Separation of free virus particles from sediments in aquatic systems
Full Citation: Danovaro, R., and M. Middelboe. 2010. Separation of free virus particles from sediments in aquatic systems, p. 74-81. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. [DOI 10.4319/mave.2010.978-0-9845591-0-7.74]
ABSTRACT: The number of benthic viruses per unit of volume, at all depths (from shallow down to abyssal sediments), exceeds water column abundances by orders of magnitude. The need of methods for the determination of viral counting in aquatic sediment is becoming increasingly urgent along with the increasing evidence of the relevance of viruses in the benthic domain. The procedures used for determining viral abundances in sediments require specific modifications to release the viruses from sediment particles and to minimize the physical and chemical interferences of sedimentary matrix with the analysis. Dislodging viruses from sediment samples is the first crucial step for the analyses of viral abundance in benthic samples. Here we present the results of several tests aimed at optimizing the protocol for viral counts based on (i) the chemical treatment (type and modality of the use of surfactants), (ii) mechanical treatment (ultrasounds), (iii) cleaning of the samples (by enzymatic digestion of the extracellular DNA by means of DNases), and (iv) the limitations associated with viral recovery from the sediment (by serial washing steps). Sediment texture and composition vary considerably along horizontal and vertical gradients, and here we compare shallow sandy sediments with more silty deep-sea sediments. We found that the use of the surfactant tetrasodium pyrophosphate (final concentration 5 mM for 15 min), followed by ultrasound treatments (3 times for 1 min with 30 s intervals) and by the addition of an enzymatic cocktail composed of DNase I, nuclease P1, nuclease S1, and esonuclease 3, increased the detectability by staining with fluorochrome, thus resulting in significantly higher and more accurate viral counting, determined by epifluorescence microscopy. Our results also indicate that sediment samples processed using this optimized protocols displayed a significantly lower coefficient of variation, thus making sufficient the counting of a lower number of optical fields. Centrifugation of sediment samples after extraction procedures could underestimate viral counting, and we recommend here an accurate check of the potential loss or an alternative procedure based on sediment dilution prior to quantification by epifluorescence microscopy.