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Offlinefossilshark
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The OFFICIAL Stasis Storage Thread * 21
    #28161262 - 01/28/23 12:06 PM (11 months, 23 days ago)

I've been waiting a long time to make this thread as this is something I am very interested in. The goal of this thread is to share the many different methods of storing cultures in stasis. I will be posting my method and I would love for everyone to contribute their method of storing cultures in stasis. I in NO way came up with stasis storage and it has been around in many different forms for decades.

What is stasis storage?
Stasis storage is storing a sterile sample of mycelium in a no-oxygen no-nutrient environment causing the mycelium to go into stasis ceasing all metabolic activity.

Why stasis storage?
There are multiple advantages of stasis storage over other long term culture storage methods.

#1: The biggest advantage of stasis storage is the complete inaction of metabolic activity. Other long term culture storage methods (slants, cryo) only slow the metabolic activity down. In theory this can lead to changes in the culture that is not desired. Furthermore this means that unlike slants you do not have to "wake up" the mycelium at any point to continue to preserve it. Stasis has been shown to be effective for 30 years in https://sci-hub.st/10.1139/w08-049 and thats just when they stopped the study. In theory stasis storage is indefinite.

#2: Stasis storage is super simple and can be done with solid media or liquid or just straight up agar wedges. Very universal technique for anyone's style.

#3: My favorite part, stasis storage works at room temperature. Absolutely no need to take up valuable space in your moms fridge. :wink:

My method:

My initial trials 2 years ago were done to preserve Enigma x Rusty Whyte F2 from Solipsis using paper strips. I plan on switching over to wood skewers to make things easier to handle. First I hydrated paper strips by dunking paper strips in distilled water for a few seconds and sterilizing them for 20 minutes in my trusty presto along with some vials of distilled water.

After the paper strips and water were prepped I laid the strips on colonized germ plates (more strains more better baby) and let the mycelium colonize them for about a week.



After the paper strips were colonized I transferred them into vials of distilled water (along with some fat plate pins to try and clone them in the future).



And done. Thats the whole tek. Stasis storage is stupid simple. I ran the cultures about a year after I made them and they bounced back very quickly and I plan on doing a 2 year test next month when I have time.

NOTES: While the original stasis vials I made are useable its apparent that my sterile technique back then was sub par. One of the vials had a scoby on top but the mycelium was just fine.

For coprophilic species paper strips, wood skewers, toothpicks ECT work just fine with no issues. I theorize that if you want to store lignophilic species this would be too much available nutrients and effectively make the solution into LC. This would still work as once LC runs out of nutrients it goes into stasis, but to me that does not seem ideal. A better solution would be to use SFD discs like in the original paper.

I appreciate any questions or discourse on this thread on the topic. I think this method is an amazing tool for a mycology enthusiast. I appreciate those of you who take the time to read through this post. :mushroom2:


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InvisiblefahtsterM
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Re: The OFFICIAL Stasis Storage Thread [Re: Nextcontestant] * 4
    #28161534 - 01/28/23 03:33 PM (11 months, 23 days ago)

Lids for drying out colonized grains for dry stasis

Ppl have also just left some kernels in the bottom of their qts (after securing the lid back on) and let them dry out that way but I hear it can take quite a while. 

I’m going to doing a lot of drying of cultures out in the future and probably simplify my lid set up so it’s just a small SHIP and use only two layers of tyvek with a widemouth 1/2 pint.. putting that in a bag of desiccant (damprid) with the larger lid and less barriers should speed things up by another week I’m thinking.  Right now I’m at about 3 weeks from hydrated to dry where I can store as is or load into swab packs to take up less space


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Re: The OFFICIAL Stasis Storage Thread [Re: bakedbeings] * 2
    #28162509 - 01/29/23 08:32 AM (11 months, 22 days ago)

Quote:

fossilshark said:
This method is really interesting, so you think the mycelium goes into stasis despite the presence of oxygen? So basically it isn't exclusively no-oxygen no-nutrient but can also just be caused by a lack of water?




Well there’s no visible growth after many months.. if there is cell division, it’s happening at a speed that isn’t noticeable after a certain point of dryness.  I just revived this mak 118 jar that’s looked exactly like this since the end of February




I think if you remove any part of those three, oxygen, food, water, you can get stasis or at least a degree of it.

That mak 118 is just about start knotting/pinning so we’ll see how it does.  You’d think after being dried out for 10 months it’d slow it down or wear out.  We shall see.  Ime, it does not


I actually already revived the clone once this season using a separate dried out jar but I was trying to laymix in a bag… didn’t work out so well and I had to remove the block from the container so fruit out the side pins.


So I kept it normal this time around as far as sub construction and I think it’ll be even better

But I also revived this fp+ culture that was dried for 8 months

Before..


After revival..


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Offlinenektar61S
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Re: The OFFICIAL Stasis Storage Thread [Re: fossilshark]
    #28173923 - 02/05/23 07:50 PM (11 months, 15 days ago)

Fascinating stuff. Thank you.

I'm curious why this will grow and old spores won't seem to. I know someone revitalized a 20 plus year old print for Redboy, but I've seen people fail to grow spores that are under 5 years old. I believe stasis works but don't know why it does and storing spores seems to not work well.

Would removing all oxygen help? What about storing cultures without the water, and in argon or similar? I know that's complicating something simple, but I'm wondering if a tiny bit of oxygen is needed.

I looked up that arctic seed vault where Bill Gates stores his seeds to grow on all the farm land he's buying after he and his friends wreck the world (just kidding, maybe), this is the tek. It's seeds, not growing culture, and plants, not fungus, but interesting:

"The seeds are stored in sealed three-ply foil packages and then placed into plastic tote containers on metal shelving racks.[51] The storage rooms are kept at −18 °C (−0.4 °F). The low temperature and limited access to oxygen will ensure low metabolic activity and delay seed ageing. The permafrost surrounding the facility will help maintain the low temperature of the seeds if the electricity supply fails."

https://en.wikipedia.org/wiki/Svalbard_Global_Seed_Vault#Seed_storage



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Offlineside-eye
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Registered: 09/01/22
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Re: The OFFICIAL Stasis Storage Thread [Re: bakedbeings] * 4
    #28181951 - 02/11/23 08:50 AM (11 months, 9 days ago)

I dig it so I tried it.

I got some 5mL tubes with a rack, and wood stir sticks. Each one breaks into three or four pieces that fit in the tubes. Then I filled a bunch of the bottles with distilled water.



I soaked the stir sticks in the bottle for about 30 minutes, figured they needed a little more time than paper. Poured water out of the stir stick bottles, then PC'd everything in my IP for 45 minutes. Stir sticks onto a few plates. These pictures were taken two days apart, myc is already starting to get into the wood. Be sure all the broken pieces are ripped apart from each other, you can see how the Reishi plate has a big V shape that I didn't want to risk pulling apart.


Once this grows into the wood I'll drop em into the distilled water. I'll report back in 30 years to check if your tek actually works.


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Edited by side-eye (02/11/23 08:51 AM)


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InvisiblefahtsterM
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Re: The OFFICIAL Stasis Storage Thread [Re: fahtster] * 4
    #28182962 - 02/11/23 07:45 PM (11 months, 9 days ago)

Quote:

fahtster said:
That mak 118 is just about start knotting/pinning so we’ll see how it does.  You’d think after being dried out for 10 months it’d slow it down or wear out.  We shall see.  Ime, it does not






Bag did aight.



The question is if it’s able to go 10 months dried out at room temp and come back and do that ^^^ what’s stopping it from going 10 more, or even more than that? 

I have dried out cultures in jars that are over a year old.  I can revisit this thread in a couple years and give those cultures a try at reviving.. see ya then :lol:


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Offlineside-eye
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Re: The OFFICIAL Stasis Storage Thread [Re: fahtster] * 1
    #28190459 - 02/16/23 04:09 PM (11 months, 4 days ago)

1

Alright so I got two plates' sticks in water.


I ran into a few problems on this one. First was I let the plates colonize the sticks a little too much, especially on the Reishi plate. I tried picking it up with tweezers, but it was so stuck to the plate that I lifted up the whole puck of agar out of the dish. I tried cutting it out but it still grabbed any agar underneath the stick. Reishi was by far the worst, but all of the sticks lifted a bunch of myc off the plate. Here's how colonized the plates were:


Second, the wood sticks float. These tubes are 5mL so pretty short, and when I let go of the stick it floated about halfway out of the tube. I used the lid to push them down as I capped em. Longer tubes/sticks may not be as bad, and paper may not be as bad either. I'm worried they picked up some bac hitting the rim of the tube, so I will give this another go soon with paper vs sticks.

Lastly, my fault, I dropped some sticks in the wrong tube of water. Not sterile so I had to toss those. Don't work high, kids.


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Edited by side-eye (02/16/23 04:10 PM)


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OfflineSoFlown99
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Re: The OFFICIAL Stasis Storage Thread [Re: fossilshark] * 1
    #28299254 - 04/28/23 07:24 PM (8 months, 26 days ago)

Stasis has been achieved :mushroom2:

Thank you for this wonderful writeup and all your help on stasis!



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Offlineside-eye
potate


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Re: The OFFICIAL Stasis Storage Thread [Re: SoFlown99] * 1
    #28299273 - 04/28/23 07:39 PM (8 months, 26 days ago)

Put a few more cultures into stasis today. PC'd 18 of these tubes, cut some long wedges, and dropped em in. Wrap, label, into the rack.



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OfflineRushing
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Re: The OFFICIAL Stasis Storage Thread [Re: SporeJockey] * 1
    #28354239 - 06/10/23 08:57 AM (7 months, 14 days ago)

Started putting some culture into the stasis library







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OfflineDredgeMyEyes
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Re: The OFFICIAL Stasis Storage Thread [Re: fossilshark]
    #28360491 - 06/15/23 06:43 AM (7 months, 9 days ago)



What do you guys think about packing a microfuge tube with  boiled hydrated wood pieces, wrapped in foil and sterilized in a jar with a sfd and then innocoulated with a few drops of known to be good LC or tiny sample from  a plate for long term storage?

Pull out a twig and toss it on a plate to revive

Gonna try it


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Offlinefossilshark
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Re: The OFFICIAL Stasis Storage Thread [Re: fossilshark] * 3
    #28405356 - 07/23/23 03:55 PM (6 months, 2 days ago)

Threw some Gymnopilus Purperatus and Gandalf in stasis today, decided to switch over to wood sticks as they are way easier to handle then paper.

I wonder how necessary the water even is?





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Offlineastra
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Re: The OFFICIAL Stasis Storage Thread [Re: fossilshark] * 3
    #28410278 - 07/27/23 11:43 AM (5 months, 29 days ago)

dH₂O 2ml cryovials






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Offlineside-eye
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Re: The OFFICIAL Stasis Storage Thread [Re: fossilshark]
    #28419036 - 08/03/23 04:50 PM (5 months, 22 days ago)

Quote:

fossilshark said:
Threw some Gymnopilus Purperatus and Gandalf in stasis today, decided to switch over to wood sticks as they are way easier to handle then paper.

I wonder how necessary the water even is?








How did you like this compared to paper, other than handling? Guess we won't know til revived. Any issue with the sticks floating when you drop em in?


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Edited by side-eye (08/03/23 04:57 PM)


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InvisibleBigwormS
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Re: The OFFICIAL Stasis Storage Thread [Re: bongoman] * 2
    #28426269 - 08/08/23 08:22 PM (5 months, 17 days ago)

Cryo storage works well. I can't say I would trust anything but distilled water with my cultures but I'm sure it's worth a try. I also made mini slants with boiled toothpicks and agar for a shorter storage method compared to the distilled cryo storage.


Edited by Bigworm (08/08/23 08:22 PM)


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Re: The OFFICIAL Stasis Storage Thread [Re: Screwup] * 3
    #28486326 - 09/29/23 12:01 AM (3 months, 28 days ago)

Getting to be about that time of year for me.  Started reviving some dried out grain cultures 8 days ago.  I GLC the dry grains and inject them into fresh grains.  I can (and sometimes do) go straight to qts but I like to test the cultures first in small jars just to be sure. 

Claketi (dried out April 2023)



PE x RW f7 clone (dried out March 2023)



Mak TP (this one is from March 2021)



Fp+ f8 clone (dried out April 2023)



Shit amazes me every time.  These new jars will be used to glc to qts for tubs.  When I made these new jars, I made a second one of each that will be dried out again for further storage


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Invisiblethe_chosen_one
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Re: The OFFICIAL Stasis Storage Thread [Re: 3.A.M] * 4
    #28609668 - 01/04/24 04:40 PM (23 days, 8 hours ago)

How is it a person can be here for years and all the sudden something like this pops up and it was started a year ago? :confused:

I've become a huge fan of the 2ml cryovials and the Castellani method.


OR

Just forget some syringes in the fridge for 13 years. :lol: We'll see these in a decade or so.


Works for spores though. :shrug: I've labelled these Tamp 13 for just that reason lol.


Thanks to Tree Frog for the link!
:manofapproval:


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OfflineSeventhMushroom
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Re: The OFFICIAL Stasis Storage Thread [Re: the_chosen_one] * 1
    #28609792 - 01/04/24 06:23 PM (23 days, 6 hours ago)

Just adding my experience here too:

I used 20ml glass vials  using 10ml of a low-nutrient solution (0.1% lme + tap water) to store several cultures. This was in April of last year, so 8+ months now.

(this kind, with rubber stoppers, coincidentally the exact same stoppers used for SHIPs on jar lids.)


As far as I can tell, these worked amazing for "stasis"! I even did a simple experiment when I first started using these vials:

I filled 3 vials with a different amount of liquid. (5ml, 10ml, and 15ml) Then added a drop of LC, and watched what happened. The 5ml of liquid had the *most* growth, with the 15ml vial stopping growth much earlier. This would make sense if the oxygen is the limiting factor. I went with 10ml for the rest of my work though, as that's just enough to fill a 10ml syringe! wow! :laugh:

Last week I got to see my first confirmation that it worked! I took two samples out (lions mane, and some blue oysters) and put some on plates. No fancy "revival" procedure, just opened the vial, used a syringe to drop some on the plate. The plates are doing great, quickly colonized, no contamination (that I can tell). *and* the liquid left in the vial started growing thicker again!! After that confirmation, I went ahead and dumped the rest of the vials into some grain jars a few days ago, they are also doing great.


All this to say, I think using wooden sticks, or paper, and stressing about a nutrient-deprived medium is over complicated and unnecessary. See you in 5 years to confirm ;P


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