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InvisiblemushboyMDiscord
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #24789280 - 11/17/17 01:29 PM (3 years, 11 months ago)

Quote:

Josex said:
by the fifth gen of plates I'm gonna let them pin out and take clones to test. The genetics should be quite slimmed down by them and I prefer a clone taken from a culture with less of a genetic hodge-podge.




:takingnotes: ill be sharing something similar with texas goliad:cool:


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InvisibleASliceofPI

Registered: 01/29/03
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Re: The Biopsy Method: A different way of doing LC. [Re: mushboy]
    #24789957 - 11/17/17 06:50 PM (3 years, 11 months ago)

responding to Josex from another thread but im putting it here because more relevent. i ended up trying some of the insulin syringes with 31g fixed tips. i had a problem keeping the water in the syringe. i cooked them standing upright in foil but had em in there for an hour while cooking LCs. so the picture i posted was made with a regular 18g needle. im gonna try cooking more of the 31g needles for a a shorter amount of time and see if i can get the water to stay inside. any tips would be appreciated.

as a side note i havent had medical insurance in several years and i purchase my insulin and syringes at walmart on the cheap no questions asked they just make me sign a book for the syringes.


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Invisibleherrenvolk


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Re: The Biopsy Method: A different way of doing LC. [Re: ASliceofPI]
    #24789971 - 11/17/17 07:00 PM (3 years, 11 months ago)

I noticed that I lose about 1/2ml of water after the pressure cooking. I used 3ml syringes, PC 17psi for 30 minutes or so.

Quote:

Josex said:
For me a bad LC is a contaminated LC, and it doesn't sound like yours was contaminated or you would've noticed by now. Your issue could be a weak culture, simple as that, and the fact that you only used 5ml per jar, which can be enough if the culture is vigorous. Another cause could be your grains being bacterial as hell and myc not wanting to leap off. Next time pour that bitch, gay things like syringes bring gay results, unless you're bod.




Yeah, I was thinking that if it was contaminated, I would be able to see some signs of that. IDK if the culture is weak, but it's only maybe 3 or 4 transfers away from spores, so its not old. I thought 5ml was already on the higher end, most people on I read say they do 2-3cc, but if more is the way to go I can do that too. As for grains being bacterial, I don't know how to tell if they are, but the grains (wheat) were freshly PC'ed for 2 hours, so most of the bacteria should have been dead. I did a 12 hr soak on those grains, if it matters.

I would actually prefer to pour, but the reason I am experimenting with syringes is that currently I am trying to use bags for spawn, and since I only have a still air box and no flowhood, I have no choice but to preseal my bags, and inoculate them with LC using a syringe and needle.


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Offlinetombosley8
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Re: The Biopsy Method: A different way of doing LC. [Re: herrenvolk]
    #24790039 - 11/17/17 07:26 PM (3 years, 11 months ago)

ok finally actually doing this and am sterilizing the lc broth and syringes with water as we speak. Really happy about this thanks again josex.

going to be cleaning some ms plates with your biopsy method as well.


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InvisiblemushboyMDiscord
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Re: The Biopsy Method: A different way of doing LC. [Re: tombosley8] * 2
    #24790052 - 11/17/17 07:31 PM (3 years, 11 months ago)

this plate was made with 2 'poke transfers' no blade. then scalpeled onto a fresh plate.

pinning rather quick.

i poked the other culture i took and put it to brf broth today as well. and more ape broth. lots of ape:hamletmonkey:


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InvisibleJosex
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Re: The Biopsy Method: A different way of doing LC. [Re: mushboy]
    #24790159 - 11/17/17 08:20 PM (3 years, 11 months ago)

brf is the shiz niz for this too Mush, but even nastier. :hehehe:

About the syringes losing water, pc'ing them separately for a short time is such a bummer I know, ain't nobody got time for that. They always lose a little of water but it isn't noticeable if you pc them for a short time, like 10 minutes. well hell they won't even need 5 min at 15psi to be sterilized. I said 15 min at 15psi in the OP just because I like the number 15. :shrug:

I usually pc them with the gw lc jars for 1 hour and they can maybe lose 1/4 ml of water.

@ASliceofPI Maybe the size matters here and bigger syringes can cope better with the cycle, dunno.

You're gonna kill it Tom, as usual. :rockon:


--------------------
LC's are easy
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Edited by Josex (11/17/17 08:26 PM)


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Offlinetombosley8
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #24790181 - 11/17/17 08:28 PM (3 years, 11 months ago)

what is the problem with losing a 1/4 of a ml of water?
Like how is that going to change anything?:shrug:


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InvisibleJosex
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Re: The Biopsy Method: A different way of doing LC. [Re: tombosley8]
    #24790195 - 11/17/17 08:38 PM (3 years, 11 months ago)

Exactly, it isn't a problem. But if you lose water that means there'll be trapped air inside the syringe, which isn't an issue either but in that case I recommend squirting water in the SAB before flaming the needle to break the vacuum created by the trapped air. Otherwise the plunger will be hard to push after flaming and you will end up squirting more water than needed to cool the needle and this usually makes the needle drip water ike crazy.


--------------------
LC's are easy
Go big with No-pours
Clean yo shit
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Offlinetombosley8
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #24790201 - 11/17/17 08:40 PM (3 years, 11 months ago)

kool thanks for the tipz


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InvisibleASliceofPI

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Re: The Biopsy Method: A different way of doing LC. [Re: tombosley8]
    #24790311 - 11/17/17 09:41 PM (3 years, 11 months ago)

yeah the problem i run into is that these syringes are tiny as hell. they only hold about 1/3 ml total. im gonna try to cook some by themselves for a shorter amount of time just because i want to see this work and im stubborn. i honestly dont see much more of an advantage as 21g needles are already pretty small but itll be cool to see this work. thanks for the tek btw.


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InvisibleMateja
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Re: The Biopsy Method: A different way of doing LC. [Re: ASliceofPI]
    #24790408 - 11/17/17 10:29 PM (3 years, 11 months ago)

I poured my LC in the toilet today when I saw that the top of the SHIP was wet even tho the jar is inside a plastic bag. When I wipe  the SHIP with paper, 3 sec later it's wet again.
Somehow presssure is forcing liquid out trough the SHIP even tho I have a Watman in the lid. And I did this LC with the only lid that still had the old SHIPs I made with silicone that is not heat resistant. I have since installed new heat resistant silicone on all new lids, I wasn't even gonna use this LC just wanted to see if it would work: Got almost full colonization in 8 days so I'm happy! Looking forward to making LC with the new lids :smile:


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InvisibleJosex
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #24790412 - 11/17/17 10:29 PM (3 years, 11 months ago)

Quote:

Josex said:

If there's bacteria you'll begin to notice after ~24-48 hours that the water you squirted is no longer clear and will usually adopt a whitish tinge that can be subtle to distinguish at first but it will become glaringly obvious if you give it more time. Also, you won't be able to move that 'dirty' water around the agar if you tilt the plate, it just won't budge.





--------------------
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: Josex]
    #24790436 - 11/17/17 10:47 PM (3 years, 11 months ago)

Hey Mateah that's a pitty man, you can still make it work I'm sure.

In my not humble at all opinion, inoculating through the SHIP is like 2 times riskier than inoculating by simply lifting the lid. You could be doing this for a year and never get a single contaminated LC.
If you can do agar, you sure as heck can lift the lid to inoculate.

When handling a scalpel to do agar work your chances of getting contams are already pretty slim if your technique is decent, but inoculating with a syringe is far easier and carries very little risk.


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

PHENO HUNTERS


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InvisibleMateja
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #24790523 - 11/17/17 11:21 PM (3 years, 11 months ago)

You're totally right, squirting in, lid off. Aspirating to make BRF inoculant and pouring  for grain jar inoculant!


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InvisiblebodhisattaMDiscordReddit
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Re: The Biopsy Method: A different way of doing LC. [Re: Mateja]
    #24790545 - 11/17/17 11:41 PM (3 years, 11 months ago)

All silicone is heat proof to far higher temps than a pressure cooker


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Re: The Biopsy Method: A different way of doing LC. [Re: bodhisatta]
    #24790557 - 11/17/17 11:52 PM (3 years, 11 months ago)

is there a reason you warm the water before adding LME?

I've found it dissolves better/more quickly into a cold liquid not hot.


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Re: The Biopsy Method: A different way of doing LC. [Re: tombosley8]
    #24790563 - 11/17/17 11:56 PM (3 years, 11 months ago)

What are about SHIPs for aspirating? I feel that SHIPs will be safer for aspirating than open lid, especially with the big 60cc syringes.


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InvisibleJosex
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Re: The Biopsy Method: A different way of doing LC. [Re: tombosley8]
    #24790579 - 11/18/17 12:07 AM (3 years, 11 months ago)

Quote:

tombosley8 said:
is there a reason you warm the water before adding LME?

I've found it dissolves better/more quickly into a cold liquid not hot.



Mine won't dissolve in cold water, it will clump. Dissolving the LME in hot water is pretty standard, never heard of doing it with cold water.


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

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Offlinetombosley8
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #24790634 - 11/18/17 12:31 AM (3 years, 11 months ago)

:shrug: Mine is opposite
just did both cold and hot and the hot had clumps that were harder to stir in. Had to use a wisk to get them stir in.

although possibly the malt I weighed for the hot had a couple clumps in dry form that caused the problem.

but I've used malt extract in lcs for a while and I've always found it easier to stir into cold. well actually I add the dry malt then pour the water over it.


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Re: The Biopsy Method: A different way of doing LC. [Re: herrenvolk]
    #24790636 - 11/18/17 12:32 AM (3 years, 11 months ago)

Well I used 500ml of LC on 6 bottles :facepalm:
Gotta work on my pour technique. Hard to gauge how much I'm pouring.... I should also probably be inoculating sober :lol:


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