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 37C.

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 37C.

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 37C.

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.


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.

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