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 jason9086
Plant Pathologist
Registered: 04/30/18
Posts: 61
Loc: California 
Last seen: 3 years, 6 months
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 Re: Best long term storage "strategy" for mushroom cultures & proper matienence  [Re: Burbles]  2
#25203450 - 05/14/18 02:32 PM (5 years, 10 months ago) |
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I don't have extensive experience with long term storage of basidiomycetes, but I have been working with a nonsexual ascomycete for my research for years and I will share with you what I know regarding this:
All my different isolates are stored in a spore solution in 10 percent glycerol in a -80c freezer. Now, because these are conidia, they all have the same genotype. This is not true of basidiospores, but the storage method should still be highly effective. I get germination years later
One thing I notice is that when the fungus is constantly being transferred in vitro to different plates, it starts losing its vigor and pathogenicity vigor (my fungus is a pathogenic bark fungus)
In order to keep it vigorous, I pass it theough host tissue every so often and reisolate from the bark lesion. Using that isolate, i grow it out on pda, collect more spores, and replace the old spores with new spores.
One lesson you can take from this is that to maintain good stock, you should not only culture in vitro, but also pass through other media. If it is a woodlover, inoculate wood and reisolate from that. If you want clones, you can isolate from the fruiting bodies and update your agar stocks every so often.
You can also continue to collect spores from the fruiting bodies and try to improve upon your stock genetics by breeding. Get spores from mushrooms showing favorable cultivation qualities, and attempt to get isolates of single spores or monokaryotic mycelium. Allow them to go through plasmogamy, and reisolate from the dikaryotic mycelium.
Test your new genotypes by fruiting them and continue cloning from the best ones.
The point is, you should not be set on providing the exact same genotype every time unless you pass through different medias to keep the organism vigorous. (Using the same media will reduce vigor over time through epigenetic alterations.
And i dont think the growers whom you hope to market to wouldnt mind if you are breeding and improving your stock for favorable traits for cultivation.
Good luck!
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lmao
Stranger
Registered: 07/24/17
Posts: 232
Last seen: 1 year, 2 months
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Re: Best long term storage "strategy" for mushroom cultures & proper matienence [Re: Burbles] 1
#25273791 - 06/17/18 06:18 AM (5 years, 9 months ago) |
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The professional institutes that store up to 30.000 different cultures use various methods.
Spores get mostly freeze dried and they can store them for up to 30 years.
Mycelium can be submerged in paraffin oil and stored for years.
And it can also be cryopreserved at -120°C or even liquid nitrogen. Depending on the fungi they may freeze them down slowly, resting for a while at certain temperatures.
https://www.sciencedirect.com/science/article/pii/B9780124325555500154
You can access the paper here: http://sci-hub.tw/10.1016/B978-012432555-5/50015-4
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 TDog
Buddy
Registered: 06/23/18
Posts: 227
Loc: Corner of Laydown and Sta...
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Re: Best long term storage "strategy" for mushroom cultures & proper matienence [Re: Burbles]
#25310921 - 07/06/18 12:33 AM (5 years, 9 months ago) |
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(*Edit 1 - Glad Mini Rounds will NOT fit in 1/2 gallon Mason Jar opening/mouth. Slants with wood are better for long-term storage. I was way out of my depth with this post... and probably all of them in this thread. :/ My bad...)
Look at how to make the containers for the "tiger drop" by elasticaltiger.
They store hella long. (I'm putting some away in the fridge stored in half gallon mason jars.)
Just my lead for you is all. I'm sure someone is going to knock it.
There are people saying that they've used them waaaaaaay later with no problems. Just saying.
Good luck.
L8
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"Laugh, and the world laughs with you. Cry, and they will kick you in the fuc**ng teeth." -TDog
Edited by TDog (07/08/18 03:33 PM)
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drake89
Mushroom Magnate
Registered: 06/26/11
Posts: 4,168
Loc: TN
Last seen: 5 years, 1 month
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Re: Best long term storage "strategy" for mushroom cultures & proper matienence [Re: jason9086]
#25311243 - 07/06/18 08:12 AM (5 years, 9 months ago) |
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Quote:
jason9086 said:
I don't have extensive experience with long term storage of basidiomycetes, but I have been working with a nonsexual ascomycete for my research for years and I will share with you what I know regarding this:
All my different isolates are stored in a spore solution in 10 percent glycerol in a -80c freezer. Now, because these are conidia, they all have the same genotype. This is not true of basidiospores, but the storage method should still be highly effective. I get germination years later
One thing I notice is that when the fungus is constantly being transferred in vitro to different plates, it starts losing its vigor and pathogenicity vigor (my fungus is a pathogenic bark fungus)
In order to keep it vigorous, I pass it theough host tissue every so often and reisolate from the bark lesion. Using that isolate, i grow it out on pda, collect more spores, and replace the old spores with new spores.
One lesson you can take from this is that to maintain good stock, you should not only culture in vitro, but also pass through other media. If it is a woodlover, inoculate wood and reisolate from that. If you want clones, you can isolate from the fruiting bodies and update your agar stocks every so often.
You can also continue to collect spores from the fruiting bodies and try to improve upon your stock genetics by breeding. Get spores from mushrooms showing favorable cultivation qualities, and attempt to get isolates of single spores or monokaryotic mycelium. Allow them to go through plasmogamy, and reisolate from the dikaryotic mycelium.
Test your new genotypes by fruiting them and continue cloning from the best ones.
The point is, you should not be set on providing the exact same genotype every time unless you pass through different medias to keep the organism vigorous. (Using the same media will reduce vigor over time through epigenetic alterations.
And i dont think the growers whom you hope to market to wouldnt mind if you are breeding and improving your stock for favorable traits for cultivation.
Good luck!
For gourmet cultivation I think institutions use cryo tissue preservation. I've always wondered how much variation you'd get in meiosis if the culture youre starting with has already been back crossed once or twice. Any idea?
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TDog
Buddy
Registered: 06/23/18
Posts: 227
Loc: Corner of Laydown and Sta...
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Re: Best long term storage "strategy" for mushroom cultures & proper matienence [Re: TDog]
#25315017 - 07/08/18 01:15 PM (5 years, 9 months ago) |
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Got in touch with The Tiger himself (elasticaltiger).
He said:
"If you really want to preserve cultures long term I would definitely look into making culture slants in test tubes. I've noticed people really like the centrifuge tubes for this."
https://www.shroomery.org/forums/showflat.php/Number/25018245/fpart/1/vc/1
https://www.shroomery.org/forums/showflat.php/Number/12968316
GG's Broseph,
-TDog
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"Laugh, and the world laughs with you. Cry, and they will kick you in the fuc**ng teeth." -TDog
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TDog
Buddy
Registered: 06/23/18
Posts: 227
Loc: Corner of Laydown and Sta...
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Re: Best long term storage "strategy" for mushroom cultures & proper matienence [Re: Burbles]
#25315060 - 07/08/18 01:50 PM (5 years, 9 months ago) |
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(*Edit 1 - Added hypothetical sterilization method for cellulose paper.)
(*Edit 2 - Screwed some stuff up. Lol.)
(*Edit 3 - Note: read the post after this one... apparently I was 180 degrees in the wrong direction... *cringe* - I refuse to delete all of this though... 3+ years from now someone will read it all. LMAO.)
Last thing for now:
#2 Is a BALLER train of thought. I would AVOID FREEZING ENTIRELY. That's a tough one to test because it's like a time capsule, lol. I really think you hit a main vein though bro. That's next level "thinkering" fo' sho'. I likes me dat... Maybe look in to a glycol suspension? You don't want to freeze anything you want to keep alive... or bring back to life... not if it has any water in it to speak of. (I would lean towards dehydrating and rehydrating before freezing and thawing... I don't know though...)
#3 Is what everyone does but with additions you may be on to something new...
#4 I personally would shy away from. I just get a feeling that your rate of spoilage will be higher, sterile or not... and also, when taking "worst case scenarios" in to consideration... power out for 24 hours = problems with that one. JS. I don't really know enough to be here talking either though. Lol.
#1 I don't really know what to think about that...
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You are WAY beyond where I'm at... kinda showed my a$$ with that "Tiger Drop" post. LMAO.
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Anyway man, I'm SO happy to see that someone is pushing SOMETHING just in general. Everyone out there is like "THIS IS HOW IT'S DONE, SO SHUT UP, STUPID!" I like that you are pushing a boundary.
Anyway, that's my 2 cents.
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So, it's been my experience in working with food that water and freezing gives some "cracked out results". That is to say that it can burst cells with ease. It's like erosion. A pothole in the road fills with water. That water freezes. It expands. It pushes the sides of the pothole. It pushes until it BREAKS the sides. Things thaw. The pothole is bigger.
Soda can in the fridge is a cell. The liquid inside freezes. It expands. It BUSTS the cell wall (can).
So, with food, this is why you don't freeze vegetables by just putting them in the freezer unless you want a soggy consistency when you thaw them back out to cook with them (for whatever reasons you have). (<<<<< OH, SNAP! Something that plays in to that though... FLASH FREEZING... that may end up being a dead end for what you are trying to do though.)
This was also the problem with "suspended animation" back in the 90's-2000's. They tried all that "freeze them slowly" stuff. Never worked. The cells bust no matter what.
(^^^^^ What they ended up going with, when it fell out off the radar [<<<<< telling me they probably got picked up by a military contract] was H2S gas to slow down the biological processes and thus prolong the life of EVERYTHING... by slowing down the rate at which that life was lived...)
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What I was "thinkering" when I rolled up though... is this:
Using (sterilized) cellulose (dissolving) paper to make spore prints with...
Hear me out on this, bro.
Sterilize the clear cellulose paper by placing sheets in a box with a bank of germicidal UV lights (within 1-2 inches of the light bank) and let them hang out for 30+ minutes (overkill the time just to be safe).
Take those prints and place them in sterilized TYVEK sheeting (sterilize using the same UV bulb method). Vacuum seal those. Wrap those up in sterilized foil and place in a UV blocking (black) baggie. Vacuum seal them. Just overkill the $hit out of the packaging, lol. Flip any of that around how you want to. I was thinking that it would be best to avoid any possible hydration of the base cellulose paper though. That is to say that if the power goes out for a few hours there will probably be condensation if just folding them up in foil. Anyways... Label those. Put them in the freezer.
(It would be funny if those were placed in a FoodSaver type package where you had a row of them, like 4 or whatever, with some space between them... then use an impulse sealer to seal between the gaps... they would come out looking like condoms! ROFL.)
I think it could at least add to what you are doing already as a "backup" just in case something goes down.
I like to have a few plans. I do not like relying on one thing only. I always have two things at once just in case one fails and I'm usually working on a third thing just as an experiment (#3 becomes #2 if #1 fails and a new #3 is started, etc... just rotating the rotation stations. ;P)
Check it though... those cellulose prints can later be taken out, rolled up, stuffed in to the barrel of a (sterilized) syringe with (sterilized) water in the barrel. Let 'em set a while, shake, and they are ready to use.
I didn't think to package and freeze them like that until I read what you wrote again just a few minutes ago.
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My only (humble) suggestion would be to take those prints in that fashion, on cellulose, and tape them to the jar of the lid that you store the un-freezable (possibly glycol) suspension in.
Hell, maybe even do some regular prints on foil and put them with the other prints and the jar. Then slap some wooden slants in the fridge.
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I get that you are trying to make a "culture library". The prints are just a failsafe is all. It sounds like you probably already have all that on lock though if you are making an "extensive culture library" because you would need an extensive collection of base material (spores) to do that. Just my guess is all.
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I did some more reading on other stuff and I was finding that people were knocking honey for slowing down growth when too much was used. I think that could actually help your slants with the wood in them. Maybe.
It would slow the growth of the culture, thus prolonging the life of the culture. It acts as an antibiotic (to my knowledge the sterilization doesn't effect the antibiotic properties... or else people would not have the slow spreading of the culture they have reported when pushing the honey up past normally accepted [low] levels... that is JUST MY GUESS THOUGH... I don't KNOW that for a FACT).
If you slow the growth down, in the presence of (presumed) antibiotics, in a culture media that has extended nutritional capacity (malt extract, carbs/sugars from the honey, wood), and crank you fridge down to the 35F-ish range... you could be on to something new as well.
Everyone I see talking about life expectancy for the standard way of doing the wood slants is saying 1 year until you have to redo things.
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I wish I knew more before I started posting to this thread. Sorry about that. You were well beyond anything I previously put up and most of what I just did. Maybe all of it. Either way, thanks for not chewing me a new a$$hole. Lol.
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I realize that probably none of this is helpful. LMAO.
Thanks for letting me talk though.
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GL.
L8.
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(Pondering... can mycelium reform after cellular rupture involved in freezing? Flash freezing? Dehydrating? Dehydrating and vacuum sealing? Then Flash freezing? Then storing IN a vacuum? In a freezer? <<<< LET'S MAKE MYCELIUM ASTRONAUT PACKS FOR USE IN THE YEAR 3000!!! #mushroomsonmars)
Back to cooking rye berries... *sigh*. LMFAO.
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L8R.
Edited by TDog (07/13/18 02:03 PM)
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TDog
Buddy
Registered: 06/23/18
Posts: 227
Loc: Corner of Laydown and Sta...
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Re: Best long term storage "strategy" for mushroom cultures & proper matienence [Re: Burbles]
#25323412 - 07/13/18 02:36 AM (5 years, 9 months ago) |
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From what I'm reading people are saying that mycelium can be frozen and still used.
https://mycotopia.net/topic/53454-can-mycelium-survive-below-freezing-temps/
Last post:
Posted 06 January 2010 - 12:27 AM
Yes, 100% cubensis mycelium can. And still fruit.
Although, the vitality of the mycelium was reduced. They did survive.
Last winter during an overnight dunk, bag was left outside. Turned out is was cold enough to freeze. And it did. Thawed and still got fruits.
BUT everything I am running in to on mycelium being COMPLETELY dried out... that's a "NO" on being able to rehydrate/revive it.
Now, as for how long the mycelium can be frozen... IDK. It "shouldn't matter", but I do not KNOW.
Anyhoo… there's that.
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"Laugh, and the world laughs with you. Cry, and they will kick you in the fuc**ng teeth." -TDog
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AndyHinton
Registered: 12/05/16
Posts: 434
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Re: Best long term storage "strategy" for mushroom cultures & proper matienence [Re: TDog]
#25331683 - 07/17/18 02:01 PM (5 years, 8 months ago) |
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I've been away for a while, but ChemBioWiz kindly suggested that assaying cell wall proteins for evidence of signal transduction may help show how different "inert" substrates affect cell metabolism.
CBW, sorry to not respond directly to you first. I promise a real reply is forthcoming! Thinking more about saline and glycerol, not necessarily frozen.
Recommended: Signal transduction cascades regulating fungal development and virulence.
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Edited by AndyHinton (07/17/18 02:04 PM)
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