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OfflineManifoldPrime
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Re: The Biopsy Method: A different way of doing LC. [Re: teladi]
    #25448934 - 09/10/18 01:40 AM (5 years, 6 months ago)

Maybe your needle is still too hot, your transfer donor plate has some issue, or you're using too small gauge a needle?


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Offlineteladi
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Re: The Biopsy Method: A different way of doing LC. [Re: ManifoldPrime]
    #25456474 - 09/13/18 01:06 AM (5 years, 6 months ago)

I suspect what I'm doing wrong is taking biopsy from too thin part of my tissue. A2A transfers can be done on the edge, but once clean, biopsies can be taken from anywhere on the donor plates.

The thinness of the leading edge of mycelium combined with a (relatively) tiny cross sectional interior of a needle means I'm likely missing tissue.


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InvisibleJosex
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Re: The Biopsy Method: A different way of doing LC. [Re: teladi] * 1
    #25456569 - 09/13/18 04:26 AM (5 years, 6 months ago)

That could be it. I remember Comebackkid having the same problem and reporting it in this thread a few months ago.

You should be poking parts of the agar that are thouroughly colonized, not the thin leading edge.

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Offlineteladi
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #25459699 - 09/14/18 12:06 PM (5 years, 6 months ago)

Yeh, I reckon that's exactly right. I did a few jars earlier today, and did the biopsy right through some thicker tissue from test plates. I'm hopeful. I'm still catching up in this thread, and haven't yet come across CBK's messages.

Is there any reason to adjust the nutrients in LC to act as a bridge between agar and grains? In my case, my main agar nutrient is potato-dextrose, while grains are oats. LC made from milled oats. I always have a hesitation of a 3-10 days (temp dependent) before the colonization starts. I understand this hesitation as the fungus adapting to a new food source. Would there be any overall benefit to adjusting the LC to have less of each, but both nutrients?


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InvisibleJHOVA
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #25460826 - 09/14/18 08:35 PM (5 years, 6 months ago)

Hope your summer is going well Josex.:cookiemonster:


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OfflineMooseShroom
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Re: The Biopsy Method: A different way of doing LC. [Re: JHOVA]
    #25468565 - 09/17/18 10:46 PM (5 years, 6 months ago)

About to try out the grain water LC. Doing 2 jars of LGT and 2 of redboy. I'll post some updates in this thread and probably start a log of my own. :cheers:

1 Redboy and 1 LGT made it through. Prepped grain yesterday for the LGT and I'm prepping grain today for the Redboy.

Edited by MooseShroom (10/09/18 01:11 PM)

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InvisiblemushboyMDiscord
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Re: The Biopsy Method: A different way of doing LC. [Re: MooseShroom] * 1
    #25468568 - 09/17/18 10:49 PM (5 years, 6 months ago)

:popcorn:post results mang

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OfflineManifoldPrime
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Re: The Biopsy Method: A different way of doing LC. [Re: mushboy]
    #25468849 - 09/18/18 04:17 AM (5 years, 6 months ago)

I'm gonna pour my LC's today (feels weird saying it, sounds so risky)

I got 200mls per bottle, and I'm noccing up 7 1litre jars of WBS/Millet, bout 2/3rds full. How much LC should I use? Some of these jars look like they have a massive clump of myc in the center like an Island , bobbing up and down. Should I just let that monster ball fall in ? can I use the whole jar? 1 LC 1 inoculation right?


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Offlineteladi
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #25477461 - 09/21/18 08:47 AM (5 years, 6 months ago)

Quote:

ManifoldPrime said:Should I just let that monster ball fall in ? can I use the whole jar? 1 LC 1 inoculation right?




Shake the fuck out of that guy, and use it to 'noc a bunch of jars. 10-20 ml per jar. I have a few LC jars with broken glass as per bod to act as an agitator and help cut up the myc.

Hope it all went well. The warm weather is fantastic for growth. I'm having record grain colonization times.

Quote:

Josex said:
That could be it. I remember Comebackkid having the same problem and reporting it in this thread a few months ago.

You should be poking parts of the agar that are thouroughly colonized, not the thin leading edge.




My efficiency has shot up to 100% now that I'm getting decent amounts of tissue now.


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Offlineanahanini
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Re: The Biopsy Method: A different way of doing LC. [Re: teladi]
    #25484572 - 09/24/18 07:37 AM (5 years, 6 months ago)

thanks.

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OfflineWoolyelephant
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Re: The Biopsy Method: A different way of doing LC. [Re: anahanini] * 2
    #25487065 - 09/25/18 01:40 AM (5 years, 6 months ago)

So I followed everything in this Tek besides a 18 gauge needle and low and behold 7 days later...
Will test on agar before knocking up grain.
PE Clone


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OfflineMooseShroom
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Re: The Biopsy Method: A different way of doing LC. [Re: Woolyelephant]
    #25490561 - 09/26/18 01:17 PM (5 years, 5 months ago)

One week in and 3 of my jars still look okay to me. They're forming myc balls, but they seem to sink. Kind of hard to see them since they're still pretty cloudy. Relatively slow growth, but I used a 23 guage needle and it's been 64-70 degrees in my house.

Is it normal for the growth to sit at the bottom or should it be throughout the solution like other pictures I see posted?

Edit: Forgot to take pictures as it went, but I got two good LC's out of the 4 that I made. Not the best success rate, but I was pretty sloppy while doing it. Got 20 jars going now that I can G2G. Excited to see how this goes!

Edited by MooseShroom (10/16/18 08:23 PM)

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Invisiblestareatclouds
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Re: The Biopsy Method: A different way of doing LC. [Re: ManifoldPrime]
    #25593005 - 11/05/18 02:59 AM (5 years, 4 months ago)

Quote:

ManifoldPrime said:
I'm gonna pour my LC's today (feels weird saying it, sounds so risky)

I got 200mls per bottle, and I'm noccing up 7 1litre jars of WBS/Millet, bout 2/3rds full. How much LC should I use? Some of these jars look like they have a massive clump of myc in the center like an Island , bobbing up and down. Should I just let that monster ball fall in ? can I use the whole jar? 1 LC 1 inoculation right?




How'd the pour go? I'm late to the party, but about to noc up 1000ml with some clones tonight and am hoping to use these on some bags.

Anyone else besides mushboy pour his LCs into bags in a SAB? Looking to avoid the syringe altogether if possible.

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Offlineteladi
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Re: The Biopsy Method: A different way of doing LC. [Re: stareatclouds]
    #25593174 - 11/05/18 06:01 AM (5 years, 4 months ago)

I pour my grain jars. Of the last 60 jars I poured, I've only lost 2 to contamination. Colonization is so much faster than agar wedges. Probably around the same as LI.

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Invisiblestareatclouds
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Re: The Biopsy Method: A different way of doing LC. [Re: teladi]
    #25593193 - 11/05/18 06:12 AM (5 years, 4 months ago)

I'm asking if anybody pours their LCs from the jar into bags, not about pouring LC into grain jars. I've always done the latter, but am now switching to bags, so I'm just curious.

Anybody who uses liquids for bags, please chime in with any tips or advice. I'm working with an SAB, but LFH folks opinions are welcomed as well. Hoping for any info on inoculating bags, whether pouring or using syringes.

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InvisiblemushboyMDiscord
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Re: The Biopsy Method: A different way of doing LC. [Re: stareatclouds]
    #25593348 - 11/05/18 08:24 AM (5 years, 4 months ago)

its kind of a pain in the ass. make sure you have a decent size SAB. mine is only 80qt and its kinda cramped.
i would lay the bag on its side and roll it open like ... bubblegum tape?.. hard to describe. havent done it in months tho.

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Invisiblestareatclouds
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Re: The Biopsy Method: A different way of doing LC. [Re: mushboy]
    #25594561 - 11/05/18 06:25 PM (5 years, 4 months ago)

How long do you consider too long to go between sterilizing and using the syringes, Josex? Also for those of who you aspirate, what's your method of capping the syringe? I have the little black plastic stoppers, but they're kind of annoying. Y'all got the long needle sheathe bits so you can just keep the needle on?

And how many of y'all use the same plate twice in a session for LC?

Edited by stareatclouds (11/05/18 09:33 PM)

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InvisibleJosex
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Re: The Biopsy Method: A different way of doing LC. [Re: stareatclouds]
    #25599805 - 11/07/18 11:15 PM (5 years, 4 months ago)

Quote:

stareatclouds said:
How long do you consider too long to go between sterilizing and using the syringes, Josex? Also for those of who you aspirate, what's your method of capping the syringe? I have the little black plastic stoppers, but they're kind of annoying. Y'all got the long needle sheathe bits so you can just keep the needle on?

And how many of y'all use the same plate twice in a session for LC?




I wrap the syringes in foil and put them in a filtered blender blottle or a tall enough glass jar.They'll be good a long time with the lid shut. I just don't like to keep them around for more than a month but you could.


I cap the syringes with a plain needle and its needle cap but I never keep LC syringes around for very long. If I did I'd probably wrap them with cling wrap for some extra peace of mind though it may not be necessary. Plastic stoppers seem like a pita as you said.

You can use the same plate to noc several LC's, never had a problem with that. Good luck keep us posted.

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Invisiblestareatclouds
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #25599907 - 11/08/18 01:31 AM (5 years, 4 months ago)

Unfortunately I'll have to use the damn stoppers. But I'll update the thread when I can. Cheers to an awesome tek!

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OfflineSeverance13
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #25614478 - 11/14/18 01:49 PM (5 years, 4 months ago)

If you're gonna use a PP5 blender bottle, why not set up the bottle to grow the LC culture? Set up a hole to receive a whatman filter, prep and sterilize the LC in the bottle, and inoculate by popping the flip lid and squirting the biopsy is?

If you keep the blender ball in when you sterilize, or add a PP5 version of the blender ball, you basically guarantee the ability to homogenize the mycelium before pouring, too.

Edited by Severance13 (11/14/18 01:51 PM)

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