Dec 09, 2015

Public workspaceSpot-on-lawn (halo) assay for screening enrichment cultures and isolates for viruses

DOI
dx.doi.org/10.17504/protocols.io.eagbabw
  • Kenneth M. Stedman1,
  • Kate Porter1,
  • and Mike L. Dyall-Smith1
  • 1Manual of Aquatic Viral Ecology
  • VERVE Net
Ken Stedman
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DOI: dx.doi.org/10.17504/protocols.io.eagbabw
External link: http://www.aslo.org/books/mave/MAVE_057.pdf
Protocol Citation: Kenneth M. Stedman, Kate Porter, and Mike L. Dyall-Smith 2015. Spot-on-lawn (halo) assay for screening enrichment cultures and isolates for viruses. protocols.io https://dx.doi.org/10.17504/protocols.io.eagbabw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: December 09, 2015
Last Modified: March 28, 2018
Protocol Integer ID: 2088
Abstract
This protocol is based on Schleper et al. (1992) as modified by Stedman et al. (2003).

This is a protocol from: 

Stedman, K. M., K. Porter, and M. L. Dyall-Smith. 2010. Chapter 6: The isolation of viruses infecting Archaea. Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7

Please see the published manuscript for additional information.
Guidelines
Authors: Kenneth M. Stedman1, Kate Porter2, and Mike L. Dyall-Smith3
1Department of Biology, Center for Life in Extreme Environments, Portland State University, P.O. Box 751, Portland, OR 97207, USA
2Biota Holdings Limited, 10/585 Blackburn Road, Notting Hill Victoria 3168, Australia
3Max Planck Institute of Biochemistry, Department of Membrane Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany


Fig. 1: Pictorial overview of isolation of Sulfolobus viruses.
(A) Wolfram Zillig sampling at a typical Sulfolobus-containing pool in Yellowstone National Park, USA, September 2000 (inset shows anaerobic tubes with samples).
(B) 80°šC incubator with long-necked growth flasks (detail in inset).
(C) Singlecolony isolates of Sulfolobus solfataricus on a Gelrite® plate. This plate contains a mixture of S. solfataricus containing (blue colonies) and lacking (brown) a vector expressing the lacS gene from S. solfataricus and was sprayed with 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) (see Jonuscheit et al. 2003).
(D) Lawn of S.solfataricus strain P1 with halos of growth inhibition due to virus production by 2-µl spots of virus-infected strains. Spots labeled SV2P1 and SV2P2 are from S. solfataricus 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 S. solfataricus strain P2 as a negative control.
Gelrite plates are preincubated ca. 10 min at 80˚C to dry.
Duration00:10:00
10 mL of Sulfolobus medium with ca. 0.2 % (w/v) Gelrite is boiled to dissolve the Gelrite.
This “softlayer” is allowed to cool slightly (to ca. 80˚C).
Approximately 3 mL of softlayer are added to ca. 0.2 mL of exponentially growing host cells, generally Sulfolobus solfataricus, and spread on a plate by swirling.
After the Gelrite solidifies, 1–2 µL of culture or supernatant to be screened is spotted on the plate.
For a positive control, 1 µL of a 0.01% (v/v) Triton X-100 solution is spotted.
Plates are incubated as above for 2–3 d and plates examined for clearing around spots (Fig. 1D in guidelines).
Duration72:00:00