Long term storage of mycelium utilizing cryo preservation and lyophilization techniques

[@guymontag2948]

There's also a lot of research into preserving yeasts and filamentous fungi in sterile distilled water, showing viable cultures after multiple decades.

[@FreshfromtheFarmFungi]

yes this might be worth exploring as well

[@MartynasShnaresys]

Lichen can survive full desiccation, and that even increases odds in extreme conditions. So with mycelium cultures should work under right procedure. Even tardigrade can deliberatedly dehydrate to increase survival chances. Thank You for captivating explorations.

[@BadddDoggg-id4po]

I saw a guy on another channel that would take pieces of mycelium on agar and drop them in a 50 mL tube of sterile water. He would store them in the fridge. He claimed that they would keep for at least 10 years with no growth or degradation, he would pull out a piece after a few years, place it on agar and it would immediately colonize the plate. Seems like the easiest way. I bet a cryo freezer takes tons of power. Freeze drying seems good too.

[@josephpatton1757]

Hmm i actually was reading the atcc studies, which led me here. You’ve done well, enjoyed the video!

[@jesnoggle13]

When you said “ long time ago in the 1900’s , 1991” I had huge lol. TY

[@geinstein8407]

Interesting seen as I had a brain fart a year ago to try and dry out some grain and try to regrow it, the dryde grain is still setting its almost time for it to go to agar

[@neogeo8267]

Gary, perhaps pre freeze using liquid nitrogen or even dry ice to get initial freeze dry temp down closer to the target temp faster to shrink crystal sizes. Best, George

[@bengreene7563]

Even using liquid nitrogen you still must use glycerol

[@bengreene7563]

Penn state mycology department has a whole write up on using liquid nitrogen and specific glycerol recipes for long term culture storage

[@DFBrews]

Awesome thanks for the content. I’m local might try and snag some morels on Saturday.

[@FreshfromtheFarmFungi]

Cool! we are doing pre-orders for pickup at our house just fyi they might go fast

[@DFBrews]

@@FreshfromtheFarmFungi oh email? Or website

[@DFBrews]

@@FreshfromtheFarmFungi website ordering now

[@Marlene5018]

One of antique ways to preserve mushrooms is by drying it. Making mycelium out of dried mushrooms is not complicated and I think that is great alternative for those tropical varieties that not survive in winter. ☺️

[@aidennymes6335]

how do you bring dryed mycelium back to life ?

[@Marlene5018]

@@aidennymes6335 Just mix it with subtract depending of the variety, remember that dried mushrooms are not dead at all because their spores are similar to virus morphology.

[@aidennymes6335]

I know that the spores have no problem being dried but I've never had any success reviving dried mushroom tissue (rehydrated and put on agar). maybe it depends on how it was dried and stored because I know that you can cryo preserve tissue/mycelium in a dried form but it has to be prepared in a special way with glycerol stock.

[@Marlene5018]

@@aidennymes6335 I successfully made mycelium out of dried mushrooms and fruited. Try blending some dried mushrooms and put it directly to substrate, wait and see. It works if you put it directly to the Agar but usually this way get easily contaminated I don’t know why (you may do everything clean or sterile but bacteria or mold usually grow) that’s why I think that is better if you do it on substrate.

[@terrysobkowich2084]

You should post the type of glycerol used and source, Would standard glycerin work ? its mostly Glycerol.

[@emoryolsoff96]

Hey Gary, do you have a reference for the DNase tidbit mentioned? I'd like to learn more about this!! Thanks.

[@aidennymes6335]

will there be a follow up to this video soon? I'm really interested in how this turned out

[@FreshfromtheFarmFungi]

Ive done 6 month recovery with about 10% success I want to wait a bit longer but it is promising with larger volumes

[@moisesaramos3229]

Genius, I couldn’t help but to think of alien covenant while watching this, like that final scene of entering into Valhalla.

[@official-kin]

hi garry, is there already a second video about retesting the cultures on your chan? i didnt find it.

[@signalmanpotter]

Gary, great thought process but I can't help but notice that you aren't flame sterilizing between cryo tubes nor are you using alcohol. Are you having contam issues with your tubes?

[@FreshfromtheFarmFungi]

no I switch blades instead of flames - now I also have an induction heater which is more efficient

[@kereyc5751]

"back in the 19 hundred's"...thanks for making me feel like an ancient person

[@FreshfromtheFarmFungi]

I was born in the 80’s lol

[@kereyc5751]

1988 here

[@vincentwu2848]

Hi Gary! Thanks for another great video, it's super helpful to see the ATCC papers actually being performed. I was looking around your channel to see if you had any updates on the viability of the 3 preservation methods; not sure if I overlooked it or you haven't had any meaningful results to share yet. What I'm most curious about is why you would be doing the glycerol formulations that call for cryofreezing when you have a freeze drier that allows for room temperature storage. Isn't the lyophilization unequivocally better in every way, except for the fact that it requires a freeze drier? And even so, if I had to choose between an expensive freezer and an expensive freeze-drier the choice seems really obvious. Am I missing some benefit that cryopreservation offers? Thanks again!

[@FreshfromtheFarmFungi]

the glycerol is for the freezing period - so far they have had decent success after a year or so but need the glycerol to preserve them during the freeze drying process

[@vincentwu2848]

@@FreshfromtheFarmFungi Ooooh of course, that makes sense, thank you! I didn't realize that the glycerol was also required for freeze drying. Did you observe much difference between the 10% concentration vs the 30%? I don't have a freeze dryer or a cryo freezer but I was thinking a glycerol solution could at least help extend storage times in a normal freezer. Also IDK if you've experimented with other long term storage methods, but I found a paper called "Preservation and distribution of fungal cultures" that had a method that seemed very practical and economical that was basically serial dilution using sterile water and then dropping a small amount into a vial of silica gel. Reviving the culture is then a matter of sprinkling some of the gel on agar. Just food for thought. Thanks again, mush love!

[@trinityharton1284]

How much glycerol do I need to add to those little tubes or to larger liquid cultures

[@FreshfromtheFarmFungi]

I have always gone for a 50/50 ratio but you could get away with less it will just be lower success and Im not sure if more would help. the standard procedure is in the article

[@donnbialik9085]

Columbo-type question: if you've isolated great genetics, would transferring monocultures, storing them for a safe amount of time, then transferring again, over time result in an even stronger genetics? Is there a way to vary the agar media to amplify this effect if it exists?

[@FreshfromtheFarmFungi]

typically over time they will degrade but it is best practice to transfer to fresh media every so often (6-12 months) store in a slant, or deep freeze/freeze dry. It is probably best to do a few things just in case and changing media recipes can help long term as well 👍

[@andybowman9697]

Have you further validated using a freeze dryer in particular? I couldn't finder any other update vids.

[@FreshfromtheFarmFungi]

I did a 4 month with 10% success now waiting for a year

[@andybowman9697]

@@FreshfromtheFarmFungi Wow. I guess that's not very viable. Wonder if spores would fare better, maybe freeze drying the whole mushroom then hydrate and sample from stem..

[@kimberlybenton4246]

Why can't we use DMSO it's a byproduct of tree ligdin? they use it in organ transplant to protect the organs cells from freezing. why wouldn't it work to preserve the integrity of the cells of the mycelium?

[@FreshfromtheFarmFungi]

it may work Im not sure

[@karlpittman9925]

Hey awesome vids! Just a quick question about sawdust spawn. Do you inoculate that the same way as grain spawn, with liquid culture?

[@FreshfromtheFarmFungi]

kzbin.info/www/bejne/pIivk6mNed54ZpI here is a comparison I inoculate with 4-5 mL of LC

[@restotech9445]

question - for your tops - are they just drilled to the correct size to fit the FAE port and SH port or is there some kind of sealant?

[@FreshfromtheFarmFungi]

The first lids I did I used silicon rbv but now my friend has a drill press with the correct bits to just fit them perfectly 👍

[@restotech9445]

@@FreshfromtheFarmFungi awesome im going to need to figure out the right sizes, just was wondring if contam can go through a pressfit :D

[@Tomer179]

1) if you are reusing the same syringe its better to first add the glycerol to all vials then add the culture. adding glycerol first prevents the possibility of cross contamination between cultures. also, make a glycerol aliquot and don't use directly from the bottle especially after dispensing into liquid culture and reusing the same syringe, you could easily contaminate your glycerol bottle. 2) its important to mix the glycerol after adding it to water as its dense and sinks to the bottom and wont dissolve quickly. so mix the 10 and 30% glycerol stocks before use and also mix by inverting several times after dispensing the liquid culture to the glycerol stock.

[@FreshfromtheFarmFungi]

thanks for the critique this process will definitely be improved upon 🍄❤️🙏🏻

[@justwhistlinpixie]

Hey, Gary! Super informative as always. Are you going to have yellow and pink oyster LC on your etsy soon, now that summer is approaching? I'll buy some day one. Mush love.🍄

[@FreshfromtheFarmFungi]

yes I have them ready to go up now! : )

[@zacharyswain7034]

Do you spray plates that you've taken from storage with isopropyl or wipe them with isopropyl?

[@noneya96]

As in befor transfer He also most probably has pre sterilized plates

[@TheCommuted]

Can you replace honey with a disaccharide like table sugar?

[@FreshfromtheFarmFungi]

maybe

[@MrExtremefisherman]

and when isolating sectors to form a monoculture from ms how many transfers can I do before it becomes weak is the staments p value system hold any merit

[@FreshfromtheFarmFungi]

I think it varies from strain to strain some senesce quickly some last a while after years of subculturing

[@MrExtremefisherman]

@@FreshfromtheFarmFungi thank you Gary ☺️ & sorry one more thing mate when I’m taking out my culture slant to re slant after few months should I change the media like if I had a culture slanted in mea should the next slant be pda ?

[@louisw5858]

I once kept a culture in the fridge at 5 degrees and it seemed to got shocked and took so long to grow and due to poor growth it got contaminated. Have you ever had that before

[@FreshfromtheFarmFungi]

I have heard of that with more sensitive varieties but overall it shouldn’t effect them if they are kept sterile

[@louisw5858]

@@FreshfromtheFarmFungi maybe because I once open when it hit full colonization and after refrigeration i remember i shook the grains with a spoon which slowed the mycelium but drew those contams from the top, anyways thanks Gary now I feel it's common. Mush love 🍄

[@bradmcclure4945]

what is the shelf life of liquid culture in a refrigerator

[@FreshfromtheFarmFungi]

it depends on the variety some may last decades some tropical species will only last a few months

[@noneya96]

My cold trap can only reach -40°c can i just dabble the run time or will i need to be able to get to negative 80°c any help would be so appreciated

[@noneya96]

Also love your stuff can't stop watching i keep coming back

[@FreshfromtheFarmFungi]

I think it will still work at that temp but may reduce storage times slightly - I haven’t done the validations for that though there might be a reason that I am unaware of

[@noneya96]

I may have to do some live research my self then, will follow you further befor i go on with any thing Mushlove mate viewing from Australia

[@tarheelgarden]

couldn't you have sterilized a .2 micron spawn or grow bag and put the entire cryo vial rack into it and seal it? or a container with plenty of micro filters on it so the vacuum doesn't rupture it for the freeze drying process.

[@FreshfromtheFarmFungi]

I will try with a sacO2 bag that may help thanks!

[@noneya96]

Any update on this as being told it will kill mycelium

[@FreshfromtheFarmFungi]

I recovered some samples in the video and am waiting a few more months to recover the rest - it seems to work relatively well (50-60% recovery) just make sure you account for the potential losses. It is a standard procedure by ATCC - also described in the video - I did not come up with this protocol but am seeing the limits 👍

[@zacharyswain7034]

how do you store pink oysters in the fridge without them dying

[@FreshfromtheFarmFungi]

37F and keep rotating on slants/plates - some may be sensitive to these temps

[@zacharyswain7034]

Awesome. I think the strain I have is sensitive as I put the first culture I had in the fridge and wasn’t able to get anything from them so I had to get a replacement. How often do you rotate them from petri and slant

[@MrExtremefisherman]

hey Gary love the new vid mate 🙏🏼 just quick question is there a limit on how many time I can agar to agar transfer before it degrades or looses vigour, how many times is safe ?

[@noneya96]

F

[@MoltenSamurai]

F

[@jesnoggle13]

Another reason to get an expensive freeze dryer.

[@emartin8907]

wooooo another use for my freeze dryer! I have heard honey and chocolates do not do well in freeze drying, maybe a LC recipe with no honey for preservation uses would be better, not sure if it matters much. I just ordered those vials off amazon, can't wait to try this out! any updates yet? :) I'm curious if the glycerol makes any difference at all, and I'm curious if loosening the lids poses any real contamination risk here as the trip from flow hood to the freeze dryer as well as all the time in the freeze dryer before the vaccuum pump kicks on, is not a sterile environment and all. did you have any contam at all? check this cool thing out for laboratory uses- kzbin.info/www/bejne/jZmwpayjfqakl9E too bad we don't have a port of some kind on the door of the harvest right, although I'm sure something could be done about that if someone really wanted to... im just hoping contam won't really be a big deal here.

[@FreshfromtheFarmFungi]

so far about 7% success rate after almost a year - no contams and I agree maybe a different lc could help! I will be exploring this more next winter at the new setup hopefully 🙏🏻

[@rickyd1126]

When he said cryo I knew this video was not for me. 😅

[@KirillVasilev-production]

Dope

[@thestig560]

When he said "Back in the Nineteen hundreds" it felt like it was a thousand years ago, and then he says 1991 lol. Guess I'm just old 🥹