Enumeration of virus particles in aquatic or sediment samples by epifluorescence microscopy

Microbes and microbial processes are crucial and quantitatively important players in aquatic environments, and viruses as major agents of microbial mortality and nutrient cycling are a key component of aquatic systems. These roles have led to the need to routinely quantify viral abundance as the part of many investigations. Electron microscopy was first used to demonstrate high viral abundances in aquatic samples; by the mid-1990s, the greater accuracy and higher precision of estimates of viral abundance made by epifluorescence microscopy (EFM) were evident. Initially, DAPI (6-diamidino-2-phenylindole) was the stain used to enumerate virus particles in natural samples, but this dye was soon superseded by a new generation of much brighter fluorochromes. This article outlines detailed protocols for enumerating virus particles in aquatic or sediment samples using SYBR Green, SYBR Gold, and Yo-Pro-1. Each of these stains has advantages and disadvantages, but for natural water samples they produce indistinguishable estimates of viral abundance when the appropriate protocols are