Reviving Sulfolobus acidocaldarius Cultures for Temperature Evolution Experimentation

To begin, use a bunsen burner to carefully heat a blunt steel wire. Pierce it into the lid of a two-milliliter microcentrifuge tube to create a one-millimeter-wide hole. Place the pierced tubes in an autoclavable vessel for sterilization.

Once the tubes have been autoclaved and cooled, fill them with 1.5 milliliters of prewarmed BBM+Now use a pipette tip to scrape about 50 to 60 milligrams of the Sulfolobus acidocaldarius culture from a glycerol stock. Transfer the inoculum into the BBM+filled tubes. Keep an uninoculated tube with BBM+as the negative control.

To prevent any aerosols from escaping from the pierced tube lid, place a heat-tolerant gas permeable membrane on the top of the lid. Place the inoculated tubes in a thermomixer for inoculation. After the cultures have reached the required optical density, centrifuge the tubes at 5, 000 G for two minutes at room temperature.

Then, pipette out the supernatant to discard it. Add 1.5 milliliters of warm BBM+to the pellet and resuspend the cell pellet in the medium. The growth was found to be similar when comparing incubation using thermomixers with that in conventional incubators.