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Freezing- Cryoprotection

Need: 2 ml cryotubes (sterile)- 2 per isolate, holding rack, sterile pipet tips ( 1 ml volume ), P 1000 pipetman, sterile 10% glycerol, freezer storage box, styrofoam container with ice, 1 cm cork borer.

  1. Place 1 ml 10% Glycerol in each tube .
  2. Cut plugs with cork borer from a 1-2 week old culture. Use a fresh plate ( never opened) and take plugs 1 cm from outer edge of mycelia.
  3. Stack 5 plugs in each tube ( make sure Glycerol covers everything- remove 1 plug if necessary).
  4. Label tubes with isolate number and date and put on ice.
  5. Transfer tubes to freezer box and place immediately in the - 80 C freezer for 30 min. ( up to 24 hrs. if necessary).
  6. Put tubes in assigned locations in - 135 C freezer.

infected tubers

infected tomatoes