Protocol for Determining Mutation Rate to Phage (T5) resistance Day -2: Prepare Media/Revive/Titer Phage 1. If necessary, revive the frozen stock of each strain in 10 ml of LB in a 50 ml flask by scraping the top of the strain to be tested. Grow the primary culture 16-24 hours at 37 degrees Centigrade. 2. Prepare 27* MG plates per condition to be tested. Day -1 : Precondition Transfer a 10,000 fold dilution** from the primary culture into a flask of 10 ml of DM25*** (or the medium that will be used for the fluctuation test) that will serve as the secondary culture. Grow 16-24 hours at 37 degrees Centigrade. Day 0: Growth of Independent Cultures Transfer between 100-1000 cells into 24 replicate lines using a 96-well plate. Transfer ~1000 fold dilution into 3 separate flasks (to serve as the control). Grow between 16-24 hours at 37 degrees Centigrade. Day 1: Plating 1. Plate the entirety of each separate replicate line in the 96-well plate onto an MG + phage plate (the amount of phage you will plate will differ depending on the titer; use an excess amount relative to the expected final density of the culture). Plate a ~10^6 dilution (or a dilution to get a reasonable colony count) of the control lines on MG plates. Grow ~48 hours. Day 3: Counting After ~48 hours of growth, count the number of colonies on each plate. *Each strain needs to be plated ~3 times on non-selective medium to determine final density. **In this case, we preconditioned with LB media, which grows to 100 times the density of DM25 media. To get 100 fold growth, we did a 10,000 fold dilution. ***The appropriate growth medium should be used such that the cultures can grow to the correct final density in ~100-200 microliters of medium.