An Advanced Protocol for the Quantification of Marine Sediment Viruses via Flow Cytometry

Viruses are highly abundant, diverse, and active components of marine environments. Flow cytometry has helped to increase the understanding of their impact on shaping microbial communities and biogeochemical cycles in the pelagic zone. However, to date, flow cytometric quantification of sediment viruses is still hindered by interference from the sediment matrix. Here, we developed a protocol for the enumeration of marine sediment viruses by flow cytometry based on separation of viruses from sediment particles using a Nycodenz density gradient. Results indicated that there was sufficient removal of background interference to allow for flow cytometric quantification. Applying this new protocol to deep-sea and tidal-flat samples, viral abundances enumerated by flow cytometry correlated well (R(2) = 0.899) with counts assessed by epifluorescence microscopy over several orders of magnitude from marine sediments of various compositions. Further optimization may be needed for sediments with low biomass or high organic content. Overall, the new protocol enables fast and accurate quantification of marine sediment viruses, and opens up the options for virus sorting, targeted viromics, and single-virus sequencing.