Fluctuation Tests

This protocol is for doing a Luria-Delbrück fluctuation test to measure the rate at which mutations occur that enable growth on selective agar. It can be used for determining whether a strain is a mutator.

Fluctuation tests commonly used on REL606-derived strains

For these experiments we generally use 12 selective plates and 3 count plates per test strain. This is sufficient for resolving differences in mutation rates on the order of 10-fold. For measuring differences in mutation rate that are only 2- to 3-fold, we generally scale this up to 48 selective plates and 12 count plates per test strain. Making accurate comparisons of mutation rates on this scale is difficult. When comparing two strains and looking for very small changes, fluctuation tests for all strains should be done at the same time to avoid any number of confounding factors such as subtle differences in media, how long plates are incubated, whether very small colonies are counted as mutants, etc. Freshly prepared antibiotic and phage stocks should always be used.

Day -2: Revive Test Strains / Prepare Media

• Start LB cultures of each E. coli strain to be tested by inoculating a scrape of ice or 5 µl from the freezer stock into 10 ml of LB in a 50ml Erlenmeyer flask.
• Grow cultures overnight at 37°C with orbital shaking at 120 rpm.
• Prepare and pour 12-48 selective plates and 4-12 nonselective plates for each strain to be tested. See media recipes and antibiotic stock solutions

Day -1: Precondition Test Strains

• Transfer 100 µl of the overnight culture into 10 ml of saline. Mix by vortexing. Transfer 100 µl from dilution in saline to 10 ml of DM25 in a 50 ml Erlenmeyer flask. (This is a total 10,000× dilution from the LB culture.)
• Grow cultures overnight at 37°C with orbital shaking at 120 rpm.

Day 0: Growth of Independent Cultures

• Determine what concentration of glucose you should supplement into DM to give a high enough number of cells to produce mutants in a 200 µl culture.
  • As a general rule of thumb, the expected average number of mutants per plate will be the reciprocal of the mutation rate, and you need at least a few plates with mutations to get a reasonable estimate of the mutation rate.
• of DM supplemented with glucose.
  • Yields of REL606 in DM glucose media are approximately linear with the limiting glucose concentration between DM25 (0.0025% glucose) and DM2000 (0.2% glucose). DM25 gives 5×10⁷ cells / ml and DM2000 gives 4×10⁹ cells / ml. Evolved strains from the long-term evolution experiment generally yield half these densities, even as early as 2,000 generations.
• Prepare 150 mm × 17 mm test tubes with 200 µl. Use a repeat pipettor if there are many samples.
• Plate 40 µl of each cell mixture on the nonselective media to measure how many cells were actually in your inocula.

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 or 48 hours at 37°C.

Day 2 or 3: 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.

Calculating Mutation Rates

References

1. Rosche WA and Foster PL. (2000) Determining mutation rates in bacterial populations. Methods 20: 4-17.
2. Hall et al. Fluctuation analysis CalculatOR: a web tool for the determination of mutation rate using Luria-Delbruck fluctuation analysis. Bioinformatics (2009) vol. 25 (12) pp. 1564-5