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

SOP – Preservation of Morels and Other Select Fungal Strains

Use the following procedure to preserve morel strains (Morchella), as well as other fungal strains that do not survive LN2 preservation in glycerol.  This may be more likely for Zygomycetes strains of Mortierella, Basidiobolus, Coemansia, or Lobosporangium (syn. Echinosporangium).  Some zoospore-forming (Basidiomycetes, Oomycetes) & non-sporulating fungi may also require this method of preservation[1].

Method for Preserving Strains in LN2[2]:

• Prepare cryoprotectant (10% skim milk + 1% DMSO):
  • Dissolve 10g skim milk powder (BD Difco, catalog #232100) in 100mL diH₂O & autoclave for 15 minutes. (Place the bottle in a beaker of water such that the water just covers the skim milk to prevent burning during autoclaving.)
  • Allow the skim milk to cool to room temperature, then add 1mL DMSO in the biological safety cabinet & mix well. Store the bottle at 4°C wrapped in aluminum foil to prevent light degradation of DMSO.
• Prepare LN2 stocks by adding 4-8 agar plugs of the strain to be preserved to each cryo vial using a transfertube harvester (prepare the number of stocks needed plus one additional stock per strain for a viability check). Then, using sterile transfer pipets, add enough 10% skim milk + 1% DMSO to each cryo vial to just cover the top of the agar plugs.  Place the tubes at 4°C for 30-60 min. to allow the cryoprotectant to penetrate the cells.
• Prepare the Nalgene Mr. Frosty Cryo 1°C Freezing Container per the manufacturer’s instructions (Thermo Scientific, catalog #5100-0001).
• Add the cryo vials to the Mr. Frosty container, then place the container in a -80°C freezer. (Alternatively, the Mr. Frosty can be placed in an insulated bucket with dry ice surrounding it.)
• Store the vials at -80°C for at least 4 hr., then move them to the appropriate -80°C holding box.
• After one week of freezer storage, perform a viability check of the new stocks by thawing one stock per strain in lukewarm water and plating the entire stock on the appropriate medium.

 

[1] Ozerskaya, SM, Ivanushkina, NE, Kochkina, GA, Eremina, SS, Vasilenko, AN, Chigineva, NI.  (2013)  Long-term preservation of fungal cultures in All-Russian Collection of Microorganisms (VKM): Protocols and results.  In: VK Gupta, MG Tuohy, M Ayyachamy, KM Turner, A O’Donovan (eds), Laboratory Protocols in Fungal Biology: Current Methods in Fungal Biology, pp. 17-65.  Springer Science+Business Media, New York.

[2] Based on the method used by the Mushroom Spawn Laboratory at Pennsylvania State University