Abundance of viruses in marine waters: assessment by epifluorescence and transmission electron microscopy (original) (raw)
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Spatial distribution and activity of viruses in the deep-sea sediments of Sagami Bay, Japan
Deep Sea Research Part I: Oceanographic Research Papers, 2006
The vertical and horizontal heterogeneity in benthic viral and bacterial abundance were investigated at spatial scales from centimetres to hundreds of meters at 1450 m water depth in Sagami Bay, Japan. In the surface sediment layer (0-1 cm), viral and bacterial abundance ranged from 3 to 23 Â 10 8 virus-like particles (VLP) cm À3 and 3 to 13 Â 10 7 bacteria cm À3 , with an average virus-bacteria ratio (VBR) of 16.875. The variability in surface viral abundance at the investigated sites (n ¼ 38) almost covered the entire range of published VLP abundances for benthic environments, and a detailed statistical analysis showed no pattern in the spatial variability at scales between 5 mm and 150 m. Viral and bacterial abundance decreased gradually with depth to 0.7-3 Â 10 8 VLP cm À3 and 0.7-3 Â 10 7 cells cm À3 at 10 cm. A compilation of all abundance data (n ¼ 89) showed significant correlation between viral and bacterial abundance (po0.01), and an average area density of viruses in the upper 16 cm sediment of 5.172.3 Â 10 13 VLP m À2 seafloor. Investigations at a ''cold seep'' location showed significantly lower viral abundances (o1 Â 10 8 VLP cm À3 ) and VBR (o3) suggesting the existence of ''cold spots'' with reduced viral activity in an otherwise virus-rich environment. Viral production and microbial respiration were measured in homogenized, undiluted, anaerobic sediment incubations, which were manipulated with respect to temperature and organic carbon (OC) addition. Elevated temperature (14 1C) and OC addition stimulated the VLP production to a maximum of 3.6 Â 10 6 74.2 Â 10 5 VLP cm À3 h À1 (population doubling time: 2.7 days). Viral production in the sediment enclosures was positively correlated to the heterotrophic dissolved inorganic carbon (DIC) production suggesting that the production was regulated by the bacterial metabolism. Virus-induced bacterial mortality constituted 7-48% of bacterial production with a decrease in the relative importance of viral lysis with increasing bacterial production. The estimated input of viral lysates could explain o10% of total bacterial metabolism, thus suggesting that viruses had only minor impact on benthic carbon cycling at the investigated deep-sea sites. r
Mortality of marine bacteria in response to enrichments of the virus size fraction from seawater
Marine Ecology Progress Series, 1992
The potential for viral lysis of marine bacteria in seawater enriched with the virus size fraction from seawater was investigated in seawater samples from Long Island Sound, USA, the eastern Pacific Ocean and the Caribbean Sea. Ultrafiltration was used to concentrate material from seawater in the >0.05 pm to ~0. 2 2 pm size fract~on. Electron microscopy counts of virus-like particles in the high molecular weight concentrate (HMWC) correlated closely to epifluorescence microscopy counts of <0.22 pm DAPI-positive, DNase-resistant particles of the HMWC. Acridine orange direct counts of bacterial abundances significantly declined (often to 1/2 or less of controls) when seawater was incubated with 4-to 16-fold enrichments of HMWC. Microwave treatment of the HMWC before addition to seawater virtually eliminated the declines in bacterial abundance. The combined evidence of the size range of particles, the heat lability of the HMWC and the presence of <0.22 pm DAPI-fluorescent, DNase-resistant particles by epifluorescence microscopy and abundant virus particles by electron microscopy suggests that a dominant bacterial mortality agent in the seawater concentrate was bacteriophage, although we could not rule out an effect of high molecular weight proteins.
Determination of Virus Abundance in Marine Sediments
Applied and Environmental Microbiology, 2001
In this study, we optimized procedures to enumerate viruses from marine sediments by epifluorescence microscopy using SYBR Green I as a stain. The highest virus yields from the bulk of the sediments were obtained by utilizing pyrophosphate and 3 min of sonication. The efficiency of extraction benthic viruses by pyrophosphate-ultrasound treatment was about 60% of the extractable virus particles. Samples treated with nucleases had increased virus counts, suggesting a masking effect of extracellular DNA. No significant differences were observed between virus counts obtained by epifluorescence microscopy and transmission electron microscopy. Both formaldehyde and glutaraldehyde gave significant reductions of virus counts after only 24 h of sediment storage, but no further loss occurred after 7 days.
1997
ABSTRACT: Transmission electron microscopy (TEM) and epifluorescence microscopy of DAPI and Yo-Pro-l stained samples were used to estimate viral abundance in natural communities along a transect from the oligotrophic central Gulf of Mexico to the productive near-shore waters at Port Aransas, Texas (USA). Estimates of viral abundance based on TEM averaged only 66%(range 26 to 108%) of those made uslng epifluorescence microscopy and the cyanine-based dye, Yo-Pro-l.
Abundance of viruses in deep oceanic waters
Marine Ecology Progress Series, 1996
Vertical distributions of bactena and viruses were Investigated in the oceanic stations located in subarctic (Stn A) and subtropical (Stn B) areas of the Paciflc using the direct count technique and transmission electron microscopy. Small DAPI-positive, virus-like particles (VLP) were found to be distributed throughout the water column down to 5000 m a t both of the stations. The abundance of VLP ranged from 38 X 10' ml-' a t 50 m depth to 0.6 X 10' ml-' at 5000 m depth a t Stn A. The ratio of VLP to bacteria-like particle (BLP) ranged from 1.1 to 7.4 a t Stn A and 1.0 to 8.7 at Stn B in the entire water column. The maximum ratio was recorded at Stn B from the deepest sample, collected at a depth of 5000 m. The electron microscopic investigation Indicated that the major proportion of VLP were probably viruses.
Microbial Ecology, 2001
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.
Dynamics of virus abundance in coastal seawater
FEMS Microbiology Ecology, 2000
The short term dynamics of virus abundance in coastal sea water was investigated by frequent sampling of open ecosystems and of water incubated in bottles in situ. Sampling intervals were 6-10 min. The viral abundance showed significant temporal fluctuations both in situ and in the bottles and it changed in some cases by a factor of 2-4 within lo-20 min. Laboratory incubations showed that production and release of viruses were not induced or stimulated by nutrient addition, high light intensities or transient increase in temperatures ca. 10°C. Our interpretation of these results is that they result from synchronous lysis and release of virus particles from bacterial hosts and a rapid disintegration of these particles when released in sea water.
Nature protocols, 2007
Viruses are the most abundant biological entities in aquatic environments, typically exceeding the abundance of bacteria by an order of magnitude. The reliable enumeration of virus-like particles in marine microbiological investigations is a key measurement parameter. Although the size of typical marine viruses (20-200 nm) is too small to permit the resolution of details by light microscopy, such viruses can be visualized by epifluorescence microscopy if stained brightly. This can be achieved using the sensitive DNA dye SYBR Green I (Molecular Probes-Invitrogen). The method relies on simple vacuum filtration to capture viruses on a 0.02-microm aluminum oxide filter, and subsequent staining and mounting to prepare slides. Virus-like particles are brightly stained and easily observed for enumeration, and prokaryotic cells can easily be counted on the same slides. The protocol provides an inexpensive, rapid (30 min) and reliable technique for obtaining counts of viruses and prokaryotes...