Isolation of Stress-Adapted Bacteria through Single Colony Selection

Take a bacterial culture that has been grown under a stress condition for an extended period.

The long-term stress results in spontaneous mutations, natural selection, and the proliferation of stress-adapted bacteria.

To isolate these bacteria, plate a small volume of the culture on an agar plate containing the same stressor and incubate.

The stressor ensures the growth of only stress-adapted bacterial colonies.

Pick a single colony to maintain genetic purity and inoculate liquid media with the stressor.

Incubate with shaking to facilitate the growth of stress-adapted bacteria.

Use a small volume of the culture to inoculate fresh media with the stressor.

Simultaneously, inoculate another medium containing the stressor with wild-type or non-stress-adapted bacteria.

Measure the absorbance at regular intervals as an indicator of bacterial growth.

Compare the growth profile to ensure successful isolation of stress-adapted bacteria.

Prepare 1.6% agar plate medium containing the same stressor at the same concentration as in the medium. Plate 0.1 milliliters of the outlet culture from the chemostat and incubate at 37 degrees celsius for 16 hours. Following incubation, pick single colonies from the plate using a sterile toothpick and inoculate them in 15 milliliter test tubes containing the same stressor and at the same medium concentration as in the chemostat.

Incubate for six hours. Transfer one milliliter of the culture broth into a 250 milliliter erlenmeyer flask containing 50 milliliters of medium. Harvest 0.5 milliliters of the culture broth every hour and measure the optical density at 600 nanometers.

Compare the growth rate of the adapted strain to that of the wild type strain given the stressor