Abstract:
: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.
journal_name
Appl Environ Microbioljournal_title
Applied and environmental microbiologyauthors
Bettarel Y,Sime-Ngando T,Amblard C,Laveran Hdoi
10.1128/aem.66.6.2283-2289.2000keywords:
subject
Has Abstractpub_date
2000-06-01 00:00:00pages
2283-9issue
6eissn
0099-2240issn
1098-5336journal_volume
66pub_type
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