Un Protocolo Para La Enumeración De Los Virus Acuáticos

Budinoff et al. BMC Microbiology 2011, 11:168 http://www.biomedcentral.com/1471-2180/11/168

METHODOLOGY ARTICLE

Open Access

A protocol for enumeration of aquatic viruses by epifluorescence microscopy using Anodisc™ 13 membranes
Charles R Budinoff, Star N Loar, Gary R LeCleir, Steven W Wilhelm and Alison Buchan*
Abstract
Background: Epifluorescence microscopy is a common method used toenumerate virus-like particles (VLP) from environmental samples and relies on the use of filter membranes with pore sizes < 0.02 μm; the most commonly used protocols employ 25 mm Anodisc™ membranes with a built-in support ring. Other filters with small pore sizes exist, including the 13 mm Anodisc™ membranes without a support ring. However, the use of these membranes for viral enumeration has notbeen previously reported. Results: Here we describe a modified protocol for 13 mm Anodisc membranes that uses a custom filter holder that can be readily constructed in individual investigators’ laboratories from commercially available Swinnex® filter holders. We compared VLP concentrations obtained from phage lysates and seawater samples using both Anodisc membranes, as well as Nuclepore™ smallpore-size membranes (0.015 or 0.030 μm). The 13 mm Anodisc membranes gave comparable estimates of VLP abundance to those obtained with the 25 mm Anodisc membranes when similar staining methods were employed. Both Nuclepore membranes typically gave an order of magnitude lower VLP abundance values for environmental samples. Conclusions: The 13 mm Anodisc membranes are less costly and require smallersample volumes than their 25 mm counterpart making them ideal for large-scale studies and sample replication. This method increases the options of reliable approaches available for quantifying VLP from environmental samples.

Background Viruses are an important component of aquatic food webs. They contribute significantly to the mortality of marine microorganisms and consequently alter speciescomposition and influence the flow of carbon and energy within an ecosystem [1]. As such, accurate and reproducible estimates of virus abundance from environmental samples are essential to our understanding of aquatic biology and biogeochemistry. The earliest estimates of virus-like particles (VLP) in aquatic samples relied on transmission electron microscopy (TEM) [2,3]. However, the high cost,limited availability, and laborious nature of TEM quickly led investigators to switch to epifluorescence microscopy approaches [4-6] using Nuclepore™ track-etched polycarbonate membranes (pore sizes 0.015 or 0.030 μm, Whatman North America) [4,5,7] and methods originally
* Correspondence: abuchan@utk.edu Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37996, USAdescribed for enumerating bacteria [8]. Due to slow flow rates, Nuclepore membranes were subsequently replaced by Anodisc™ inorganic (Al2O3) membranes (pore size 0.02 μm, Anodisc™, Whatman) (refer to Table 1) [9,10]. Anodisc membranes are available in 13 and 25 mm diameters. The 25 mm membrane with a built-in support ring is commonly used to determine VLP abundances in natural systems and is recommendedin several published protocols [11,12]. However, the establishment of a protocol using the 13 mm membranes, lacking a support ring, has the advantages of significantly reducing processing costs (by 50% or more; Table 1) and the amount of sample required.

Results and Discussion A practical limitation of the 13 mm Anodisc membranes is the lack of a peripheral support ring to facilitate handlingof the membranes. To alleviate this limitation, we constructed custom filter holders and used modifications of

© 2011 Budinoff et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium,...