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.

2. Grow the primary culture 16-24 hours at 37°C.

3. Prepare 27¹ MG plates per condition to be tested.

Day -1 : Precondition

• Transfer a 10,000 fold dilution² from the primary culture into a flask of 10 ml of DM25³(or the medium that will be used for the fluctuation test) that will serve as the secondary culture. Grow 16-24 hours at 37°C.

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°C.

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).

2. Plate a ~10⁶ dilution (or a dilution to get a reasonable colony count) of the control lines on MG plates.

3. Grow ~48 hours.

Day 3: Counting

• After ~48 hours of growth, count the number of colonies on each plate.

Notes

¹Each strain needs to be plated ~3 times on non-selective medium to determine final density.

²In 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 10⁴ dilution.

³The appropriate growth medium should be used such that the cultures can grow to the correct final density in ~100-200 microliters of medium.