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Applied and Environmental Microbiology, January 1999, p. 45-52, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Enumeration of Marine Viruses in Culture and Natural Samples by Flow Cytometry

Dominique Marie,1,* Corina P. D. Brussaard,2 Runar Thyrhaug,2 Gunnar Bratbak,2 and Daniel Vaulot1

Station Biologique, CNRS, INSU and Université Pierre et Marie Curie, 29682 Roscoff cedex, France,1 and Department of Microbiology University of Bergen, N-5020 Bergen, Norway2

Received 26 May 1998/Accepted 6 October 1998

Flow cytometry (FCM) was successfully used to enumerate viruses in seawater after staining with the nucleic acid-specific dye SYBR Green-I. The technique was first optimized by using the Phaeocystis lytic virus PpV-01. Then it was used to analyze natural samples from different oceanic locations. Virus 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°C in the presence of detergent before analysis. Virus counts obtained by FCM 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 profile from the Mediterranean Sea revealed that the abundance of viruses displayed the same vertical trend as that of planktonic cells. FCM permits us to distinguish between at least two and sometimes three virus populations in natural samples. Because of its speed and accuracy, FCM should prove very useful for studies of virus infection in cultures and should allow us to better understand the structure and dynamics of virus populations in natural waters.

* Corresponding author. Mailing address: Station Biologique, Place Georges Tessier, BP 74, F-29680 Roscoff, France. Phone: 33 (2) 98 29 23 14. Fax: 33 (2) 98 29 23 24. E-mail: marie{at}sb-roscoff.fr.

Applied and Environmental Microbiology, January 1999, p. 45-52, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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