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InvisibleJosex
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Re: The Biopsy Method: A different way of doing LC. [Re: bongoman]
    #27064859 - 11/30/20 05:16 PM (10 months, 9 days ago)

Quote:

bongoman said:


I suspect this is probably answered elsewhere in the thread but how important is stirring/agitation with LME-based broth? I don’t have a stir-plate and can manually swirl a few times a day I guess.





Very important yes. You need to swirl at least a couple times a day to break the colony some and help disperse it, it speeds things up.

Quote:

bongoman said:


Also, do you get a clean pour from a mason jar when pouring into grain jars? I’ve been using media bottles for LC which pour nicely but need to keep some aside for agar prep and so will try out mason jars as my LC containers. I would have assumed that mason jars give a “sloppy” pour.




No, I never ever got a clean pour from mason jars, shit was always a bit messy for me. What I used to do is in the tek somewhere. I used to pour the whole LC into a sterile blender bottle and poured from there.
I don't pour anymore these days tho.

Quote:

bongoman said


Also, for some reason I feel tempted to ignore the test step where you squirt to agar and then grow it out then poke and squirt to the broth - simply my impatience I guess. How important is this step in your view?





Not important at all, if you're confident in your ability to tell when a culture is clean.


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LC's are easy
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Clean yo shit
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Offlinekarri0n
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27064952 - 11/30/20 06:06 PM (10 months, 9 days ago)

Josex, are you using a SHIP these days? I see you said you aren't pouring.


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

Anything written above is just as likely to be accurate as it is to be corrected by someone smarter than me two posts from now.


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Re: The Biopsy Method: A different way of doing LC. [Re: karri0n]
    #27065009 - 11/30/20 06:51 PM (10 months, 9 days ago)

I made a few of these a week ago, I think I can see myc starting from a sediment clump in one but that’s it so far.


Gonna give it another week before a redo. One of the plates was poured extremely thin so I don’t even know if there was enough myc in/on the needle. Or if it got wiped off going through the ship.


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Re: The Biopsy Method: A different way of doing LC. [Re: A.k.a]
    #27065513 - 11/30/20 11:41 PM (10 months, 8 days ago)

First cup is a from a poke 11/13. Made another LC from it the 27th. Both looking good.



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Re: The Biopsy Method: A different way of doing LC. [Re: karri0n]
    #27065771 - 12/01/20 06:50 AM (10 months, 8 days ago)

Quote:

karri0n said:
Josex, are you using a SHIP these days? I see you said you aren't pouring.




No, never liked them. I use a no tilt lid build of mine hat allows me to aspirate with big 60ml syringes and then use thick 14g needles. I inoculate with biopsy through a poly filter. I posted the jars many times on different threads, a quick search should yield a bunch of results if you're interested. Never mind the explanation I did on how to build them right here in this thread time ago, that build failed eventually and had to redo it again using better materials.


--------------------
LC's are easy
Go big with No-pours
Clean yo shit
LAGM '21

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InvisibleJosex
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Re: The Biopsy Method: A different way of doing LC. [Re: A.k.a]
    #27065774 - 12/01/20 06:56 AM (10 months, 8 days ago)

Quote:

A.k.a said:
I made a few of these a week ago, I think I can see myc starting from a sediment clump in one but that’s it so far.


Gonna give it another week before a redo. One of the plates was poured extremely thin so I don’t even know if there was enough myc in/on the needle. Or if it got wiped off going through the ship.




Poking through a SHIP isn't going to be a  problem, the tissue you squirt is inside the needle.

A week is a lot though, you should have seen life by now. A high strength broth can make the liquid very cloudy and with lots of sediment, making it increasingly harder to see stuff, what nute ratio did you use? The sweet spot is 0.1-0.2% strength imo/e.

Maybe give it a couple days and a couple swirls before reinoculating.


--------------------
LC's are easy
Go big with No-pours
Clean yo shit
LAGM '21

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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27066245 - 12/01/20 02:28 PM (10 months, 8 days ago)

Quote:

Josex said:
what nute ratio did you use? The sweet spot is 0.1-0.2% strength imo/e.






Is there any way to know or control for this type of stuff with grain water, or reason to care? Is grain water even recommended anymore or should I just buy LME and be done with it?

The cube LC I did is pretty easy to see and distinguish from the sediment in GW broth.

Chicken of the woods and lion's mane not so much, and I'm starting to hate sediment.


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Re: The Biopsy Method: A different way of doing LC. [Re: karri0n]
    #27066299 - 12/01/20 03:02 PM (10 months, 8 days ago)

I just use BRF Agar cause its cheap and easy. Waiting for more input.


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Re: The Biopsy Method: A different way of doing LC. [Re: Adrenognome]
    #27066496 - 12/01/20 04:36 PM (10 months, 8 days ago)

I think I screwed up my LC recipe somehow. Meant for this to be 3% LME.

Guess I cooked it.

Also... in the future I’ll filter.



Poke inoculated anyway. We’ll see what happens.


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Re: The Biopsy Method: A different way of doing LC. [Re: Sold Out Online]
    #27066518 - 12/01/20 04:47 PM (10 months, 8 days ago)

3% will make your broth look amber and will create sediment, .1% will make it clear like a glass of water with no sediments :thumbup:


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Re: The Biopsy Method: A different way of doing LC. [Re: Sold Out Online]
    #27066529 - 12/01/20 04:50 PM (10 months, 8 days ago)

3% LME is a lot, that's where you "screwed" if we can call it that.
I use 0,2% LME and that's already more than you need.

You used 15 times more LME, so that's why your LC is so cloudy and full of sediment, which is not an issue besides not letting you see clearly.


--------------------
LC's are easy
Go big with No-pours
Clean yo shit
LAGM '21

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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27066578 - 12/01/20 05:14 PM (10 months, 8 days ago)

Yeah. Reading helps...

I’m going for a do-over. There’s too much weird stuff in there for me to know what’s what.

Tomorrow morning I’ll give these a squirt.



(Froth from vigorous shaking)


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Re: The Biopsy Method: A different way of doing LC. [Re: Sold Out Online]
    #27066584 - 12/01/20 05:17 PM (10 months, 8 days ago)

I used a pinch of lme in mind but they’re like 125ml prob.


I just doubled checked and the one definitely has growth now. Surprisingly it’s from the plate that was extremely thin but I also poked it a couple times and wiggled it around to make sure there was some myc.


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Re: The Biopsy Method: A different way of doing LC. [Re: A.k.a]
    #27066600 - 12/01/20 05:24 PM (10 months, 8 days ago)

Nice AKA. A friend of mine that has done already lots of LC's also wiggles the needle because he's adamant poking ain't enough. :lol:
I believe wiggling does absolutely nothing, it's the poke what gets the tissue inside the needle, a super quick in an out is all that's needed.
Sometimes if I see that the culture is a little wispy I may poke a couple times for good measure, but that's about it.


--------------------
LC's are easy
Go big with No-pours
Clean yo shit
LAGM '21

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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27066705 - 12/01/20 06:32 PM (10 months, 8 days ago)

Yeah normal plates I would totally agree but this one 1/20” lol. I wasn’t even sure the tip of the needle would be covered.

Luckily it’s the one I most wanted to run so that’s cool.


I hate the type of sediment that’s like a super fine dust that swirls up and makes the broth cloudy when you move it. It’s too bacteria like for me.


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Re: The Biopsy Method: A different way of doing LC. [Re: A.k.a]
    #27066726 - 12/01/20 06:45 PM (10 months, 8 days ago)

Yea I know what you mean, I've had broths coming out of the PC looking like they were totally fucked with bacteria.
What does the trick for me is using a low LME ratio and bringing the mix to a simmer prior to sterilizing.

Also, a 25 minutes cycle at 15 psi seems to be the sweet spot for sterilizing LME broths, more than that and the liquid start to get increasingly darker and with more sediment. Taking the jars hot as soon as the pressure drops also helps.


--------------------
LC's are easy
Go big with No-pours
Clean yo shit
LAGM '21

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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27067332 - 12/02/20 12:02 AM (10 months, 7 days ago)

When you say “poke a couple of times”, is that:

poke - poke - squirt

Or

poke - squirt - poke - squirt


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Re: The Biopsy Method: A different way of doing LC. [Re: bongoman]
    #27067468 - 12/02/20 03:04 AM (10 months, 7 days ago)

First.


--------------------
LC's are easy
Go big with No-pours
Clean yo shit
LAGM '21

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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27068340 - 12/02/20 04:22 PM (10 months, 7 days ago)

My dirt nasty cooked up, over concentrated, sediment-filled LC started 11/27 has a globby boy in there.

It’s my first LC so I have no confidence whatsoever that it’ll turn out clean, but I guess I’ll try a flamed-loop to test it out on some agar.



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Re: The Biopsy Method: A different way of doing LC. [Re: Sold Out Online]
    #27068486 - 12/02/20 05:34 PM (10 months, 7 days ago)

:rockon:

It looks like you used very little water to make the LC, wise man. At this point you can swirl it several times a day to speed things up, should be ready real soon.


--------------------
LC's are easy
Go big with No-pours
Clean yo shit
LAGM '21

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