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URL: https://dx.doi.org/DOI:10.17504/protocols.io.j8ncrve

Authors: Kathryn Kauffman, Martin Polz

Summary: This protocol simplifies current approaches for agar overlay plaque assays by eliminating the use of tubes for premixing of agar, hosts, and viruses, in favor of pipetting each of these directly onto the bottom agar. The benefits of this approach include simplified experimental set-up, and reductions in preparation and clean-up times and material requirements. In addition, by eliminating tube-based steps, the duration of exposure of cells and viruses to heat is reduced and the need for potentially damaging vortexing or agitation of virus-host mixtures is also eliminated. We note that Hershey et al. [1] mentioned the possibility of such an approach in their 1943 description of the tube-based overlay procedure, stating: “A further slight improvement may be made by mixing the sample directly on the phage with only 3 ml 0.7 per cent agar, but the mixing is difficult.” By using a lower percentage agar, which is advantageous for other reasons (see Additional Information section in the associated publication), we find that this approach works very well with as low as 2 ml of top agar.Using this streamlined, tube-free, plating method, >45 samples can readily be plated per hour from a common or pre-prepared virus stock, without the need for individual tubes of molten agar or multiple transfers of bacteria and virus. When deployed for isolation of novel viruses, or quantification of environmental viruses, this translates to a potential for screening hundreds of potential host strains per day using a single bottle of molten agar rather than hundreds of tubes. When used for routine bench assays, a single bottle of top agar can be used over multiple days by simply re-microwaving.[1] A. Hershey, G. Kalmanson, J. Bronfenbrenner "Quantitative methods in the study of the phage-antiphage reaction" J. Immunol., 46 (1943), pp. 267-279

Affiliations: Massachusetts Institute of Technology, Massachusetts Institute of Technology

External URL: https://doi.org/10.1016/j.mex.2018.01.007

Version: 1

Publication Date: 2018

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