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Offlineblueboy353
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can you clone from dried mushrooms
    #4360062 - 07/01/05 02:50 PM (18 years, 10 months ago)

i have dried mushrooms, can youclone them some how. or is their no luck b/c their dried.

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Offlineshirley knott
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Re: can you clone from dried mushrooms [Re: blueboy353]
    #4360176 - 07/01/05 03:16 PM (18 years, 10 months ago)

it is possible to reanimate the host if the mushrooms was dried with mature spores on the gills. you could try selecting a piece of gill and putting it on agar, then crossing your fingers and transferring to a clean plate if a white fluffy growth emerges.
if you're lucky, someone like workman, eontan or rogerrabbit will happen by with details.


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Re: can you clone from dried mushrooms [Re: shirley knott]
    #4360237 - 07/01/05 03:31 PM (18 years, 10 months ago)

that's not really a clone though. you have to use tissue for cloning.


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InvisiblePrisoner#1
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Re: can you clone from dried mushrooms [Re: Dragonaut]
    #4360272 - 07/01/05 03:41 PM (18 years, 10 months ago)

no but it's the best he can expect

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Re: can you clone from dried mushrooms [Re: Prisoner#1]
    #4360416 - 07/01/05 04:23 PM (18 years, 10 months ago)

Check out this thread, and please try it yourself as well.

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Offlineshirley knott
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Re: can you clone from dried mushrooms [Re: Pinback]
    #4360546 - 07/01/05 04:58 PM (18 years, 10 months ago)

that was a great read, thanks for 'reviving' it, Pinback  :wink:

seems like the evidence that spores are NOT responsible for the reanimation RR and others proclaim possible, is impossible to disprove. you'd need to electron-microscope the sample before growth attempts, but this process of EM-preparation would surely itself render the tissue dead. maybe it's possible within a limited timeframe, maybe it's possible indefinitely, maybe it's impossible. i noted with interest roger's assertion that peroxide exposure limits the tek's success.


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OfflineRogerRabbitM
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Re: can you clone from dried mushrooms [Re: shirley knott]
    #4363162 - 07/02/05 01:28 PM (18 years, 10 months ago)

Many of the edible culture banks store mycelium in its dry form. The commercial mushroom farms routinely buy dry mycelium to restart on agar. It helps if there are no imperfect mold spores on the tissue. Bacteria usually isn't a problem as it is killed by drying.

Perhaps a segment in my upcoming video series can be to take a piece of mycelium from the inside of a fresh mushroom stipe as though I were going to clone it, but instead slip it inside a petri dish with desiccant rather than agar. I could let the tissue dry for a few months, under sterile conditions, then retrieve and grow it out. This is how the tissue banks do it. Dry mycelial tissue will grow again, I assure you. I've switched over to all legal edibles and medicinals, so I'd have to do it with oyster or something, but you'd get the point. The procedure would be the same.
RR


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Re: can you clone from dried mushrooms [Re: RogerRabbit]
    #4368584 - 07/04/05 02:55 AM (18 years, 10 months ago)

That would be good to see.
How long does tissue last in H2O2.
I picked a fruit in open air, tore it up with dirty hands and tossed it in a jar of H2O2 for about 10mins, I then sucked up some tissue in a syringe through a self healing hole and injected it in a jar of grain. 2 days later I have growth started in the jar. It was 3% H2O2. Do you think it will last? Am I better leaving it in sunlight to speed up the degradation into H2O2.

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OfflineRogerRabbitM
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Re: can you clone from dried mushrooms [Re: blackout]
    #4368975 - 07/04/05 09:01 AM (18 years, 10 months ago)

Peroxide will kill some of the bacteria, but there's way more contaminants than just bacteria, especially working with dirty hands in open air. Unfortunately, I give your experiment little chance of success. I hope it succeeds, but the odds are against you.
RR


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"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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Offlineblueboy353
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Re: can you clone from dried mushrooms [Re: RogerRabbit]
    #4371531 - 07/05/05 07:51 AM (18 years, 10 months ago)

allready molded :frown:

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Offlinemattymonkey
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Re: can you clone from dried mushrooms [Re: blueboy353]
    #4372463 - 07/05/05 02:23 PM (18 years, 10 months ago)

great thread, nice read pinback thanks...

so is this a way to store strains long term?

would it be better to use dried myc from an agar plate to get a strain thats a little closer to its origins?

im not really understanding the exact process... if placing a piece of mushroom tissue, or a agar wedge, in an empty plate.. would you seal the plate with parafilm still? would it dry out otherwise? how would one add desiccant to the plate, wouldnt the desiccant itself be contaminated? is it possible to sterilize it?
thanks

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Re: can you clone from dried mushrooms [Re: mattymonkey]
    #4377507 - 07/06/05 08:32 PM (18 years, 10 months ago)

The way I do it is in front of my flowhood, I cut a piece of mycelium from the very center of a thick stipe. This gets 'virgin' tissue. I place this small piece of tissue on a piece of wax paper and place in a clean petri dish with desiccant under the wax paper. I then seal it up with parafilm, desiccant and all. I've pulled two year old cracker dry tissue out this way and it began growing within two weeks after placing on agar. Mix the agar a bit weak, so it's wetter than normal. Make a transfer as soon as you can see mycelium growing.
RR


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Re: can you clone from dried mushrooms [Re: RogerRabbit]
    #4382422 - 07/08/05 01:08 AM (18 years, 10 months ago)

Great thread, it inspired me to try this with a Pleurotus eryngii plate that dried up completely.

I prepared some MYA using a little bit less agar than usual to make the solidified medium a bit more moist. I inoculated 5 dishes with chunks of dry colonized agar.

Two days later I saw new growth in 3 dishes.

I am very happy with this tek because I thought I had lost this species permanently. It shows that these perfect fungi are amazingly resilient organisms.

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OfflineRogerRabbitM
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Re: can you clone from dried mushrooms [Re: Owl]
    #4386246 - 07/09/05 07:30 AM (18 years, 10 months ago)

Transfer some of that growth as soon as you see it. If it dried up on a plate, it was a slow-ride into stasis and contaminants are probably right behind the mycelium. Just a suggestion.
RR


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"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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OfflineOwl
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Re: can you clone from dried mushrooms [Re: RogerRabbit]
    #4389486 - 07/10/05 10:28 AM (18 years, 10 months ago)

Good suggestion thanks.

I will make some transfers today...

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Re: can you clone from dried mushrooms [Re: Owl]
    #4394958 - 07/12/05 03:26 AM (18 years, 10 months ago)

From TMC
"KOH Potassium Hydroxide, an agent commonly used to revive dried mushroom material for microscopic study at a concentration of 2.5%"

Anybody use this?

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OfflineWorkmanV
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Re: can you clone from dried mushrooms [Re: blackout]
    #4398112 - 07/12/05 11:14 PM (18 years, 10 months ago)

"Revive" in this context is not reanimate but more like rehydrate for viewing structures under the microscope. You can't slice or microscopically view dried material directly. It must be plumped up with water. KOH is caustic (think drain cleaner) and wouldn't be helpful in cultivation. The addition of KOH causes some color changes in certain microscopic features that can help in identification, but I have never been clear on what else it is for. It must make the structures clearer for viewing or aid in the reconstitution in some way since it is used for all dried mushroom tissue destined for the microscope. I purchased a supply of KOH but have yet to make a solution with it. NaOH is supposed to be a reasonable substitute.


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Re: can you clone from dried mushrooms [Re: Workman]
    #4398721 - 07/13/05 02:55 AM (18 years, 10 months ago)

Makes sense now you say it. I am so used to seeing the term "microscopic study" in a disclaimer context that it didn't even register with me that it may mean that its not intended for growing.

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Offlinered914
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Re: can you clone from dried mushrooms [Re: blackout]
    #4403207 - 07/14/05 11:02 AM (18 years, 10 months ago)

I have heard of using "Ice Melter" from Home depot to dry stuff. It is some kind of salt, a little easier on your concrete if you actually deal with ice at all. Ya use it in crystal form, not solution to dry stuff. It is so dry where I live that trying to fruit a 10 lb. sawdust block of namekos will dry out compleatly in 24 hrs. Misting? Waist of time. Hose! Ya have to rethink your procedures around here.
Will most mushrooms rehydrate to working myc after drying? Ya mean all those agar dishes I throw out because they looked like potatoe chips could have been saved? I had to use short widemouth jars instead of petri dishes to control moisture loss even in a closed box. Anything you can add to rehydrating tissue would be very useful.

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Re: can you clone from dried mushrooms [Re: red914]
    #4405320 - 07/14/05 07:45 PM (18 years, 10 months ago)

If you live in a desert climate, you'll need to run a humidifier in the rooms you grow in. In fact, running a huge humidifier in your house would be a good idea. That will make it much easier to keep your grow areas humidified. It will also make it easier for you to breathe and keep your skin from drying out. Petri dishes should be wrapped with parafilm to hold in moisture and keep out contaminants.
RR


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Invisiblemycogirl
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Re: can you clone from dried mushrooms [Re: Pinback]
    #4406775 - 07/15/05 02:28 AM (18 years, 10 months ago)

That is a great thread. Fungi are so interesting (to me) because they don't seem to follow a lot of the rules that we tend to apply to 'life'. Any the vitality and growth patterns are extraordinary. When thinking about these organisms, I think its important not to try to compare them to plants.


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Offlinered914
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Re: can you clone from dried mushrooms [Re: mycogirl]
    #4407436 - 07/15/05 10:49 AM (18 years, 10 months ago)

They DO follow a lot of rules. That's the problem. There are so many variables, all with pitfalls and all very subtle and all take time to prove out so if you are not very organized, like me, and don't remember to write it down, evey stinking time, you end up with a basement full of contams. It is kind of like the old Weird Science movie where things go wrong because they forgot to put the bra on their head. You are right though, that is exactly why it is so interesting. Ya keep hearing Nubies say they want to make a business of growing mushrooms. Ya gotta grow em first, THEN say, OOOH look, I have more than I can use (that haven't molded) I better sell some! (more likely "help me... I have black mold in my underwear!")

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Re: can you clone from dried mushrooms [Re: red914]
    #4413425 - 07/16/05 11:24 PM (18 years, 10 months ago)

I've got some Maitaki dishes goin from a dried specimen. It's actually not as hard as you'd think. All samples were taken from the inside of the super tiny stalk and then cleaned with a 3% H202 and then a vinegar dip.

Edited by hotnutz (07/16/05 11:36 PM)

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Offlinered914
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Re: can you clone from dried mushrooms [Re: Hotnuts]
    #4424429 - 07/19/05 04:44 PM (18 years, 9 months ago)

Vinegar dip? how strong? Whats it do?

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Re: can you clone from dried mushrooms [Re: red914]
    #4425597 - 07/19/05 09:26 PM (18 years, 9 months ago)

Quote:

red914 said:
Vinegar dip? how strong? Whats it do?




I got some tips from Mycota or known as Agar here a while back on the sanatization purposes of H202 and vinegar. The combo, one used right after the other, are fantastic sanatizers. Far better than bleach and other cleaners for this purpose. The only prob is the vinegar is very acidic. Molds love it. In this case for cloning the vinegar was used last. Specimen into H202 for cleaning and hydration, then into white vinegar, and last into sterile H20 for a cleanse of the vinegar. Then on to cardboard for a growth medium. It took 9 days to see a growth start. Pretty slow..

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Offlinered914
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Re: can you clone from dried mushrooms [Re: Hotnuts]
    #4425657 - 07/19/05 09:37 PM (18 years, 9 months ago)

I have heard of the h2o2 and vinegar cleaning for tables and the like, but tissue! who'd a guessed. Im gonna try it, havn't had any luck lately with contams.

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Re: can you clone from dried mushrooms [Re: Hotnuts]
    #4426012 - 07/19/05 11:00 PM (18 years, 9 months ago)

Definately will sanitize. What's happening is that vinegar (acetic acid) and peroxide (h202) residues are combining to form peroxyacetic acid. It's a labeled healthcare/greenhouse sanitizer. Excellent oxidizer, can burn your eyes.

But since many fungi can overcome acid environments better than bacteria, and fungi can deal with peroxides, maybe they are less affected. Peroxyacetic acid is used as an antifungal, anti-algae, antibacterial, antiviral, antiwhatever. I guess it makes contaminant ceviche (mexican food reference).

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Re: can you clone from dried mushrooms [Re: mushrx1]
    #4427161 - 07/20/05 05:21 AM (18 years, 9 months ago)

I personally don't think vinegar is necessary at all in cleaning up cloning tissue, I have never used anything besides peroxide myself with excellent results and h2o2 has little detrimental effect to the myc itself, I'm sure any form of acid would be detrimental to myc, but great for killing off contams/anything as a sanitizer. This is just my personal opinion/experience.


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Re: can you clone from dried mushrooms [Re: hyphae]
    #4428405 - 07/20/05 01:03 PM (18 years, 9 months ago)

Quote:

hyphae said:
I personally don't think vinegar is necessary at all in cleaning up cloning tissue, I have never used anything besides peroxide myself with excellent results and h2o2 has little detrimental effect to the myc itself, I'm sure any form of acid would be detrimental to myc, but great for killing off contams/anything as a sanitizer. This is just my personal opinion/experience.




Do you think the vinegar was the culprit for it's slow start of growth? Or are edible isolations slow in the first place?

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Offlinehyphae
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Re: can you clone from dried mushrooms [Re: Hotnuts]
    #4429295 - 07/20/05 04:30 PM (18 years, 9 months ago)

Peroxide retards growth somewhat even, I'm sure acid would have even more of an affect. All tissue cultures that I have done were a bit slow starting out but I contribute that to the sample getting a foothold in the agar before uptake of nutes could be established.


--------------------
Getting the most out of your casings!, A pinning strategy.
Oyster Shell "Flour" $2 for 1lb. a hell of a deal :wink:
Not what is overlay but rather what overlay is
Gas Exchange vs. FAE

"We all have priorities. I used a closet once setup a nice little lab trouble was all the shit that was in there ended up in the bedroom that pissed off the GF then I ended up dumping her as she was getting in the way of my sterile culture technique! Ya I got priorities too!!!"

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Re: can you clone from dried mushrooms [Re: hyphae]
    #4431974 - 07/21/05 02:30 AM (18 years, 9 months ago)

I tore up a bit of a shroom in dirty air with dirty hands and dumped it in 3% H2O2. I swirled it around and it fizzed up. It is now 1 or 2 weeks on and the H2O2 is now brown with the tissue not looking great. However some tissue clinged to the side of the jar and it is showing signs of growth. I presume the H2O2 on it decomposed into water and oxygen so it grew well. It is a thin strip covered in fuzz. I will try and transfer it to a agar plate at some stage.

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Re: can you clone from dried mushrooms [Re: blackout]
    #4432179 - 07/21/05 06:05 AM (18 years, 9 months ago)

I only do a 20-30 min. dunk then transfer. GL blackout


--------------------
Getting the most out of your casings!, A pinning strategy.
Oyster Shell "Flour" $2 for 1lb. a hell of a deal :wink:
Not what is overlay but rather what overlay is
Gas Exchange vs. FAE

"We all have priorities. I used a closet once setup a nice little lab trouble was all the shit that was in there ended up in the bedroom that pissed off the GF then I ended up dumping her as she was getting in the way of my sterile culture technique! Ya I got priorities too!!!"

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Offlinered914
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Re: can you clone from dried mushrooms [Re: hyphae]
    #4432654 - 07/21/05 09:41 AM (18 years, 9 months ago)

So, would it be better to just place the sample in the agar, no dip, and use agar technique to seperate the new myc? Agar tek is my worst skill. Still working on a pressure glove box.

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Re: can you clone from dried mushrooms [Re: red914]
    #4433049 - 07/21/05 11:17 AM (18 years, 9 months ago)

No way! Clean it up in H2O2 and then transfer to agar, besides the dip helps with rehydration of the dried tissue.


--------------------
Getting the most out of your casings!, A pinning strategy.
Oyster Shell "Flour" $2 for 1lb. a hell of a deal :wink:
Not what is overlay but rather what overlay is
Gas Exchange vs. FAE

"We all have priorities. I used a closet once setup a nice little lab trouble was all the shit that was in there ended up in the bedroom that pissed off the GF then I ended up dumping her as she was getting in the way of my sterile culture technique! Ya I got priorities too!!!"

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Re: can you clone from dried mushrooms [Re: hyphae]
    #4433084 - 07/21/05 11:26 AM (18 years, 9 months ago)

If you were to skip some kind of cleaning for the tissue, you'd have a horrible mold problem that couldn't be transfered away from.

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Offlinered914
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Re: can you clone from dried mushrooms [Re: Hotnuts]
    #4436120 - 07/21/05 11:17 PM (18 years, 9 months ago)

Thats my problem! thanks guys

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Re: can you clone from dried mushrooms [Re: red914]
    #4447543 - 07/24/05 05:10 PM (18 years, 9 months ago)

Personally, I never dip in H202 when making transfers. The key is to take a very small piece of mycelium, whether you're cloning fresh tissue or dry. A piece of wet tissue half the size of a grain of rice will grow out fine, and dry tissue half that size will work. Remember, the larger the chunk of tissue you transfer, the more contaminants you transfer. You can drop a very small piece of dry or fresh tissue into distilled water and shake it up. That will wash it very well without stalling the mycelium with something toxic to it. The distilled water will also hydrate dry tissue. A second rinse in clean distilled water may be called for if the sample is especially dirty.

Transfer the now clean piece of mycelium to agar under asceptic conditions with sterile tools. Always wear a surgical mask, hairnet and latex gloves. Use a glovebox if the tissue is contaminated, not a flowhood. If the tissue is not contaminated, a flow hood will give a better success rate. Watch it daily and transfer the very first mycelial growth you see to new petri dishes. Don't wait for contamination to show up. The mycelium will recover much faster if it hasn't had its ass kicked with hydrogen peroxide, and you'll usually make that first transfer from fresh cloned material within 48 to 60 hours. If dry tissue is used, usually within a week you'll see enough growth to make a transfer to a new petri dish. Save the peroxide for those cobweb outbreaks on your casing material. Good luck.
RR


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