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Preservation of agar specimen


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#1 raymycoto

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Posted 02 December 2018 - 07:00 PM

I've read a few opinions on long term storage of a specimen. I have a specimen or two on agar I want to keep mainly to experiment with the technique but also in order to propagate again in 6 mo or so if I choose. They are right now on agar.

 

Another thought on preserving a specimen is that it might be a while before one gets fruit from the specimen on agar. By that time, agar specimens are getting old unless you keep transferring and then there is the question of senescence from prolonged growth.

 

So . . . it seems that it's best to keep the specimen cool and free of nutrients and oxygen for preservation.

 

Here is my plate BTW. It's a bit old and past its prime but these are monoclonal at this point and the subsequent transfer and LC from agar did well (so far).

 

Additional question btw. Note the dark, tall growth on the plates. I have seen this on other old plates. Is this some sort of primordia or pinning perhaps? Don't think its contamination.

 

20181202_173131.jpg

 

What I had planned to do was to scrape some myc off the agar then insert into a small, sterile specimen cup with sterile water. 

 

My concern is that I'll likely not get all the agar off the specimen so it will have some nutes with it in the water. But I'll use a #10 blade and just scrape it off, I guess.

 

Does this sound like a good plan?

 



#2 Billcoz

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Posted 03 December 2018 - 04:17 PM

 I haven't stored cultures before but I've read about using wood, like tongue depressors/popsicle sticks or toothpicks in agar slants, the myc will colonize the wood and somehow it will keep for years.

 

 You sterilize the wood by boiling and then put slivers of it into your culture slants, this is described on the "Let's Grow Mushrooms" video.

 

 I can't find a specific tek about it right now, just search "long term culture storage", I'm sure you'll find some way.



#3 raymycoto

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Posted 03 December 2018 - 07:23 PM

Yes, I've seen that. Mine is not a wood loving creature. Probably would not hurt to add some wood in any case.

 

I ended up just scraping off some myc and putting it into the water. Wow, what a small scrap of tissue it is (myc on agar) when you scrape it off. It doesn't really embed itself into the agar but just grows on top, it seems.I probably should, at the same time just take some agar wedges and drop them into the same or different bottle. This is, after all, a sort of experiment. At least the water won't allow them to dry out.

 

Could also do the same with some stipe or cap tissue, I imagine.


Edited by raymycoto, 03 December 2018 - 07:24 PM.


#4 425nm

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Posted 04 December 2018 - 01:57 AM

I've heard of people storing mycelium in water. Never tried it myself but I imagine it could work.

Slanting I have done. It can be a bit of a pain to get capped test tubes but if you can its a very space efficient method. I don't think the addition tongue depessor will help for non-woodlovers as you've already observed. What can help with slants is your choice of media. Cornmeal glucose agar is often used for slants because its shitty and nutrient poor. You want your fungi to grow slowly in the slants. But regular old MEA will work fine too.

 

Additionally once your slants are colonized you can fill your test tube with sterilized mineral oil. This will limit O2 further slowing growth.


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#5 Billcoz

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Posted 04 December 2018 - 02:00 AM

Yes, I've seen that. Mine is not a wood loving creature. Probably would not hurt to add some wood in any case.

 

I ended up just scraping off some myc and putting it into the water. Wow, what a small scrap of tissue it is (myc on agar) when you scrape it off. It doesn't really embed itself into the agar but just grows on top, it seems.I probably should, at the same time just take some agar wedges and drop them into the same or different bottle. This is, after all, a sort of experiment. At least the water won't allow them to dry out.

 

Could also do the same with some stipe or cap tissue, I imagine.

 For culture slants ANY myc will colonize the wood, at least that's exactly what the guy on "Let's Grow Mush's", He said even non- wood lovers would colonize in the vid, I've read about people doing it too.

 

 Here's a lnk to someone asking about wood in cultures -https://www.shroomer...Number/13224156


Edited by Billcoz, 04 December 2018 - 02:04 AM.


#6 coorsmikey

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Posted 04 December 2018 - 08:36 AM

Honestly I don’t care about what some guy that makes a video says, nor many of the what is considered normal at the place of the above link.

Even non wood loving species will colonize the wood in slant, especially when soaked in agar. That’s what helps with the long term storage. After the mycelium has dried up on the agar on can often still get a slice of the colonized wood for a viable transfer. It’s a common practice that was adopted long before YouTube videos and even the OMC if one were to read some of the older books written by the pioneer mycologists.


Edited by coorsmikey, 04 December 2018 - 08:39 AM.

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#7 425nm

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Posted 04 December 2018 - 02:42 PM

Ah right, I hadn't considered that some of the the nutrients will probs penetrate into the wood during PC'ing. Storing under mineral oil eliminates the drying out as a concern though.


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#8 Billcoz

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Posted 04 December 2018 - 08:36 PM

Honestly I don’t care about what some guy that makes a video says, nor many of the what is considered normal at the place of the above link.

Even non wood loving species will colonize the wood in slant, especially when soaked in agar. That’s what helps with the long term storage. After the mycelium has dried up on the agar on can often still get a slice of the colonized wood for a viable transfer. It’s a common practice that was adopted long before YouTube videos and even the OMC if one were to read some of the older books written by the pioneer mycologists.

LOL, RRs not too popular here I guess, I'm pretty sure it's in the book "Growing Gourmet and Medicinal Mushrooms" too, does that count? 


Edited by Billcoz, 04 December 2018 - 08:37 PM.


#9 coorsmikey

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Posted 04 December 2018 - 10:43 PM

Well Bilcoz, my comment was in agreement with you, but yes It’s in a lot of books that the wood in slants work with just about all species.

RR is alright but this Site was actually founded with a falling out with Hippie3 and RR and a couple of others. We don’t necessarily have that attitude with others in the OMC now. We just don’t agree with some of the information that is passed on. But there is tons of conflicting info here and other sites. That’s what make threads and conversations like this one important, we get to talk about it and hash out reasoning with current situations instead of watching videos of what worked for someone else. So yeah no hard feelings toward our sister sites. Actually the more info that they give that doesn’t work for people, the more people come here and and hang out. Gosh darn we could use some more traffic. If peeps don’t like our bad attitude and vibe, the are more than welcome to go over there. I particularly just love everyone everyone, Including RR. Even if I don’t agree with some of his advice, he is still helping people find medicine and explore themselves. Good peeps in my book and I still hate SGFC’s


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#10 raymycoto

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Posted 05 December 2018 - 01:06 AM

Cool. Thanks for the discussion. 

 

Agree that at the worst, wood is simply a wick for the nutrients that it was soaking in. So eliminating nutrients is necessarily part of storage or even possible unless you are scraping it off the medium or placing a chunk of fruit into storage.

 

Cold storage and eliminating access to O2 - good. Mineral oil storage noted above. Wondering about a nitrogen flush prior to sealing the vial or bottle. There would be a bit of dissolved O2 in the medium and a repeat flush could be done after a few days of storage. 

 

I have a couple of general reference mushroom books. I'll take another look there as well.


Edited by raymycoto, 05 December 2018 - 01:07 AM.


#11 raymycoto

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Posted 05 December 2018 - 01:30 AM

Info from Stamets "The Mushroom Cultivator" Copyright 1983

 

The discussion here involves duplicate, slant, screw top test tubes with standard agar, incubate and grow x 1 week, then cold storage, possibly with mineral oil coverage and occasional testing of your slants for viability, prorogation and sharing.

 

stamets myc storage.jpg

 

These pathology specimen containers are type 5 plastic and PC well at up to 250 C (20 PSI). They have a lid that seals nicely and seem to be the perfect size. They come with a bit of formaldehyde solution that washes out completely. I've used them for aqueous sterile solutions. Perhaps I'll do some agar in them and report on how it does. I'm sure it will work.

 

tube storage.jpg


Edited by raymycoto, 05 December 2018 - 01:47 AM.

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#12 Billcoz

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Posted 05 December 2018 - 08:03 AM

 It's all cool, I personally think the guys over at shrmry are more harsh towards noobs than here and I see way too many people answering/replying to questions with "RR said..." lol, almost any search you do you'l see thatin the replies.

 

 Funny about hating SGFCs, after doing 2 sgfc & three mini bulk grows, I'm done with the fanning of the SG 4+ times a day.

 

 Hey Raymycoto, this is kinda off topic but since you mentioned cold storage, mycelium can survive freezing, would old cakes tossed out in the snow & frozen have any chance of fruiting in the spring when they thaw?

 

 Most of them were tossed out after 2 or 3 flushes to make room for new ones so they definitely were still gonna pop out some fruits if I kept em in the FC.

 

 They're in a pile by my compost in my garden, in the fall I had tossed all the wild mushrooms I found(non actives) in that spot just to see if I get a patch, so maybe they would compete.

 

 Just a thought I had when throwing some out, oh yeh, I did have some fruit after tossing out when the weather was warm.


Edited by Billcoz, 05 December 2018 - 08:05 AM.


#13 raymycoto

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Posted 05 December 2018 - 11:28 AM

Interesting point . . . would freezing be better than cold storage? Don't know but probably worthy of an experiment although it would be a long time to learn the answer, like maybe many years.

 

Problem I see with just popping something into the freezer is the problem with 'freezer burn' which is desiccation of the item while the water accumulates elsewhere in the container, a sort of slow freeze drying of the specimen. Although this would seem to be a good application for the mineral oil.

 

About the cakes - don't know but one would certainly give it a go. I have put some old bulk into a bit of mulch once and then covered with about a foot of dry and moist leaves. Forgot about it and a few weeks later there were some nice fruits, dried and black with spores under the leaves. Have tried but not duplicated it successfully. I think you would be best to break up the cakes into the mulch a bit rather than just throw them in  --- much like you would seed a bulk bin.


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#14 Billcoz

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Posted 05 December 2018 - 05:08 PM

 It would be cool if I find some cubes popping up in my garden in the spring, I did crumble some of them.

 

 But yeah in the freezer it seems like mineral oil would keep the cells from being destroyed by the water in em expanding, since mineral oil has a lower freezing point than h2o, it should keep it from freezing & epanding in the cells of myc, though I've only just now read a lil bit about using it like this so I'm not sure hw well it works.



#15 Billcoz

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Posted 05 December 2018 - 05:26 PM

Good peeps in my book and I still hate SGFC’s

 I like to make small FCs for single cakes and I just kinda make em like SGs, but I make the holes smaller and further apart since it's a smaller container, but if there's a better design for cakes than a SG I would like to try, so do you recommend any other FC for brf cakes than a SG, or just to not even make cakes? 

 

 After seeing how much better and less maintenance bulk subs are, the fruiting has been way more uniform, more yield, and I don't need to fan it every few hours, can't wait till I can spawn my p-corn  pints(to coir). 


Edited by Billcoz, 05 December 2018 - 05:26 PM.


#16 raymycoto

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Posted 05 December 2018 - 09:42 PM

I've only done bulk so I can't advise on the best tech for fruiting cakes. With my (limited) experience fruiting consolidated bulk bins I'm still a bit uncertain of whether mist and fan is really necessary or simply a constant humidity level with just a bit of FAE and maintenance of water content. I've tried side by side identical bins, some with lots of fanning and some with limited and not really noted a difference. I did do some misting to the limited fanning bin to keep up its water weight. 


Edited by raymycoto, 05 December 2018 - 09:43 PM.

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#17 425nm

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Posted 05 December 2018 - 10:57 PM

I wouldn't assume just mineral oil is enough of a cryo-protectant for freezers. Yes the mineral oil won't freeze but that doesn't mean the water in the media and/or fungal cells won't.

I'm not 100% sure if this holds true for fungi but with other cells/tissues you usually have to go through a series of increasingly high concentrations of glucose washes with time between each to allow the cells to adapt to the new osmolarity of the solution they are in (basically you're drying to dehydrate your cells ever so slowly without killing them). I think glycerol is also used as a cryoprotectant. Might be worth googling for a protocol. Or if you want to go hell for leather just don't use a culture you care about.


Edited by 425nm, 05 December 2018 - 10:57 PM.

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