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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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OfflineHotnuts
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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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OfflineHotnuts
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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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Invisiblemushrx1
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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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Offlinehyphae
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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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Getting the most out of your casings!, A pinning strategy.
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OfflineHotnuts
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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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Offlineblackout
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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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Offlinehyphae
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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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Offlinehyphae
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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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OfflineHotnuts
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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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OfflineRogerRabbitM
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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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