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NecNomen
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Registered: 10/15/13
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Last seen: 8 years, 7 months
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Positively identifying culture on agar Need Help! TLC?
#19192098 - 11/26/13 01:29 PM (10 years, 2 months ago) |
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Hey guys, first post here but I've been studying/lurking these forums for a while. I have a situation and need some advice but I haven't found a good solution yet on the forums as it's a little unique. This might be long, so I appreciate your time and advice.
I recently obtained a specimen that I thought had disappeared years ago. It was a limited sample and can't be obtained again. The exact species is unknown. I wanted to preserve and replicate it in perpetuity, so that meant culturing it myself. The sample was dried and frozen/unfrozen repeatedly, as well as assumed greatly contaminated. I prepared 2 dozen agar plates to have the best chance of recovering viable spores or tissue and inoculated them from various parts of the original sample. Methods included swabs from powdered material in the original container, cap fragments, and biopsies from inside of stems. Very careful sterile procedures were uses to minimize further contamination. Some agar was made with an untested antibiotic used for farm animals.
Surprisingly only a few samples showed clear contamination after three weeks. The large majority have clearly visible colonies of some type of fungus. It is a brilliant pure bright white in color. It is fluffy and spreads evenly from the point of inoculation across the surface. I don't see any clear network of larger mycelium forming or penetrating the agar. The fuzz is thin and not towering, and it is spreading by faintly visible mycelium
My understanding is this presentation is possible when species are cultured on agar. However since the sample must be considered uniformly contaminated, and possibly not viable, it doesn't seem diligent to assume that the culture is the desired fungus. Because of the large investment of time and money all ready involved I would prefer to make a positive identification before attempting fruiting. It seems to me the best way would be to test for the presence of psilocybin as that would distinguish it from a common contaminate.
I have read up on the use of Ehrlich's reagent for TLC and will if necessary purchase the DMACA. It is however a bit expensive and a little more elaborate than I was hoping for. I also will not have a known sample to compare bands. It's my understanding that since tryptamine reacts similarly to psilocin and psilocybin and is present in all or most fungi I would need a known example to compare it to.
Is there an easier method for a positive identification with little/no risk of a false positive?
I will post pictures just to be thorough, and any speculation is welcome, however a positive confirmation is what I'm hoping for. I look forward to being able to contribute to the forum when I have more experience, and appreciate the good faith assistance.

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shopdropper
Professional Psychonaut


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Re: Positively identifying culture on agar Need Help! TLC? [Re: NecNomen]
#19192549 - 11/26/13 03:17 PM (10 years, 2 months ago) |
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try a small sample fruiting batch, or reagent test it.
-------------------- DON'T TRY THIS AT HOME: no guarantees can be made about the accuracy of the information herein. The information dicussed in these posts is purely hypothetical, and for intelectual purposes only. Any similarity between internet chat and real life is pure coincidence.
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NOS4A2
This is the way


Registered: 07/22/04
Posts: 572
Loc: -tite
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Re: Positively identifying culture on agar Need Help! TLC? [Re: shopdropper]
#19195192 - 11/27/13 06:13 AM (10 years, 2 months ago) |
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Just from what I've witnessed recently, I would say with relative certainty, that bottom pic is viable and you should transfer it to three or four spawn jars while the agar gets suspended in the fridge and see if you can fruit it. There's nothing to lose from the sounds of it except your time. GL
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sharpshroomer98
Student of the Universe.



Registered: 08/30/11
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Re: Positively identifying culture on agar Need Help! TLC? [Re: NOS4A2]
#19195863 - 11/27/13 10:50 AM (10 years, 2 months ago) |
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That top pic looks like mold,,not any worth of growth. It's stretched out farther and has kept the same consistency throughout. It looks really light airy, not sure if that's what your want.
-------------------- No, I wasn’t born in 98’
 
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NecNomen
Stranger

Registered: 10/15/13
Posts: 12
Last seen: 8 years, 7 months
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Re: Positively identifying culture on agar Need Help! TLC? [Re: sharpshroomer98]
#19211200 - 12/01/13 04:29 AM (10 years, 1 month ago) |
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It's not looking good. Two of the cultures have turned a pinkish hue. It's not the most developed ones and it's where its touching the edge so I'm not certain if it's mold showing it's true colors or a separate contamination.
Thanks for the input guys, that bottom one does look like it radiates out like mycelium but it still hasn't colonized into the agar. I appreciate the educated speculation though.
What I really was hoping for was confirmation as to whether or not there is a way to test it for psilocybin without getting a false positive. Shopdropper you suggested reagent testing which is what I'd like to do, but what reagents do I use? Won't it react to all the reagents whether it's what I want or not. All the reagents I've researched will react to any indole, including tryptophan (tryptamine?), which all mushrooms produce. Or at least that's my understanding. I was thinking of using TLC but I'm still not sure how I would identify the bands without a reference.
Thanks guys, I really appreciate the help, and if I didn't really need some results soon I wouldn't be bugging you.
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RogerRabbit
Bans for Pleasure



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Re: Positively identifying culture on agar Need Help! TLC? [Re: NecNomen]
#19211223 - 12/01/13 04:47 AM (10 years, 1 month ago) |
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There will be little to no psilocybin in mycelium on a petri dish.
How are you culturing it if you don't know the species? RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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NecNomen
Stranger

Registered: 10/15/13
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Last seen: 8 years, 7 months
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Re: Positively identifying culture on agar Need Help! TLC? [Re: RogerRabbit]
#19211263 - 12/01/13 05:26 AM (10 years, 1 month ago) |
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Hey Roger Rabbit, thanks for the reply. I've read a LOT of your posts in my research, I appreciate all your help. I just finally found an archived article on fanaticus that explained the indols don't develop until pinning begins, as you've indicated.
I received the original tissue out of the blue from a friend. He couldn't get any more but I was able to identify it as a type I had a lot of experience with many years ago. It was superior to anything I had found since, so I want to try to revive it. I'm all but certain it's a psilocybe from visual identification, but I have no information first hand and little experience in identification.
I had tried just putting samples straight into LME agar. I'm hoping that will work for this strain well enough to get it started, then do some trial and error refinements. It isn't looking promising though, I have 12 dishes with the fungus pictured above, but it could eaily be an environmental contaminate.
I think I'm going to try soaking the tissue for several hours in plain water, then putting it on agar. Maybe dunk a few in H2O2 in case the soaking encourages a bacterial bloom, although only 6 of 24 showed yeast or bacteria the first time. I'm also going to order some syringes to culture concurrently just in case, I'm invested in this project at this point.
Any advice from you RR will be appreciated as gospel, lol. Any idea what spore suppliers have a quick turn around right now? I have several open in the other windows. No idea what I want.
Thanks
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fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Positively identifying culture on agar Need Help! TLC? [Re: NecNomen]
#19305920 - 12/21/13 12:46 AM (10 years, 1 month ago) |
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There are no actives present until the hyphal knot stage. Even at that point you'd need a far more sensitive test or a lot more material to test for anything.
Another point is that you should have transferred those cultures long ago judging from the pics. When using a questionable source you only want to wait long enough to get a clean piece of tissue. If you let them go too long they will most likely all contaminate.
-FF
-------------------- It drinks the alcohol and abstains from the weed or else it gets the hose again. -Chemy The difference between the substances doesn't matter. This is a war on consciousness, on our right to the very essence of what we are. With no control over that, we have no need to speak of freedom or a free society. -fireseed "If we are going to have a war on marijuana, the least we can do is pull the sick and the dying off the battlefield." -Neal Levine (MPP) I find the whole "my drug should be legal but yours should be illegal" mindset disgusting and hypocritical. It's what George Bush and company do when they drink a cocktail and debate the best way to imprison marijuana users. -Diploid
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Amanita virosa
botanist by day


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Re: Positively identifying culture on agar Need Help! TLC? [Re: fastfred]
#19309304 - 12/21/13 07:54 PM (10 years, 1 month ago) |
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Impossible to confirm on agar. You are gonna have to suck it up and put it to grain and try to fruit the bitch out. Rather than making a shitload of transfers, just make two slants from what you got and use the rest of the plate to make a couple of grain masters. I suspect if it is so rare and wonderful it ain't a cube so it isn't gonna be so easy.
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