Quote:

Five-hundred ninety-four strains of fungi were studied. They were found being preserved with Castellani's method with distilled water during 1 to 20 years. 62% of the strains (n = 368) did grow when subcultured and maintained their main morphological features. 90% of the 20 years old strains of different species were viable.
https://link.springer.com/article/10.1007/BF00437084

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In a low-air low-nutrient environment, the mycelium goes dormant until you transfer it to another environment with air and nutrients. It's nice when the best way to do something is also the easiest.


Supplies:
* Water
* Syringe
* Some kind of cryotube. I use 5ML (link).
* Colonized agar
* Scalpel
* Tweezers (optional, for later retrieval)

Water and equipment should be properly sterilized. I pressure cook for 40 minutes to be on the safe side.


Open your plate and slice it into appropriately sized chunks (these are on the small side).


Rather than lifting the chunks out whole, I slice underneath at a very shallow angle to only get the top layer. This cuts down on bulk so that more slices fit in the tube. In theory it should also help it go dormant faster since it runs out of nutrients sooner.


Drop it in the tube. Repeat until you're either out of agar or out of tube.


Grip the syringe from the back (away from the water), and suck up enough sterile water to fill the tube.


Squirt squirt bb. I like to drum the tube with my fingers and lightly tap it on something solid a few times to dislodge air bubbles trapped between wedges. Be mindful of movement if in a SAB. The water level in a "full" tube will sometimes drop as much as 25%. Fill back to the top so the air level inside stays as low as possible.


Close it up, label it, you're done. Truly, that's it.


If I'm saving a culture like this it was probably a happy plate, so I try to record what kind of agar the myc seemed to appreciate, or other things worth knowing.


Store at room temperature with no special considerations. I designed a 3d printed stackable storage rack that comes in multiple tube sizes. You can download it and print your own in the 3d printing thread.

To revive your culture, just pull out an agar chunk with scalpel or tweezers, plop it on a plate, and let 'er rip.

Now go forth, preserve your cultures, and fugeddaboudit.

bongoman said:

Do you think there’s an optimal vial size for this method? I’ve been using small 2ml vials but not sure if I should be going bigger.

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bongoman said:

Also, do you think it matters how much nutrition from the agar wedges ends up in the vial? I’ve seen one implementation of this method where only a small agar wedge was used to minimise nutrition in the vial. I’ve also seen it done where the vial is packed full of wedges.

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Generic said:

why is air bad in the top of the tube?

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Generic said:

seems easier to sterilize slant-centrifuge-tubes WITH the water in them, like i do with agar for making slants (inside of a quart jar with a filter, quarter-turn opened).

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Mashedpotatoes said:

Do you think if you scrape just the mycelium into water, store the myc water in the vial, would it last longer?

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phytophthora said:

Not sure why everyone uses cryovials though. I use Eppendorf 1.5 ml.

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phytophthora said:

Cheap and easy, just like me.

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Range Spacer said:

Since I started playing with this hobby 5 years ago, I've been trying different methods of storage. Almost everything I tried failed. Most likely, cleanliness in the beginning. Agar slants always contammed, Penicillin vials the same. I think it was air contamming me out in the vials and maybe the household fridge being used for the agar slants, as I had drilled holes in thew lids and put SFD's on them. I met another old codger on a fishing trip, that told me to do this, and said he'd stored samples for 20 years doing this. I take no credit for it, it's not my "tek", but I've had 99% luck doing this, since I learned how to wake up dormant mycelium. I threw a bunch of tries away before accidentally forgetting some plates on a shelf for a few weeks only to find them all doing well. Over the last 6 months, I played around and found it was much better to put the sample on very weak nutrients before going to MEAP. This too, was an accident, as I had some weak MEA plates left over from an attempt at getting a strain out of tomentose and into rhizo. The recipes, WA tek and sandwich method, I learned from here. Actually almost everything I know I learned from here. I made this up off my notes, so bear with me and my way of wording things. I don't get in here much, so I'm not a very good contributor and figured maybe this could help others, so here goes. I'm placing it here because this is where I come the most and figured Nichrome wouldn't chase me down and beat me up for not posting in the proper place. LOL. If a TC feels it's useful, place it where it belongs.

Slant Vial Fungal Preservation
Long Term Storage 5 years or more.

Phase 1: Prepare the Water Agar (WA)
Water agar provides a solid physical structure for the mycelium to anchor to, but contains zero sugars to feed it.
• The Recipe: Combine 500 ml distilled water and 10 grams pure agar-agar powder. Do not
add malt, yeast, potato starch, or honey.
Sterilization: Autoclave or pressure cook at 15 PSI for 20 minutes. Allow the vials to cool completely to room temperature inside a sterile field (laminar flow hood or near a Bunsen burner).
• Pouring: Pour into petri dishes (the largest you can buy), in front of your flow hood. Because there are no nutrients, these plates are highly resistant to contamination while sitting on your shelf. I pour as thin as I possibly can (1/8th”) because it makes the “sandwich” in a later step much easier to do.

Phase 2: Create the "Starved" Culture. You must transition your genetics from a nutrient-rich environment to a nutrient-free environment.
Identify the active, outer edge of your 2-inch fungal colony on the water agar plate.
Flame-sterilize your scalpel or inoculation loop and let it cool.
• The Transfer: Take a tiny speck of clean mycelium from your standard Malt Extract Agar (MEA) plate and place it onto the center of a fresh Water Agar plate.
I like a ¼” piece and I cut the mycelium off the top so it isn’t so thick to cover with the “sandwich” piece of water agar. Otherwise it’s hard to get a seal on the sandwich. Take another Water Agar plate and cut a square section large enough to cover your transfer by at least ½” on each side and place it on top. Press it down so that it sticks to the transfer all around.
• The Growth: Incubate at room temperature. The mycelium will grow outward very slowly, looking like a faint, transparent, ghostly web. It is searching for food that isn't there.

The Harvest: Let it grow out about 1 ½ - 2”. This is why I use the largest plates I can buy. The mycelium on this plate has completely emptied its internal nutrient reserves. It is now ready for long-term dormancy.

Phase 3: Prep the Storage Vials
• The Setup: Fill your glass screw-cap vials 7/8 full with pure distilled water.
• The Cook: Keep the screw caps one full turn loose and cover the tops with aluminum foil. Pressure cook at 15 PSI for 45 minutes to ensure the water is completely sterile.
• The Cooling Rule: Let the pressure cooker drop to 0 PSI naturally. Do not open it until the cooker is sitting directly in front of your running flow hood. Tighten the caps completely only while they are bathed in the sterile airflow.


Phase 4: Inoculation for Multi-Year Storage

The Action: In front of your flow hood, sterilize your scalpel. Cut small 3mm x 3mm squares from the outer edge of your faint water agar plate.
• The Plunge: Open a water vial, drop 2 to 3 squares into the water, fill to brim with more PC’d water and screw the cap down tightly. Wrap top in Parafilm, dip top 1/3rd in beeswax. I label with sharpie and Label Maker.
• The Result: Because the water agar and distilled water contain zero nutrients, the myceliums metabolism flat lines into true cryptobiosis. It stops consuming oxygen and stops producing CO2, resolving the suffocation issue.

Phase 5: Storage and Future Revival

Storage: Store the vials in a dark box, place in dresser drawer at stable room temperature (60°F–75°F). Do not refrigerate refrigerate water vials, as temperature swings in a fridge can crack the glass or cause condensation issues over a 5-year span.

The Old Vial Wake-Up Plan

1. Prep a "Soft Wake-Up" Plate: Do not use full-strength MEAP. The sudden blast of sugar can kill or permanently stall starving cells. Instead, pour a very weak plate: 1% Malt Extract Agar (MEA) with no peptone (about 10 grams of malt extract and 15 grams of agar per liter of water). I failed a few times before learning this.

2. The Extraction: In front of your flow hood, open an old vial. Use your long scalpel or a sterile loop to fish out the water agar wedge. I now put 3 pieces in each vial and put one on three different plates, as it betters my odds.

3. Face-Down Placement: Place the old wedge face-down onto your weak MEA plate. This forces the dormant mycelium to make direct contact with the new moisture and mild nutrients.

Be Patient (The 2-Week Window): Because the cells are waking up from a multi-year slumber, you might see absolutely zero growth for the first 5 to 10 days. Do not throw the plate away thinking it failed. It can take up to two full weeks just for the first tiny, fuzzy white hairs to creep out of the old wedge. Because those cells have been starving in a dresser drawer for 3 years, their internal clock is completely reset, or that’s my guess.
• Day 1 to 5: Expect absolutely nothing to happen. The wedge will look dead.
• Day 7 to 10: The cells will finally finish rehydrating and absorbing the micro-nutrients from your low-nutrient plate.
• Day 14+: You should see those first pristine, white explorer threads creep out onto the agar.

4. The Jump to Vigor: Once that weak plate shows a clean circle of new growth, take a piece from that fresh leading edge and transfer it to your standard MEAP plate. That is when the mycelium will realize it is fully safe, digest the peptone, and explode back into its normal, aggressive self.

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