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USDA ARS PGRU Management of Fungal Specimens and Isolates Generated through Hemp Research – Appendix II

SOP – ARS Culture Collection Cryopreservation Protocol

Use the following procedure to cryopreserve strains for long-term storage.  Strains should only be preserved using fresh culture growth with sufficient cell/spore production.

 

  1. Label 2 mL cryo vials with the strain number & date of stock preparation. Make enough stocks for each strain such that the collection inventory will contain 2 vials per strain.
  2. Prepare LN2 stocks by adding 4-8 agar plugs of the strain to be preserved to each cryo vial using a transfertube harvester. Then, using sterile transfer pipets, add enough 20% glycerol to each cryo vial to just cover the top of the agar plugs.  Place the vials at 4°C for 30-60 min. to allow the cryoprotectant to penetrate the cells.  (For Morchella & other hard to preserve strains, see the SOP – Preservation of Morels and Other Select Fungal Strains.)
  3. After 30-60 min. at 4°C, move the LN2 to the appropriate holding box in the -80°C freezer.
  4. After one week, perform a viability check of the new LN2 stocks by plating a loopful of the frozen stock on the appropriate medium. The LN2 stock may also be thawed just enough to remove an agar plug to plate.  (Note: Repeated freeze/thaw cycles will decrease the viability of LN2)
  5. After a viability check of the new stocks has been confirmed by the Collection Technician and the Designated Advisory Scientist, submit the new (and any old issued) stocks to the Collection Administrator for addition (and return) to the collection.