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MonkeyJesusFresco
am i suspended in agar?



Registered: 10/09/12
Posts: 3,306
Loc: South East USA
Last seen: 4 hours, 11 minutes
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Wild Tissue Culturing
#21696589 - 05/18/15 10:58 PM (8 years, 8 months ago) |
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1. So I've been fooling around with these little agar containers. 2. I have a tendency to collect wild fruit-bodies and then culture them.
Long story short:
I read something somewhere, in a book by Paul Stamets and I came up with this:
 
So, what you see there is a 3.5 fl. oz. polypropylene container, with about 2 tablespoons of light-malt-extract-agar and a coffee-stir-stick cut down to size, sticking up.
I came across some wild fruit-bodies that I wanted to culture, unfortunately they had been out there for a while...and they sat in the refrigerator for some time, as I simply never got around to fooling with them and... needless to say that, in the end, clean inner-stipe tissue was not gonna be an option for culturing 
So what I did was:
-Chop up a stem
-Soak stem pieces in a diluted dishsoapy-water for 15-20 mins
-in a still air box, with tweezers, I picked each stem piece out of the soapy water, and rinsed it off in a 1:10 bleach:water solution for an unknown amount of time (I did a double-blind study on myself, a.k.a. I didn't take lab notes)
Anyways, in the end I had 6 of the containers that you see pictured above, each with a "washed" piece of mushroom stem laying on or near the intersection of the stick&agar. Each piece was washed off for different unknown times in the bleach solution; (no longer than 1 minute)
So, the idea was to "leave all the contaimination behind" on the agar as the "wood-loving" mycelium from the mushroom stipe, crawled up the stick, in the hopes that, at a later date, I could come back and retrieve a clean culture. (From what I recall, Paul Stamets version of this used whole pieces of straw)
In conclusion:
- I desperately need to get my hands on some Gentamycin Sulfate, as 5 out of the 6 containers that I made were over run with bacteria, not to mention MOLD!
- Oddly enough, one of the containers grew mycelium and no bacteria or mold (the "pride&joy" pictured above), at the moment I'm contributing this "low success rate" to the varied wash times in the bleach-water bath, and possibly the small amount of soap used in the soap bath.
And finaly:
Does anyone have anything to contribute to this topic? Experience? Ideas? Anything???
I can't remember where I got the idea to run the mycelium up a stick like that, and last time I mentioned something like it (on a different website) I was very heavily ridiculed for it 
So hopefully this post will open a line of discussion and informative input.
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
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Skip the soap and bleach use those and anti biotic agar as a last resort try it the normal way first. Inner tissue biopsy. Then the mycelium is less injured will recover faster and you have a fighting chance of it over running bacteria and if your sterile technique is good you should get 0 mold.
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MonkeyJesusFresco
am i suspended in agar?



Registered: 10/09/12
Posts: 3,306
Loc: South East USA
Last seen: 4 hours, 11 minutes
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Re: Wild Tissue Culturing [Re: bodhisatta]
#21697710 - 05/19/15 09:53 AM (8 years, 8 months ago) |
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Yeah, I had samples collected from two different patches; the first patch/sample, the mushroom had been plucked from the ground, the entire fruit body was intact, so inner-stipe tissue was clean and there was no problem culturing that; the second batch was old/weathered, had been cut and neglected in the fridge for some time, so I rigged this up, basically washing cut-up stipe pieces off; either way, I got clean cultures from both patches 
and yes, all that non-sense should be left as last resort  especially the "washing", took about a week-and-a-half to two to recover 
edit: also, the circumstances I'm working under, resources are TIGHT. no petris dishes, much less antibacterial agar
Edited by MonkeyJesusFresco (05/19/15 09:56 AM)
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