Efficiency of plaquing (EOP) assay for C. jejuni phagesJessica Sacher (with help from Stephanie Guerra)For each strain, streak jejuni cells from frozen onto NZCYM 1.5% agar plates, grow 2 days in microaerobic incubator.Restreak cells onto a fresh NZCYM plate and grow 1 day.Harvest cells by flooding plate with 5 mL NZCYM broth, mixing with a spreader and collecting resultant suspension.Mix well by inversion and set OD600 for each strain to 0.35 (or to 0.0035 \u2013 this has also worked well) in 5 mL NZCYM broth in an empty Petri dish (regular size).Incubate at 37\u00b0C for 4 h at 100 rpm (place Petri dishes in a large glass beaker inside of an autoclave bag and fill with trigas mixture \u2013 beaker should also contain ~50 mL water in a flask which is taped to the side of the beaker, which helps to keep atmosphere moist).During 4-h incubation: Melt NZCYM 0.6% agar (microwave) and keep at 55\u00b0C until ready to harvest cells (let sit at least ~30 mins at 55C prior to use in order to ensure it is not too hot).\n Serially dilute phages (10-fold dilutions, usually down to 10-5 or 10-6) and store on ice until needed.\n Label (including spaces to spot dilutions: 0, -1, -2, -3, -4, -5) and pre-dry (open inside biosafety cabinet for ~30 min) NZCYM plates (one per phage\/strain combination).\n After the 4-h incubation, mix cultures by swirling and measure OD600. Set OD of each strain to 0.3 and add 250 \u00b5L of this suspension to a 15-mL Falcon tube (0.5 OD x 200 \u00b5L has also worked well).Add 5 mL molten agar to each tube, mix thoroughly but gently (avoid bubbles) by inversion. Immediately pour the entire contents of each tube onto an NZCYM plate.Let solidify ~10-20 mins.Spot dilutions of phages (10 \u00b5L x 2 spots per dilution) as well as undiluted phage stock and let plates dry with lids off until spots are absorbed (~15-20 min).Incubate plates upside down in microaerobic incubator for 18-24 h. Check plates for plaques and image using an iPhone (or appropriate substitute). Count isolated plaques and record.To convert plaque numbers to PFU\/mL, multiply the number of isolated plaques by the inverse of the dilution factor (ie. If 5 plaques are recorded on the -4 dilution spot, multiply 5 by 104, then multiply this number by 100 to account for pipetting only 10 \u00b5L \u2013 this would give 5 x 106 PFU\/mL). Shortcut: number of plaques (5, in this example) x 10(dilution number plus 2) = 5 x 10(4+2) = 5.0 x 106 PFU\/mL.Graph PFU\/mL vs. jejuni strain to compare plaquing efficiency of a phage on multiple strains. Alternatively, convert each PFU\/mL value to a percentage of a reference (e.g. wild type) strain.