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The following methods are basically method &ldquo;A&rdquo; of Zillig et al. (1994) and were described recently in detail by Prangishvili (2006). These methods consist of enrichment cultures followed by host isolation and screening for virus production in both these isolates and enrichment cultures. These techniques are very similar to those used for bacteriophages, with the major exception being the extreme growth conditions (80˚C, pH 3).<br /><br />This is a protocol from:&nbsp;<br /><br />Stedman,&nbsp;K. M., K. Porter, and M. L. Dyall-Smith. 2010. Chapter 6:&nbsp;The isolation of viruses infecting Archaea.&nbsp;Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7<br /><br />Please see the <a href="http://www.aslo.org/books/mave/MAVE_057.pdf" target="_blank">published manuscript</a> for additional information.

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<strong>Authors:</strong> Kenneth M. Stedman<sup>1</sup>, Kate Porter<sup>2</sup>, and Mike L. Dyall-Smith<sup>3</sup> <br /><sup>1</sup>Department of Biology, Center for Life in Extreme Environments, Portland State University, P.O. Box 751, Portland, OR 97207, USA <br /><sup>2</sup>Biota Holdings Limited, 10/585 Blackburn Road, Notting Hill Victoria 3168, Australia <br /><sup>3</sup>Max Planck Institute of Biochemistry, Department of Membrane Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany<br /><br /><strong>Alternative sample collection</strong>&mdash;If the laboratory is relatively close to the sampling location, water and sediment samples are collected as above, but instead of an anaerobic tube, a sterile screw-cap vial or centrifuge bottle is completely filled so that very little air is present. Samples can then be transported at ambient temperature and should be enriched within 8&ndash;10 h of collection (Rice et al. 2001).<br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/2xc2xe.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/2wc2xe.png" data-ofn="MAVE ch 6 fig 1.png" /><br /><strong>Fig. 1:</strong> Pictorial overview of isolation of <em>Sulfolobus</em> viruses. <br />(A) Wolfram Zillig sampling at a typical <em>Sulfolobus</em>-containing pool in Yellowstone National Park, USA, September 2000 (inset shows anaerobic tubes with samples). <br />(B) 80&deg;C incubator with long-necked growth flasks (detail in inset). <br />(C) Singlecolony isolates of <em>Sulfolobus</em> <em>solfataricus</em> on a Gelrite&reg; plate. This plate contains a mixture of <em>S. solfataricus</em> containing (blue colonies) and lacking (brown) a vector expressing the <em>lacS</em> gene from <em>S. solfataricus</em> and was sprayed with 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) (see Jonuscheit et al. 2003). <br />(D) Lawn of <em>S.solfataricus</em> strain P1 with halos of growth inhibition due to virus production by 2-&micro;l spots of virus-infected strains. Spots labeled SV2P1 and SV2P2 are from <em>S. solfataricus</em> strains P1 and P2 infected with SSV-I2 respectively (Stedman et al. 2003). Spot labeled C is a detergent-positive control. Spot labeled P2- is uninfected <em>S. solfataricus</em> strain P2 as a negative control. <br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/2rc2xe.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/2rc2xe.png" data-ofn="MAVE ch 6 fig 2.png" /><br /><strong>Fig. 2:</strong> TEM of <em>Sulfolobus</em> viruses and VLPs. <br />(A) <em>Sulfolobus</em> spindle-shaped virus SSV-I2 particles. <br />(B) <em>Sulfolobus</em> turreted icosahedral virus (STIV). <br />(C) Three different VLPs from an enrichment culture from Amphitheater Springs, Yellowstone National Park, USA. Note end of a <em>Sulfolobus islandicus</em> rod-shaped virus (SIRV)-like particle in upper right of image). <br />(D) Virus-like particles from Amphitheater Springs. All scale bars 200 nm. Negative stain with uranyl acetate. <br /><br />

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