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Offlinecutemushie
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Another long term culture storage method? grain, resurrection, mycelium water
    #8885159 - 09/06/08 01:56 AM (16 years, 4 months ago)

I’m thinking if we let mycelium fully colonise a thin layer of grains inside a jar and then let the grains dry out so that the mycelium goes into dormancy, we could store it for many years?  When we need it, we just inject some sterile distilled water to rehydrate the grains and to reanimate the dried mycelium. (Or should we use sterile nutrient solution instead?)  It should be easy since the reanimation is done in sterile condition inside the jar.  Once it is growing nicely again, we inject some sterile distilled water and shake the grains to dislodge the mycelium.  We then use the syringe to suck out all the mycelium water and use it to inoculate new grains or to start new cultures.  The original jar is then left to dry so the mycelium goes into dormancy again for further storage.  Any comments on whether this method is workable and how long do you think we could store the culture?

Cutemushie


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HAVE: Paneolus cyanescence, Paneolus cambodgiensis, Psilocybe cubensis, Flammulina velutipe (white, and brown Korean strain), Hericium erinaceous, Coprinus atramentarius, Coprinus comatus, Agrocybe aegerita, Pleurotus eryngii, Psilocybe subaeruginosa, Ganoderma lucidum, Pleurotus nebrodensis, Lepista nuda, Agaricus augustus, Pleurotus cystidiosus, Volvariella speciosa.

LOOKING FOR: Those that I don't have.

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Invisiblewisp

Registered: 04/13/08
Posts: 5,304
Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: cutemushie]
    #8885173 - 09/06/08 02:01 AM (16 years, 4 months ago)

I believe that drying mycelium is a proven method for long-term storage already. I think what you are suggesting could work. You can do the same with petri dishes - let the agar dry out, then at some point in the future, cut a piece of the dried, colonised agar out and place onto a new dish.

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Offlinecutemushie
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Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: wisp]
    #8885193 - 09/06/08 02:16 AM (16 years, 4 months ago)

I have thought of using agar.  However, the issue with agar is when it is dried, it becomes very leathery and hard to cut up.  Also you can't shake agar to dislodge the mycelium.  With agar plate, you need to open it in sterile environment to prevent contamination and to cut a piece out.  But with jar and grains, you don't open the lid at all.  You use a needle and don't require a very clean environment.  I'm hoping that this could be an alternative method that might suit some people.


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HAVE: Paneolus cyanescence, Paneolus cambodgiensis, Psilocybe cubensis, Flammulina velutipe (white, and brown Korean strain), Hericium erinaceous, Coprinus atramentarius, Coprinus comatus, Agrocybe aegerita, Pleurotus eryngii, Psilocybe subaeruginosa, Ganoderma lucidum, Pleurotus nebrodensis, Lepista nuda, Agaricus augustus, Pleurotus cystidiosus, Volvariella speciosa.

LOOKING FOR: Those that I don't have.

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Invisiblewisp

Registered: 04/13/08
Posts: 5,304
Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: cutemushie]
    #8885198 - 09/06/08 02:23 AM (16 years, 4 months ago)

You have valid points there. To make things more practical, maybe you could use small vials (5ml - 10ml). Sterilise the grain in them as you usually you, then drop a small wedge of agar or a single drop of LC onto the grains. Make sure the hole in the lid is large enough to facilitate drying. Then once the colonised grain are dry, you would only have to store 5ml or 10ml vials, rather than bulky jars. Making mycelium from them would be pretty simple too.

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Offlinecutemushie
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Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: wisp]
    #8885227 - 09/06/08 02:43 AM (16 years, 4 months ago)

Yes I can use vials.  At the moment I don't mind the space issue.  I am experimenting using jars because they are free.  Also, the lids are bigger and I can make a very very big hole and the grains can dry much much faster.  Later I'll try to move on to smaller jars and then to vials.  But for experimenting, I prefer to use free stuff than buy vials.  I'm thinking there might be a problem with rehydrating the dried grains.  You know the issue of overlay.... when the mycelium has completely covered the grain or agar (similar to an overlay situation), it forms a waterproof barrier to rehydrating the grain or agar.  Probably more so for grain than for agar.  Hence I pose the question of whether or not to use nutrient soln because then the mycelium has direct contact with a souce of food and water.


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HAVE: Paneolus cyanescence, Paneolus cambodgiensis, Psilocybe cubensis, Flammulina velutipe (white, and brown Korean strain), Hericium erinaceous, Coprinus atramentarius, Coprinus comatus, Agrocybe aegerita, Pleurotus eryngii, Psilocybe subaeruginosa, Ganoderma lucidum, Pleurotus nebrodensis, Lepista nuda, Agaricus augustus, Pleurotus cystidiosus, Volvariella speciosa.

LOOKING FOR: Those that I don't have.

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Invisiblewisp

Registered: 04/13/08
Posts: 5,304
Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: cutemushie]
    #8885250 - 09/06/08 02:56 AM (16 years, 4 months ago)

Using what is already available is always preferable. Do you think overlay would be a problem with dehydrated grains? I think it would be fine, providing you leave the grains sufficient time to rehydrate. Using a nutrified solution would probably help though, as I think it would help catalyse the mycelium back into growing.

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Invisibleroquet
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Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: cutemushie]
    #8885419 - 09/06/08 05:35 AM (16 years, 4 months ago)

Quote:

cutemushie said:
The original jar is then left to dry so the mycelium goes into dormancy again for further storage.  Any comments on whether this method is workable and how long do you think we could store the culture?



I'm interested in long term storage of mycelium, so I hope this would work. But I suspect once you re-animate the dry mycelium you won't be able to use that jar again. I say that because I've tried the tek where you inject water into a jar, shake it about and aspirate it back out again for myc water. I found the 'donor' jar didn't recolonise with mycelium. Probably because it got too waterlogged; it's impossible to get all the water back out again.

But I might try out your idea. I can't grow for about 5 months of the year over winter. On a related point, I've found LCs last a surprisingly long time at room temperature, like 9+ month old LC colonises grain fine. I've yet to conclude whether the fruits suffer.

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Offlinecutemushie
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Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: roquet]
    #8886948 - 09/06/08 01:39 PM (16 years, 4 months ago)

Quote:

roquet said:
Quote:

cutemushie said:
The original jar is then left to dry so the mycelium goes into dormancy again for further storage.  Any comments on whether this method is workable and how long do you think we could store the culture?



I'm interested in long term storage of mycelium, so I hope this would work. But I suspect once you re-animate the dry mycelium you won't be able to use that jar again. I say that because I've tried the tek where you inject water into a jar, shake it about and aspirate it back out again for myc water. I found the 'donor' jar didn't recolonise with mycelium. Probably because it got too waterlogged; it's impossible to get all the water back out again.

But I might try out your idea. I can't grow for about 5 months of the year over winter. On a related point, I've found LCs last a surprisingly long time at room temperature, like 9+ month old LC colonises grain fine. I've yet to conclude whether the fruits suffer.




I tried a quick test run with P suberuginosa (using grain spawn previously exposed to non sterile air) and the mycelium grew back quickly, covering all the grains again.  I think you just need to think of a way or just tweak it to get most of the water out.  It will be a bit wet inside the jar but not sogging wet.  The amount of grains in the jar influences how much water stays in there due to surface tension and surface for evaporation.  Hence I proposed using a thin layer of grains.  With LC, I suspect that due to the favourable conditions inside, ie. with food and water, metabolic wastes will build up if the growth is not slowed by refrigeration.  Another issue with continued growth is senescence.  Alternatively you can syringe out all the nutrient solution and replace it with sterile distilled water.  This way you can store in room temperature and still halt growth and metabolic build up.  But still, there is a problem with accidental wetting of the lid filter (resulting in contamination) if the jar is accidentally knocked over.  I read that some species eg Pleurotus do not store well in water.


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HAVE: Paneolus cyanescence, Paneolus cambodgiensis, Psilocybe cubensis, Flammulina velutipe (white, and brown Korean strain), Hericium erinaceous, Coprinus atramentarius, Coprinus comatus, Agrocybe aegerita, Pleurotus eryngii, Psilocybe subaeruginosa, Ganoderma lucidum, Pleurotus nebrodensis, Lepista nuda, Agaricus augustus, Pleurotus cystidiosus, Volvariella speciosa.

LOOKING FOR: Those that I don't have.

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Offlinecutemushie
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Loc: Aus
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Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: cutemushie]
    #8886958 - 09/06/08 01:43 PM (16 years, 4 months ago)

I should point out that with the above post, I have yet to dry out the grains completely.  I'm gonna replace the lid with a bigger hole.


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HAVE: Paneolus cyanescence, Paneolus cambodgiensis, Psilocybe cubensis, Flammulina velutipe (white, and brown Korean strain), Hericium erinaceous, Coprinus atramentarius, Coprinus comatus, Agrocybe aegerita, Pleurotus eryngii, Psilocybe subaeruginosa, Ganoderma lucidum, Pleurotus nebrodensis, Lepista nuda, Agaricus augustus, Pleurotus cystidiosus, Volvariella speciosa.

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Offlinewillowmp
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Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: cutemushie]
    #8887019 - 09/06/08 02:02 PM (16 years, 4 months ago)

very interesting post i didn't know you could do this.


i have some dried out mycelium covered coir. like on little chunk, maybe i could spawn it to some wbs and use that with some extra coir i have to make a plug for an outdoor grow.


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"Fuck it too much Im outta wittbhjkhbhkjsrglhkrgl g I DEE JEUD."
-Because it made sense on xanax and booze...

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Offlinecutemushie
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Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: willowmp]
    #9170517 - 11/01/08 11:28 PM (16 years, 2 months ago)

Just an update here:

The mycelium seems to colonise the grains well.  The grains were boiled with coffee solution, left to soak for 24 hrs and then steamed dried.  I put a thin layer of the grains and pc'd them in jars with a big hole cut out on the lid.  The hole is covered with three layers of micropore tape.

The problem that often crops up is after shaking, some jars failed to recover and some recovered slowly but formed mycelium exudate which is likely  an indication of bacteria contamination.  Dunno how the bacteria got in.  I suspect it might be linked to the micropore tape.  Perhaps it was due to the micropore tape being wet after sterilization (so bacteria being wicked in) or being in 100% humidity during mycelium colonization, since I had to cover part of it with plastic cling wrap as it was drying out too fast?  Or could it be due to bacteria endospores within the grains?  Or could it be when the grains were shaken, some touched the micropore tape?  I know in other threads people mentioned that mycelium often stalls after the jars were shaken.  Any suggestions what I should do?

Tripsis, I know you have deliberately let your agar cultures dry out.  But I find that my agar plates take a very long time to dry and during this time, mycelium continues growing and so is senescence.  The main reason I'm using grain and large micropore taped hole is so I could dry it out extremely fast, especially later on when I have to squirt distilled water in to get mycelium water (bearing in mind that it would be impossible to get all the distilled water back out).  But because it dries so fast, I have to cover part of the hole with plastic cling wrap during mycelium run.  Would appreciate comments from you.  Thanks.

I would also appreciate any comments from other experienced growers.  Thank you.

Cheers,
cutemushie


--------------------
HAVE: Paneolus cyanescence, Paneolus cambodgiensis, Psilocybe cubensis, Flammulina velutipe (white, and brown Korean strain), Hericium erinaceous, Coprinus atramentarius, Coprinus comatus, Agrocybe aegerita, Pleurotus eryngii, Psilocybe subaeruginosa, Ganoderma lucidum, Pleurotus nebrodensis, Lepista nuda, Agaricus augustus, Pleurotus cystidiosus, Volvariella speciosa.

LOOKING FOR: Those that I don't have.

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Offlinewedaft
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Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: cutemushie]
    #9170676 - 11/02/08 12:35 AM (16 years, 2 months ago)


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Offlinecutemushie
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Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: wedaft]
    #9170817 - 11/02/08 01:37 AM (16 years, 2 months ago)

Quote:

wedaft said:
It's all right here homie:
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/6886717




Thank you Wedaft.  That's where I got the idea from but I am running into grain stalling after shaking.  Is tyvek waterproof?  Would it wick in contamination if wet?

Cheers,
cutemushie


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HAVE: Paneolus cyanescence, Paneolus cambodgiensis, Psilocybe cubensis, Flammulina velutipe (white, and brown Korean strain), Hericium erinaceous, Coprinus atramentarius, Coprinus comatus, Agrocybe aegerita, Pleurotus eryngii, Psilocybe subaeruginosa, Ganoderma lucidum, Pleurotus nebrodensis, Lepista nuda, Agaricus augustus, Pleurotus cystidiosus, Volvariella speciosa.

LOOKING FOR: Those that I don't have.

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OfflineRogerRabbitM
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Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: cutemushie]
    #9171513 - 11/02/08 08:08 AM (16 years, 2 months ago)

Sterilization of grains is never complete, because it would take 24 hours or more at 15 psi, which would turn them to mush.  I'd suggest long term storage on master culture slants with a small piece of hardwood inserted into the agar.
RR


--------------------
Download Let's Grow Mushrooms



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Offlinecutemushie
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Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: RogerRabbit]
    #9172661 - 11/02/08 02:13 PM (16 years, 2 months ago)

Thanks RR.  Appreciate your comment and the link.  That bacteria problem is driving me nuts.  Wasted so much of my time on all the preparation work only to see them fail after extended periods of time.

Cheers,
cutemushie


--------------------
HAVE: Paneolus cyanescence, Paneolus cambodgiensis, Psilocybe cubensis, Flammulina velutipe (white, and brown Korean strain), Hericium erinaceous, Coprinus atramentarius, Coprinus comatus, Agrocybe aegerita, Pleurotus eryngii, Psilocybe subaeruginosa, Ganoderma lucidum, Pleurotus nebrodensis, Lepista nuda, Agaricus augustus, Pleurotus cystidiosus, Volvariella speciosa.

LOOKING FOR: Those that I don't have.

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Invisiblewisp

Registered: 04/13/08
Posts: 5,304
Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: cutemushie]
    #9181086 - 11/04/08 03:12 AM (16 years, 2 months ago)

I've actually found similar problems with old grain jars which I haven't got around to using due to moving house. It's good to know why the problem arises, that is is from the fact that the grains are not 100% sterile.

Having said that, I've made a couple of jars with only 1cm or 2cm of grain at the bottom and inoculated that. They've dried out over time with no contamination and no modification to the lids either. I would think that if you use a minimal amount of grain, inoculate with a liquid culture and use a lid modified to allow quick drying, colonisation and drying would be able to occur reliably before bacterial contamination set in.

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Invisiblebillyboy36
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Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: cutemushie]
    #9182334 - 11/04/08 12:06 PM (16 years, 2 months ago)

bacteria is more than likely getting in from your inoculation holes via the tape.  If you're looking for immediate one time use, the tape works fine.  But if you are looking for longtime storage, make those lids that fatster makes in that link wedaft posted.  The lids have double sided silicone inoculation ports with three layers of tyvek.  I use them and they're pretty F'ing sweet.  Going on close to a year with various master jars with no problems.  You get tremendous control over how much drying out you can achieve with the large exposed tyvek that you can tape up to whatever amount you need.  After you suck up your myc water, let the jars sit with all the tyvek exposed until it dries back up to a preferred colonization moisture level and retape.  This will help with fighting bacteria.  I haven't had a problem with bacteria as you describe using those lids.

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Offlinecutemushie
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Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: billyboy36]
    #9187396 - 11/05/08 06:21 AM (16 years, 2 months ago)

Quote:

billyboy36 said:
bacteria is more than likely getting in from your inoculation holes via the tape.  If you're looking for immediate one time use, the tape works fine.  But if you are looking for longtime storage, make those lids that fatster makes in that link wedaft posted.  The lids have double sided silicone inoculation ports with three layers of tyvek.  I use them and they're pretty F'ing sweet.  Going on close to a year with various master jars with no problems.  You get tremendous control over how much drying out you can achieve with the large exposed tyvek that you can tape up to whatever amount you need.  After you suck up your myc water, let the jars sit with all the tyvek exposed until it dries back up to a preferred colonization moisture level and retape.  This will help with fighting bacteria.  I haven't had a problem with bacteria as you describe using those lids.




Thanks, Billyboy.  I suspect the micropore tape too and I'm also hoping the problem is with the tape.  I recently got my hands on a tyvek coverall.  According to RR or Agar (not sure which) the tyvek used in coverall is thicker.  So I only use one layer.  I'll test it out.  We don't have the tyvek envelopes here in Melbourne, so I don't have much choice.  By the way, what is the reason behind the fact that we cannot 100% sterilize grains?  Even with fractional sterilization before pcing?  Is tyvek much drier than micropore tape?  I'm hoping the problem is with the micropore tape.  After pcing, the micropore tape was soaking wet!  If the problem is with the grains as RR suggested, then I'll have to think of other alternative to using grains.  I really like fatster's method.  Problem with culture slants is the need for refrigeration.  I lost a few cultures before through accidental freezing and limited spece in the fridge.  Do you shake your master jars often?

Cheers,
cutemushie


--------------------
HAVE: Paneolus cyanescence, Paneolus cambodgiensis, Psilocybe cubensis, Flammulina velutipe (white, and brown Korean strain), Hericium erinaceous, Coprinus atramentarius, Coprinus comatus, Agrocybe aegerita, Pleurotus eryngii, Psilocybe subaeruginosa, Ganoderma lucidum, Pleurotus nebrodensis, Lepista nuda, Agaricus augustus, Pleurotus cystidiosus, Volvariella speciosa.

LOOKING FOR: Those that I don't have.

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Invisiblebillyboy36
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Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: cutemushie]
    #9187890 - 11/05/08 09:39 AM (16 years, 2 months ago)

once they are dry?  no, You should shoot them up with water every six months or so so you don't fossilize them.  Hit them with about 10 mls of water.  The grains will suck that water back up and the myc will start growing again.  Then remove the tape and let them dry back out.  Again, I haven't had any problems with bacteria or any contam's due to non 100% sterilization.  I suspect it has more to do with your lids than that.  I do use those 1/4 pint jars though.  There isn't a whole lot of grain in there.  Using a whole qt jar like in Agar's method might be a different story with the sterilization over time.

Tape is just, well, tape.  There is a lot of room for error in the adhesive, especially when it gets wet.  The lids don't get wet at all after the initially pc'ing with fatster's method.  You swoosh, not shake; no need with that small amount of grain.

Edited by billyboy36 (11/05/08 09:42 AM)

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Offlinecutemushie
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Re: Another long term culture storage method? grain, resurrection, mycelium water [Re: billyboy36]
    #9189025 - 11/05/08 01:18 PM (16 years, 2 months ago)

Quote:

billyboy36 said:
once they are dry?  no, You should shoot them up with water every six months or so so you don't fossilize them.  Hit them with about 10 mls of water.  The grains will suck that water back up and the myc will start growing again.  Then remove the tape and let them dry back out.  Again, I haven't had any problems with bacteria or any contam's due to non 100% sterilization.  I suspect it has more to do with your lids than that.  I do use those 1/4 pint jars though.  There isn't a whole lot of grain in there.  Using a whole qt jar like in Agar's method might be a different story with the sterilization over time.

Tape is just, well, tape.  There is a lot of room for error in the adhesive, especially when it gets wet.  The lids don't get wet at all after the initially pc'ing with fatster's method.  You swoosh, not shake; no need with that small amount of grain.




Hi Billy,  I knocked up some grains again last night but using tyvek instead of micropore tape.  See how it goes.  My lid is not as elaborate as that of fatster.  I'm a bit lazy.  So I tend to cut corners or make things simpler.  The hole is about a square inch.  Not sure how fast this will help the grain dry next time.  That's as big as I could get it for my situation.  So far I like tyvek.  It doesn't get too wet. 

My aim is to give them only a little water every year or two.  Just enough to get them surviving but not growing.  Since you don't have contam issue, maybe you should try it since mushrooms can be resurrected from bone dry tissues after many years. Ultimately it'd be good if we could leave it untouched in a dark place for like 10 years and then resurrect them when we need it.

I use only about an inch deep of grain, but my container is a bit short so even when shaking or swooshing, sometimes it touches the tyvek or micropore, esp when the grains are stuck too strongly together by the mycelium.

I have some old jars but when i let them grow too much (hole was too small and so couldn't dry them fast enough, the grains clumped together and stuck together so much so that even vigorous shaking couldn't separate them.  I was using P. subaeruginosa.  With some of my other jars with other spp. however, the grains got coated with white mycelium and remained as separate grains.  To get this I had to shake it more often.

Is the 1/4 pint jar the same size as those that people normally used to make brf cakes?


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HAVE: Paneolus cyanescence, Paneolus cambodgiensis, Psilocybe cubensis, Flammulina velutipe (white, and brown Korean strain), Hericium erinaceous, Coprinus atramentarius, Coprinus comatus, Agrocybe aegerita, Pleurotus eryngii, Psilocybe subaeruginosa, Ganoderma lucidum, Pleurotus nebrodensis, Lepista nuda, Agaricus augustus, Pleurotus cystidiosus, Volvariella speciosa.

LOOKING FOR: Those that I don't have.

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