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OfflineJohn in WI
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best way to clean stalk for making a "biopsy"
    #19347528 - 12/30/13 05:34 PM (10 years, 1 month ago)

I read the TEK on making a liquid culture by using a syringe to cut a plug out of a mushroom stem.  The TEK made it sound like being completely OCD about sterility was not required.

I'm nearly done with a pretty nice glove box and have no problem going the extra mile if it will help ensure success. 

So I'm wondering how you take something as filthy as a mushroom stem that's been exposed to humid air and dirty vermiculite, and get it clean enough that you can pure culture something with it?

The TEK said to just swab down the stalk with isopropyl, then run the (flame sterilized) needle through it.  A buddy of mine in the biology department said a safer bet might be to carefully whittle the stalk down to remove the outer skin and then do the biopsy. 

I was planning to give this method a try if (or hopefully WHEN) I get some fruit.  I had planned on doing a couple of LCs from a couple of fruits, just to plan for a couple of them failing to contamination.  Still, I'd like to stack the odds in my favor.


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Offlinemylfgur
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Re: best way to clean stalk for making a "biopsy" [Re: John in WI]
    #19347571 - 12/30/13 05:41 PM (10 years, 1 month ago)

Quote:

John in WI said:
I read the TEK on making a liquid culture by using a syringe to cut a plug out of a mushroom stem.  The TEK made it sound like being completely OCD about sterility was not required.

I'm nearly done with a pretty nice glove box and have no problem going the extra mile if it will help ensure success. 

So I'm wondering how you take something as filthy as a mushroom stem that's been exposed to humid air and dirty vermiculite, and get it clean enough that you can pure culture something with it?

The TEK said to just swab down the stalk with isopropyl, then run the (flame sterilized) needle through it.  A buddy of mine in the biology department said a safer bet might be to carefully whittle the stalk down to remove the outer skin and then do the biopsy. 

I was planning to give this method a try if (or hopefully WHEN) I get some fruit.  I had planned on doing a couple of LCs from a couple of fruits, just to plan for a couple of them failing to contamination.  Still, I'd like to stack the odds in my favor.




Very simple really:

Take a very sharp blade

Sterilize the blade with alcohol inside your glove box

Sterilize the outside part of the stem that you are going to be cutting through with alcohol

Slice through the stem lengthwise with your sterile blade.

You now have two halves of perfectly sterile mushroom flesh with pristine flesh in the middle. Take a small biopsy from the inside of the stem and voila.


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OfflineJohn in WI
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Re: best way to clean stalk for making a "biopsy" [Re: mylfgur]
    #19347607 - 12/30/13 05:49 PM (10 years, 1 month ago)

You just made a light bulb come on! 

I could sterilize a blade like you say, and wipe down the stem.  Then I could cut a piece ~1cm long from the stem (sort of like cutting a piece of firewood out of a log).  That would leave very clean end grain, and a semi-clean outer skin.  Then I could push the needle in from the bottom to the top, cutting out a nice sterile core from the center of the stalk without having to go through anything dirty first. Then put it into the LC jar through a silicone plug.


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InvisibleMushmitch
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Re: best way to clean stalk for making a "biopsy" [Re: John in WI]
    #19349720 - 12/31/13 02:45 AM (10 years, 1 month ago)

Flame sterilize blade after alcohol wipe...


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OfflineRogerRabbitM
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Re: best way to clean stalk for making a "biopsy" [Re: mylfgur] * 1
    #19349792 - 12/31/13 03:24 AM (10 years, 1 month ago)

Quote:

mylfgur said:




Very simple really:

Take a very sharp blade

Sterilize the blade with alcohol inside your glove box

Sterilize the outside part of the stem that you are going to be cutting through with alcohol

Slice through the stem lengthwise with your sterile blade.

You now have two halves of perfectly sterile mushroom flesh with pristine flesh in the middle. Take a small biopsy from the inside of the stem and voila.




No.

Never slice a mushroom stem with a knife for cloning because you'll push contaminants from the surface down into the flesh.  Always tear the stem from one end to the other.  This opens up tissue which has never been exposed to the air.

Alcohol doesn't sterilize anything.  Use flame sterilization.  Don't tell people to pour alcohol on a mushroom stem. :facepalm:
RR


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InvisibleEnigma1
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Re: best way to clean stalk for making a "biopsy" [Re: RogerRabbit]
    #19352913 - 12/31/13 09:45 PM (10 years, 1 month ago)

:takingnotes:


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Offlinedrake89
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Re: best way to clean stalk for making a "biopsy" [Re: Enigma1]
    #19355634 - 01/01/14 05:49 PM (10 years, 1 month ago)

light the outside on fire after spraying it with alcohol if youre dealing with something like a wild clone.  break it open with your hands and without anything touching the inside.


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InvisibleGanzig
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Re: best way to clean stalk for making a "biopsy" [Re: drake89]
    #19356000 - 01/01/14 07:38 PM (10 years, 1 month ago)

Quote:

drake89 said:
light the outside on fire after spraying it with alcohol if youre dealing with something like a wild clone.  break it open with your hands and without anything touching the inside.




If the mushroom is not a species that is brittle enough to break you can
tear it or pull it apart to expose the clean flesh inside.

But yeah, don't cut the mushroom in half.


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OfflineMo_Mo
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Re: best way to clean stalk for making a "biopsy" [Re: Ganzig]
    #19407088 - 01/12/14 06:35 AM (10 years, 1 month ago)

Always tear or break never cut.  I don't clean the outside of the stem before biopsy.


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Invisible36fuckin5
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Re: best way to clean stalk for making a "biopsy" [Re: Mo_Mo]
    #19409772 - 01/12/14 07:25 PM (10 years, 1 month ago)

I always wipe my stems down with IPA. Maybe not necessary, but I like to do it.

When I go to open the mushroom up I rip it. Even with brittle species with thin stems, you can tear it straight up and get a little piece of flesh out of the cap.


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If you're offended I don't give a shit and don't come see me no more.

Pat The Bunny said:
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bodhisatta said:
i recommend common sense and figuring it out.

These are the TEKs I use. They're all as cheap and easy as possible, just like your mom.


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Invisiblefastfred
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Re: best way to clean stalk for making a "biopsy" [Re: 36fuckin5] * 1
    #19411092 - 01/13/14 03:19 AM (10 years, 1 month ago)

Dip samples in peroxide then 1:10 bleach.  About 10-20 sec. each.

Works well for wild samples.  Shouldn't really be needed for working with clean samples.


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OfflineRauhfasertapete
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Re: best way to clean stalk for making a "biopsy" [Re: fastfred]
    #19411119 - 01/13/14 03:35 AM (10 years, 1 month ago)

When I find something interesting in the forest, I usually tear the stalk apart, cut a big piece out with the scalpel (without touching any surface tissue that had been exposed to the environment before), and throw it into peroxide. then i put the dish with the peroxide into the glovebox, where i cut off the peroxide-drenched outer surfaces of the piece with another sterile scalpel, and transfer it inro a petri dish.
however, a few times i was too lazy for all the effort with the glovebox, so i transferred on antibiotic agar in the open air, that also worked fine.


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OfflineYuri.Pono
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Re: best way to clean stalk for making a "biopsy" [Re: Rauhfasertapete]
    #19414118 - 01/13/14 07:58 PM (10 years, 1 month ago)

:takingnotes:


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InvisibleMushmitch
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Re: best way to clean stalk for making a "biopsy" [Re: Rauhfasertapete]
    #19436148 - 01/18/14 02:12 AM (10 years, 1 month ago)

Quote:

Rauhfasertapete said:
When I find something interesting in the forest, I usually tear the stalk apart, cut a big piece out with the scalpel (without touching any surface tissue that had been exposed to the environment before), and throw it into peroxide. then i put the dish with the peroxide into the glovebox, where i cut off the peroxide-drenched outer surfaces of the piece with another sterile scalpel, and transfer it inro a petri dish.
however, a few times i was too lazy for all the effort with the glovebox, so i transferred on antibiotic agar in the open air, that also worked fine.




that seems like bad info to post even if it worked out..


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OfflineRogerRabbitM
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Re: best way to clean stalk for making a "biopsy" [Re: Mushmitch]
    #19436432 - 01/18/14 05:18 AM (10 years, 1 month ago)

It's bullshit, so don't believe it.  Antibiotic agar won't do anything to stop mold and there's mold spores in the air everywhere.  Nobody does open air inoculations on agar outdoors and succeeds.

Nobody soaks cultures in peroxide or bleach for very long either.  None of the chemicals we have available will replace proper procedure with lots of rapid transfers to separate the desired mycelium from everything else.  Bleach and peroxide are ineffective against mold mycelium, so people claiming success that way are making shit up.

Further, with outdoor wild mushrooms, the mold mycelium and bacteria isn't just on the surface, but infused right into the interior flesh of the mushroom.  This is why it takes a series of transfers to get a clean monoculture.
RR


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OfflineRauhfasertapete
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Re: best way to clean stalk for making a "biopsy" [Re: Mushmitch]
    #19436820 - 01/18/14 08:45 AM (10 years, 1 month ago)

Quote:

Mushmitch said:
Quote:

Rauhfasertapete said:
When I find something interesting in the forest, I usually tear the stalk apart, cut a big piece out with the scalpel (without touching any surface tissue that had been exposed to the environment before), and throw it into peroxide. then i put the dish with the peroxide into the glovebox, where i cut off the peroxide-drenched outer surfaces of the piece with another sterile scalpel, and transfer it inro a petri dish.
however, a few times i was too lazy for all the effort with the glovebox, so i transferred on antibiotic agar in the open air, that also worked fine.




that seems like bad info to post even if it worked out..




I´m also surprised how well that works sometimes, especially because i live in a moist and moldy old house. However, I attribute it to a method to which I have switched after I had broken several petri dish tops and was left with the bottoms: I wrap the dish bottom in saran wrap, wind a strip of transarent adhesive tape around and sterilise it. Agar is injected through the tape from a clyster syringe with a wide veterinary cannula. The injection hole should be closed with another piece of tape. Then I make the biopsy (which I did in the open air, so there should be some air contaminants on the surface of the stalk piece) and poke it through a hole that I had molten into the saran with a hot wire the moment before. Then the hole is closed.
the dish usually looks pretty shitty at the end, but as the agar itself only had minimum air contact, and the stalk piece overgrows any air contam sticking to its surface pretty quickly, it usually still works out. The antibiotic agar is necessary because bacteria would be a much bigger problem than molds if sterility isn´t 100%.
However, it´s still a botch-up and I wouldn´t do it that way if it was my last isolate, but it worked out for me. I do it the same way when I want to put some spores on agar, as prints usually aren´t really clean anyway.


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OfflineRogerRabbitM
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Re: best way to clean stalk for making a "biopsy" [Re: Rauhfasertapete]
    #19439427 - 01/18/14 07:27 PM (10 years, 1 month ago)

Autoclavable transparent tape and saran wrap?  This is getting a bit deep for advanced mycology dude.  Most of us aren't wearing boots.
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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Invisiblefastfred
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Re: best way to clean stalk for making a "biopsy" [Re: RogerRabbit]
    #19441163 - 01/19/14 04:55 AM (10 years, 1 month ago)

I agree with you RR, but you'd make a better case if you didn't always argue against everything that you don't approve of.

Bleach and peroxide dips are standard practice in explant culture.  It works find with mushroom tissue too.  It doesn't change the quick transfers strategy but it can help with dirty samples.


-FF


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OfflineJohnTheRipper
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Re: best way to clean stalk for making a "biopsy" [Re: fastfred]
    #19450258 - 01/20/14 10:23 PM (10 years, 1 month ago)

:takingnotes:

I always tear the stem in half when taking some mycelium to clone. Never knew about some of the other techniques in here.


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Offlinemycopanda
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Re: best way to clean stalk for making a "biopsy" [Re: JohnTheRipper]
    #19450437 - 01/20/14 10:58 PM (10 years, 1 month ago)

Shit just got real! :goodluckwiththat2:


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