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 FishLevelMidnight
Aquaman
 
Registered: 09/01/17
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 Glycerol for long term preservation of stocks 
#24706836 - 10/13/17 10:43 AM (6 years, 6 months ago) |
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Hey guys,
Just curious if any cultivators have used or herd of making glycerol stocks for long term mycelium preservation?
In theory you can make a vial/eppendorf with mycelium in distilled water and a 15-25% final glycerol concentration and store at -80 C. When ready for use, the mycelium can be plated on agar to recover and then a clean culture without glycerol can be used for whatever purpose.
I know this is done routinely for bacteria and the stocks can be kept in this way for decades. Anyone tried his with active/gourmet mycelium or think it would/wouldn’t work?
I have the supplies to try it myself (although a non-active specimen donation would be helpful hehe) but don’t want to waste the time and effort if it’s a definitive no.
Fisherman
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 bodhisatta  
Smurf real estate agent
Registered: 04/30/13
Posts: 61,891
Loc: Milky way
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You have a fridge that goes to -80c?
Glycerol storage is decades old. Been used a long time
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FishLevelMidnight
Aquaman
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 Re: Glycerol for long term preservation of stocks [Re: bodhisatta]
#24706892 - 10/13/17 11:08 AM (6 years, 6 months ago) |
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I work in a research lab and we have a few -80 freezers. I could easily sneak some samples in there without alarm.
I did a quick search on here and google and didn’t see anything or any protocols, I’ll have to check around more for glycerol concentrations and what not.
Alternatively feel free to post anything you know or links!
Fisherman
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Homesteader
Student of shroomery
Registered: 01/17/16
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Hey fisherman, the technique you're referring too is definitely used for long term storage of mycelium but the ultra-low cryo freezer really isnt necessary. Cell divisions will be at near zero in just a regular freezer. The Glycerol is used to prevent freezing of the mycelium's cell walls which would otherwise kill it.
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FishLevelMidnight
Aquaman
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Re: Glycerol for long term preservation of stocks [Re: Homesteader]
#24721418 - 10/19/17 09:15 AM (6 years, 5 months ago) |
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Thanks for the reply, so a 0°C freezer is plenty?
Also, do you have any ideas on final glycerol concentration used?
I've seen as low as 10% and as high as 25.
I'm assuming the mycelium will only need to be refreshed every few years this way? I know parafilmed plates can last like 6-12 months at 4°C and then refreshed and stored again.
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Homesteader
Student of shroomery
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A standard freezer is much colder than 0C they are usually anywhere from -10 to -20C. I've heard of dilutions in that range yes, 20% or so. Also, Test tube slants closed tight and parafilmed can easily store for 1.5-2 years at 4C, your average fridge temp. Glycerol is usually reserved for long, long term storage of extremely high value strains - think 10-50 years easily
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Drayce
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Re: Glycerol for long term preservation of stocks [Re: Homesteader]
#24725737 - 10/20/17 08:09 PM (6 years, 5 months ago) |
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Shroomery is awesome...
I learn new things everyday just cruizin' posts.
I have quarts of glycerol or vegetable glycerin for e-juice, and to be able to stock live cultures in freezer seems better than the fridge to me.
Don't be harsh if this sounds lame. I just became beshroomed when bestowed with boomers a few months ago.
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AndyHinton
Registered: 12/05/16
Posts: 434
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Hi fishermansjc,
Have you read Homolka's 2014 paper? He reviews all the major protocols for basidiomycete storage and includes an extended discussion of cryopreservation.
I'm reluctant to make recommendations because I'm just getting started with –80 °C myself. The citation below has lots of useful information.
PM me if you need a copy of the full text. Also, many home freezers have an auto-defrost mechanism that fluctuates the temperature, which isn't ideal.
Code:
@article{homolka2014,
title = {Preservation of live cultures of basidiomycetes---Recent methods},
journal = {Fungal Biology},
volume = {118},
number = {2},
pages = {107--125},
year = {2014},
issn = {1878-6146},
doi = {https://doi.org/10.1016/j.funbio.2013.12.002},
url = {https://www.sciencedirect.com/science/article/pii/S1878614613001803},
author = {Ladislav Homolka},
keywords= {Fungi, Maintenance methods},
}
Edited by AndyHinton (01/09/18 01:42 PM)
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FishLevelMidnight
Aquaman
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Last seen: 8 months, 10 days
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Re: Glycerol for long term preservation of stocks [Re: AndyHinton]
#24900871 - 01/09/18 02:09 PM (6 years, 3 months ago) |
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Very interesting read, thanks for that!
That perlite method is pretty dang cool!
I will have some edible master liquid cultures made up shortly; I plan on making a 40% glycerol solution (in water) and mixing it 1:4 with the liquid culture for a final concentration of 10%.
I believe that at that low of a concentration the culture can be taken from cold storage and used directly on agar/ in LC to revive the culture. With the glycerol and at -80, I doubt that the small amount of LC media would negatively impact things.
Maybe I'll make up 10% glycerol masters while I have access to a -80, and then just do yearly transfers from slants for any cultures I want to keep.
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AndyHinton
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Posts: 434
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I agree, perlite soaked in colonized saline or LC may be a convenient alternative to agar slants. I also like the frozen agar plugs in sealed drinking straws. 
For cryovials, it seems important that the samples are cooled slowly and revived quickly. Stepping down hourly as in 0, –20, –40, –80 seems okay compared to –1 °C/min.
Update: I'm starting the soaked perlite method soon and worked out some numbers. I'll ferment the mycelium with 0.33g perlite in 5 mL LB broth with 10% glycerol. For 25 samples in 2 mL cryovials, this is 125 mL dH2O, 12.5 mL glycerol, and 2.5g LB powder. I may add an antibiotic to the broth. Will bump later with results and hopefully also pics.
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Edited by AndyHinton (01/27/18 02:55 PM)
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Burbles
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Re: Glycerol for long term preservation of stocks [Re: AndyHinton]
#24964309 - 02/03/18 04:23 PM (6 years, 2 months ago) |
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I've played with it a bit. You can also use sucrose, but that is more difficult since there is risk it will hydrolyze during PC into glucose and fructose which I don't think work as well.
The key to this is to very slowly freeze the solution; you want large ice crystals to form as it will slowly concentrate the glycerol, resulting in a progressively higher and higher concentration of glycerol into the mycelium which is what protects it from freezing/dying from the formation of ice crystals within the cell.
If I were to do it I would take a LC syringe with 10ml of LC solution in it, suck up another 2ml for 20% glycerol, and then I would put it, along with the other syringes I want to preserve into a Styrofoam box with a bag of 20% glycerol. Then place into a freezer.
The bag of 20% glycerol would slowly release heat as it froze, slowing down the rate at which the LC syringes freeze, and the insulation of the foam box would slow it down further. I would opt to leave it in the box with the bag to protect against temperature fluctuation which might cause ice crystals to form in unwanted ways.
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leschampignons
Biochemistry + Mycology
Registered: 08/30/13
Posts: 1,584
Loc: NY/NJ/ME 
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Re: Glycerol for long term preservation of stocks [Re: Burbles]
#24966550 - 02/04/18 10:37 AM (6 years, 2 months ago) |
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ChemBioWiz
Laboratory Explorer
Registered: 01/27/18
Posts: 39
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Quote:
fishermansjc said:
...Just curious if any cultivators have used or herd of making glycerol stocks for long term mycelium preservation?...
...I know this is done routinely for bacteria and the stocks can be kept in this way for decades. Anyone tried his with active/gourmet mycelium or think it would/wouldn’t work?
Yes, it should work, but it might not work as effectively in liquid media v. solid media.
As you're reading through the literature, you might see a common theme: fungal cultures frozen using cryopreservatives and solid media (e.g. sawdust) tend to perform better in the deep-freeze than those cells using liquid media. Biochemists preserve bacterial and human cell lines using a ≈20-60% glycerol suspension, but their success has to do with the nature/structure of the cells they're working with. Fungal cells are different structurally and don't preserve as effectively using the same method. Keep an eye on this as you do your search and begin to experiment. Different spp. will perform differently using the same protocol, so you're going to have to find what works for your target cells.
Quote:
fishermansjc said:
...I did a quick search on here and google and didn’t see anything or any protocols, I’ll have to check around more for glycerol concentrations and what not.
Sometimes Google isn't always the best bet, especially when it comes to the literature on fungi. There's a lot out there, but much of it is behind a paywall.
If you're working in a research lab you should see if you have access to a journal article database. If you're in a University lab you'll definitely have access. Use that academically-focused network to conduct your keyword/term search and you'll have much better luck.
If you're working in a smaller, private lab that doesn't have database access, email/call your nearest university/college library staff and biology faculty to try and coordinate for access. Just be honest and tell them that you're a hobbyist interested in articles related to cryopreservation of fungal cells, but can't afford the fees for access. IF you meet them, don't forget to bring a flash drive; those files can be large.
If you have a large university in your region, scope out their Biology/biochemistry department's webpage and look for graduate students to email; they're paid poorly and would probably kill someone's dog for a free lunch. They will probably help you out if you send them a voucher for a pizza, especially if all they have to do is request a few articles via email and forward them to you.
If that doesn't work, check your local library to see if they can request access/articles via Inter-Library Loan. Your local librarian will probably enjoy helping you with such an unusual request.
Your local commercial growers are also an excellent resource. If they don't cryopreserve cells, they will have a network of colleagues they can get in touch with to help with your search. There are also the North American Mycology Association (NAMA) and the Mycology Society of America(MSA). NAMA is more about the community and MSA is more professionally-oriented. Both of those avenues might help to point you in the right direction and can assist you with a community of other mycologists in your region moving forward.
Lastly, when you find an article, check its citations; those will help direct you to additional resources.
Here are a few articles to get you on your way:
Kitamoto, Y., Suzuki, A., Shimada, S. et al. Mycoscience (2002) 43: 0143. https://doi.org/10.1007/s102670200021
http://www.biomed.cas.cz/ccbas/Methods%20of%20Cryopreservation%20in%20Fungi.pdf
https://pdfs.semanticscholar.org/80fc/668a42263c96aea22cc3f3404f0a6371a621.pdf
http://zoologia.biologia.uasnet.mx/protozoos/protozoa41.pdf
https://www.researchgate.net/file.PostFileLoader.html?id=579caf4f217e200a4c11ccc9&assetKey=AS%3A389545477591041%401469886287648
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Mycology is a cool community of people. Try not to add any bad vibes to the neighborhood.
Finding mushrooms in the woods and growing them at home brings me a lot of joy.
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AndyHinton
Registered: 12/05/16
Posts: 434
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Re: Glycerol for long term preservation of stocks [Re: ChemBioWiz]
#24999844 - 02/16/18 08:44 PM (6 years, 1 month ago) |
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A quick update on the perlite protocol. Search Ladislav Homolka 2001 and 2006 for details (5 and 442 basidiomycetes tested, respectively).
The media is 5 mL Lennox LB (5 g/L NaCl), 10% glycerol, 25 μg/mL chloramphenicol, and 0.5g dry perlite per vial. I'll ferment these on a stir plate for 1 week with Parafilm wrapped over loose lids. Then I'll discard the LC and progressively freeze the perlite in cryovials.
I feel that a version of Sabouraud's formula (antibiotics, peptone, and double sugar) would perform well: 30 g/L malt extract, 10 g/L peptone, and green tea instead of water (2 g/cup steeped 5 min).
Update: The first batch of cryovials is done! I inoculated the above vials and fermented them at 28 °C and 150 RPM for 10 days. Then I made some representative slides and verified the presence of hyphae and conidia. No microscopy pictures, unfortunately; the RPi HDMI output wasn't working.
Getting the perlite into the cryovials was a pain in the ass. I pipetted out as much broth as possible, working against the small perlite crystals that blocked the way. Then I shoveled out the perlite with the wide end of a disposable spatula, nestled it into the cryovial, and tapped it in. I did this about three times per vial.
They're currently in the –20 °C freezer, and I'll move them to –80 °C tomorrow. I can't wait to try and revive some of them soon.
Edited by AndyHinton (03/02/18 08:25 AM)
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Spindlymass
wafer thin.
Registered: 09/25/17
Posts: 957
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Interesting thread and lots of good information. 
Quote:
fishermansjc said:
Maybe I'll make up 10% glycerol masters while I have access to a -80, and then just do yearly transfers from slants for any cultures I want to keep.
I understand the impetus is experimentation and 'see for yourself' with freezing cultures, but I thought this might be helpful:
As a perhaps more accessible storage solution and not dependent on freezers is the distilled water method. At the least it would save you the trouble of yearly transfers on the cultures not frozen. Basically, filling glass dram vials etc. with distilled water and small amounts of culture.
Certainly not a new idea but one that bears repeating.
The instructions can be found HERE. It rates multiple methods of long term storage. Distilled water method towards the bottom of page.
* The instructions state to "Put plenty of inoculum into each vial to insure success". However, I had read elsewhere the volume of water must be at least 40 times greater than the volume of inoculum.
I'm very interested to hear how the reanimating fairs!
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leschampignons
Biochemistry + Mycology
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Re: Glycerol for long term preservation of stocks [Re: Spindlymass]
#25037932 - 03/04/18 10:22 AM (6 years, 1 month ago) |
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I made some dH20 backups a few months ago but haven't revived any yet. mine definitely had at least 40x more water than inoculum.
the inoculum is just mycelium from the surface of an agar dish, no agar included. Since the smallest tube I have is roughly 1ml, that is about a gram of water which corresponds to 1/40th of a gram of mycelium. 1/40th of a gram of myc doesn't seem like much but it actually is a fair amount because myc is so fluffy and light once it's separated from the agar.
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AndyHinton
Registered: 12/05/16
Posts: 434
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Re: Glycerol for long term preservation of stocks [Re: leschampignons]
#25038501 - 03/04/18 03:17 PM (6 years, 1 month ago) |
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Spindlymass and leschamps, I'm sorry to report that dH2O doesn't work. The hypertonic solution floods the cells and bursts them. Benedek's 1962 report recommends saline to fix this. Please see my journal thread for a discussion with pacmanbreed.
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Edited by AndyHinton (03/04/18 03:51 PM)
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Spindlymass
wafer thin.
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Re: Glycerol for long term preservation of stocks [Re: AndyHinton]
#25038720 - 03/04/18 05:03 PM (6 years, 1 month ago) |
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Well poop.
Thanks for that Andy!
It's frustrating, the scholarly articles and books regarding that method mention YEARS of storage before successful reanimation.
Much appreciated, I'll read further and adjust my methods.
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enough knowledge to get into trouble. Not nearly enough to get out again.
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 Stupendous-Yappi
Anomaly XB-311394
Registered: 09/23/13
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Re: Glycerol for long term preservation of stocks [Re: Spindlymass]
#25039309 - 03/04/18 09:27 PM (6 years, 1 month ago) |
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I've received cultures in dH20 that were a few years old and most revived quickly without issue. He let the culture grow on a non-nutritious (agar+water) plate and put a wedge of that in dH2O.
I believe the agar helps protect the culture from the osmotic pressure.
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leschampignons
Biochemistry + Mycology
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Re: Glycerol for long term preservation of stocks [Re: Stupendous-Yappi] 1
#25073488 - 03/18/18 03:22 PM (6 years, 1 month ago) |
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I just made up some agar with 20% glycerol. I'm PCing it now. I am planning on loading it into microcentrifuge tubes and making tiny slants, then putting those in the freezer. I might also try putting some in the fridge and keep some at room temp to compare.
I feel like any method for long term storage has to involve very small amounts of material such as microcentrifuge tubes or 2ml cryovials or something. I don't have a fridge/freezer shelf that I can afford to spare to store my cultures, but a small corner could fit my entire culture library if I get it into micro tubes sucessfully.
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