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OfflineJohnLothe
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Micro Grow and Agar Cloning
    #21814223 - 06/16/15 11:25 AM (8 years, 7 months ago)

The following is a collection of images I have taken documenting a “micro-grow” and cloning attempt. I have about run out of spore syringes so I thought I’d try my hand at propagating what I had left. So I inoculated a few ½ pint brown rice flour jars and set them to go. Of all 12 jars I inoculated only 2 showed any signs of growth. I assume the other syringes had already expired or had been duds as the only jars which showed any growth where from the same syringe.

Moreover I inoculated 3 ½ pint agar jars with some spores from each of the syringes and they all showed contamination. The only jars which did not where those which showed grown within the ½ pint brown rice flour jars(BRF).

Finished BRF cakes





Mushroom samples




After fruiting one of the BRF jars inside the jar (I let it go the entire first flush within the jar to insure no contamination and safe samples to clone from) I transferred 3 samples of the mushrooms to 3 ½ pint agar jars. Each of which has exploded with healthy looking growth. The ground work being laid out by a very rhizomorphic looking grown with a much fluffier mycelia mat coming in behind it.

Agar media Colonized From Mushroom Samples







I am very pleased with the agar tek I used, very simple, very easy to acquire the ingredients and it worked for me in a still air box. I have taken the liberty of updating my jars, having done a good deal more research into their construction; I have included poly fill filters in the newest batch. The next batch will use the suggested tyvek. The poly fill is what I had on hand, and I figured it wouldn’t hurt to test a batch to see how it worked out. I did half with one air transfer hole and the other half with two.

Agar Tek results:





Updated Jars







I’ll post updates to this thread until I have completed the entire grow; my goal is to successfully clone these guys and restart the strain with spore prints and spore syringes. I intend to isolate the most promising looking mushrooms clone them and reboot my cultures. 

Agar Tek Used: http://www.shroomery.org/9427/Grocery-Store-Agar-Tek


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OfflineJohnLothe
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Re: Micro Grow and Agar Cloning [Re: JohnLothe]
    #21820880 - 06/17/15 09:05 PM (8 years, 7 months ago)

Cake update






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OfflineSksoul
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Re: Micro Grow and Agar Cloning [Re: JohnLothe]
    #21821168 - 06/17/15 10:19 PM (8 years, 7 months ago)

you are on your way to self-sustainability. keep it up, everything is looking good.


--------------------
Like all great travellers, I have seen more than I remember, and remember more than I have seen.


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Offlineivangrozny2
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Re: Micro Grow and Agar Cloning [Re: Sksoul]
    #21822884 - 06/18/15 09:35 AM (8 years, 7 months ago)

you go wit yo bad self


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Invisiblemushmagic
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Re: Micro Grow and Agar Cloning [Re: ivangrozny2]
    #21823379 - 06/18/15 12:21 PM (8 years, 7 months ago)

A little confused on your tub tho..Wats with all the poly in the holes?

Trying to put your own spin on the SGFC?

I'm assuming you have holes on the underside of the tub as well, correct?


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OfflineJohnLothe
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Re: Micro Grow and Agar Cloning [Re: JohnLothe]
    #21825278 - 06/18/15 08:03 PM (8 years, 7 months ago)

Cake Update







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OfflineMachiavelliavore
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Re: Micro Grow and Agar Cloning [Re: JohnLothe]
    #21826053 - 06/18/15 11:04 PM (8 years, 7 months ago)

I believe you can make some liquid cultures without a pressure cooker.  LI or slurries are also an option.  Making one of those out of a kickass clone while spore printing as a backup might be a good option for you.

LC seems like an especially good option if you already have clean agar samples.

PF cakes will be massively faster with any of those inoculation options.


--------------------


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


No, this does not look right...


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OfflineJohnLothe
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Re: Micro Grow and Agar Cloning [Re: Machiavelliavore]
    #21826752 - 06/19/15 05:03 AM (8 years, 7 months ago)

Quote:

Machiavelliavore said:
I believe you can make some liquid cultures without a pressure cooker.  LI or slurries are also an option.  Making one of those out of a kickass clone while spore printing as a backup might be a good option for you.

LC seems like an especially good option if you already have clean agar samples.

PF cakes will be massively faster with any of those inoculation options.




What Tek would you suggest using for the LC?


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OfflineMachiavelliavore
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Re: Micro Grow and Agar Cloning [Re: JohnLothe]
    #21827372 - 06/19/15 09:39 AM (8 years, 7 months ago)

Since I started with grains, I have always done grain to grain transfers, and had no need to use lc, especially given it can contaminate with less obvious signs, however I don't have agar and do have a pc.

http://www.shroomery.org/forums/showflat.php/Number/5238137#5238137

Another guide from a tc that's similar
http://www.shroomery.org/forums/showflat.php/Number/5874305


This guide looks solid to me, but the important takeaway is the steam treatment time.  Honeymaple also seems like an easy starting point.

Instead of using your spore syringe, you can make a liquid inoculant (mycellium suspended in water) by doing something like injecting some sterile water from a clean syringe into the agar jars, swirling it over the mycellium, then sucking it back up.  Look up a guide because you may need a certain gauge needle, or to do more than swirl.  This in itself will give you a good inoculant, but also a really clean way to inoculate your lc, which will be even faster than li.  You could also probably scrape myc off the agar into the lc, or dump the whole wedge in, but opening the lc even in still air seems risky to me.

Anyway, you should be able to jack up an army of cakes with that lc easily.  You can also make clone or isolate cakes with li or lc.  It's about as hightec as you can get without a pc.  Check out the slurry guide as well, Though the blender attachment seems a bit involved, it sounds pretty badass.

Again, Im not experienced with these tecniques, just aware of then.  Li may be closer in speed to lc than I'm giving it credit for.


--------------------


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


No, this does not look right...


Edited by Machiavelliavore (06/19/15 09:53 AM)


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OfflineJohnLothe
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Re: Micro Grow and Agar Cloning [Re: Sksoul]
    #21828084 - 06/19/15 01:00 PM (8 years, 7 months ago)

Cake Update







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Offlinespacechildo
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Re: Micro Grow and Agar Cloning [Re: JohnLothe]
    #21828341 - 06/19/15 02:17 PM (8 years, 7 months ago)

trim the foil and remove the polyfill for better FAE and fatter nicer fruits!

next time roll the cakes in dry verm before placing in the FC also.


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OfflineJohnLothe
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Re: Micro Grow and Agar Cloning [Re: spacechildo]
    #21832206 - 06/20/15 12:18 PM (8 years, 7 months ago)

Cake Update




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OfflineMachiavelliavore
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Re: Micro Grow and Agar Cloning [Re: spacechildo]
    #21832918 - 06/20/15 04:13 PM (8 years, 7 months ago)

Quote:

spacechildo said:
trim the foil and remove the polyfill for better FAE and fatter nicer fruits!

next time roll the cakes in dry verm before placing in the FC also.




I agree.  FAE is on the low side.  You can tell when the size of the pin head stays the same and the stipe just keeps getting longer.  It seems these conditions cause lots of aborts also IME.  Propper pinning will see the formation of the pin, then it will get fatter and bigger and fatter and bigger, eventually stopping and elongating as it becomes a mature fruit.


--------------------


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


No, this does not look right...


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OfflineJohnLothe
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Re: Micro Grow and Agar Cloning [Re: Machiavelliavore]
    #21834024 - 06/20/15 09:12 PM (8 years, 7 months ago)

Quote:

Machiavelliavore said:
Quote:

spacechildo said:
trim the foil and remove the polyfill for better FAE and fatter nicer fruits!

next time roll the cakes in dry verm before placing in the FC also.




I agree.  FAE is on the low side.  You can tell when the size of the pin head stays the same and the stipe just keeps getting longer.  It seems these conditions cause lots of aborts also IME.  Propper pinning will see the formation of the pin, then it will get fatter and bigger and fatter and bigger, eventually stopping and elongating as it becomes a mature fruit.





Interesting, so if the air transfer isn't adequate it will stunt their growth. So here is what my plan is moving forward.

I'm going to lower the amount of Poly fill I am using and replace it with medical tap, the porous kind. I'll see if this will work better. In the mean time, this humble harvest will be converted into agar clones. The other Agar jars are fully colonized and I have just sterilized a few more jars for inoculation. These have poly fill Gas transfer ports so I expect them to colonize much quicker.

I have one question, how does one transfer a slice of cloned mycelium into a pf cake with vermiculite on top? I know it is to be done in a SAB but what is the most full proof way? I have a plan in which I am going to fill a syringe with sterilized water, with a thick needle head and then stab said syringe into the agar jars squirt the water in and mix it around, and then pull it back in. Would anyone advise a different approach?

So far this has been a very fulfilling and educational grow. I can't wait to get these under my microscope.


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OfflinePsilicon
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Re: Micro Grow and Agar Cloning [Re: JohnLothe]
    #21834334 - 06/20/15 10:18 PM (8 years, 7 months ago)

:kaneclap:

Always happy to see someone else getting into agar.  Looking good so far!


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Agar - what, why and how?  Everything a beginner needs to know.
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OfflineMachiavelliavore
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Re: Micro Grow and Agar Cloning [Re: JohnLothe]
    #21834834 - 06/21/15 12:13 AM (8 years, 7 months ago)

From what I've heard, it's difficult to inoculate PF cakes with any solid innoculant.

Stabbing method from the LC tek I linked you is more like converting the syringe into a piston.  You hydrolically eject the piece of mushroom tissue into the LC which will then be colonized by it.  It will most likely will result in poor inoculation for a PF cake.

PF tek doesn't work very well unless it's permiated by inoculant.

Don't be too hasty to clone before you have a nice cluster to work with.  Around 3-6 mushrooms of decent size without too many dead pinheads or tiny mushrooms surrounding it.

I think liquid culture (lc) made with a nice clone will be the next logical step for you.  Despite your nice agar setup, agar is really much more useful with grains, and the best thing you can do with it now is make isolates from clones, colonize LC with those isolates, then inoculate cakes with the isolate LC.  You could also verify your clone tissue is clean, but then getting to the agar plate into the LC cleanly would be more difficult than the piston method you described above.


--------------------


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


No, this does not look right...


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OfflineJohnLothe
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Re: Micro Grow and Agar Cloning [Re: Machiavelliavore]
    #21839510 - 06/21/15 09:52 PM (8 years, 7 months ago)

Quote:

van der griegen said:
:kaneclap:

Always happy to see someone else getting into agar.  Looking good so far!




How would you take the cloned agar medium and transfer it into a syringe?
Quote:

Machiavelliavore said:
From what I've heard, it's difficult to inoculate PF cakes with any solid innoculant.

Stabbing method from the LC tek I linked you is more like converting the syringe into a piston.  You hydrolically eject the piece of mushroom tissue into the LC which will then be colonized by it.  It will most likely will result in poor inoculation for a PF cake.

PF tek doesn't work very well unless it's permiated by inoculant.

Don't be too hasty to clone before you have a nice cluster to work with.  Around 3-6 mushrooms of decent size without too many dead pinheads or tiny mushrooms surrounding it.

I think liquid culture (lc) made with a nice clone will be the next logical step for you.  Despite your nice agar setup, agar is really much more useful with grains, and the best thing you can do with it now is make isolates from clones, colonize LC with those isolates, then inoculate cakes with the isolate LC.  You could also verify your clone tissue is clean, but then getting to the agar plate into the LC cleanly would be more difficult than the piston method you described above.





I see. Which LC Tek would you suggest using? There are a few I am looking at but one requires I build a Blender Jar. (Tek in question) I don't currently posses the means to do such a thing so I was planning to do what is suggested in the Grocery Agar Tek and just use the needle to mix it around and inject sterile water into it. I am also planing to start this LC Tek unless anyone has any suggestions contrary to going with it.

Cake update:





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InvisiblePastywhyteMDiscord
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Re: Micro Grow and Agar Cloning [Re: JohnLothe]
    #21839818 - 06/21/15 11:00 PM (8 years, 7 months ago)

Biopsy from a fruit to LC is risky as hell. Just cut a small colonized wedge from an agar plate and drop it into the LC. Easy and much cleaner.


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OfflineMachiavelliavore
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Re: Micro Grow and Agar Cloning [Re: JohnLothe]
    #21840271 - 06/22/15 01:05 AM (8 years, 7 months ago)

http://www.shroomery.org/forums/showflat.php/Number/5238137#5238137 looks like a nice LC tek to me.  I haven't ever made one though.  Prolly gonna give ^ a shot soon.

Dropping a piece or entire agar wedge in would be the fastest.

Squirting water ontop of the agar, swirling, and sucking up, then injecting into the LC the cleanest, but slower.

Pasty is a smart dude, I've read many of his posts, so I'd just do what he said.


--------------------


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


No, this does not look right...


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OfflineJohnLothe
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Re: Micro Grow and Agar Cloning [Re: Machiavelliavore]
    #21843412 - 06/22/15 08:54 PM (8 years, 7 months ago)

Quote:

Machiavelliavore said:
http://www.shroomery.org/forums/showflat.php/Number/5238137#5238137 looks like a nice LC tek to me.  I haven't ever made one though.  Prolly gonna give ^ a shot soon.

Dropping a piece or entire agar wedge in would be the fastest.

Squirting water ontop of the agar, swirling, and sucking up, then injecting into the LC the cleanest, but slower.

Pasty is a smart dude, I've read many of his posts, so I'd just do what he said.



Quote:

Machiavelliavore said:
http://www.shroomery.org/forums/showflat.php/Number/5238137#5238137 looks like a nice LC tek to me.  I haven't ever made one though.  Prolly gonna give ^ a shot soon.

Dropping a piece or entire agar wedge in would be the fastest.

Squirting water ontop of the agar, swirling, and sucking up, then injecting into the LC the cleanest, but slower.

Pasty is a smart dude, I've read many of his posts, so I'd just do what he said.





Just made some LC jars and I'm about to transfer some cuts from the agar jars I made. I didn't harvest the Mushrooms at the right time, left them for to long and they released their spores all over the terrarium.  :godfuckingdammit:  Completely bumble fucked that portion of the experiment. Moving forward I will be more careful. Also I'm modifying my Terrariums from what you saw previously to one in which I free up the air transfer holes. I am also building a containment center which has a florescent light and small desk fan. I'll post some images of it when I'm done.


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