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Triggering pinning using primordial hormone extract


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#1 peacefrog

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Posted 08 January 2017 - 06:52 AM

I have been reading a lot about this and decided to try some experimentation with it. So I am starting with an old, known culture of Psilocybe cubensis Golden Teacher variety. The truth be known, it is about 6 years old, hasn’t been rejuvenated since then, so I hope it’s still decent but it is the only cube culture I still have. And since cubes are great and easy candidates for agar pinning I will revive my slant, make a few plates, re-slant and go to work.

The basic premise is to use the hormones found in primordia to trigger faster and more controlled pin sets. Using these principles, one could possibly control where he or she wants the surface of the colonized substrate to pin. I do not have access to a lab or equipment, so as we amateur cultivators tend to do, I will improvise.

The tek I am following comes from “Organic Mushroom Farming and Mycoremidiation” by Tradd Cotter and a small amount from good old TMC. Tradd Cotter called for primordia to be harvested, cleaned, surface sterilized with ethanol, blended in sterile water to make a slurry, vacuum filtered, 5 minutes in a centrifuge, sterilized using a syringe filter, then finally diluted and applied to a fully colonized substrate.

I have decided to keep it simple at first and only work with agar. If I get any results, I will move to bulk grows. My plan is to grow out several agar jars and use roughly half for a control, i.e. no hormone treatment, and the other half, I will apply tiny amounts of hormone extract in a calculated pattern on the agar surface.

I do not own a vacuum filter system, Buchner funnel, centrifuge, or syringe filters, so here comes the improvisation.

My homemade vacuum filter:
Filter.jpg
It works okay, a little slower than a lab grade system would, but it did the trick in my test trials with some LI I didn’t mind wasting.

I read about someone publishing an article where he used an egg beater to separate plasma from blood. I did not have one, so I decided to use the beater attachment on my Kitchen Aid. Hopefully it should work but I will spin on high speed for 10 minutes, not 5. It took that long to separate mycelium particulates from the same LI in my equipment test trials.
My make shift centrifuge:
Centrifuge.jpg

And the syringe filter is the easy part. I found some for pretty cheap on Amazon. Ordered as of yesterday.

Below is a pic of my slant:
Culture.jpg
Notice how matted it looks, but only after 12 hours out of cold storage there are signs of new growth emanating from previously un-colonized agar. I hope it can be seen with my cheap camera (lower middle of pic). It goes to show how resilient mycelium truly is.

Wish me luck here and if anything good or promising comes from this, I will most definitely update as I go.

Edit: I just realized after I posted this, it may need to be in the Mycolab or Mad scientist forum? If so I apologize and any mod feel free to move.

Edited by peacefrog, 08 January 2017 - 09:39 AM.

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#2 CatsAndBats

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Posted 08 January 2017 - 08:03 AM

I have been reading a lot about this and decided to try some experimentation with it. So I am starting with an old, known culture of Psilocybe cubensis Golden Teacher variety. The truth be known, it is about 6 years old, hasn’t been rejuvenated since then, so I hope it’s still decent but it is the only cube culture I still have. And since cubes are great and easy candidates for agar pinning I will revive my slant, make a few plates, re-slant and go to work.

The basic premise is to use the hormones found in primordia to trigger faster and more controlled pin sets. Using these principles, one could possibly control where he or she wants the surface of the colonized substrate to pin. I do not have access to a lab or equipment, so as we amateur cultivators tend to do, I will improvise.

The tek I am following comes from “Organic Mushroom Farming and Mycoremidiation” by Tradd Cotter and a small amount from good old TMC. Tradd Cotter called for primordia to be harvested, cleaned, surface sterilized with ethanol, blended in sterile water to make a slurry, vacuum filtered, 5 minutes in a centrifuge, sterilized using a syringe filter, then finally diluted and applied to a fully colonized substrate.

I have decided to keep it simple at first and only work with agar. If I get any results, I will move to bulk grows. My plan is to grow out several agar jars and use roughly half for a control, i.e. no hormone treatment, and the other half, I will apply tiny amounts of hormone extract in a calculated pattern on the agar surface.

I do not own a vacuum filter system, Buchner funnel, centrifuge, or syringe filters, so here comes the improvisation.

My homemade vacuum filter:
attachicon.gifFilter.jpg
It works okay, a little slower than a lab grade system would, but it did the trick in my test trials with some LI I didn’t mind wasting.

I read about someone publishing an article where he used an egg beater to separate plasma from blood. I did not have one, so I decided to use the beater attachment on my Kitchen Aid. Hopefully it should work but I will spin on high speed for 10 minutes, not 5. It took that long to separate mycelium particulates from the same LI in my equipment test trials.
My make shift centrifuge:
attachicon.gifCentrifuge.jpg

And the syringe filter is the easy part. I found some for pretty cheap on Amazon. Ordered as of yesterday.

Below is a pic of my slant.
attachicon.gifCulture.jpg
Notice how matted it looks, but only after 12 hours out of cold storage there are signs of new growth emanating from previously un-colonized agar. I hope it can be seen with my cheap camera (lower middle of pic). It goes to show how resilient mycelium truly is:

Wish me luck here and if anything good or promising comes from this, I will most definitely update as I go.

Edit: I just realized after I posted this, it may need to be in the Mycolab or Mad scientist forum? If so I apologize and any mod feel free to move.

 

Oh you KNOW that I'm in on this! Where's microbe at?! :biggrin: Good stuff as usual @peace!


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#3 Newmusher

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Posted 08 January 2017 - 08:40 AM

This sounds incredibly interesting, I'm not sure the chair I usually pull up is gonna be comfy enough for how long i wanna watch you do this.


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#4 peacefrog

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Posted 08 January 2017 - 08:57 AM

Yea man I don't know if anything will come of this experiment but it should be fun. Tell Microbe to join the party lol.
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#5 Microbe

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Posted 08 January 2017 - 04:23 PM

Interesting. However i need to work out my beginners mistakes at the moment, i cant even get a tub to pin at the moment so until then i will just watch lol.

Im getting some new cultures going this week at starting over from spore. Be back soon!
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#6 ethnobotanist420

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Posted 08 January 2017 - 08:23 PM

Very interesting stuff! I'll be anxiously watching your progress!
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#7 MLBjammer

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Posted 15 January 2017 - 06:27 AM

I finally found your new thread, lol.  

 

Are you gonna try some AA+ with this?  Or is the GT responding better?


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#8 Arathu

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Posted 15 January 2017 - 05:07 PM

This is awesome Peace.........I'm diggin it!


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#9 Nsnail

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Posted 16 January 2017 - 02:39 PM

What exactly are you doing when you vacuum filter it? Boiling off the water or pulling only the fine particles through the filter? I would be willing to give this a try as I have a vacuum pump and 3D printer files for a centrifuge.

Edited by Nsnail, 16 January 2017 - 02:40 PM.

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#10 peacefrog

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Posted 17 January 2017 - 05:26 AM

Jammer,

The votes still out on the old GT culture. I rehydrated that poor mycelium and inoculated via syringe onto 6 agar plates to see if I can rejuvenate that neglected culture. I have 2 AA+ cakes fruiting invitro as back up just incase. My other AA+ mini trays are knotting up as we speak so either way I hope I'm covered lol. But I hope the GT makes it, so the experiment can be better controlled since I know that culture used to be good and consistent. But I do have 3 AA+ pure cultures but they are untested at the moment.

Nsnail,

From what I understand from the research I have done, the vacuum filter is supposed to isolate the liquid from the solid particulates but then the centrifuge does as well. Perhaps this tek calls for pure liquid hormone extract with no particulates present. This is a new experiment for me and I have not done anything like it in the past. With my very low tek design, I'm sure just letting gravity take over would do the trick as well, but I assume one would loose some valuable liquid to evaporation. The vacuum sucks the h2o in seconds leaving pretty dry particulates in the filter.
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#11 Nsnail

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Posted 18 January 2017 - 09:50 AM

Hmm so the idea here is blend primordia with water, vacuum filter, separates in a centrifuge to maybe separate the hormone, then run through something called a filter syringe? Then mist onto a sub to see if we can trigger pins?

Once I get my agar up to speed and get an isolate I'll give this a shot and see if I can really push a strain to its limits.

What kind of progress have you made with this so far?

Edited by Nsnail, 18 January 2017 - 09:50 AM.

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#12 peacefrog

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Posted 22 January 2017 - 05:32 AM

Yes that is the theory from my research. I don't know though if it will work, or if it will even be worth the time and effort.

And no experience yet except testing my make shift equipment. This may be a long experiment lol.
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#13 CatsAndBats

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Posted 22 January 2017 - 10:53 AM

This may be a long experiment lol.

 

We're good at those! I gotta a couple too. :tongue:



#14 Microbe

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Posted 17 April 2017 - 03:37 PM

Update?

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#15 peacefrog

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Posted 01 May 2017 - 06:26 PM

Wow I just saw this. Sorry, Microbe for the late response.

Sadly, no updates yet. I became more interested in reviving my GT culture and my other 3 cube substrains for my spore bank. Now I have moved on to the Viva's and trying to get a few fruits.

I will come back to this experiment though.
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