Protocol For Determining Mutation Rates to Antibiotic Resistance

Day -2: Prepare Media / Revive

Prepare 100 LB-agar plates with the antibiotic to be tested and 13 LB-agar plates with no antibiotic. This requires about 2 L of media.

Start a 10 ml culture in rich media from ice scraped from the top of a freezer stock of the bacterial strain to be tested. Grow 16-24 hours at 37°C.

Day -1: Precondition

Transfer an appropriate dilution of the overnight starter culture to the 10 ml of the medium that will be used during the fluctuation test. At least 100-fold growth should occur during this preconditioning step. Grow 16-24 hours at 37°C.

Day 0: Growth of Independent Cultures

Prepare a 96-well plate. Reserve well A1 for a blank (no cells added). Innoculate the remaining 95 wells with approximately 10³ cells in the growth medium.

Grow microplate exactly 24 hours at 37°C.

Day 1: Plating of Independent Cultures

Plate the entire volume (now slightly less than 200 µl) from 86 of the 96 wells onto LB-antibiotic plates. Make an appropriate dilution of the remaining 9 wells to count the number of viable cells.

Day 2: Counting Plates

Streak out any questionable colonies on the remaining antibiotic plates to verify that they are resistant. Also streak out any colonies that you might want to save.

-- Main.JeffreyBarrick - 18 Sep 2007