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Applied and Environmental Microbiology, November 2003, p. 6628-6633, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6628-6633.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Sampling Natural Viral Communities from Soil for Culture-Independent Analyses

Kurt E. Williamson,1 K. Eric Wommack,1* and Mark Radosevich2

Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware 19716,1 Department of Biosystems Engineering and Environmental Science, University of Tennessee, Knoxville, Tennessee 379962

Received 3 April 2003/ Accepted 12 August 2003

An essential first step in investigations of viruses in soil is the evaluation of viral recovery methods suitable for subsequent culture-independent analyses. In this study, four elution buffers (10% beef extract, 250 mM glycine buffer, 10 mM sodium pyrophosphate, and 1% potassium citrate) and three enumeration techniques (plaque assay, epifluorescence microscopy [EFM], and transmission electron microscopy [TEM]) were compared to determine the best method of extracting autochthonous bacteriophages from two Delaware agricultural soils. Beef extract and glycine buffer were the most effective in eluting viable phages inoculated into soils (up to 29% recovery); however, extraction efficiency varied significantly with phage strain. Potassium citrate eluted the highest numbers of virus-like particles from both soils based on enumerations by EFM (mean, 5.3 x 108 g of dry soil-1), but specific soil-eluant combinations posed significant problems to enumeration by EFM. Observations of virus-like particles under TEM gave confidence that the particles were, in fact, phages, but TEM enumerations yielded measurements of phage abundance (mean, 1.5x108 g of dry soil-1) that were about five times lower. Clearly, the measurement of phage abundance in soils varies with both the extraction and enumeration methodology; thus, it is important to assess multiple extraction and enumeration approaches prior to undertaking ecological studies of phages in a particular soil.

* Corresponding author. Mailing address: Delaware Biotechnology Institute, 15 Innovation Way, Newark, DE 19711. Phone: (302) 831-4362. Fax: (302) 831-3447. E-mail: wommack{at}dbi.udel.edu.

Applied and Environmental Microbiology, November 2003, p. 6628-6633, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6628-6633.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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