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Re: The Biopsy Method: No Mercy for Bacteria. [Re: pacmanbreed]
    #24989352 -

pacmanbreed said:
I have a pinning coir puck innoc 21 days ago thats fast.
Is there a possibility bacteria can be enmeshed within the pin body?
Im plannin to clone this guy.


1 out of 10 did pin, some stalled, some are fully colonized but with something piggy backing bothering the mycleium enough. Man my samples are bacterial AF.



Try pf-tek sub next time, I think I told you coir is not nearly as effective to deal with the bitch? can't remember if it was you. And yea clone that pin. :thumbup:

Jhova, glad to hear that man. Now to an awesome grain prep and lotta shrooms. :cheers:

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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Josex]
    #24990424 -

It was me brother. Spot on.
Nothing beats pf subs. Coir as last resort.
Im so happy after 3 weeks. One of the Biopsy produced pin.
Most wedge inoculated puck didnt.


Man this are fast in coir pucks. Double in size in 8 hrs.
Hope still have time to transfer. Im whipping my no-pour agar now.

Edited by pacmanbreed (02/13/18 03:05 AM)

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Re: The Biopsy Method: No Mercy for Bacteria. [Re: pacmanbreed]
    #24992429 -

can this work on grain pucks?

:smile:

brf is a pita to get where I'm based...


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Fermented Mushrooms!!
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Re: The Biopsy Method: No Mercy for Bacteria. [Re: pablokabute]
    #24993347 -

pablokabute said:
can this work on grain pucks?

:smile:

brf is a pita to get where I'm based...



You can try it but I don't think it'd be remotely as effective.

I'd like to clarify that there's nothing antibacterial about BRF (that answers Logical's question a few pages back, sorry for the delay) and that the whole point of using PF-tek substrate is to create an unfavorable environment for bacteria, capable of hindering its growth or slowing it down to a near halt, while allowing optimal growth for cube mycelium.

The texture, consistency and also probably the high nutritional value of the BRF+verm substrate (bacteria won't fucking know what to do with so much food in one single spot) are the things that make this possible.
Any other substrate with similar characteristics will probably do the job just fine.

All this talk just to tell ya that you can probably substitute BRF for any grain flour to get the job done. So get a coffee grinder and grind whatever grain you have handy into a flour (just don't do wheat) and use it to make the PF tek sub.
Also, take into account that other flours might not be as clean as BRF is, so you'd need to up the PC times to make sure the pucks are completely sterile out of the PC.

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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Josex]
    #25031832 -

Holy shit, this might actually make isolation far faster and cleaner. Good shit.

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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Severance13]
    #25101624 -

Amazing tek. I believe my " Easiest Liquid Culture for the incredibly lazy" tek could use your info.
Some people have been mentioning your methods on my thread.

Hope you don't mind I add a link of your tek in my thread.

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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Dr. Freeman]
    #25101755 -

Check out josex’ LC thread for more info this thread is the precursor to using his LC thread.


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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Josex]
    #25180594 -

Hi! I'm having issues with a syringe from a vendor that somehow got some bacteria up in it after my first grain run, and now every dish of agar I try winds up with this weird milky bacterial growth before any spores have a chance to germinate. I posted the issue in the cultivation forum and someone gave me a link to your biopsy tek with the thoughts I might be able to have better luck with brf pucks.

Great write up btw! As I was reading it though, it seemed as if the method was meant to take microscopic pieces of tissue from an agar plate that though has contams, still has mycelium growing, and to then take that and inject it onto a brf puck. Was I understanding it correctly, or can I take the contaminated syringe and use on the brf to help save the 5 cc's of spores I have left? I snagged a few pieces of the colonized grain and stuck them on some agar so if the syringe needs to be tossed, all is not lost.

Your input would be greatly appreciated!


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Remember... It's not whether the glass is half-full nor half-empty, but that it is always refillable.

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Re: The Biopsy Method: No Mercy for Bacteria. [Re: FungiCurious]
    #25180629 -

I happened to read the thread you made where you were explaining the problems you were having and saw that you wiped the needle after flaming, never do that.
Everything on the needle gets killed when you flame, wiping after flaming is a good way to make the needle unsterile again.

That could explain why you're having issues with bacteria, although it could also be that the syringe is dirty, but you'll never know for sure till you quit doing that bad practice.

Quote:
As I was reading it though, it seemed as if the method was meant to take microscopic pieces of tissue from an agar plate that though has contams, still has mycelium growing, and to then take that and inject it onto a brf puck. Was I understanding it correctly, or can I take the contaminated syringe and use on the brf to help save the 5 cc's of spores I have left?



Yes you understood correctly, but since you didn't get any growth on the plates you can also use the syringe on some pucks. Spore solution germinates easy on pf-tek substrate without the mess you can get on agar. Just flame the needle, let it cool for a few seconds and squirt a couple drops in each puck, you don't need to flame between inoculations, you could do 2 or 3 pucks before flaming again. When you get growth drop by with any questions you might have on what to do next.

Quote:
I snagged a few pieces of the colonized grain and stuck them on some agar so if the syringe needs to be tossed, all is not lost.




That's actually a very good idea. :thumbup:

Edited by Josex (05/02/18 09:57 PM)

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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Josex]
    #25180748 -

Josex said:
I happened to read the thread you made where you were explaining the problems you were having and saw that you wiped the needle after flaming, never do that.
Everything on the needle gets killed when you flame, wiping after flaming is a good way to make the needle unsterile again.

That could explain why you're having issues with bacteria, although it could also be that the syringe is dirty, but you'll never know for sure till you quit doing that bad practice.

Quote:
As I was reading it though, it seemed as if the method was meant to take microscopic pieces of tissue from an agar plate that though has contams, still has mycelium growing, and to then take that and inject it onto a brf puck. Was I understanding it correctly, or can I take the contaminated syringe and use on the brf to help save the 5 cc's of spores I have left?



Yes you understood correctly, but since you didn't get any growth on the plates you can also use the syringe on some pucks. Spore solution germinates easy on pf-tek substrate without the mess you can get on agar. Just flame the needle, let it cool for a few seconds and squirt a couple drops in each puck, you don't need to flame between inoculations, you could do 2 or 3 pucks before flaming again. When you get growth drop by with any questions you might have on what to do next.

Quote:
I snagged a few pieces of the colonized grain and stuck them on some agar so if the syringe needs to be tossed, all is not lost.




That's actually a very good idea. :thumbup:



Thank you Josex, and I for sure will! I've definitely stopped wiping my needles. For the past decade growing cannabis, when taking cuttings, or anything that has to do with touching plant tissue 91 - 99% ISO has been my friend. Not in this hobby though. lol

Anyways, thanks again!


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Remember... It's not whether the glass is half-full nor half-empty, but that it is always refillable.

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Re: The Biopsy Method: No Mercy for Bacteria. [Re: FungiCurious]
    #25228817 -



Wedge>pfpuck


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Re: The Biopsy Method: No Mercy for Bacteria. [Re: JHOVA]
    #25229102 -

I've done agar MS wedge (non-bacterial) to pucks lots of times in the past just to see what'll pop up to clone, but biopsy to pucks almost always ends up being stronger looking and faster myc (I've seen bios achieving 100% colonization faster than pucks inoculated with wedges the same day) and pins more heavily on pucks most of the times.

I swear I'm not trying to sell you anything, trust me I couldn't care less and I've always hated stating things that I can't really prove or explain but it's what I've observed after having done countless pucks, the difference is huge.

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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Josex]
    #25229142 -

That’s interesting.


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Re: The Biopsy Method: No Mercy for Bacteria. [Re: JHOVA]
    #25229245 -

Would this also help with a yeast or mold contam? I've been battling something in my agar for weeks now and I'm at the point where I need to find something that will slow down the contam so I can grab some clean myc. I thought it was bacterial but others have posted that it sounds like its yeast or mold contam. My plates dont look contaminated they are all white but a little on the fluffy side I think this fluff is the contams growing right along with the myc. The only reason why i know it is contaminated is when I open up the agar it dont smell right it has sorta fermented smell to it and when I transfer to grain it stalls out and wont inoculate the jar fully then after a week the fluffiness takes over and turns light grey.

So would your PF puck help in this attempt to clean whatever the fuck I got going on? Is there any other types of subs I could use to help the myc grow faster than whatever is intertwined with it?

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Re: The Biopsy Method: No Mercy for Bacteria. [Re: more4u2c]
    #25229266 -

A pic would speak volumes, do you have a spare plate of this culture that you can sacrifice? It could also be meshed-in mold.

Shit intertwined with the culture is another beast and can really pose a problem.
You could try poking the culture to some pucks but I can't guarantee anything, it's a roll of the dice at best.

I was able to separate meshed-in mold from a culture once. Somehow when I squirted the bio myc separated from mold and it was cool to see myc growing on one part of the puck and some kind of white mold on the other.

Try it out and let us know. Do a bunch of pucks to up your chances.

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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Josex]
    #25229374 -

Here are the probably 2nd or 3rd transfer dishes not very good pics I'm now 2 or 3 transfers away from these pics I'll get pics of the most recent ones when I get home. This is a PE strain and one contaminated vtek actually gave me like 6 little penises I should of cloned them but didnt realize I had a contam issue until afterwards


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Re: The Biopsy Method: No Mercy for Bacteria. [Re: more4u2c]
    #25229410 -

I can't see anything iffy in those pics, myc looks healthy to me. Maybe contamination slipped in at a later time than those pics, let's see the current plates then.

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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Josex]
    #25229417 -

That's what I'm saying these look fine but they dont smell right and when going to grain they stall out and dont colonize the grain fully (grain is simple WBS) when I go to toss the grain it has the same odd scent that the agar dishes have so I know it's coming from the agar dishes... I'll post new agar pics in a few hours when I get off work

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Re: The Biopsy Method: No Mercy for Bacteria. [Re: more4u2c]
    #25229430 -

Here is one that when I was grabbing wedges a drop of soapy bleach water from the top of the SAB dripped into the damn plate so I closed it up and put it to the side to toss and noticed that it turned the rizo part blue but the fluffy parts not hurt so I'm assuming the fluffy stuff is my contam I'm fighting

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Re: The Biopsy Method: No Mercy for Bacteria. [Re: more4u2c]
    #25229447 -

And do you happen to have any pics of those wbs jars? I doubt it's yeast like you said it could be because yeast is one of the easiest contams to identify on grains. It grows like little dots (usually white) on the grains and spreads fast.

more4u2c said:
Here is one that when I was grabbing wedges a drop of soapy bleach water from the top of the SAB dripped into the damn plate so I closed it up and put it to the side to toss and noticed that it turned the rizo part blue but the fluffy parts not hurt so I'm assuming the fluffy stuff is my contam I'm fighting




Hard to tell from that pic but I think I see like a strange ring of growth on the edges of the culture. It could be meshed-in mold.
Looks something like this?

In the pic myc stopped growing and meshed-in mold got ahead and started colonizing the edge of the plate, eating the food the color in the process.

Does it usually show up when the plate is more than 50% colonized?
Have you noticed any weird shapes of non-colonized agar, like perfect little triangles?
Like this:

Mateah said:
Check out this weird growth on this Mazatapec culture






Edited by Josex (05/26/18 01:23 PM)

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