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Applied and Environmental Microbiology, February 2001, p. 539-545, Vol. 67, No. 2
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.2.539-545.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Application of Digital Image Analysis and Flow Cytometry To Enumerate Marine Viruses Stained with SYBR Gold†

Feng Chen,1,* Jing-rang Lu,2 Brian J. Binder,2 Ying-chun Liu,2 and Robert E. Hodson2

Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202,1 and Department of Marine Sciences, University of Georgia, Athens, Georgia 306022

Received 29 June 2000/Accepted 16 November 2000

A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.

* Corresponding author. Mailing address: Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 East Pratt St., Suite 236, Baltimore, MD 21202. Phone: (410) 234-8866. Fax: (410) 234-8896. E-mail: chenf{at}umbi.umd.edu.

dagger Contribution no. 542 from the Center of Marine Biotechnology, University of Maryland Biotechnology Institute.

Applied and Environmental Microbiology, February 2001, p. 539-545, Vol. 67, No. 2
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.2.539-545.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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