Hennes, K.P. and C.A. Suttle. 1994. The use of cyanine dyes for quantifying free viruses in natural water samples by epifluorescent microscopy. Abstracts American Society Limnology and Oceanography, and Phycological Society American, Miami, FL, June 1994.
We developed a method to quantify viruses in aquatic samples using epifluorescence microscopy and the monomeric cyanine dye "Yo-ProTM-1". The dye has high binding affinity for nucleic acids and a high quantum yield. It fluoresces green when bound to double or single stranded DNA or RNA, and yellow when bound to detritus. Viruses were filtered onto 0.02-µm pore-size AnoporeTM membranes, stained with Yo-Pro, and quantified by epifluorescence microscopy using a standard Acridine Orange filter set. Small bacteria and viruses could be easily discriminated as they differed markedly in fluorescence intensity. Prepared samples could be stored indefinitely at -20°C. The average coefficient of variation ranged from 0.10 to 0.20 for counts on samples from virus cultures and natural virus communities, respectively. A comparison of our method with TEM-based protocols showed that electron microscopy underestimated the abundance of viruses in pure cultures by ca. 20%. For natural samples containing a relatively high concentration of detrital particles our results suggest that as few as 12% of the viruses counted using Yo-Pro could be enumerated by TEM. We also compared our method with DAPI-based protocols and found that the Yo-Pro method was far superior for enumerating viruses. The high counting efficiency, ease of preparation and modest equipment requirements makes this method ideal for ship-board use and routine environmental analysis.