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Applied and Environmental Microbiology, May 2004, p. 2801-2805, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.2801-2805.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Double-Layer Plaque Assay for Quantification of Enteroviruses

Laura Mocé-Llivina, Francisco Lucena, and Juan Jofre*

Department of Microbiology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain

Received 13 November 2003/ Accepted 14 January 2004

We describe here a double-layer plaque assay for the quantification of enteroviruses, combining a monolayer plaque assay and a suspended-cell plaque assay. The double-layer assay provides significantly greater counts than other methods of virus quantification of both suspensions of pure culture viruses and naturally occurring viruses. The counts obtained by this method are approximately one order of magnitude greater than those obtained with the more commonly used method, the monolayer plaque assay. We conclude that the methods available for quantifying viruses rank in efficiency as follows: double-layer plaque assay >= suspended-cell plaque assay > counting cytopathogenic virus adsorbed to cellulose nitrate membrane filters >= most probable number of cytopathogenic units > monolayer plaque assay. Moreover, the double-layer plaque assay allows the use of two different cell lines in the two layers. Using the human colonic carcinoma cell line CaCo2 facilitates the recovery of a greater number and diversity of naturally occurring enteroviruses in water than the monolayer agar method. In addition, the pretreatment of cells with 5-iodo-2'-deoxyuridine (IDU) prior to the quantification of enteroviruses by the double-layer plaque assay provides significantly higher recoveries than the use of IDU does with the other methods of quantification.

* Corresponding author. Mailing address: Department of Microbiology, Faculty of Biology, University of Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain. Phone: 34 93 402 14 87. Fax: 34 93 411 05 92. E-mail: jjofre{at}ub.edu.

Applied and Environmental Microbiology, May 2004, p. 2801-2805, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.2801-2805.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Moce-Llivina, L., Lucena, F., Jofre, J. (2005). Enteroviruses and Bacteriophages in Bathing Waters. Appl. Environ. Microbiol. 71: 6838-6844 [Abstract] [Full Text]  


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