Phage Titering

The number of plaques will increase linearly with added phage only when the multiplicity of infection (MOI) is much less than 1 (i.e., E. coli cells are in considerable excess). For this reason, it is recommended that phage stocks be titered by diluting prior to infection, rather than by diluting cells infected at a high MOI. Plating at low MOI will also ensure that each plaque contains only one DNA sequence.

  1. Day culture. Inoculate 5–10 ml of LB with E. coli K12 ER2738 (NEB #E4104S) from a plate and incubate with shaking 4–8 hrs until mid-log phase, OD600 ~ 0.5.

  2. Prepare top agar and LB/IPTG/Xgal plates. While cells are growing, melt Top Agar in microwave and dispense 3 ml into sterile culture tubes, one per expected phage dilution. Maintain tubes at 45–50°C. Pre-warm, for at least 30 minutes, one LB/IPTG/Xgal plate per expected dilution at 37°C until ready for use.

  3. Phage Dilutions. Prepare 10 to 103-fold serial dilutions of phage in LB; 1 ml final volumes are convenient. Suggested dilution ranges: for amplified-PEG/NaCl concentrated phage culture supernatants, 108–1011; for unamplified panning eluates, 101–104. Use aerosol- resistant pipette tips to prevent cross-contamination and use a fresh pipette tip for each dilution.

  4. Pre-plating Infections. When the culture in Step 1 is turbid, dispense 200 μl into microfuge tubes, one for each phage dilution. To carry out infection, add 10 μl of each phage dilution to each tube of cells, vortex quickly, and incubate at room temperature for 1–5 minutes.

  5. Plating. Transfer the infected cells one infection at a time to culture tubes containing warm Top Agar. Vortex briefly and IMMEDIATELY pour culture onto a pre-warmed LB/IPTG/Xgal plate while maintaining sterile conditions. Gently tilt and rotate plate to spread top agar evenly. Allow the plates to cool for 5 minutes, invert, and incubate overnight at 37°C.

  6. Count plaques the next day. Count blue plaques on plates. Multiply each number by the dilution factor for that plate to calculate phage titer in plaque forming units (pfu) per 10 μl. For example, if there are 91 pfu on a 109 dilution plate, the titer result would be 9.1 x 109 pfu/μl for the stock.