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ARS Home Northeast Area Geneva, New York Plant Genetic Resources Unit (PGRU) Docs HDM Versioning







fungal_appx5headline bar **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.


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