Efficient method to isolate and purify viruses of bacteria from marine environments (original) (raw)
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International Microbiology, 1999
A method for concentrating, enumerating and isolating viral particles from marine water samples was developed and evaluated. The method consists of a concentration step by a tangential flow filtration (TFF) system, ultrafiltration by centrifugal concentrator, and visualization by transmission electron microscopy (TEM). This procedure allows to reduce volumes of ca. 2 l of seawater to 10-20 µl, which can be dispensed on electron microscopy grids to count total viral particles. This method allows the recovery of small numbers of viral particles from oligotrophic seawater samples, in which viral numbers ranged from 10 5 to 10 6 viral particles/ml. The tangential flow filtration system was evaluated as quantitative technique using suspensions of two different bacteriophages (T6 and φX174) in autoclaved seawater. Recovery rates varied depending on both the viral morphology and flow rate; recovery percentages reached 117.4% for T6 and 60.6% for φX174 using low flow rate.
Scientific reports, 2015
Free living viruses are ubiquitous in marine waters and concentrations are usually several times higher than the bacterial abundance. These viruses are capable of lysing host bacteria and therefore, play an important role in the microbial loop in oligotrophic waters. However, few studies have been conducted to compare the role of viruses in regulating bacterial abundance and heterotrophic activities between natural oligotrophic waters and anthropogenic influenced eutrophic waters. In this study, we examined viral effects on bacterial functions of four single bacterial species incubated with natural viral assemblages in seawater samples from eutrophic and oligotrophic waters. The viral-lysis of bacteria was significantly higher in eutrophic than oligotrophic waters. This suggests that viruses were capable of controlling bacterial abundance, respiration and production in the eutrophic waters. Cellular bacterial respiration and production was higher with viruses than without viruses, w...
Impact of viruses on bacterial communities in marine systems
2009
Memoria de tesis doctoral presentada por Julia Anna Boras para optar al grado de Doctora por la Universitat de Barcelona (UB), realizada bajo la direccion de la Dra. Ma Dolors Vaque Vidal y la Dra. Maria Montserrat Sala Farre del Institut de Ciencies del Mar (ICM-CSIC) y la Dra. Montserrat Vidal.-- 222 pages
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.
Journal of Virological Methods, 2008
A novel and simple procedure for concentrating adenoviruses from sea water samples is described. The technique entails the adsorption of viruses to pre-flocculated skimmed milk proteins, allowing the flocs to sediment by gravity, and dissolving the separated sediment in phosphate buffer. Concentrated virus may be detected by PCR techniques following nucleic acid extraction. The method requires no specialized equipment other than that usually available in routine public health laboratories, and due to its straightforwardness it allows the processing of a larger number of water samples simultaneously. The usefulness of the method was demonstrated in concentration of virus in multiple seawater samples during a survey of adenoviruses in coastal waters.
Viruses in Marine Planktonic Systems
Oceanography, 1993
The last 10-15 years have seen major changes in our views of marine planktonic food webs, primarily from the realization that prokaryotic microorganisms and small eukaryotes are responsible for a significant fraction, often 50% or more, of the primary production and heterotrophic consumption of organic matter in these systems (Williams, 1981: Azam et al., 1983: Stockner and Antia, 1986: Fuhrman, 1992).