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

Protocol for Cyropreservation of Fungi at -80 C in Glycerol. Bushley Lab (12/2021)

 

  1. Obtain sterile cryovials (able to withstand -80 C temperatures)
  2. A solution of 20-40% glycerol is typically used. In the Bushley lab, we use a final concentration of 25% glycerol, in either sterile water or a medium (e.g. PDA, SDA, etc.). For some fungi, addition of dried milk power (~3.5% weight/volume) to this solution will help with cryoprotection.
    1. To make a spore suspension stock (for sporulating cultures). Spore stocks are often easier to replate because only a small piece of frozen sample is needed:
  1. In a sterile biosafety cabinet pipet 2 mL of the glycerol solution onto an agar plate grown for not more than 3-4 weeks (fresh cultures make the best stocks).
  2. Carefully scrape the surface of the plate with a sterile pipet or plate spreader to dislodge both mycelia and spores. Avoid disrupting agar.
  3. Use a sterile Pasteur or plastic pipet with the tip cut to allow chunks, transfer up 1 mL of the glycerol slurry on the plate (and any spores and mycelia) to each of two replicate cryovials.
  4. Repeat steps 3-5 with another 2mL of glycerol solution.

Mycelial plug stock: You can also cut small agar pieces with mycelia from the edge of a growing colony to put into glycerol directly. How many plugs you put will determine how many samples you can revive from this stock. Stocks that contain spores may be much easier to revive.

Slowly freeze the samples to -80. For many fungi, it can be important to have a controlled slow freeze at least to -50 C. Cryovials can be placed in a device such as Thermo Fischer Mr. Frosty (https://www.thermofisher.com/order/catalog/product/5100-0001#/5100-0001) or similar Either: Nalgene Cyro 1°C Freezing Container (Nalgene Cat. No. 5100-0001 with Isopropyl alcohol (See Burdsall, H.H., Jr., and E.B. Dorworth, Mycologia 86: 275-280, 1994) or a Coolcell device (Corning CoolCell Freezing System for Cryogenic Vials, purple, 12 unexposed vials; 1/ea. Corning Mfr #432000 (Item UX-04392-00). This will allow the fungi to cool at roughly -1 degree C per minute and helps prevent ice crystals.

  1. Place cryovials directly into the -80 for a slow controlled cooling (Best option) or you can place in -20 C overnight and then into -80 if this option is not available.

 

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