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Mycoplex
Sporocarp



Registered: 10/09/21
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'Cryovial Tek': Long-Term Storage of Cultures with Cryogenic Vials 13
#27778467 - 05/15/22 02:32 PM (1 year, 8 months ago) |
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This is a tek that has been shared throughout the years here on the Shroomery; there is an old Tribe Tek writeup of this method as well as numerous other accounts of members using this strategy for preserving cultures in mycology.
I want to provide a writeup of my own method for following this tek, including some additional details about how this strategy is distinct from other long-term storage methods like slants, etc.
Cryovial Tek
This tek seeks to take an existing culture and preserve it over a long period of time in nutrient-free distilled water. Placing a culture in this state effectively stalls it until nutrients become available at some point later in time.
Tools
- Cryogenic Vials
- Existing clean mycelium culture
- Scalpel
- Inoculation loop (optional)
Preparing the Vials
This tek centers around using sterilized polypropylene vials filled with distilled water to store a culture over a long period of time. The following two photos show examples of different vials that can be used with this tek. The color of the caps is inconsequential to this tek, the important thing is labeling the vial.

Simply fill the vials with enough distilled water to leave a small gap under the cap. Don't fill the vials all the way to the top, they'll need to be pressure-sterilized and leaving a small amount of space will keep the vial intact and also let you place the donor mycelium into the vial without displacing water.
Once the vials are full, sterilize them at 15psi for 30 minutes.
This is how I like to lay out the vials during sterilization. I place them in a small half-pint jar and surround the vials with a paper towel so they don't roll around in the jar. Try to align the vials in a way where they don't lay on their sides.
I've found that using sterilization pouches can be problematic with these because the vials tend to leak during pressure sterilization if aligned on their sides.

Preserving and Storing the Culture
This part is straightforward. Simply take a sterilized instrument (scalpel, etc.) and transfer a tiny piece of the mycelium of interest. You don't need a lot, just cut a small piece that can fit easily into the vial. The mycelium chosen for storage should be a culture that has already been cleaned up, isolated, and tested for cleanliness.

Once the donor mycelium is in the vial, close the lid and gently agitate the vial is that the mycelium is totally enveloped in the distilled water.
Label the vial with an identifier for the culture, along with the date it was preserved.
That's it. You can vacuum-seal these vials in plastic if you'd like or just leave them as-is.
There is no need to refrigerate these vials or store them in some kind of special manner. Just keep them in a cool, dark place away from light and temperature swings.
"Resurrecting" the Mycelium
When you'd like to make use of a culture that has been stored in this way, simple unscrew the vial and place the donor mycelium on an agar plate.
You can also place it directly into a liquid culture if you're confident in the cleanliness of the mycelium but this is riskier and should not be done if there are no other "backups" of the culture. The safest option is to place it back on agar just in case and transfer from the agar plate.
Tek Theory
How does this vary from using slants?
Slants are a method of taking an existing culture and allowing it to expand out over a long period of time on an agar medium that is deeper than usual. Preserving cultures in vials is different from keeping slants because with vials we're not actively growing out a culture over time: We're taking a specific culture and stalling it out for a long period of time by removing all nutrition.
How does this vary from preserving spores?
When preserving spores (via spore prints or spore syringes), the end result is the preservation of largely random genetics over time. Preserving cultures in vials is different from keeping spores because with vials we're not preserving random genetics: We're taking a specific culture and stalling it out for a long period of time by removing all nutrition.
Edited by Mycoplex (08/30/22 10:09 PM)
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Baba Yaga
โฅ coir grower

Registered: 09/13/20
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Loc: Hyperspace Chicken Coop
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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: Mycoplex] 2
#27778519 - 05/15/22 02:59 PM (1 year, 8 months ago) |
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Nice work man, always good to see how others showing their method. I transferred much bigger pieces to dH20, will try it your way next time.
Any particular technique to get this tiny piece onto agar or does enough mycelium float around to just dunk and streak a inoculation loop?
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hazyhorse
scoobin



Registered: 03/19/19
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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: Baba Yaga] 1
#27778537 - 05/15/22 03:06 PM (1 year, 8 months ago) |
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funny you posted this, i just recently started trying it out. havenโt resurrected anything yet but it seems like other had success with it in the past. i just got some 1/4 pint jars for the sake of dropping larger wedges in, that way i could use a loop & take a small bit out while leaving the wedge in for future use. the tiny vials are probably much easier to work with, though
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Mycoplex
Sporocarp



Registered: 10/09/21
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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: Baba Yaga] 3
#27778553 - 05/15/22 03:13 PM (1 year, 8 months ago) |
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Hey Baba, hazy, thanks for checking it out. If the mycelium chunk has been in the vial long enough, I've found that the inoculation loop method should work just fine: inevitably the vial will eventually contain small pieces of mycelium that have broken off (like you described).
If the chunk hasn't been in there long enough to where the water looks like it has chunks broken off, it can be done by sterilizing a dental pick and lifting the chunk out of the vial by poking it.
My favorite part of this tek is that it lets us create multifold backups of our cultures in a way that doesn't require refrigeration, upkeep costs, or long-term effort.
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hazyhorse
scoobin



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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: Mycoplex] 1
#27778556 - 05/15/22 03:14 PM (1 year, 8 months ago) |
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i loved the idea when i first read about it. i never really wanted to get into slants (i know they work well & arenโt that hard but just seemed annoying to me) & im glad there are solid alternatives. canโt get much easier than dropping a wedge in water lol
-------------------- you're not the first to set foot here, just another =================================== i love glass petris & you can too!! posts i constantly refer back to new to mushroom cultivation?? read this!! ===================================
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Baba Yaga
โฅ coir grower

Registered: 09/13/20
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Loc: Hyperspace Chicken Coop
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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: hazyhorse] 2
#27778606 - 05/15/22 03:36 PM (1 year, 8 months ago) |
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No refrigeration is the main incentive for me and not having to refresh every year or two.
My 30ml vials are just over 1 year old now. Will let them sit for another year or two before checking for viability.
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hazyhorse
scoobin



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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: Baba Yaga] 1
#27778617 - 05/15/22 03:39 PM (1 year, 8 months ago) |
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Quote:
Baba Yaga said: No refrigeration is the main incentive for me and not having to refresh every year or two.
same here, i like the idea of room temp long term storage better in this case. itโs just so easy
-------------------- you're not the first to set foot here, just another =================================== i love glass petris & you can too!! posts i constantly refer back to new to mushroom cultivation?? read this!! ===================================
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Rotnpins
๐คฎ Rotten-Pins ๐



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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: Mycoplex] 1
#27778637 - 05/15/22 03:47 PM (1 year, 8 months ago) |
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very cool!
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SlugWorthx1


Registered: 03/16/20
Posts: 219
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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: Mycoplex] 1
#27778711 - 05/15/22 04:17 PM (1 year, 8 months ago) |
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Myco, have you tested bringing back any cultures? I was under the impression that only the mycelium is supposed to be stored in the dh20 phials, as transferring pieces of agar would defeat the purpose of a nutrient free environment
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Mycoplex
Sporocarp



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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: SlugWorthx1]
#27778731 - 05/15/22 04:25 PM (1 year, 8 months ago) |
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Quote:
SlugWorthx1 said: Myco, have you tested bringing back any cultures? I was under the impression that only the mycelium is supposed to be stored in the dh20 phials, as transferring pieces of agar would defeat the purpose of a nutrient free environment
My experience with this is that the mycelium will finish colonizing whatever amount of agar is attached to it at the time of storage (like in my photo above with the scalpel). Eventually, once that agar residue is depeted, the end state is the same. Eventually there will be a total lack of nutrition, and absolutely no FAE or gas exchange from the get-go. As long as the agar and culture are both clean I don't see this being an issue.
You can try scraping off the mycelium without getting any agar involved at all, but it might come at the risk of accidentally damaging the mycelium.
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SlugWorthx1


Registered: 03/16/20
Posts: 219
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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: Mycoplex] 1
#27778809 - 05/15/22 05:07 PM (1 year, 8 months ago) |
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I see, thats what i assumed as well. Although, im having trouble understanding if the purpose of the phials is to prevent & stall growth - wouldn't the agar wedge suspended in the dh20 , remain in a dormant state rather than colonize the rest of the agar?
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Mycoplex
Sporocarp



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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: SlugWorthx1]
#27779028 - 05/15/22 06:53 PM (1 year, 8 months ago) |
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Quote:
SlugWorthx1 said: wouldn't the agar wedge suspended in the dh20 , remain in a dormant state rather than colonize the rest of the agar?
It wouldn't remain in a dormant state because you're taking it out of the sterilized distilled water and exposing it to nurtient agar.
The wedge becomes dormant in the vial because apart from whatever leftover agar is present on the wedge, there is no other nutrition, FAE, or gas exchange at all in the vial to facilitate any development.
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ReverendMyc

Registered: 03/29/19
Posts: 1,494
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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: Mycoplex]
#27780492 - 05/16/22 03:50 PM (1 year, 8 months ago) |
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-------------------- LAGM 2.024Stoned Gummys tek (Gummies from sclerotia or mushrooms) *Not just for stones any moreHow to succeed in mycology (and life) - know nothing, read everything, try something, and accept advice. Don't Panic   
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shuna


Registered: 03/07/21
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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: ReverendMyc] 1
#27921679 - 08/27/22 06:32 PM (1 year, 4 months ago) |
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This is the TEK I wish I found when first starting out. Props for the clear thought out write up Mycoplex!
The vials and caps need to be Polypropylene PP5 to survive the PC cycle, often cheap ones have caps that aren't.
Made some up and stored them for a few months before emptying them without even trying them on agar . I'm always trying to get rid of stuff, lol.
Between spore prints and grain jars in the fridge, reckon I'm covered for long term storage though.
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Mycoplex
Sporocarp



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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: shuna]
#27921691 - 08/27/22 06:45 PM (1 year, 4 months ago) |
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Thanks for the words shuna, and for checking out the writeup
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Psicomb


Registered: 01/13/18
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Loc: the womb
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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: Mycoplex] 1
#27943261 - 09/10/22 06:55 AM (1 year, 4 months ago) |
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Bumping this for awesomeness
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When we constantly pull things apart trying to see how it works, we may end up with only an understanding of how to destroy something - nick sand
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Rotnpins
๐คฎ Rotten-Pins ๐



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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: Psicomb] 1
#27943262 - 09/10/22 06:59 AM (1 year, 4 months ago) |
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Quote:
Psicomvb said: Bumping this for awesomeness 
Nice bump I ordered some slants and Cryovials yesterday.. Going to give these a try later today after they get here ๐ฌ
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Mycoplex
Sporocarp



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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: Rotnpins]
#27943614 - 09/10/22 12:01 PM (1 year, 4 months ago) |
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Quote:
Rotnpins said:
Quote:
Psicomvb said: Bumping this for awesomeness 
Nice bump I ordered some slants and Cryovials yesterday.. Going to give these a try later today after they get here ๐ฌ
Let us know how it goes, Pins. I couple weeks back I tested the resurrection of a Cosmoplex F3 culture stored in one of these and it was super simple and I had no contam issues at all.
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Rotnpins
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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: Mycoplex] 1
#27943626 - 09/10/22 12:09 PM (1 year, 4 months ago) |
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Quote:
Mycoplex said:
Quote:
Rotnpins said:
Quote:
Psicomvb said: Bumping this for awesomeness 
Nice bump I ordered some slants and Cryovials yesterday.. Going to give these a try later today after they get here ๐ฌ
Let us know how it goes, Pins. I couple weeks back I tested the resurrection of a Cosmoplex F3 culture stored in one of these and it was super simple and I had no contam issues at all.
Will do.. I'm planning on making a handful of extras and reviving them periodically to test then out.
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bongoman
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Re: Tek: Long-Term Storage of Cultures with Cryogenic Vials (Cryovials) [Re: Rotnpins] 1
#27951927 - 09/15/22 07:34 PM (1 year, 4 months ago) |
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Do you think the nutrient content in the actual agar wedges makes a difference in terms of longevity here? My understanding is that the lack of nutrition is what puts the mycelium into suspended animation.
I did read of someone placing a wedge of colonised nutrient agar on a plate of water agar and then taking wedges from the colonised water agar to the vials of distilled water so that no nutrition found it's way into the distilled water...
Also, would these vials be ok?
Edited by bongoman (09/15/22 08:06 PM)
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