Enumeration of marine viruses in culture and natural samples by flow cytometry

Flow cytometry (FCM) was successfully used to enumerate viruses in seawater after staining with the nucleic acid-specific dye SYBR Green-I. The techni que was first optimized by using the Phaeocystis lytic virus PpV-01. Then i t was used to analyze natural samples from different oceanic locations. Vir us samples were fixed with 0.5% glutaraldehyde and deep frozen for delayed analysis. The samples were then diluted in Tris-EDTA buffer and analyzed in the presence of SYBR Green-I. A duplicate sample was heated at 80 degrees C in the presence of detergent before analysis. Virus counts obtained by FC M were highly correlated to, although slightly higher than, those obtained by epifluorescence microscopy or by transmission electron microscopy (r = 0 .937, n = 14, and r = 0.96, n = 8, respectively). Analysis of a depth profi le from the Mediterranean Sea revealed that the abundance of viruses displa yed the same vertical trend as that of planktonic cells. FCM permits us to distinguish between at least two and sometimes three virus populations in n atural samples. Because of its speed and accuracy, FCM should prove very us eful for studies of virus infection in cultures and should allow us to bett er understand the structure and dynamics of virus populations in natural wa ters.