1. In biohood, inoculate strain into yeast-malt broth for cells/mycelium for DNA extraction. Yeast-malt (YM) broth: yeast extract 3 g, malt extract 3 g, peptone 5 g, glucose 20 g per liter.
2. Cryopreservation from agar plate. If a culture arrive on an agar plate, use sterile individually wrapped Pasteur pipette to punch out about 20-30 plugs from colony to place in a cryotube labeled with the accession #. Then add about 1.5 mLs of 10% glycerol [sterile] with a Pasteur pipette. Place cryotube(s) in LN2 freezer. Accession information [species name (if known), equivalent #, LN2 position, depositor, host/substrate].
3. Cryopreservation from YM broth. All non-agar plate Fusarium culture accessions are first inoculated into a 250-300 mL flask containing ~50 mLs of sterile yeast-malt (YM) broth. Grow on rotary shaker at ~150 rpm typically for 2-3 days to obtain abundant growth.
a. Remove flask to biohood and place a few drops/colonies into a cryotube labeled with accession # and already filled with ~ 1 mL 15% glycerol and resuspend by shaking briefly. Check bottle of 15% glycerol to make sure that it is not contaminated with mold.
b. Place cryotube in LN2 using location information on LN2 accession card.
4. Preparation of 15% glycerol [sterile] cryogenic medium: Add 850 mLs milliQ water to a liter [L] graduated beaker [stored by sink] and bring volume up to one L with glycerin and stir till completely resuspend. Dispense ~100 mLs into each milk dilution bottle and autoclave at 250oF for 15 min (i.e., regular cycle). Place autoclave tape on one bottle to check autoclave cycle. Tighten caps after cooled to room temp. and store in biohood if room permits.
5. When starting cultures from LN2 storage, place cryovial in ~45oC water from faucet till just thawed. In biohood, place 1-2 drops onto a PDA agar plate [60 mm] and wrap with ~ 2 cm wide strip of Parafilm.
