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Offlinesuperwhitedolemite
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n00b's first agar and clone!
    #26331366 - 11/19/19 02:46 PM (4 years, 2 months ago)

I started here with my first PF Tek grow and it's still going well. On my way to the third and fourth flushes. Getting some great results! I'm ready to give a few tubs a try. :smile:

So, I did some research, got all the supplies, made my first batch of agar dishes, and did my first round of clones. So far, so good and the mycelium is starting to colonize!

Now, these dishes are messy as hell, so forgive my n00b-ness. A couple of the tissue samples are a bit oversized as well. Working inside of an awkward little SAB made it tough to get it right, but it's an honest attempt.

I'm a little worried about some of the spots I'm seeing but wanted to get feedback.



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Offlinesuperwhitedolemite
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Re: n00b's first agar and clone! [Re: superwhitedolemite]
    #26338333 - 11/22/19 02:53 PM (4 years, 2 months ago)

OK, maybe I just didn't ask the right questions. Here's where I'm at a few days later. I can't tell what might or might not be contamination. If these plates are coming along well or if they're trash.

Any feedback would be greatly appreciated. My first attempt!

Each is numbered so the different shots of the same plates can be tracked.

Thanks!



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OfflineChardRich
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Re: n00b's first agar and clone! [Re: superwhitedolemite]
    #26338348 - 11/22/19 03:02 PM (4 years, 2 months ago)

All trash


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Offlinefootpath
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Re: n00b's first agar and clone! [Re: superwhitedolemite]
    #26338352 - 11/22/19 03:03 PM (4 years, 2 months ago)

It's all contamination. Just bacteria, that little yellow speck might be yeast.
You might have a tiny bit of mycelium pointing at 11 o'clock in picture 3. But your chances of transferring it successfully are very low.


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OfflineFreeCanadianHugs
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Re: n00b's first agar and clone! [Re: superwhitedolemite]
    #26338353 - 11/22/19 03:03 PM (4 years, 2 months ago)

Did you clone clean tissue? When you clone you want to use your sterile scalpel  to trim the outside tissue to expose the tissue that was not exposed. If you didn't then you will get whatever contaminate was on the outer tissue.


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Offlinesuperwhitedolemite
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Re: n00b's first agar and clone! [Re: FreeCanadianHugs]
    #26338371 - 11/22/19 03:13 PM (4 years, 2 months ago)

Well...shit. Thought it looked weird.

Yes, flame-sterilized a fresh, sterile scalpel, sliced the mushroom open, and sliced some tissue from the inside.

Should I have sterilized again, in between cutting the stem open and cutting the tissue sample from inside?

Followed what I learned from a few YouTube vids, to a T. Clean environment, hands, gloves, inside a SAB. I went overboard cleaning everything with alcohol ahead of time. Bummer.


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Offlinefootpath
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Re: n00b's first agar and clone! [Re: superwhitedolemite] * 1
    #26338403 - 11/22/19 03:26 PM (4 years, 2 months ago)

What I do is:
Cut about a 3" piece of the stipe (stem) - take it inside my SAB, peel it in half - hold it inside the SAB with my left hand while my right sterilizes my blade - take that in the SAB and cool it on agar - then cut out a very small (grain-sized) piece of one of the inner parts.

Taking smaller biopsies allows less possibility for you to transfer something extra.

Here's one I took on the 20th, it's a little big, even.


But you can see it's only growing its own tissue.


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OfflineShroomerquest
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Re: n00b's first agar and clone! [Re: superwhitedolemite]
    #26338431 - 11/22/19 03:36 PM (4 years, 2 months ago)

Woah, nasty


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Offlinesuperwhitedolemite
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Re: n00b's first agar and clone! [Re: footpath]
    #26338466 - 11/22/19 03:56 PM (4 years, 2 months ago)

Quote:

footpath said:
What I do is:
Cut about a 3" piece of the stipe (stem) - take it inside my SAB, peel it in half - hold it inside the SAB with my left hand while my right sterilizes my blade - take that in the SAB and cool it on agar - then cut out a very small (grain-sized) piece of one of the inner parts.

Taking smaller biopsies allows less possibility for you to transfer something extra.

Here's one I took on the 20th, it's a little big, even.


But you can see it's only growing its own tissue.





Awesome, I’ll try that. Thanks!


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Offlinecurious.psychonaut
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Re: n00b's first agar and clone! [Re: footpath]
    #26338521 - 11/22/19 04:23 PM (4 years, 2 months ago)

Quote:

footpath said:
What I do is:
Cut about a 3" piece of the stipe (stem) - take it inside my SAB, peel it in half - hold it inside the SAB with my left hand while my right sterilizes my blade - take that in the SAB and cool it on agar - then cut out a very small (grain-sized) piece of one of the inner parts.





I'm yet to make clones (waiting for first harvest, shroom gods willing) but I was planning on doing something similar, except for the blade cooling part. Opening and closing the receiving petri, esp. with stem in that hand (or keeping it open during the whole process) seems to introduce contam opportunities. Isn't cooling the blade while cutting out a grain-sized sample good enough?


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My LAGM2020 grow log



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Offlinefootpath
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Re: n00b's first agar and clone! [Re: curious.psychonaut]
    #26338564 - 11/22/19 04:52 PM (4 years, 2 months ago)

Likely.
I grip the stipe between my index and middle finger while I lift/replace the lid with my thumb and pinky. I also have a 'cooling plate', hence why you don't see any deformities in the above pictured plate. Once the biopsy is on my blade, I then have my left hand free.
It might be an unnecessary practice in dexterity. Letting it cool in the air or when you insert it into the tissue might do well enough. Just not the way I've found success.

Cooling plates are great because they'll show you if your technique has any flaws, I've been using this one for over a week:


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Offlinecurious.psychonaut
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Re: n00b's first agar and clone! [Re: footpath]
    #26338600 - 11/22/19 05:09 PM (4 years, 2 months ago)

Quote:

footpath said:
Cooling plates are great because they'll show you if your technique has any flaws, I've been using this one for over a week:





Oh wow, I thought of using a cooling plate before but decided it's unwise to dip the scalpel in anything other than the receiving plate due to the extra contam chance. You do it as a challenge, nice!


--------------------
My LAGM2020 grow log



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Offlinesuperwhitedolemite
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Re: n00b's first agar and clone! [Re: curious.psychonaut]
    #26353437 - 11/30/19 09:19 AM (4 years, 1 month ago)

Great info here - many thanks to everyone! Will take it to heart and do a better job next time. Glad I only used 5 of 20 plates.

Just started my first tub from my first-run of cakes, and will clone again if/when another big boy pops up from that.


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InvisibleAyePlusS
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Re: n00b's first agar and clone! [Re: superwhitedolemite]
    #26353467 - 11/30/19 09:40 AM (4 years, 1 month ago)

Rip it open instead of cutting, when you cut thhe blade contacts the dirty outside of the fruit and drags contam into the middle. And flame between every cut.


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Offlinesuperwhitedolemite
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Re: n00b's first agar and clone! [Re: AyePlus]
    #26353504 - 11/30/19 10:11 AM (4 years, 1 month ago)

Yep, that seems to be the takeaway here, overall. Thanks!


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