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_appx3headline bar **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)  Obtain sterile cryovials (able to withstand -80 C temperatures)
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.
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: 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).
Carefully scrape the surface of the plate with a sterile pipet or plate spreader to dislodge both mycelia and spores. Avoid disrupting agar.
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.
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. 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.  ​


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