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Offlinevitaminz
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Registered: 08/29/22
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Claviceps Identification
    #27923881 - 08/29/22 10:59 AM (1 year, 4 months ago)

Greetings, its been about ten years since I have been active on this site. Long lost my old account and any email it may have been associated with. So here's my first post back! Hope you are all doing well!

I was going through a large bag of rye berries a few weeks ago and picked out about 50 full sclerotia of ergot. Being the tinkerer that I am, I decided to look up a few papers on its cultivation in the lab, and was able to find a few good threads here that helped as well. Based on the literature, I washed the dried sclerotia with a salt solution, as well as a solution of butanol, in an attempt to obtain a clean culture. I made some T25 agar, which a lot of folks used to cultivate claviceps in petri dishes. After breaking up the sclerotia into a few pieces per plate, I let it do its thing. At first many of the sclerotia oozed this clear brown/yellowish liquid that turned cloudy after a few days, and than developed into mycelial growth about a week later. It has now been about three weeks and the growth is starting to slow, I am unsure if this is because my plates are drying out (they appear to be, I am quite new to this, should I wrap them in parafilm?), although I have also read that claviceps will not colonize to the edge of the plate. I am wondering if the fungus released some sort of honey dew that has developed into a mycelial culture. Anyone here have experience to possibly confirm the ID on these plates? I am in the market for a good scope, but do not have one currently. Pictured are a few different plates, one of which I am quite confident is a mold contam. It should be noted that all plates have been cultured in the dark, and I put a few in the light by a windowsill a few days ago, and they are still white. I would have thought that the light exposure would cause the fungus to produce some colored byproducts, but perhaps that is only if it is a heavy alkaloid producing strain? I have thought about making a LC with rye soak water, if yall are confident this is claviceps perhaps ill inoculate a standard grain jar with some and see what happens. Any and all help is appreciated!













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OfflineAlan RockefellerM
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Re: Claviceps Identification [Re: vitaminz]
    #27923958 - 08/29/22 11:42 AM (1 year, 4 months ago)

I believe that the puddles you saw were bacteria, and that the fungus outgrew it, fortunately.

You should wrap the edges of the dishes with parafilm or saran wrap, or put the whole thing in a new ziplock sandwich bag - both to keep them from drying out and to keep contamination out.

The best way to figure out which fungus is growing is ITS sequencing.  The easiest way to do this is to send a sample to http://alvalab.es, though you can also set up a small DNA lab at home following https://wiki.counterculturelabs.org/wiki/DNA_sequencing.

You also might be able to get a bit of information by shining ultraviolet light on the culture - ergot alkaloids are fluorescent.  Should work but is untested as far as I know.  If some glow more than others they are likely more potent alkaloid producing strains.

What I see in the photo is white mycelium, and there are millions of species of fungi that look like this on agar.  The chance that it is Claviceps is probably somewhere around 50%.


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Offlinevitaminz
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Re: Claviceps Identification [Re: Alan Rockefeller]
    #27924274 - 08/29/22 03:11 PM (1 year, 4 months ago)

Thanks for the reply, I tried the UV light, and had no discernible luck with either UVA or a lower 254nm bulb. I also tried dissolving some sclerotia into DCM and shining the light on the solution, but also was not able to see anything that I could say for sure was fluorescence (than again, Im not exactly sure what it would look like, I have only seen pictures). Bacteria  would certainly make sense, I will set up a home DNA lab asap and report back! Lots to learn, which is why Im doing it! I am looking at purchasing a good 1000x scope this week, and will upload pictures of the samples as soon as I have them.


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Invisiblefantanyl
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Registered: 12/14/21
Posts: 58
Re: Claviceps Identification [Re: vitaminz] * 1
    #27931824 - 09/03/22 08:10 AM (1 year, 4 months ago)

I bet letting them develop stroma and trying to get the culture from cloning the tiny stem or from the spores would be a more viable way.
check out this thread:

https://www.shroomery.org/forums/showflat.php/Number/14762126/fpart/all

I've read the purpurea sclerotia is inactive when fully developed and therefore difficult to clone.
But who knows, i whish you best luck your myc actually is the claviceps :super:

And i don't think the culture not glowing under blacklight says it cant be calviceps, because the small ones that grow on wild grases and not on barley or another big cereal, cut in half, the inside doesnt even glow under blacklight.
It seems only the big ones have already enough alks to glow.
and in culture I've read they won't produce many alks before they nearly run out of phosphates or sulfates (not sure which ones)
and therefore not glow before this happens.

:hatsoff:


Edited by fantanyl (09/03/22 08:12 AM)


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