Cryopreservation in glycerol at -80°C

  1. Cut three plugs from the border of a growing agar colony and put the plugs in a cryogenic vial containing 1 ml 10 % sterile glycerol
  2. Place the vials in a Cryo 1°C Freezing container, “Mr. Frosty” (Nalgene Co; Rochester, NY). Mr. “Frosty” ensures a 1°C /min cooling rate
  3. Set the “Mr. Frosty” at 5°C for 4 hours and then in -80°C for 24 hours
  4. After 24 hours transfer the vials to standard freezer boxes
  5. Store at -80°C until use
  6. Upon use, thaw the vial in a 34°C water bath and place the hyphal plugs on new agar plates