Changing Environment Long Term

General Procedures

Inoculating Tubes

To start an experiment, obtain 98 test tubes. Split the test tubes into two racks, 49 in each. Label tubes 1-16, and fill them with 3 ml 0.01% glucose. Inoculate the tubes with 5 µl of frozen stock, starting with 1-1, with a red (Ara-) strain. In the next tube, 1-2, inoculate with a white (Ara+) strain. Continue this pattern in each row of both racks, leaving one tube blank. Grow for two full days at 37°C and 120 rpm.

In order to fill the test tubes with 3 ml of media, use the repeat pipettor with a 50 ml syringe. Set the pipettor to setting 3 to aliquot 3 ml each time. To use the 50 ml syringe, the gray plastic extending piece of the repeat pipettor must be attached to the syringe.

Preparing Media

Growth media should be made by adding the necessary nutrients to 500 ml bottles of DM0 and can be stored indefinitely. For 0.01% Glucose or Galactose, add 125 µl of 40% Glucose or Galactose to 500 ml DM0. Fill tubes according to the schedule.

Check bottles of media for contamination by swirling them before filling new tubes. Contamination is marked by cloudiness or discoloration of the media. Also be sure to use new troughs each time filling new tubes.

Transferring Tubes

Every 24 hours, transfer 11.7 µl from each tube into 3 ml of fresh growth medium using the P-20 pipette. This is a 256x dilution. Be sure to break the meniscus of the fresh media with the pipette tip before expelling the sample. This one day cycle of dilution and growth is one time increment in the experiment.

Every 16 transfers, freeze all 96 samples for later evaluation.