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OfflineEdmunter
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Re: The Biopsy Method: A different way of doing LC. [Re: Edmunter]
    #27182689 - 02/02/21 01:04 PM (8 months, 20 days ago)

Oh and by the way you were right all 3 bottles of LC I wasn't sure I had achieve inoculation are growing cultures.


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OfflineEdmunter
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27182691 - 02/02/21 01:05 PM (8 months, 20 days ago)

Quote:

Josex said:
Quote:

how long would you expect full colonisation to take and do you shake to get rid of the moisture at the base of the jar?




I'm currently not a fan of pooling LC. To avoid that as much as possible, each jar gets 5ml max of LC and I currently add 0.1% agar (by volume) to all my LME broths (love it), that way the liquid is more viscous and doesn't pool nearly as much. I shake them jars as soon as myc recovers and starts jumping off on the grains (3-4 days).






So 1/2 g per 500ml


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Invisiblecoversall
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Re: The Biopsy Method: A different way of doing LC. [Re: Edmunter]
    #27182693 - 02/02/21 01:06 PM (8 months, 20 days ago)

I just had a thought, is there ever a point with LC where it's 'fully colonised' and more contam resistant? Or does that not apply due to it being liquid?


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

..:: E V E R Y  ::..

..:: New? Start here. ::..
..:: How I Panaeolus. From Agar to Tea ::..


Edited by coversall (02/02/21 01:06 PM)


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InvisibleJosex
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Re: The Biopsy Method: A different way of doing LC. [Re: Edmunter]
    #27182695 - 02/02/21 01:09 PM (8 months, 20 days ago)

Quote:

Edmunter said:
Quote:

Josex said:
Quote:

how long would you expect full colonisation to take and do you shake to get rid of the moisture at the base of the jar?




I'm currently not a fan of pooling LC. To avoid that as much as possible, each jar gets 5ml max of LC and I currently add 0.1% agar (by volume) to all my LME broths (love it), that way the liquid is more viscous and doesn't pool nearly as much. I shake them jars as soon as myc recovers and starts jumping off on the grains (3-4 days).






So 1/2 g per 500ml




That's right.

@coversall, I'm pretty sure that doesn't apply to LC. I feel they can get  contaminated easily even when they're fully colonized.


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

PHENO HUNTERS


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Invisiblecoversall
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27182696 - 02/02/21 01:10 PM (8 months, 20 days ago)

I thought as much, but you never know!


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

..:: E V E R Y  ::..

..:: New? Start here. ::..
..:: How I Panaeolus. From Agar to Tea ::..


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Re: The Biopsy Method: A different way of doing LC. [Re: coversall]
    #27183261 - 02/02/21 07:10 PM (8 months, 19 days ago)

Quote:

coversall said:
I just had a thought, is there ever a point with LC where it's 'fully colonised' and more contam resistant? Or does that not apply due to it being liquid?




I used food coloring in an LC to try to see if it could indicate "full colonization" because the mycelium always ate the food coloring in agar plates. It never ate it all, so that makes me think it doesn't "fully colonize" liquid the way it does surfaces.


--------------------
Micropore tape? More like myco-poor tape, right? HAR HAR


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InvisibleJosex
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Re: The Biopsy Method: A different way of doing LC. [Re: karri0n]
    #27183272 - 02/02/21 07:20 PM (8 months, 19 days ago)

Yea the colony never really colonizes the broth fully.
When I used to pour I used to give the leftover LC a sniff outside the SAB after the sesh and sometimes I would forget to toss it and mold would grow on the remaining LC.


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

PHENO HUNTERS


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27186223 - 02/04/21 02:40 PM (8 months, 18 days ago)

I am finally giving LC another try had terrible luck so far. I used this method with 250ml of water varying degrees of newts. these where inoculated on 1-29-20 by the poke method used here. I use unmodified 1 pint jars with original lids and bands. PC 15-18 psi for 30 minutes.

.1g and 250ml water. 3 days after inoculation I start to use magnetic stirrer on this jar. this jar gets spun for 10 minutes every hour or 2 except when asleep.


.25g and 250ml water. 3 days after inoculation I start to swirl this jar. this jar gets swirled for 10 second when 1-3 times a day.


.50g and 250ml water. 3 days after inoculation I start to swirl this jar. this jar gets swirled for 10 second 1-3 times a day.


.75g and 250ml water. 3 days after inoculation I start to swirl this jar. this jar gets swirled for 10 second when 1-3 times a day.


1g and 250ml water. 3 days after inoculation I start to swirl this jar. this jar gets swirled for 10 second when 1-3 times a day.


Edited by kanemush (02/04/21 02:42 PM)


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OfflineEdmunter
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Re: The Biopsy Method: A different way of doing LC. [Re: kanemush]
    #27187697 - 02/05/21 11:27 AM (8 months, 17 days ago)

Is this now the only way you do cultivation?


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Re: The Biopsy Method: A different way of doing LC. [Re: Edmunter]
    #27187810 - 02/05/21 12:44 PM (8 months, 17 days ago)

Yea it's always been, too fucking easy man. Easy expansion of a colony from a single poke from a single plate, quicker colonization of grain and not having to be a slave to culture work makes it a no-brainer for me.

Some people don't like LC because they feel it's an unnecessary step and there's some that think it's risky. For me LC is not an extra step, I make it to be the step and having 100% success with it is not only doable but easy. :shrug:


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

PHENO HUNTERS


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27187956 - 02/05/21 02:32 PM (8 months, 17 days ago)

I will get into lc someday when I have the supplies. But for right now I love g2g


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OfflineEdmunter
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27187966 - 02/05/21 02:36 PM (8 months, 17 days ago)

Quote:

Josex said:
Yea it's always been, too fucking easy man. Easy expansion of a colony from a single poke from a single plate, quicker colonization of grain and not having to be a slave to culture work makes it a no-brainer for me.

Some people don't like LC because they feel it's an unnecessary step and there's some that think it's risky. For me LC is not an extra step, I make it to be the step and having 100% success with it is not only doable but easy. :shrug:




Ive been having contamination issues and after replacing my filter realise there was mould everywhere in my shed.  Im moving it out but in the meantime will be definitely getting into this.


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Re: The Biopsy Method: A different way of doing LC. [Re: Edmunter]
    #27193256 - 02/08/21 10:32 AM (8 months, 14 days ago)

When you use a sterile syringe, is there a risk for contaminants to land inside the syringe before you suck up LC?

For example, I open up my sterilized 60cc syringe pack. It is empty. While in my SAB getting the lid undone on my LC, if the syringe pack is already opened, is there a risk of contaminating the whole syringe if you allow the syringe to be out too long before sucking up LC?


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Re: The Biopsy Method: A different way of doing LC. [Re: shadyshroomie]
    #27193376 - 02/08/21 11:52 AM (8 months, 14 days ago)

I'm running about 40 jars of lc. and use my one 60 ml. syringe a few times everyday.

What works for me:

I keep my syringe filled with white vinegar all the time.

When I'm ready to use the syringe I empty the vinegar into a jelly jar then torch the needle and do what needs to be done then fill the syringe up again with vinegar.

When I'm doing multiple jars of lc. I purge the syringe a couple times in vinegar just to keep from cross contaminating different strains or passing a possible bad jar to a good one.

I buy my vinegar by the gallons and find It to be much cheaper than buying little bottle.

To the answer to your question, always flame your needle right before you use It and then you don't need to worry about contaminates falling into your syringe.


--------------------
     


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Re: The Biopsy Method: A different way of doing LC. [Re: micelio]
    #27193956 - 02/08/21 06:01 PM (8 months, 13 days ago)

Quote:

When you use a sterile syringe, is there a risk for contaminants to land inside the syringe before you suck up LC?




It's safe to say there's always a risk to any procedure but the risk of what you're describing is so small that it would be  negligible at most even if you happen to have really shaky hands.

Quote:

While in my SAB getting the lid undone on my LC, if the syringe pack is already opened




Are you planning on taking off the lid of the LC jar and aspirating directly from the jar? If that's the case I think you are creating far bigger vectors with that procedure alone than possibly compromising a sterile empty syringe. I'd much rather pour the LC to grain than aspirating that way.

On the other hand, I always reuse my 60ml syringes. I wrapped them individually in foil and also wrap the base of 14G needles that I put in a PP5 test tube. All gets sterilized in a filtered gallon jar and I use them as I see fit. Hope it helps.


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

PHENO HUNTERS


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OfflineEdmunter
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27194723 - 02/09/21 05:13 AM (8 months, 13 days ago)

Right!! clear up my confusion.



I have a 330ml LC and want to get the most out of it.  You say only use the culture once but is filling 3 60ml syringes flaming and capping bad practice in your opinion?  For example, I will inoculate 10 jars with 1 and have the other 2 ready for 20 more jars?

Yesterday i made 8 jars and ran out of lc,  I simply flamed the syringe and ship out some more.  Ive marked them as 2nd inoculations to watch out for differences.  Also I shook 3 of the 8 jars hard to see if there is problems doing this.  I have found a few LCs not leap and am hoping the added agar will help. 

Why would some lcs not take?

Also how much mycellium leaping off do you like to see before and shake.  Due to not using agar in the first batches there is pooling that a big shake will cure but I want success.

Do you practice inocualting a new culture into a lc from every old LC?  If so woud you do this befoere or after grain inoculation or doesnt it matter?

I have 9 new LCs shelved and you could be the saviour of my bacon.........


Edited by Edmunter (02/09/21 05:33 AM)


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InvisibleJosex
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Re: The Biopsy Method: A different way of doing LC. [Re: Edmunter]
    #27196201 - 02/09/21 09:28 PM (8 months, 12 days ago)

@Ed, lots of stuff I said in the OP were written years ago and currently I don't do anything the same way anymore. I advised in the OP to use the LC just once because I used to pour and after opening the lid I feel it's sketch to keep the remaining LC, considering how easy it is to make new ones.

Currently I aspirate as much LC as I'll be needing for a sesh and keep the remaining LC in the jar if I plan on using the culture later on again. But I don't see a problem doing what you suggest of getting most of the LC into syringes and then using those syringes when you need them, just keep them somewhere clean. LC has a lot of shelf life in the syringe itself, but the times I knew I'd get them around for a while I made sure to cap them with an actual syringe cap, not just a needle and its cap.

Quote:

Why would some lcs not take?




Weird man. I've personally never seen an LC not take but I've seen some that took way too long.
A thing that can really slow down the leap-off is a shake upon inoculation, LC don't like shook ime.
Underhydrated/dry grains can also slow shit down a lot, as well as overly wet grains.
If the LC was bacterial it might not take too, but you're also likely to notice the contamination on the first days after inoculation...

Quote:

Also how much mycellium leaping off do you like to see before and shake.  Due to not using agar in the first batches there is pooling that a big shake will cure but I want success.




Currently a little less than in the pics


Depending on temps and the vigor of the  culture, 3-5 days since inoculation.

Quote:

Do you practice inocualting a new culture into a lc from every old LC?  If so woud you do this befoere or after grain inoculation or doesnt it matter?




Never, always start new ones from a poke. 200ml LC take 7-9 days from a poke for me (that bit of agar helps to cut down on colonization times, among other things). Also, making fresh LC from a poke is less of a vector/less risky than LC to LC, for me it ain't worth the trouble at all even tho I'm aware LC to LC is also safe if you do it well, but fuck it my LC's take too little from a poke.


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

PHENO HUNTERS


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27196207 - 02/09/21 09:31 PM (8 months, 12 days ago)

I decided to try your tek for My first LC attempt thanks josex!


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OfflineEdmunter
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27196913 - 02/10/21 09:45 AM (8 months, 12 days ago)

Quote:

Josex said:
@Ed, lots of stuff I said in the OP were written years ago and currently I don't do anything the same way anymore. I advised in the OP to use the LC just once because I used to pour and after opening the lid I feel it's sketch to keep the remaining LC, considering how easy it is to make new ones.

Currently I aspirate as much LC as I'll be needing for a sesh and keep the remaining LC in the jar if I plan on using the culture later on again. But I don't see a problem doing what you suggest of getting most of the LC into syringes and then using those syringes when you need them, just keep them somewhere clean. LC has a lot of shelf life in the syringe itself, but the times I knew I'd get them around for a while I made sure to cap them with an actual syringe cap, not just a needle and its cap.

Quote:

Why would some lcs not take?




Weird man. I've personally never seen an LC not take but I've seen some that took way too long.
A thing that can really slow down the leap-off is a shake upon inoculation, LC don't like shook ime.
Underhydrated/dry grains can also slow shit down a lot, as well as overly wet grains.
If the LC was bacterial it might not take too, but you're also likely to notice the contamination on the first days after inoculation...

Quote:

Also how much mycellium leaping off do you like to see before and shake.  Due to not using agar in the first batches there is pooling that a big shake will cure but I want success.




Currently a little less than in the pics


Depending on temps and the vigor of the  culture, 3-5 days since inoculation.

Quote:

Do you practice inocualting a new culture into a lc from every old LC?  If so woud you do this befoere or after grain inoculation or doesnt it matter?




Never, always start new ones from a poke. 200ml LC take 7-9 days from a poke for me (that bit of agar helps to cut down on colonization times, among other things). Also, making fresh LC from a poke is less of a vector/less risky than LC to LC, for me it ain't worth the trouble at all even tho I'm aware LC to LC is also safe if you do it well, but fuck it my LC's take too little from a poke.




thanks man, concise and really helpful.  So from poke to fully colonised in 3 weeks?  Thats actually banging!!! 

I have 25 jars where I injected through the micropore filter after sanitising in front of the flow hood.  Im hoping this works.  I cut a square of tape and stuck it on the jar lid edge like you do when you wrap a present up, then after inoculating stick it over the hole as the syringe comes out.

Im just about to add a silicon ship which I will tape up b4 pc-ing and take of just before inoculating.  This will cut back on vectors as the MP will act as the filter from cooker to hood

If this works its a proper game changer..........


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OfflineEdmunter
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Re: The Biopsy Method: A different way of doing LC. [Re: Edmunter]
    #27196921 - 02/10/21 09:51 AM (8 months, 12 days ago)

Also im toying with messing around with one of these.

https://www.ebay.co.uk/itm/402658567051


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