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

• SOP – Preservation of Oomycetes
• Use the following procedure to preserve Oomycetes (Phytophthora, Pythium, Phytopythium, Halophytophthora, Nothophytophthora, Elongisporangium) that do not survive LN2 preservation in glycerol.
• Method for Preserving Oomycete Strains
• Isolates are grown at their optimal temperatures (16 to 24 C) for 4 to 10 days dependent on growth rate on a nutrient rich medium such as V8 juice or Pea agar.
• Three preservation methods are used for new accessions: 1) agar plugs in sterile water at room temperature in the dark for distribution; 2) agar plugs in 10% DMSO in LN2 for long-term storage; and 3) agar plugs on sterilized hemp seed in sterile water at room temperature in the dark for long-term storage.
• Agar plug in sterile water for distribution (Water Stocks)
  • Place 6-8 plugs from the actively growing culture into a 2ml screw-cap tube
  • Fill remaining space in tube with sterile water
  • Store at room temperature in the dark in Oomycete cabinet in 3118A
  • New water stock should be made each year using a plug from the hemp vial.

• Agar plugs in 10% DMSO in Liquid Nitrogen (LN2 Stocks)
  • Prepare cryoprotectant (10% aqueous solution of DMSO) - Prepare 10% aqueous solution of DMSO in the fume hood (DMSO is toxic). Filter sterilize DMSO as autoclaving may degrade. Keep at room temperature covered with aluminum foil.
  • Prepare LN2 stocks by adding 5 agar plugs (up to the 1ml mark on the cryo vial) from the leading edge of each strain to be preserved to each cryo vial using a transfer tube harvester (prepare the number of stocks needed plus one additional stock per strain for a viability check).
  • Add enough cryoprotectant (10% DMSO) to each cryo vial to just cover the top of the agar plugs. Place the tubes at 4°C for 1 hour to allow the cryoprotectant to penetrate the cells.
  • Place the prepared cryo vials in a Corning Coolcell FTS30 and place in the -80.
  • Store the vials at -80°C for minimum of 4 hours, but can be left overnight, then move them to the appropriate LN box.
  • After one week in LN storage, perform a viability check of the new stocks by thawing in 30°C water bath one stock per strain in lukewarm water and plating the entire stock on the appropriate medium.

• Agar plugs on hemp seed in sterile water (Hemp Vials)
  • Prepare hemp seed vials – Place 1/16 teaspoon of hemp seeds (~20 seeds) in a 24ml (27.75mm diameter x 70mm length) vial with 10ml water and autoclave twice at 253°F temp for 20 minutes.
  • Add 10 agar plugs of a fresh culture to the sterile hemp seed and water
  • Store at room temperature in the dark in Oomycete cabinet 3118A.
  • Viability can be checked each year when new water stocks need to be made.