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Liquid Nitrogen Freezing and Thawing Protocols

  1. Prepare mushroom culture by growing on PDYA agar plate for 1-2 weeks.
  2. Prepare Nalgene Cryo-Freezing Container by adding Isopropanol and fill up to 250 ml line on jar.
  3. To a Nunc storage tube, add 0.5 ml of Sterile 10% glycerol solution.
  4. Cut inoculation pieces out with #2 cork bore, sterilely and add two pieces of mycelium into each Nunc tube, submerged in 10% glycerol solution.
  5. Label with number and date with Cryo-Markers.
  6. Place Cryo tube into prepared Nalgene Cryo-Freezing Container, seal and place in minus 80 BC freezer. After 4 hours, transfer Cryo tube into Liquid nitrogen storage rack in Liquid Nitrogen Storage Freezer.

Thawing Protocol

Remove Cryo tube from LN Storage freezer, place in a 35 BC water bath for several minutes. Pour thawed culture onto PDYA plate. Label plate and put into small plastic bag. Then store at Room Temperature and at a slight slant to keep inoculum in center of plate, but Cryo storage fluid on bottom of plate. Watch for it to grow and replace culture and use as needed.

Cryoprotectants: A cryoprotectant is a substance that is used to protect biological tissue from damage during freezing. Common cryoprotectants include glycerol and dimethyl sulfoxide (DMSO). Both of these materials are penetrating cryoprotectants at physiological temperatures, meaning that they are able to move across cell membranes into the cytoplasm.

Cryoprotectants serve to help maintain osmotic equilibrium during the freezing process, and decrease the damage caused by intracellular ice nucleation. Glycerol is commonly used to protect mushroom cultures from freeze damage. We commonly add a 1 ml sterile glycerol solution (10%) to 1.8 ml capacity cryotubes and then submerge a few (up to 6) mycelial plugs or spawn grains into the solution.

10% Glycerol Solution: Mix 10 ml of Glycerol with 90 ml Distilled Water in a 250 ml flask. Dispense 10 ml of this solution into 10 screw cap tubes. Sterilize in an autoclave for 15 minutes on slow exhaust. Use solution once cool and store other tubes until need.

DMSO Cryogenic Solution: To make 100 ml. In a 250 ml bottle, dissolve 10 g of Skim Milk Powder in 100 ml of warm distilled water. May want to use a stir bar and stirrer, to dissolve powder. Cover bottle with a loose cap. Then sterilize the milk solution in an autoclave for 15 minutes on slow exhaust. Allow to cool to room temperature before sterile adding 1 ml DMSO to the solution with a pipet. Store this solution in the refrigerator. Note once DMSO is added, wrap the bottle with aluminum foil to keep out light. DMSO is light sensitive and will degrade.