Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy

Abstract

Epifluorescent microscopy was used to determine the abundance of viruses in samples from marine and freshwater environments and in laboratory cultures that were filtered onto 0.02‐ µ m pore‐size filters and stained with a cyanine‐based dye (Yo‐Pro‐1). Estimates of viral abundance based on Yo‐Pro‐stained samples were 1.2–7.1 times greater than estimates obtained with transmission electron microscopy (TEM). Moreover, the precision of the Yo‐Pro‐based method was much greater than that for TEM (C.V. 7% vs. 20%, respectively). DNase treatment of samples did not result in lower numbers of particles that could be stained by Yo‐Pro, suggesting that the fluorescence was not the result of nucleic acids associated with the surface of particles. These results indicate that the concentration of viruses in natural waters may be higher than previously recognized and imply that the TEM‐based method significantly underestimates virus abundance. Virus abundances ranged from 10 7 −> 10 8 ml −1 in surface waters along a transect in the western Gulf of Mexico to 10 9 ml −1 in water overlying a submerged cyanobacterial mat. High counting efficiency, ease of preparation, modest equipment requirements, and the possibility of preparing specimens for long‐term storage, make the Yo‐Pro‐based method ideal for routine environmental analysis.