A comparison of methods for counting viruses in aquatic systems

In this study, we compared different methods-including transmission electro n microscopy-and various nucleic acid labeling methods in which we used the fluorochromes 4',6'-diamidino-2-phenylindole (DAPI), 4-[3-methyl-2,3-dihyd ro-(benzo-1,3-oxazole)-2-methylmethyledene]-1-(3'-trimethyl ammoniumpropyl) -quinilinium diioide (YOPRO-1), and SYBR Green I, which can be detected by epifluorescence microscopy (EM), for counting viruses in samples obtained f rom freshwater ecosystems whose trophic status varied and from a culture of T7 phages. From a quantitative and qualitative viewpoint, our results show ed that the greatest efficiency for all ecosystems was obtained when we use d the EM counting protocol in which YOPRO-1 was the label, as this fluoroch rome exhibited strong and very stable fluorescence. A modification of the o riginal protocol in which YOPRO-1 was used is recommended, because this mod ification makes the protocol faster and allows it to be used for routine an alysis of fixed samples. Because SYBR Green I fades very quickly, the use o f this fluorochrome is not recommended for systems in which the viral conte nt is very high (>10(8) particles/ml), such as treated domestic sewage effl uents. Experiments in which we used DNase and RNase revealed that the numbe r of viruses determined by EM was slightly overestimated (by approximately 15%) because of interference caused by the presence of free nucleic acids.