TITLE: No Title
AUTHORS: 

[DESCRIPTION]
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

[BEFORE_START]
Materials to prepare in advance for agar overlay approaches

Host culture: This procedure works well with 100 ul of overnight host culture for each standard size (100 mm) petri dish.

 Virus material:
 For Isolation of Viruses the appropriate stock material can include any potential virus source, for example: an iron-chloride flocculate of filtered seawater, resuspended in oxalate solution (John et al. 2011 [2], Poulos et al. 2018 [3]); a PEG-precipitate of sewage supernatant; or soil or stool resuspended in buffer and then pelleted or filtered to remove cells. The total volume needed per plate depends on the concentration of viruses in the stock material but the agar overlay procedure can accommodate up to several hundred microliters of material; at higher volumes of stock material (greater ~ 100 ul) it is recommended that the volume of host and top agar be increased proportionately, up to 3 ml top agar.
For Quantification of Virus Titer by Direct Plating the appropriate stock material is a 10-fold dilution series of the virus stock in buffer or media, with a recommended plating volume of ≥10 ul per plate.

Bottom agar plates: Prepare media containing 1.0% agar ('bottom agar') in a glass bottle or flask with a stirbar and sterilize, pour 25 ml per standard size petri dishes, and allow to solidify. 
 
Top agar bottle: Prepare media containing 0.3% agar ('0.3% top agar') in a glass bottle with a PTFE-coated stirbar and sterilize; though each plate will require only 2ml of top agar, volumes of up to 500 ml can be prepared and re-used across multiple days of plating.

[2] John, S.G., Mendez, C.B., Deng, L., Poulos, B., Kauffman, A.K.M., Kern, S., Brum, J., Polz, M.F., Boyle, E.A., and Sullivan, M.B. (2011). A simple and efficient method for concentration of ocean viruses by chemical flocculation. Environmental Microbiology Reports 3, 195–202.

[3] Poulos, B.T., John, S.G., and Sullivan, M.B. (2018). Iron Chloride Flocculation of Bacteriophages from Seawater. In Bacteriophages, (Humana Press, New York, NY), pp. 49–57.



[GUIDELINES]
Please also refer to the original publication for a  "Microreview of additional considerations relevant to agar overlays", which highlights methodological aspects that are known or expected to impact the nature, number, and diversity of viruses recovered using agar overlay approaches.

[STEPS]
SECTION: I. Preparation of the top agar in beaker-waterbath
?. 
SECTION: I. Preparation of the top agar in beaker-waterbath
?. 
SECTION: I. Preparation of the top agar in beaker-waterbath
?. 
SECTION: II. Procedure for Tube-free Agar Overlays
?. 
SECTION: II. Procedure for Tube-free Agar Overlays
?. 
SECTION: II. Procedure for Tube-free Agar Overlays
?. 
SECTION: II. Procedure for Tube-free Agar Overlays
?. 
SECTION: II. Procedure for Tube-free Agar Overlays
?.