Competition Assays

Competition on plates

• Day 1

1. Choose strains that you can differentiate using antibiotics plates.
2. Pull aliquots of strains from the -20 freezer. Thaw on ice.
3. Take 200 ul of each and inoculate them in each in 3 ml of appropriate broth.
4. Incubate the tubes in the 37 degrees water bath.
5. Wait until strains get to O.D. 0.3; dilute strains with broth so that all reach O.D. 0.3 at approximately the same time.
6. Take x microliters from each strain and add them to (200 - #x) microliters saline, for a final volume of 200ul
7. Take 50 microliters from the 200 microliters above and plate them on the competition plate.
8. Dilute the mix 10-3 and 10-4 and plate 50 microliters on each antibiotics plate to count the initial ratio between strains.

• Day 2

1. Count the colonies on each of the antibiotics plate and write them down in the lab book (make notes of the strains, antibiotics used, dilutions!, time they stood in the incubator)
2. Add 1 ml of saline + glass beads to the competition plate. Gentiley shake to release the cells.
3. Pipette the cells in an microcentrifuge tube.
4. Dilute the cells 10-1, and plate 50 microliters to a new competition plate.
5. Dilute the cells 10-3 and 10-4 and plate 50 microliters on each antibiotics plate to count the ratio between strains.

• Day 3 onwards

1. Repeat Day 2. The more days, the more pronounced any fitness differences will be between strains.

Competition in Liquid

• Day 1

1. Choose strains that you can differentiate using antibiotics plates.
2. Pull aliquots of strains from the -20 freezer. Thaw on ice.
3. Take 200 ul of each and inoculate them in each in 3 ml of appropriate broth.
4. Incubate the tubes in the 37 degrees water bath.
5. Wait until strains get to O.D. 0.3; dilute strains with broth so that all reach O.D. 0.3 at approximately the same time.
6. Take three microliters of each strain and add to a 3 ml tube of media.
7. Immediately vortex and dilute cells; plate onto antibiotic plates to count the initial ratio between strains.
8. Incubate in the 37 degrees incubator.

• Day 2

1. Check exactly 24 hours later.
2. Vortex and dilute cells; plate onto antibiotic plates to count the initial ratio between strains.
3. If continuing, put 30ul of grown cells into 3ml of media in new test tube and incubate.