In:
Applied and Environmental Microbiology, American Society for Microbiology, Vol. 66, No. 6 ( 2000-06), p. 2283-2289
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.
Type of Medium:
Online Resource
ISSN:
0099-2240
,
1098-5336
DOI:
10.1128/AEM.66.6.2283-2289.2000
Language:
English
Publisher:
American Society for Microbiology
Publication Date:
2000
detail.hit.zdb_id:
223011-2
detail.hit.zdb_id:
1478346-0
SSG:
12
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