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TheAtlantean's - Cloning/Agar Tek * 2
    #28406306 -

Greetings Everyone,

"Nature is an endless combination and repetition of a very few laws. She hums the old well-known air through innumerable variations." - Ralph Waldo Emerson

"The Vision of God is the beginning of initiation." - Corpus Hermeticum


Agar Recipe:
7G LME
10G Lab Grade Agar
1G Nutritional Yeast
500ML Purified Reverse Osmosis Water
Coloring Gel

Today we are going to talk about tissue cloning from our favorite fruits and vegetables...... err.. wait.. MUSHROOMS. :coolcat:


In order for us to accomplish this, I will assume you have the ability to acquire the necessary tools and techniques to accomplish this wonderful method of cloning with near zero contamination. This TEK is generally good for fruits produced in our favorite monotub/martha type environments.

And with work done in front of a Flow Hood or someone skilled in a their SAB.


Here are the required tools:

- Trusty Scalpel

- Biopsy Lab Punch

- Clean Agar Plates

- Your Favorite Fruits from the Current Harvest


- Flow Hood or SAB
or



STEP 1:
Start by admiring the beauty of Nature.


Step 2:
Take your sterilized scalpel and cut off the end of the stipe of the mushroom you are wanting to clone.
Single cuts, people.
No sawing.
Just slice.
One solid motion.


Step 3:
Next, We need to get out the Biopsy Punch, these come presterilized and are wonderful for cloning.

MAKE ALL CUTS AND INCISIONS ONCE, AND WITH PRECISION

Gently, and directly, place it into the center of the stipe tissue, like so:


Step 4
I personally do the same to the other side and get two clones from the single cut of the stem/stipe.



Step 5:
Both ends should look like this, then, take your sterilized scalpel and make an incision about 1/4" / 2-4mm down the stipe in a horizontal motion and cut out the center piece:


Here is what the transfer looks like from the center of the stipe


And here are the results:






How to Clone/Biopsy Panaeolus Cyanescens with MINIMAL CONTAMINATION



First we want to take our chosen Fruit, this is TTBVI from Gordo:


Then collect our Biopsy Tool and carefully unwrap it:



Remove the Cap with either a scalpel or if you can pull it off with your fingers like so:


Use the tool to gather a bit of flesh from the top end of the fruit/stipe where you removed the cap:


Results:







Happy Cloning

Edited by TheAtlantean (05/10/24 10:30 AM)

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Re: TheAtlantean's 0% Contam - Cloning Tek [Re: TheAtlantean] * 1
    #28406340 -

Awesome, I’ll have to snag some of those punches. Are they able to be sterilized or are they single use?

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Re: TheAtlantean's 0% Contam - Cloning Tek [Re: Brundis116]
    #28406344 -

Brundis116 said:
Awesome, I’ll have to snag some of those punches. Are they able to be sterilized or are they single use?


The ones pictured are single use. But, I generally get 3-5 uses out of them as long as I have and maintain sterile technique.


--------------------
Astra said:

“Peace River has a gentle personality, yet TTBVI hit me like strong LSD with a twist of medical grade meth. Not for the faint of heart.”

Team Panaeolus

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Re: TheAtlantean's 0% Contam - Cloning Tek [Re: TheAtlantean] * 1
    #28406370 -

Nice TEK! Will get some of thes punches and give it a try :headbang:

I usually tear open the stipe of the shroom and try to scrape a piece off with my scalpel (as in RRs video about cloning). That does however tend to be a bit fidgety and can be difficult to get the stringy thread off the scalpel and onto the agar smoothly..


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

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Re: TheAtlantean's 0% Contam - Cloning Tek [Re: tittlez]
    #28406377 -

tittlez said:
Nice TEK! Will get some of thes punches and give it a try :headbang:

I usually tear open the stipe of the shroom and try to scrape a piece off with my scalpel (as in RRs video about cloning). That does however tend to be a bit fidgety and can be difficult to get the stringy thread off the scalpel and onto the agar smoothly..



Thank you.

I had the same problem and said fuck that noise.

Thought that taking tissue samples must be perfected in biology somewhere.


Et voila.


--------------------
Astra said:

“Peace River has a gentle personality, yet TTBVI hit me like strong LSD with a twist of medical grade meth. Not for the faint of heart.”

Team Panaeolus

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Re: TheAtlantean's 0% Contam - Cloning Tek [Re: TheAtlantean] * 1
    #28406421 -

:takingnotes:
My pe6 are almost ready to fruit, will def be getting biopsy punch. Lovely Write Up!


--------------------
How would you feel if you hadn't eaten breakfast?

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Re: TheAtlantean's 0% Contam - Cloning Tek [Re: FeelFighters] * 1
    #28406442 -

FeelFighters said:
:takingnotes:
My pe6 are almost ready to fruit, will def be getting biopsy punch. Lovely Write Up!



Awesome. I’m happy to know the tek has proven useful for others already.


--------------------
Astra said:

“Peace River has a gentle personality, yet TTBVI hit me like strong LSD with a twist of medical grade meth. Not for the faint of heart.”

Team Panaeolus

Extras: Filter Print Post Top
Re: TheAtlantean's 0% Contam - Cloning Tek [Re: TheAtlantean]
    #28406455 -

I’ve never used the biopsy tool, would it work if you pinched the end of the stem and peeled the shroom open, then took a biopsy from the center or would it be too thin and go all the way through the stem?


Cutting through the place the sample is coming from id worry about forcing bacteria from the skin into the center.


--------------------
LAGM2020

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Re: TheAtlantean's 0% Contam - Cloning Tek [Re: TheAtlantean]
    #28406460 -

Thoughts on the outside of the stipe compromising cleanliness of the scalpel when making the cut? Not trying to be a dick, I've just been thinking about that lately with some attempts at making cleaner prints and swabs.

Edit: just saw Aka beat me to the same thought

Edited by crabs (07/24/23 03:16 PM)

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Re: TheAtlantean's 0% Contam - Cloning Tek [Re: A.k.a]
    #28406466 -

A.k.a said:
I’ve never used the biopsy tool, would it work if you pinched the end of the stem and peeled the shroom open, then took a biopsy from the center or would it be too thin and go all the way through the stem?


Cutting through the place the sample is coming from id worry about forcing bacteria from the skin into the center.


Chances are slim IME of getting the bacteria down in the tissue if you follow the procedure properly. Using a fresh fruit and cloning it within the hour.

I place my cut clones in front of my flowhood while harvesting and immediately clone after I am finished with that tub.

Also. If you have an aggressive culture then most likely to mycelium will outgrow the bacteria and you’ll be able to see it on the bottom of the plate as metabolites

I inoculated 90 jars beginning of June with an LC produced from this method of agar work and the flushes you see in my most recent posts are from that LC.

I have done almost 100 plates contam free since beginning  this method.

Also, what you are describing is the reason why I use sterile tools only, either a new blade or one that has been freshly sterilized using an induction sterilizer. Although…🧐🤓 I suppose it could work if I’m understanding you correctly.


The amount of time needed for a bacterium to produce two daughter cells under ideal circumstances, (most medically significant bacteria) have a generation time of around 20 minutes. Some may even require 18 to 24 hours or more.

Bacteria lack complex, membrane bound organelles including a nucleus. They have much less DNA on average, and their DNA is not as complexly packaged as eukaryotic DNA is. All of these factors enable bacteria to generally divide faster than eukaryotic cells. So as long as we complete our transfers within or under an hour, the chance of bacterial replication is lowered due to overall time in exposure.

We must also consider that the fruit is alive, and also has an active immune system. These are just my personal thoughts. Also assuming your fruits were produced in a clean and sterile environment. This tek would not work for wild cultures or fruits. Of which I would be taking a spore print of anyways and working it from there due to obvious contamination.

Edited by TheAtlantean (07/24/23 03:33 PM)

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Re: TheAtlantean's 0% Contam - Cloning Tek [Re: TheAtlantean] * 2
    #28406513 -

I’m just talking about the bacteria on the skin of the stem.

If you ever tried cloning tiny mushrooms like pans or tamps it’s extremely difficult to get a sample that’s not bacterial cuz the stem is like 90% outer skin :lol:


I’ve read that cutting through the shroom can push bacteria from the skin into the clean tissue inside, but I’ve also never tried it myself. Cubes are definitely big enough to get samples that are 100% interior tissue so you don’t have to worry about it liek with pans, so if you’re getting clean samples cutting through the outside then I guess it’s not as serious as the older teks expected it would be.


--------------------
LAGM2020

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Re: TheAtlantean's 0% Contam - Cloning Tek [Re: A.k.a]
    #28406520 -

A.k.a said:
I’m just talking about the bacteria on the skin of the stem.

If you ever tried cloning tiny mushrooms like pans or tamps it’s extremely difficult to get a sample that’s not bacterial cuz the stem is like 90% outer skin :lol:


I’ve read that cutting through the shroom can push bacteria from the skin into the clean tissue inside, but I’ve also never tried it myself. Cubes are definitely big enough to get samples that are 100% interior tissue so you don’t have to worry about it liek with pans, so if you’re getting clean samples cutting through the outside then I guess it’s not as serious as the older teks expected it would be.


Well. We will soon find out for sure with Pans.

There are smaller gauged biopsy punches available.

So I will try that why I have my first Pan Harvest. Thanks for the perspective.


--------------------
Astra said:

“Peace River has a gentle personality, yet TTBVI hit me like strong LSD with a twist of medical grade meth. Not for the faint of heart.”

Team Panaeolus

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Re: TheAtlantean's 0% Contam - Cloning Tek [Re: A.k.a]
    #28406524 -

A.k.a said:
I’m just talking about the bacteria on the skin of the stem.

If you ever tried cloning tiny mushrooms like pans or tamps it’s extremely difficult to get a sample that’s not bacterial cuz the stem is like 90% outer skin :lol:


I’ve read that cutting through the shroom can push bacteria from the skin into the clean tissue inside, but I’ve also never tried it myself. Cubes are definitely big enough to get samples that are 100% interior tissue so you don’t have to worry about it liek with pans, so if you’re getting clean samples cutting through the outside then I guess it’s not as serious as the older teks expected it would be.



One thing to consider is the area where the tissue is being cut from.


It's such a small chance IME and opinion, that as long as the Fruit/culture is healthy, the mycelium will outrun the bacteria. Let the plate grow out and inoculate from T2+ plates only. This is also a wonderful way to determine sectoring of a fruit and the different strains working in concert to create the specimen.


--------------------
Astra said:

“Peace River has a gentle personality, yet TTBVI hit me like strong LSD with a twist of medical grade meth. Not for the faint of heart.”

Team Panaeolus

Extras: Filter Print Post Top
Re: TheAtlantean's 0% Contam - Cloning Tek [Re: TheAtlantean] * 1
    #28406526 -

Dude if you can biopsy a pan  this way that would be incredible. Such a pain to clone, almost always end up waiting on the myc to outrun the bacteria. Prob the most common complaint with pans


--------------------
LAGM2020

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Re: TheAtlantean's 0% Contam - Cloning Tek [Re: A.k.a]
    #28406529 -

A.k.a said:
Dude if you can biopsy a pan  this way that would be incredible. Such a pain to clone, almost always end up waiting on the myc to outrun the bacteria. Prob the most common complaint with pans


Looks like I found my next challenge!

I will for sure give this a go.


--------------------
Astra said:

“Peace River has a gentle personality, yet TTBVI hit me like strong LSD with a twist of medical grade meth. Not for the faint of heart.”

Team Panaeolus

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Re: TheAtlantean's 0% Contam - Cloning Tek [Re: TheAtlantean]
    #28541412 -





Pan Biopsy. Images are four days apart.


--------------------
Astra said:

“Peace River has a gentle personality, yet TTBVI hit me like strong LSD with a twist of medical grade meth. Not for the faint of heart.”

Team Panaeolus

Extras: Filter Print Post Top
Re: TheAtlantean's 0% Contam - Cloning Tek [Re: TheAtlantean]
    #28541521 -

TheAtlantean said:





Pan Biopsy. Images are four days apart.


What gauge punch did you use?

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Re: TheAtlantean's 0% Contam - Cloning Tek [Re: TheAtlantean] * 1
    #28541548 -

Oh hell yeah! Nice work!

I too am wondering what size you used on the pans. The bacteria is a real bitch as mentioned above

Edited by PhenoDreamers (11/13/23 04:24 PM)

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Re: TheAtlantean's 0% Contam - Cloning Tek [Re: normalperson]
    #28541555 -



It is a 1mm sterile biopsy punch.

Brand is Royaltek


--------------------
Astra said:

“Peace River has a gentle personality, yet TTBVI hit me like strong LSD with a twist of medical grade meth. Not for the faint of heart.”

Team Panaeolus

Extras: Filter Print Post Top
Re: TheAtlantean's 0% Contam - Cloning Tek [Re: TheAtlantean]
    #28541560 -

Another plate and biopsy with two pieces of tissue to see cleanliness from different places in the tiny little mushroom



--------------------
Astra said:

“Peace River has a gentle personality, yet TTBVI hit me like strong LSD with a twist of medical grade meth. Not for the faint of heart.”

Team Panaeolus

Extras: Filter Print Post Top
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