U.S. flagAn official website of the United States government
                    Here's how you know **Official websites use .gov**
                            
                            A **.gov** website belongs to an official government organization in the United States.
 **Secure .gov websites use HTTPS**
                            
                            A **lock** (                            
                              A locked padlock                                                      
                            
                            ) or **https://** means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.


Menu Agricultural Research Service
						
                    U.S. DEPARTMENT OF AGRICULTURE
 Plant Genetic Resources Unit (PGRU): Geneva, NY
 close



    Research

                    Research Projects
                                    Publications
                                    Collaborations
                                    News
                People

                    Staff Listing
                                    Careers


        


                    Diversity Collections
                        Apple
                                    Brassica
                                    Buckwheat
                                    Celery
                                    Grape
                                    Hemp
                                    Onion
                                    Radish
                                    Squash
                                    Tart Cherry
                                    Tomato
                                    Other Seed CropsSearch small
                    Search
            
            ARS Home
                                About ARS
                                Contact Us




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







fungal_appx4headline bar **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


Return to top


Connect with ARS Sign up


ARS Home
                            USDA.gov
                            Plain Writing
                            Policies & Links
                            Civil Rights Statements
                            FOIA
                            Accessibility Statement
                            Privacy Policy
                            Non-Discrimination Statement
                            Quality of Information
                            USA.gov
                            WhiteHouse.gov