Initiation of Independent Lineages of Sulfolobus acidocaldarius Cultures for Temperature Evolution Experimentation

To begin, use a spectrophotometer to measure the optical density of a sulfolobus acidocaldarius starting culture. Next, create serial dilutions of the starting culture from 10 to the 1 to 10 to the 6. Pipette 100 microliters each from the 10 to the 5 and 10 to the 6 dilutions, then transfer them into the wells of a six well plate containing solid BBM plus.

Place the plate in a box with damp cloth to a static incubator at 75 degrees Celsius for five to seven days until single colonies emerge. To establish the desired number of evolving lineages from single colonies, with an inoculation loop, pick a colony at random. Resuspend the colony in 1.5 milliliters of prewarmed BBM plus contained in a pierced two milliliter tube.

Label the tube appropriately. After repeating the resuspension for several colonies, fill a tube with 1.5 milliliters of the medium as negative control. Seal the tops of the tubes with a breathable membrane, then incubate them in a thermomixer until the cultures become turbid.

Centrifuge the clonal cultures at 5, 000G for one minute at room temperature. After discarding the supernatant, pipette 200 microliters of BBM plus into the tube and resuspend the pellet. Add 200 microliters of 50%glycerol to each tube to create glycerol stalks.

Then, store the stalks at 80 degrees Celsius until further experimentation.