Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR gold

A novel nucleic acid stain, SYBR Gold, was used to stain marine viral parti cles in various types of samples. Viral particles stained with SYBR Gold yi elded bright and stable fluorescent signals that could be detected by a coo led charge-coupled device camera or by how cytometry. The fluorescent signa l 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 digit al image analysis software. Estimates of viral concentration based on digit ized images were 1.3 times higher than those based on direct counting by ep ifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-staine d viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stai ned with SYBR Gold formed a distinct population in flow cytometric signatur es. Flow cytometric analysis revealed at least four viral subpopulations fo r a Lake Erie sample and two subpopulations for a Georgia coastal sample. F low 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 accu rate measurement of viral abundance in aquatic environments is discussed.