Cloning From Dried Tissue

[Myc]

I've seen this question posed in one fashion or another so I thought I'd shine up this little nugget I found in the vaults and share.
http://archives.mycotopia.net/discus...tml?1106008656

If you have good sterile technique, cloning from dried material is a cinch.
I started with an X style petri dish.
One section is poured with gentamycin agar
The other three poured with standard agar prepared in the usual fashion.

I did this two different ways in order to demonstrate just how easy this is.

First, I've selected material from a gourmet sample which was stored in a plastic print baggie and shipped.
During transit the bag was ruptured, the sample squeezed out into the envelope, and the sample seemingly destroyed.

Samples of this material were rescued - as cleanly as possible - and transferred to the gentamycin section of the plates.
New, clean growth was observed after only two days.


So now let's try this with a dried fruitbody sample.
This fruitbody is known to be at least one year old. Was stored in a tool box in a garage and forgotten about. Has been handled many times with no aseptic considerations at all. Sample was returned to the lab for experimentation.

First, I split the sample as best I could.

Material was selected from the cap area. I tried to get material that had previously not been exposed to open air.

Select a tiny sample and transfer to gentamycin agar.

Seal plates with parafilm and incubate

Note the blueing of the transfer as it rehydrates on the agar!


In this manner, dried mycelial tissue can be revived with ease.
I've also done this with culture plates which dried out during storage under refrigeration with excellent results. In the case where sterile tissue is being revived, the gentamycin agar is not necessary.

I hope this helps with your rescue efforts.

[hyphaenation]

Thank you sir !

Really needing to do this. For saving redboy ... i'm down to tiny dried mushrooms.

[Myc]

Now that's how you spell success!!!

Mycelial revivification after 6 days on standard (gent.) agar
From a one + year old, man-handled, filthy specimen.

Tell me that Fungi aren't survivors!!!
DAMN! Talk about will to live!

[Ben Dover]

That's awesome, thanks Myc!

I'll have to file this for later reference.

[Mad_Hatter]

Amazing. I had no idea that dry tissue was reanimatable.

[BuckarooBanzai]

I recently had success reanimating a stem that had been in a specimen vial with dessicant for 2 years. It was *BONE* dry.

At 85F on agar, I had fresh growth on the first plate within 3 days and all of them within 6. Once it got started, the myc growth was perfectly normal - but they do start kind of slow. Really mashing the tissue down into the agar seems to help a little.

I believe this technique can be used with virtually any kind of mushroom.

[eatyualive]

nice myc!

[crazy1]

great info Myc thanx!!!!

[M&M420]

Nice write up Myc.Do you think I could transfer to an LC as well?I'm going to try this with a LC as soon as it cools down.

[TastyBeverage]

Very nice! I have some very expensive imported dried shitakes that are begging for this!

[livewire420]

[hyphaenation]

Good idea tasty ! You smart ...

[BuckarooBanzai]

Originally Posted by meanman420

Nice write up Myc.Do you think I could transfer to an LC as well?I'm going to try this with a LC as soon as it cools down.

If your sterile technique is really good, you might get away with it.

Part of the purpose of using agar is identifying any contams on the flat surface. Myc grows very quickly and you can cut healthy growth out to start a new plate, usually leaving the contams behind (though sometimes you have to transfer again).

[Myc]

So here's the culture after ten more days.
No contams!

I actually took four transfers.
Two were laid on top of the agar and gently pressed into the surface. Both of these specimens are growing fine.
Two were thouroughly mashed into the agar. These two specimens failed to take off. I'm still going to experiment further and see if a transfer will cause them to grow.

As for transfer to LC, I think BB said it best.
You'll have to be very meticulous.
If you can't work with agar, I would at least add gentamycin to the LC to give a greater chance of success. Never added gent to LC but it makes sense that it would work.

[BuckarooBanzai]

Looks good, man! It is really weird the way the starter tissue browns and dies off - just like cloning fresh tissue. Some fun stuff is going on there, microscopically, as the cells hydrate, shift gears back to growing myc and then perish as the colony starts.

And I'm sorry if I misled you with "mash the tissue down" onto the agar. You want some pressure, so it gets good contact and rehydrates, but you don't want to force it down into the agar. I'm not sure why, but the really squished down tissue didn't grow well for me either (which is odd, I thought that would be better).

Just don't "perch" the tissue on the agar - it needs some solid contact with the agar or it won't rehydrate and start growing again.

Hmmm - just thought - maybe swab the dry tissue against the condensation in the edges of the plate cover? That condensation should be sterile.

There have been some term 10 and 20 year studies on reanimating dehydrated tissue from other mushrooms; very high success rates. I think it is a good way to store strains.

[Om shanti]

So mushrooms only really die if allowed to naturally decompose.. Amazing.. Very interesting thread!

[RugRat5288]

wonder if you were to introduce dry tissue to an lc if you would be able to get growth?

[MrZeus]

Some mushrooms store special sugars that allows them to resurrect themselves from the dead. I found one in the yard this year and learned this myself. Fungi is kewl!

[P4ulada]

s-u-c-c-e-s-s
Nice myc, it works ^^
Congratz man!

[Om shanti]

If I may suggest, this belongs in the vaults !

[zodd]

very nice my c. i have catalogued all of my successful projects for later reanimation because ive heard that this was possible. and now i have proof that it works

just one question though

is this gentamicin what is known as antibacterial agar

and

where can i find this gentamicin is it to be found on your average large supermarket chain shelf

thanks for sharing your results with us

[MLBjammer]

Myc, thanks for the explanation and illustration. I always appreciate your insights.