% CTC-positive cells in Apalachicola River and Apalachicola Bay sediments to high values of 25% CTC-positive cells in anoxic Gulf of Mexico sediments. After correction for abiotic CTC reduction and chlorophyll interference, estimates of cell-specific CTC reduction ranged from 0.15 to 0.55 fmol CTC(red)/active cell in the Apalachicola Bay sediments to 1.6 to 3.8 fmol CTC(red)/active cell in anoxic Gulf of Mexico sediments.     

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.     

Distribution of viruses in the Chesapeake Bay.

High virus counts were found in water samples collected from the Chesapeake Bay. Viruses were enumerated by ultracentrifugation of water samples onto grids which were visualized by transmission electron microscopy. Virus counts in September 1990, April 1991, June 1991, August 1991, and October 1991 ranged between 2.6 x 10(6) and 1.4 x 10(8) viruses ml-1 with a mean of 2.5 x 10(7) viruses ml-1. Virus counts were usually at least three times higher than direct bacterial counts in corresponding samples. Virus counts in August and October were significantly higher than at the other sampling times, whereas bacterial counts were significantly lower at that time, yielding mean virus-to-bacterium ratios of 12.6 and 25.6, respectively. From analysis of morphology of the virus particles, it is concluded that a large proportion of the viruses are bacteriophages. The high virus counts obtained in this study suggest that viruses may be an important factor affecting bacterial populations in the Chesapeake Bay, with implications for gene transfer in natural aquatic bacterial populations and release of genetically engineered microorganisms to estuarine and coastal environments.