University of Kentucky
1. Fill vials half full with silica gel granules, put caps on loosely, and sterilize by heating in 160 degree drying oven for at least 2 hours. DO NOT AUTOCLAVE THE SILICA, IT WILL BECOME WET!
2. Prepare a 7.5% solution of Difco skim milk, autoclave.
3. Grow C. graminicola on V8 juice agar or Difco potato dextrose agar plates for 1-2 weeks under the lights. Do not use plates more than 2 weeks old.
4. Add 2 or 3 mls of sterile skim milk to the culture, GENTLY scrape the surface with a sterile loop to loosen the spores.
5. Add 0.5 mls of the milk-spore suspension to a vial of chilled, sterilized silica. Add the suspension slowly and evenly. Shake the vial during and after the process of adding the suspension.
6. Tighten the cap on the vial, seal with parafilm. Label the vial!!! Use the small circular paper labels.
7. Vials can be stored at 4 degrees for a few months, or at -80 for many years (up to 25 years and counting!).
8. To revive cultures, sprinkle a few silica grains onto a potato dextrose agar plate. The cultures may take a bit longer than normal to start growing.
1. Autoclave small circles of Whatman filter paper, any of the coarser grades will do.
2. Place filter paper circles on an agar plate that has been inoculated with C. graminicola. Allow the fungus to grow over the circles.
3. Do not let the plates get too old! As soon as the fungus has grown over the whole plate, remove the circles of filter paper with a sterile forceps, and place them in sterile, labeled Petri dishes.
4. Put the papers in the vacuum dessicator for up to a month: the papers must be completely dry and brittle.
5. Place the papers in folded envelopes made from weighing paper, label, and store at 4 degrees in plastic boxes with dessicant. The papers will be viable for several years if kept cold and absolutely dry. Replace the dessicant frequently.
6. To revive cultures, tear off a small piece of the paper and place on an appropriate agar medium.
1. Fill liquid scintillation vials 1/3 full with ultrapure water. Autoclave.
2. Grow C. graminicola on agar medium for 1-2 weeks (do not use older plates).
3. Use a sterile scalpel to cut small squares of agar (1cm square) containing mycelium.
4. Put the squares into the chilled vials of water.
5. Label and store vials at 4 degrees.
6. To revive, place agar squares on an appropriate agar medium.
NOTE: we no longer recommend storage of C. graminicola in DMSO. This is because, after about 12 months of storage, cultures seemed to be degrading significantly.
Back to Protocols Page
Back to Vaillancourt Lab Home Page