Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria

Abstract

A new nucleic acid stain, SYBR Green I, can be used for the rapid and accurate determination of viral and bacterial abundances in diverse marine samples.We tested this stain with formalinpreserved samples of coastal water and also from depth profiles (to 800 m) from sites 19 and 190 km offshore, by filtering a few m1 onto 0.02 pm pore-size filters and staining for 15 min.Comparison of bacterial counts to those made with acridine orange (AO) and virus counts with those made by transmission electron microscopy (TEM) showed very strong correlations.Bacterial counts with A 0 and SYBR Green 1 were indistinguishable and almost perfectly correlated (r2 = 0.99).Virus counts ranged widely, from 0.03 to 15 X 10' virus ml-l.Virus counts by SYBR Green 1 were on the average higher than those made by TEM, and a SYBR Green 1 versus TEM plot yielded a regression slope of 1.28.The correlation between the two was very high with an value of 0.98.The precision of the SYBR Green I method was the same as that for TEM, with coefficients of variation of 2.9%.SYBR Green I stained viruses and bacteria are intensely stained and easy to distinguish from other particles with both older and newer generation epifluorescence microscopes.Detritus is generally not stained, unlike when the alternative dye YoPro I is used, so this approach may be suitable for sediments.SYBR Green I stained samples need no desalting or heating, can be fixed with formalin prior to filtration, the optimal staining time is 15 min (resulting in a total preparation time of less than 25 min), and counts can be easily performed at sea immediately after sampling.This method may facilitate incorporation of viral research into most aquatic microbiology laboratories.