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Applied and Environmental Microbiology, June 2000, p. 2283-2289, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A Comparison of Methods for Counting Viruses in Aquatic Systems

Yvan Bettarel,1 Telesphore Sime-Ngando,1,* Christian Amblard,1 and Henri Laveran2

Laboratoire de Biologie des Protistes, UMR CNRS 6023, Université Blaise Pascal (Clermont-Ferrand II), F-63177 Aubière Cedex,1 and Service Hygiène Hospitalière, Faculté de Médecine, Université d'Auvergne, F-63000 Clermont-Ferrand,2 France

Received 20 December 1999/Accepted 8 March 2000

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 (>108 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.

* Corresponding author. Mailing address: Laboratoire de Biologie des Protistes, UMR CNRS 6023, Universite Blaise Pascal (Clermont-Ferrand II), F-63177 Aubiere Cedex, France. Phone: 33 4 73 40 78 36. Fax: 33 4 73 40 76 70. E-mail: Telesphore.SIME-NGANDO{at}lbp.univ-bpclermont.fr.

Applied and Environmental Microbiology, June 2000, p. 2283-2289, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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