A comparison of methods for counting viruses in aquatic systems.
In this study, we compared different methods-including transmission electron microscopy-and various nucleic acid labeling methods in which we used the fluorochromes 4',6'-diamidino-2-phenylindole (DAPI), 4-[3-methyl-2,3-dihydro-(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 from freshwater ecosystems whose trophic status varied and from a culture of T7 phages. From a quantitative and qualitative viewpoint, our results showed that the greatest efficiency for all ecosystems was obtained when we used the EM counting protocol in which YOPRO-1 was the label, as this fluorochrome exhibited strong and very stable fluorescence. A modification of the original protocol in which YOPRO-1 was used is recommended, because this modification makes the protocol faster and allows it to be used for routine analysis of fixed samples. Because SYBR Green I fades very quickly, the use of this fluorochrome is not recommended for systems in which the viral content is very high (>10(8) particles/ml), such as treated domestic sewage effluents. Experiments in which we used DNase and RNase revealed that the number of viruses determined by EM was slightly overestimated (by approximately 15%) because of interference caused by the presence of free nucleic acids.
Bettarel
Y
Y
Laboratoire de Biologie des Protistes, UMR CNRS 6023, Université Blaise Pascal (Clermont-Ferrand II), F-63177 Aubière Cedex, France.
Sime-Ngando
T
T
Amblard
C
C
Laveran
H
H
D000818:Animals; D003851:Deoxyribonucleases / Q000378:metabolism; D017753:Ecosystem; D005456:Fluorescent Dyes / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009696:Nucleic Acids / Q000378:metabolism; D010933:Plankton / Q000821:virology*; D012260:Ribonucleases / Q000378:metabolism; D014780:Viruses / Q000254:growth & development*; D014871:Water Microbiology
D005456:Fluorescent Dyes; D007211:Indoles; D009696:Nucleic Acids; C007293:DAPI; D003851:Deoxyribonucleases; D012260:Ribonucleases