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.

• For most cells use LB media.
• For JEB1/REL606 tests use DM0 + 0.2% Glucose + 200 µM dT.

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.

• For most cells use DM25 or DM2000 media.
• For JEB1/REL606 tests use DM0 + 0.2% Glucose + 200 µM dT.

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 200 µl of the growth medium.

• For REL 606 grown in DM0 + 0.2% Glucose, the final cell density is about 4 x 10⁹cells/ml. To make 25 ml (19 ml is required for the plate) such that there are 10³ cells in each 200 µl, make a 100x dilution via 100 µl into one wet DT, then add 3 µl of this dilution to the 25 ml. (JEB1 grows to half this density, so 6 µl of the 100x dilution should be added)

Spread 40 µl of the solution remaining after each well has been filled on an LB plate. Counting this plate will verify the inoculum size. If there are 10³ cells per well, then there should be ~200 cells on this plate.

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.

• For REL 606 grown in DM0 + 0.2% Glucose, add the entire 200 µl from the well to one wet DT. Then make two further dilutions of 100 µl to a second and third wet DT. Plate 25 µl of this 5 x 10⁵ dilution. (JEB1 grows to half this density, so plate 50 µl of the same dilution.)

Grow plates exactly 24 hours at 37°C.

Day 2: Counting Plates

Count visible colonies on each plate. Streak out any questionable colonies on the remaining antibiotic plates to verify that they are resistant. Also streak out to single colonies any mutants that you are saving for further experiments or to sequence. Be aware that compensatory mutations may arise quickly during further growth when there is a fitness cost for antibiotic resistance.