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.     

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     

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.     

Does virus-induced lysis contribute significantly to bacterial mortality in the oxygenated sediment layer of shallow oxbow lakes?

Despite the recognition that viruses are ubiquitous components of aquatic ecosystems, the number of studies on viral abundance and the ecological role of viruses in sediments is scarce. In this investigation, the interactions between viruses and bacteria were studied in the oxygenated silty sediment layer of a mesotrophic oxbow lake. A long-term study (13 months) and a diel study revealed that viruses are a numerically important and dynamic component of the microbial community. The abundance and decay rates ranged from 4.3 x 10(9) to 7.2 x 10(9) particles ml of wet sediment(-1) and from undetectable to 22.2 x 10(7) particles ml(-1) h(-1), respectively, and on average the values were 2 orders of magnitude higher than the values for the overlying water. In contrast to our expectations, viruses did not contribute significantly to the bacterial mortality in the sediment, since on average only 6% (range, 0 to 25%) of the bacterial secondary production was controlled by viruses. The low impact of viruses on the bacterial community may be associated with the quantitatively low viral burden that benthic bacteria have to cope with compared to the viral burden with which bacterial assemblages in the water column are confronted. The virus-to-bacterium ratio of the sediment varied between 0.9 and 3.2, compared to a range of 5.0 to 12.4 obtained for the water column. We speculate that despite high numbers of potential hosts, the possibility of encountering a host cell is limited by the physical conditions in the sediment, which is therefore not a favorable environment for viral proliferation. Our data suggest that viruses do not play an important role in the processing and transfer of bacterial carbon in the oxygenated sediment layer of the environment investigated.     

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.     

Contrasting distributions of bacteriophages and eukaryotic viruses from contaminated coastal sediments

Viruses are ubiquitous, abundant and play an important role in all ecosystems. Here, we advance understanding of coastal sediment viruses by exploring links in the composition and abundance of sediment viromes to environmental stressors and sediment bacterial communities. We collected sediment from contaminated and reference sites in Sydney Harbour and used metagenomics to analyse viral community composition. The proportion of phages at contaminated sites was significantly greater than phages at reference sites, whereas eukaryotic viruses were relatively more abundant at reference sites. We observed shifts in viral and bacterial composition between contaminated and reference sites of a similar magnitude. Models based on sediment characteristics revealed that total organic carbon in the sediments explained most of the environmental stress-related variation in the viral dataset. Our results suggest that the presence of anthropogenic contaminants in coastal sediments could be influencing viral community composition with potential consequences for associated hosts and the environment.     

Does Virus-Induced Lysis Contribute Significantly to Bacterial Mortality in the Oxygenated Sediment Layer of Shallow Oxbow Lakes?

Despite the recognition that viruses are ubiquitous components of aquatic ecosystems, the number of studies on viral abundance and the ecological role of viruses in sediments is scarce. In this investigation, the interactions between viruses and bacteria were studied in the oxygenated silty sediment layer of a mesotrophic oxbow lake. A long-term study (13 months) and a diel study revealed that viruses are a numerically important and dynamic component of the microbial community. The abundance and decay rates ranged from 4.3 × 10⁹ to 7.2 × 10⁹ particles ml of wet sediment⁻¹ and from undetectable to 22.2 × 10⁷ particles ml⁻¹ h⁻¹, respectively, and on average the values were 2 orders of magnitude higher than the values for the overlying water. In contrast to our expectations, viruses did not contribute significantly to the bacterial mortality in the sediment, since on average only 6% (range, 0 to 25%) of the bacterial secondary production was controlled by viruses. The low impact of viruses on the bacterial community may be associated with the quantitatively low viral burden that benthic bacteria have to cope with compared to the viral burden with which bacterial assemblages in the water column are confronted. The virus-to-bacterium ratio of the sediment varied between 0.9 and 3.2, compared to a range of 5.0 to 12.4 obtained for the water column. We speculate that despite high numbers of potential hosts, the possibility of encountering a host cell is limited by the physical conditions in the sediment, which is therefore not a favorable environment for viral proliferation. Our data suggest that viruses do not play an important role in the processing and transfer of bacterial carbon in the oxygenated sediment layer of the environment investigated.     

Bacterial Diversity in Shallow Oligotrophic Marine Benthos and Overlying Waters: Effects of Virus Infection,Containment, and Nutrient Enrichment

Little is known of the factors shaping sediment bacterial communities, despite their high abundance and reports of high diversity. Two factors hypothesized to shape bacterial communities in the water column are nutrient (resource) availability and virus infection. The role these factors play in benthic bacterial diversity was assessed in oligotrophic carbonate–based sediments of Florida Bay (USA). Sediment–water mesocosm enclosures were made from 1-m diameter clear polycarbonate cylinders which were pushed into sediments to 201 cm sediment depth enclosing 80 L of water. Mesocosms were amended each day for 14 d with 10 µM NH ₄ ⁺ and 1 µM PO ₄ ^3– . In a second experiment, viruses from a benthic flocculent layer were concentrated and added back to flocculent layer samples which were collected near the mesocosm enclosures. Photosynthesis by microalgae in virus-amended incubations was monitored by pulse-amplitude modulated (PAM) fluorescence. In both experiments, bacterial diversity was estimated using automated rRNA intergenic spacer analysis (ARISA), a high-resolution fingerprinting approach. Initial sediment bacterial operational taxonomic unit (OTU) richness (236 ± 3) was higher than in the water column (148 ± 9), where an OTU was detectable when its amplified DNA represented >0.09% of the total amplified DNA. Effects on bacterial diversity and operational taxonomic unit (OTU) richness in nutrient-amended mesocosms may have been masked by the effects of containment, which stimulated OTU richness in the water column, but depressed OTU richness and diversity in sediments. Nutrient addition significantly elevated virus abundance and the ratio of viruses to bacteria (p < 0.05 for both) in the sediments, concomitant with elevated bacterial diversity. However, water column bacterial diversity (in unamended controls) was not affected by nutrient amendments, which may be due to rapid nutrient uptake by sediment organisms or adsorption of P to carbonate sediments. Addition of live viruses to benthic flocculent layer samples increased bacterial OTU diversity and richness compared with heat-killed controls; however, cluster analyses showed that the community structure in the virus-amended mesocosms varied greatly between replicates. Despite the strong effects upon eubacterial communities, photosynthesis of co-occurring protists and cyanobacteria was not significantly altered by the presence of virus concentrates. This study supports the hypothesis that nutrient availability plays a key role in shaping sediment bacterial communities, and also that viruses may regulate the abundance of the dominant competitors and allow less dominant organisms to maintain or increase their abundance in a community due to decreased competition for resources.     

Importance of Micromonospora spp. as colonizers of cellulose in freshwater lakes as demonstrated by quantitative reverse transcriptase PCR of 16S rRNA

The relative abundance of micromonosporas in the bacterial communities inhabiting cellulose baits, water columns, and sediments of two freshwater lakes was determined by quantitative PCR (qPCR) of reverse-transcribed 16S rRNA. Micromonospora spp. were shown to be significant members of the active bacterial population colonizing cellulosic substrates in the lake sediment, and their increased prevalence with greater depth was confirmed by enumeration of CFU.     

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.     

Effects of fish farming on microbial enzyme activities and densities: comparison between three Mediterranean sites

AIM: The effects of fish farming on microbial enzyme activities and heterotrophic bacterial density were investigated in three Mediterranean sites before and after the start of mariculture. METHODS AND RESULTS: Microbial activities were measured on water and sediment samples by using fluorogenic substrates specific for leucine aminopeptidase, beta-glucosidase and alkaline phosphatase (AP); bacterial counts were determined by Marine agar plates. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison of activity and abundance values obtained before and after the experiment showed that fish farming mainly affected the levels of microbial activities; they were significantly enhanced both in water and sediments, reaching an increase of 183.66 times for AP in Castellammare Gulf. After mariculture, no significant variations were recorded in heterotrophic bacterial density in the waters, while significant changes were observed in the sediments. Effects induced appeared to be extended not only to stations in which cages were located, but also to control sites far from the direct influence of fish farming.     

A disintegration method for direct counting of bacteria in clay-dominated sediments: dissolving silicates and subsequent fluorescent staining of bacteria

The masking of bacteria by abundant microparticles of the clay and silt fraction and cell losses due to sonication hampered direct enumeration of bacteria in sediments dominated by fine sediments. These problems can be circumvented by dissolving silicate fine particles using hydrofluoric acid and subsequent staining of bacteria by DTAF. The developed disintegration method partly replaces mechanical separation of bacteria from sediment particles by chemical disintegration of the silicates. Recovery efficiency ranged from 90% to 111% for different clays and clay-dominated sediments. Especially for the analysis of fine sediments and clays, this method circumvents both strong dilution of the sediment sample and harsh sonication. The method can also therefore be used in sediments where particle abundance is several orders of magnitude higher than bacterial abundance and simple dilution would not suffice in reliably counting bacteria.     

黄海海域海洋沉积物细菌多样性分析

【背景】海洋独特的环境造就了海洋生物的多样性,海洋沉积物中细菌对海洋环境具有至关重要的作用。【目的】研究陆地土壤和海洋沉积物间细菌群落相似性和差异性,以便更好地认识海洋细菌多样性,深入了解沉积物细菌在海洋环境中的潜在作用。【方法】从中国黄海海域及大连市大黑山脚下分别采集样品,以陆地土壤为对照,采用16SrRNA基因高通量测序技术分析海洋沉积物的细菌群落结构。【结果】海洋沉积物样品中芽孢杆菌纲(Bacilli)、鞘氨醇单胞菌属(Sphingomonas)和芽孢杆菌属(Bacillus)丰度高于陆地土壤样品;海洋沉积物中亚硝化单胞菌(unculturedbacterium f. Nitrosomonadaceae)和厌氧绳菌(uncultured bacterium f. Anaerolineaceae)丰度虽低于陆地土壤,但丰度值也均高于1%;样品分类学统计显示酸杆菌门(Acidobacteria)在海洋沉积物和陆地土壤样品中的序列丰度比例都较大,鞘氨醇单胞菌属(Sphingomonas)在海洋沉积物样品中的序列丰度大于陆地土壤样品。【结论】海洋沉积物细菌多样性可作为海洋环境恢复情况的重...     

Flow cytometric analysis of bacteria- and virus-like particles in lake sediments

Flow cytometry (FCM) was successfully used to analyze freshwater bacteria and viruses in lake sediments after relatively simple sample treatment and optimization of dilution/fixation/staining procedures. Biological particles from Lakes Geneva and Bourget were first separated from the sediments by using both Sodium Pyrophosphate (0.01 M final concentration) and Polyoxyethylene-Sorbitan Monooleate (10% final concentration) and sonicating for 3 min in a water bath. The best results (based on FCM signature and the highest virus and bacterial yields from the sediments) were obtained by formaldehyde fixation carried out within less than one hour (2% final concentration, vs. no fixation or using glutaraldehyde at different concentrations), SYBR-Green II staining (x1/20,000 stock solution concentration, vs. use of SYBR-Gold and SYBR-Green I dyes at different concentrations). There was a considerable loss of particles after only a few days of storage at either 4 or -22 degrees C. For FCM analysis, the samples were diluted in Tris-EDTA buffer (pH 8) and heated for 10 min at 75 degrees C after incubating for 5 min in the dark. The bacterial and viral counts paralleled those obtained using epifluorescence microscopy (EFM), but EFM always gave lower counts than FCM. Analysis of the distribution of the viruses in the water column and in the sediments of Lakes Bourget revealed a marked gradient, with larger quantities in the top layer of the sediment than in the water above it. These results are discussed, as well as the possible novel application of flow cytometry in the study of aquatic viral ecology.     

Application of a rapid direct viable count method to deep-sea sediment bacteria

For the first time, a Live/Dead (L/D) Bacterial Viability Kit (BacLight ) protocol was adapted to marine sediments and applied to deep-sea sediment samples to assess the viability (based on membrane integrity) of benthic bacterial communities. Following a transect of nine stations in the Fram Strait (Arctic Ocean), we observed a decrease of both bacterial viability and abundance with increasing water (1250-5600 m) and sediment depth (0-5 cm). Percentage of viable (and thus potentially active) cells ranged between 20-60% within the first and 10-40% within the fifth centimetre of sediment throughout the transect, esterase activity estimations (FDA) similarly varied from highest (13.3+/-5.4 nmol cm(-3) h(-1)) to lowest values below detection limit down the sediment column. Allowing for different bottom depths and vertical sediment sections, bacterial viability was significantly correlated with FDA estimations (p<0.001), indicating that viability assessed by BacLight staining is a good indicator for bacterial activity in deep-sea sediments. Comparisons between total L/D and DAPI counts not only indicated a complete bacterial cell coverage, but a better ability of BacLight staining to detect cells under low activity conditions. Time course experiments confirmed the need of a rapid method for viability measurements of deep-sea sediment bacteria, since changes in pressure and temperature conditions caused a decrease in bacterial viability of up to 50% within the first 48 h after sample retrieval. The Bacterial Viability Kit proved to be easy to handle and to provide rapid and reliable information. It's application to deep-sea samples in absence of pressure-retaining gears is very promising, as short staining exposure time is assumed to lessen profound adverse effects on bacterial metabolism due to decompression.     

The significance of viruses to mortality in aquatic microbial communities

A variety of approaches including enumeration of visibly infected microbes, removal of viral particles, decay of viral infectivity, and measurements of viral production rates have been used to infer the impact of viruses on microbial mortality. The results are surprisingly consistent and suggest that, on average, about 20% of marine heterotrophic bacteria are infected by viruses and 10–20% of the bacterial community is lysed daily by viruses. The effect of viruses on phytoplankton is less certain, but ca. 3% of Synechococcus biomass may be lysed daily. The fraction of primary productivity this represents depends upon the relative biomass and growth rate of Synechococcus. Virus enrichment experiments suggest that the productivity of eukaryotic phytoplankton would be ca. 2% higher in the absence of viruses. Overall, probably about 2–3% of primary productivity is lost to viral lysis. There is considerable variation about these estimates; however, they represent a starting point for incorporating viral-mediated processes into aquatic ecosystem models.     

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.     

Impact of hydrology on free-living and particle-associated microorganisms in a river floodplain system (Danube, Austria)

1. A floodplain segment of the Danube River downstream of Vienna was studied during the hydrologically most dynamic phase (spring–summer) to evaluate the significance of connection between the main channel and the floodplain segment for particle abundance and quality as well as for bacterial and viral parameters, both free‐living and attached to particles. 2. Hydrological connectivity between the main channel and its floodplain segment (expressed as water age) influenced particle abundance and quality. Polysaccharide‐containing particles [Alcian positive‐stained particles (ASP)] and protein‐containing particles [Coomassie positive‐stained particles (CSP)] each contributed a substantial fraction to total suspended solids and were both positively related to water age. ASP were about twice as abundant as CSP. 3. Water age influenced bacterial and viral abundance and the bacterioplankton productivity in the surrounding water. Free‐living bacterial abundance and their bacterial secondary production (BSP) increased continuously with water age, best described by a linear regression. Water age also significantly impacted BSP and per cell BSP of bacteria attached to particles. The abundance of attached bacteria and viruses was not influenced by water age. 4. Bacteria and viruses on particles were influenced by particle quality. Their abundance on particles was closely related to particle size. Particle‐attached bacteria accounted on average for 30.34% (± 3.09) of the total bacterial abundance. A variable and occasionally significant proportion of viruses, between 0.43% and 35.06%, were associated with particulate material. 5. Bacteria attached to particles were significantly more productive than their free‐living counterparts. Their per‐cell activity was on average 8.6 times higher than that of the free‐living fraction. 6. Hydrological connectivity between the Danube River and its floodplain is crucial not only for the exchange of water, sediment and nutrients, but also for microbiota, thus influencing microbial life, distribution and activity.     

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°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°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°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.