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InvisibleChanga Alchemist
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Re: The Biopsy Method: A different way of doing LC. [Re: elasticaltiger]
    #25020192 - 02/25/18 09:38 AM (3 years, 9 days ago)

It depends. If you took a biopsy to inoculate the puck that's got a pin, I'd put the whole pin on agar. Same as an agar pin.

Also, I would not squirt water into your petri dish, ever. I mean the only exception is if you have to use a MS syringe. All this does is give bacteria a head start if there is any there to begin with, and make it harder to transfer away from.


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Re: The Biopsy Method: A different way of doing LC. [Re: Changa Alchemist] * 1
    #25020232 - 02/25/18 10:09 AM (3 years, 9 days ago)

:whathesaid:
That's exactly why it's good for testing out a culture, it gives bacteria a head start if there's any. Using the bio exclusively as your only way  to do agar work is something I'd never do for a series of reasons that I mentioned in other posts but right now I don't have time to make a long-winded post.

It can be used to reduce the genetics from a plate that's maybe 2 tranfers in from a streaked plate in order to slim down the genetics (a lot) and go with the scalpel from there. All this is subjective and based on personal experience but by all means try it and see for yourself if you like it. BTW, only a qick little squirt is required, the equivalent of a few drops.

I do culture work by poking alone too, but for that I use PF tek pucks and it goes to be something like: inoculate a puck with spores> transfer from the first puck to multiple pucks using a bio > let those pin out > transfer the pins to pucks > use those pucks to inoculate LC's for a very long time. Why? because the pucks grow very slowly, you can make lots of LC's from a the same puck over time and you're gonna be working with a young culture if you do it this way. It relieves me of some of the agar workload and I appreciate that a lot in those periods where I'm busy at work. Perfectly legit way to get shit done, just a different approach.


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #25020251 - 02/25/18 10:21 AM (3 years, 9 days ago)


Hericium coralloides "poke" lc


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Offlinepacmanbreed
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #25020536 - 02/25/18 12:54 PM (3 years, 9 days ago)

Quote:

Josex said:
use those pucks to inoculate LC's for a very long time




I wonder if those puck can also be fridge up for super long term forbyoung culture? Togther with high nute content of pfpucks



Quote:

Josex said:
let those pin out > transfer the pins to pucks



Sharing my update.
Thanks brother josex. Im getting a feel and hang of the method. You rock.:rockon:

Anyway on the part of let those pin out.
Its usefull to use a weak nute concentration for rapid pinning.


I got pins excatly 18-21 days :mushroom2:. On most of my coir/brf @ 5:1 ratio.
Be sure go to the dry side or add ricehusk or perlite to the mix by 30-50% dry volume.(aim for hydeated aged dung consistency, think of how grain to bulks works in micro scale.)
Ive used it since verm in our area cost a fortune.
I have found that the problem with coirpucks @ 2:1:1 is it has mud like consistency when the rice starches is cooked & mixed with the water during pc.
This is where my few batches failed.
:dumblol:



I also feel You can even check piggybacking contams using coirpucks w/ super low nutes & super dry @ less than field capacity with minimal water content.
This has a different ratio from the one posted above.

I dont know whats up with those mutated sky pointing hypae(migth be piggy backing or morphology).


Still observing them @ the moment.


Edited by pacmanbreed (02/25/18 01:26 PM)


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Invisiblevan hatton
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Re: The Biopsy Method: A different way of doing LC. [Re: pacmanbreed]
    #25020734 - 02/25/18 02:22 PM (3 years, 9 days ago)

I'm at 2 months on some of my brf  pucks and no pins :frown:

Tiger.  I was just transferring via the poke for awhile imo the biggest downside is how it grows. And not exactly being able to see contam unless it's mold.


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Tmethyl said:
Chuck Norris once roundhouse kicked a monotub that wasn't pinning fast enough. The force of the kick rearranged the genetics of the mushrooms, we now call them Penis Envy.

Caps McGee said:
:thumbsup:
Fun part is figuring out what works best for you


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Re: The Biopsy Method: A different way of doing LC. [Re: van hatton]
    #25020777 - 02/25/18 02:39 PM (3 years, 9 days ago)

Quote:

van hatton said:
I'm at 2 months on some of my brf  pucks and no pins :frown:





PF pucks or coir+brf pucks?  PF pucks pin much quicker than coir, 1 month max, usually sooner.

Quote:

van hatton said:
Tiger.  I was just transferring via the poke for awhile imo the biggest downside is how it grows. And not exactly being able to see contam unless it's mold.



Yea it grows ugly and disorganized like you would expect spore solution to behave on agar. But I can't agree about that part of not being able to see contams. If you shoot a biopsy on agar from a bacterial culture, the first thing you're going to see on the plate is bacteria way before myc has time to recover and grow. You're giving bacteria a ~5 days window to grow if there's any bacteria at all.
If the plate is still clean by the time myc starts to grow  you can be damn sure the plate is not gonna get bacteria all of a sudden out of thin air, however ugly the culture may look as it grows out, which can make you think it's contam'd when in fact it isn't. Being paranoid about contams doesn't help here (not saying you're :lol:)


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Invisiblevan hatton
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #25020813 - 02/25/18 02:51 PM (3 years, 9 days ago)

I should have added that I was transferring from brfc plate to another brfc plate :lol: I can't see anything but mold on a brfc plate.

You just have to assume the spot that you poke is going to be free of bacteria.  It usually is if you let them grow out a bit.  But I've found that you never no where the water you actually squirt into the puck. 

Since you cant see the water you squirted and your possibly  dealing with something bacterial shit you may end up grabbing a spot where you squirted if that makes sense.

One thing as well with my clone it kept taming out.  Turned out it has some beautiful Mycogone in it.  Difficult too see on brfc plates but the second I put it too agar it was quite apparent

I think I explained that right :bongload:


Edit: another thing. If you don't care about the plate your transferring from (loads of bacteria of mold) you can dam near ensure that your not gonna snag something. I've grabbed a spot that was barely grow away from bacteria and almost touching some green mold.  Put it too another Perri and I didn't grab anything surprisingly.


--------------------
If I ever give out misinformation please inform me so I can have the correct information. :cheers:

Tmethyl said:
Chuck Norris once roundhouse kicked a monotub that wasn't pinning fast enough. The force of the kick rearranged the genetics of the mushrooms, we now call them Penis Envy.

Caps McGee said:
:thumbsup:
Fun part is figuring out what works best for you


Edited by van hatton (02/25/18 02:55 PM)


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Offlinepacmanbreed
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Re: The Biopsy Method: A different way of doing LC. [Re: van hatton]
    #25021759 - 02/25/18 09:29 PM (3 years, 8 days ago)

Quote:

van hatton said:
One thing as well with my clone it kept taming out.  Turned out it has some beautiful Mycogone in it.  Difficult too see on brfc plates but the second I put it too agar it was quite apparent





I feel Thats One of the reason my lc did go south.

I can say that mycogone are shit to isolate from. They dont show up from start. until the mycelium almost fully colonized an agar plate, pucks, or even brfc at later stages when nutes are almost depleted.
Seems its tightly enmeshed being part of mycleium.

Most of agar plates dont produce pins because of this shit.
I dont know what coirpuck did to it, but mycogone ladden culture did fruit on it.
Might be because of the porous surface allowing mycelium to fruit on top and the tams & excess water to to go down below the plate.

Im not yet sure if the tissue of fruits are clean from mycogone though?


Sidenote:
I think Pucks and agar plates has its own advantages and are two different beast for our use...
In Agar nutes tend deplete quicker.(usefull for checking piggybacks
While Pucks can produce pin even from bacterial culture.

I feel thats one problem with brfc/paste. Being high nute with low porosity.


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Edited by pacmanbreed (02/25/18 09:40 PM)


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OfflineDoc9151M
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Re: The Biopsy Method: A different way of doing LC. [Re: pacmanbreed]
    #25021828 - 02/25/18 10:00 PM (3 years, 8 days ago)

Great write up, but why not put a port on the lid that is self healing, that way you can use sterile needles to draw up only what you need?


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https://www.shroomery.org/forums/showflat.php?Cat=0&Number=26513593&page=0&vc=1#26513593


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InvisibleChanga Alchemist
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Re: The Biopsy Method: A different way of doing LC. [Re: Doc9151]
    #25021835 - 02/25/18 10:02 PM (3 years, 8 days ago)

My ports always ended up with small holes in them. They lost their "self healing" powers about 3 or 4 pulls in.

Also, pushing a needle in and out from the outside is a contam vector that some of us did away with, because we got holes in our  ports.

To each their own. Some people just squirt high temp silicon  over there hole after some ware and tear. I prefer to pour, and keep a mother dish or slant to poke for LC when I need more.


Edited by Shomann (02/25/18 10:06 PM)


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Re: The Biopsy Method: A different way of doing LC. [Re: Changa Alchemist]
    #25021867 - 02/25/18 10:11 PM (3 years, 8 days ago)

Makes sense about the wear and tear, but an alcohol wipe over the port takes care of vectors, that's how ee do it in medicine and pharmaceutical compounding.


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


Psilocybe cubensis data collection thread. please help with this project if you hunt wild cubensis.
https://www.shroomery.org/forums/showflat.php?Cat=0&Number=26513593&page=0&vc=1#26513593


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Invisiblevan hatton
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Re: The Biopsy Method: A different way of doing LC. [Re: Doc9151]
    #25021870 - 02/25/18 10:12 PM (3 years, 8 days ago)

Because it is not sterile. It's just sanitized.


--------------------
If I ever give out misinformation please inform me so I can have the correct information. :cheers:

Tmethyl said:
Chuck Norris once roundhouse kicked a monotub that wasn't pinning fast enough. The force of the kick rearranged the genetics of the mushrooms, we now call them Penis Envy.

Caps McGee said:
:thumbsup:
Fun part is figuring out what works best for you


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InvisibleChanga Alchemist
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Re: The Biopsy Method: A different way of doing LC. [Re: van hatton]
    #25021895 - 02/25/18 10:26 PM (3 years, 8 days ago)

:whathesaid: people/animals have immune systems that allow us to sanitize and not sterilize certain forms of medicine intake. Sterile water has no defense.

That's my own thoughts on it anyways, might not be 100% accurate.


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Re: The Biopsy Method: A different way of doing LC. [Re: Changa Alchemist]
    #25022139 - 02/26/18 12:56 AM (3 years, 8 days ago)

I don't understand what you mean, once an lc is put into the pc @15psi for 25-30min  it's sterile and will remain so until exposed to contamination or sits around for years at room temperature. Unless of course the pc is not accurate.


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


Psilocybe cubensis data collection thread. please help with this project if you hunt wild cubensis.
https://www.shroomery.org/forums/showflat.php?Cat=0&Number=26513593&page=0&vc=1#26513593


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Re: The Biopsy Method: A different way of doing LC. [Re: Doc9151]
    #25022142 - 02/26/18 12:58 AM (3 years, 8 days ago)

I've used sterile water in the 90's that was left over from the Vietnam conflict.


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


Psilocybe cubensis data collection thread. please help with this project if you hunt wild cubensis.
https://www.shroomery.org/forums/showflat.php?Cat=0&Number=26513593&page=0&vc=1#26513593


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Re: The Biopsy Method: A different way of doing LC. [Re: Doc9151]
    #25022159 - 02/26/18 01:07 AM (3 years, 8 days ago)

Unless you put your pressure cooker or sterilizer in fromt if your flow hood to cool the outside of the port can only be considered sanitized, not sterilized. So your sterile needle has to pass through an unsterile surface to reach into the jar, whereas if you just open the jar the needle only passes through air. Therefore the port is considered a vector, an unneccesary one that many choose to avoid.


Sanitized may be enough for medicine but we use sugar rich broth and its a breeding ground for contamination, unlike a sterile compound or medicine which would have little to no food for organisms to grow.


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Re: The Biopsy Method: A different way of doing LC. [Re: AyePlus]
    #25022174 - 02/26/18 01:18 AM (3 years, 8 days ago)

:whathesaid:

There we go. Thanks A+!


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Re: The Biopsy Method: A different way of doing LC. [Re: AyePlus]
    #25022194 - 02/26/18 01:25 AM (3 years, 8 days ago)

Quote:

AyePlus said:
Unless you put your pressure cooker or sterilizer in fromt if your flow hood to cool the outside of the port can only be considered sanitized




What if you seal the top of the container with foil and then take it out of your pc and let it cool in your sterile area?

Sure once it touches air it's considered sanitary but if you wait to take the foil off until right before inoculation that's about as good as it gets.


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Re: The Biopsy Method: A different way of doing LC. [Re: elasticaltiger]
    #25022240 - 02/26/18 02:00 AM (3 years, 8 days ago)

Quote:

elasticaltiger said:
Quote:

AyePlus said:
Unless you put your pressure cooker or sterilizer in fromt if your flow hood to cool the outside of the port can only be considered sanitized




What if you seal the top of the container with foil and then take it out of your pc and let it cool in your sterile area?

Sure once it touches air it's considered sanitary but if you wait to take the foil off until right before inoculation that's about as good as it gets.




As soon as the pressure drops to zero even the inside of your pressure cooker is technically no longer sterile, is it clean enough? Maybe. Is it sterile? No its not, but neither is the inside of your SAB, but thats full of air, not a physical surface for comtaminants to land on.


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Re: The Biopsy Method: A different way of doing LC. [Re: elasticaltiger]
    #25022243 - 02/26/18 02:00 AM (3 years, 8 days ago)

He's right as far as the sanitized vs sterilized, but then he sunk his self with the rest.

Once you open a sterile field you lose sterility. So his argument is  void. Passing a sterilized needle through a port reduces the chance of contamination exponentially compared to his reasoning.


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


Psilocybe cubensis data collection thread. please help with this project if you hunt wild cubensis.
https://www.shroomery.org/forums/showflat.php?Cat=0&Number=26513593&page=0&vc=1#26513593


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