Optimization of extraction and estimation of viruses in silty freshwater sediments

The present study focused on the optimization of procedures for the extraction of viruses from silty freshwater sediments for subsequent enumeration. Viral abundance in 2 different shallow backwater systems of the River Danube (Austria) ranged from 1.45 x 10 super(9) to 9.58 x 10 super(9) particles ml super(-1) wet sediment. The highest virus yields from the bulk of the sediments were obtained by 1 min sonication (3 x 20 s intervals, with 10 s interruptions). An increase in sonication time of up to 5 min decreased viral counts by an average of 15%. Since dissolved DNA within sediment samples could bind to the nucleic acid stain and thereby inflate viral estimates, sediment samples are often treated with DNase before the staining procedure. Moreover, they are usually centrifuged and diluted to a high extent in order to avoid interference of particulate material with virus counting. Centrifugation led to a reduction of viral numbers by 2 to 36% compared to untreated samples and did not reduce the background fluorescence; thus counting of viruses was not facilitated. Diluting 2000x with Milli-Q water always provided an average of 19% lower viral numbers than diluting 4000x. Treatment with DNase had no significant effect on virus counting, with viral numbers in untreated samples being on average 96% of those in DNase-containing samples. Additionally, 2 different nucleic acid stains were compared: viruses stained with SYBR Gold fluoresced brighter than those stained with SYBR Green I and fluorescence lasted longer, while background fluorescence was reduced sufficiently, thus facilitating virus counting. Viral numbers using SYBR Gold were on average twice of those obtained with SYBR Green I. The mean efficiency of virus extraction was 88.8% using the protocol outlined in this paper, and was thus slightly higher than that obtained in previous sediment investigations.