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OfflineEdmunter
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Re: The Biopsy Method: A different way of doing LC. [Re: Sockadin]
    #27172166 - 01/27/21 05:18 AM (1 month, 8 days ago)

Quote:

Sockadin said:
I finally read this all the way through and Boy oh Boy is it entertaining. Great write up Josex!

  I am going to order some SFD's and give this ago. None of my lids are currently compatible with this method.

Question, can you poke to plate? Why wouldn't this work for agar transfers?




Ive seen someone do something similar with a leather punch for perfectly round wedges

Thanks Josex, great info.

So do you mainly pour or use syringes.  What is you optimum quantity of LC per Jar and how mush would you use for a 1.5Kg bag of millet?,

have you shipped all your jars?

I must say this is an excitingly clean way to make spawn

Also I just got my 12 gauge needle which should help


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Re: The Biopsy Method: A different way of doing LC. [Re: Sockadin]
    #27172167 - 01/27/21 05:18 AM (1 month, 8 days ago)

Quote:

I finally read this all the way through and Boy oh Boy is it entertaining. Great write up Josex!




It's so embarrassing people have to endure the outrageous TL;DR :facepalm:
It's just what I did at the time and now I feel it's wrong to trim it down but I'm considering it.

You can poke to plate and I know people who transfer just like that on agar (I only do it on PFtek pucks routinely).

Personally I don't like squirting water on agar so I like to be a normal person there and use the scalpel for transfers. However, if you want to narrow the genetic pool fast in one single transfer, poking a nice spot and transferring that does wonders.

Growth on the receiving plate is likely to be disorganized but transferring normally with the scalpel will get you a established/organized colony in no time and you are likely to notice substantial changes in the colony's appearance from having narrowed down the genetic pool so much in one fell swoop.

Quote:

I am going to order some SFD's and give this ago. None of my lids are currently compatible with this method.




You sure? If your lids happen to have a SHIP then they're compatible with the method. You can squirt the biopsy poking through a SHIP without issues. Just make sure to find a way of keeping the SHIP sterile until you inoculate.


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27172173 - 01/27/21 05:23 AM (1 month, 8 days ago)

Quote:

So do you mainly pour or use syringes.  What is you optimum quantity of LC per Jar and how mush would you use for a 1.5Kg bag of millet?,




Always syringes these days. ~5 ml per jar. I cant speak for bags as I don't use them.


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27172178 - 01/27/21 05:33 AM (1 month, 8 days ago)

Ed I am currently using D3monics punch method. It works great and creates nice even transfers.

Josex, I wonder if you made up a light mixture of agar to water, like 2g/400ML and then loaded your syringes and PC'd if it would help to prevent adding more water to run around on top of the plate.  Im just thinking out loud here.

I am fixing to run a batch of Modified Paperclips through the PC to try a new method of transfers. I will let you guys know if it works out. Im so tired of Flaming, Im looking for a way to remove that from this hobby completely.


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Re: The Biopsy Method: A different way of doing LC. [Re: Sockadin]
    #27172221 - 01/27/21 06:36 AM (1 month, 8 days ago)

D3's tek looks bomb, def the bestest plates I ever did saw.

Quote:

Josex, I wonder if you made up a light mixture of agar to water, like 2g/400ML and then loaded your syringes and PC'd if it would help to prevent adding more water to run around on top of the plate.  Im just thinking out loud here.




You are not saying nonsense at all, been playing with the idea myself. The fact I haven't done it yet is because transferring with the scalpel is just too damn convenient.
To do what you say I'd make the thickest agar I can get in the syringe that won't set and is still runny enough.
If you ever get down to doing it please let me know how it went.


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27172242 - 01/27/21 07:17 AM (1 month, 8 days ago)

Do you grow edibles i.e. oyster mushrooms?  I still havent found the sweet spot for nutrients in LC for these yet.  Has anyone had success?


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Re: The Biopsy Method: A different way of doing LC. [Re: Edmunter] * 1
    #27173293 - 01/27/21 07:16 PM (1 month, 7 days ago)



The rest of an LC I had to chuck to make room for a new one. Thought the myc on the surface looks pretty.


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27173486 - 01/27/21 09:40 PM (1 month, 7 days ago)

Quote:

Josex said:
Just make sure to find a way of keeping the SHIP sterile until you inoculate.




How best to do this? Is it sufficient to keep the lid wrapped in foil during sterilisation and when it's removed from the PC?

I'm starting to think I might use modded lids for my LC jars and also for the lids of the mudafuka bottles that I will be inoculating.


Edited by bongoman (01/27/21 09:53 PM)


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Re: The Biopsy Method: A different way of doing LC. [Re: bongoman]
    #27179203 - 01/31/21 12:44 PM (1 month, 4 days ago)

Made five LC's about a week ago. All 1g LME:500ml water. PC'd 20 mins. Little 125ml batches in media bottles, no stirrer, un-modified lids.

Three noc'd with the needle poke and two with a wedge for insurance (I'm still unnerved by the needle poke, it really feels like you are doing nothing!)

The three needle pokes finished up before the wedges.



This tek continues to rock.


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27179316 - 01/31/21 01:57 PM (1 month, 4 days ago)

Quote:

Josex said:

Always syringes these days. ~5 ml per jar. I cant speak for bags as I don't use them.





Forgive me if this was mentioned in the thread already. I got to page 25 and said fuck it I'll just ask and if I am repeating someone else, my apologies.


I plan on using a 60cc syringe with a 3" needle through a SHIP to then inoculate grain bags through a self-healing injection port. I will probably lift the lid to inoculate the LC with my poke, but then use the SHIP to get LC into the syringe. (I see earlier in this thread you were against SHIPs but now seem to be fine with them... if I have a SHIP would you still recommend inoculating the LC with your poke through opening the lid, or would the SHIP be better?)

What is the proper method for getting the LC into the syringe? If the syringe is already sterile, can I just open it in my SAB and suck up LC as soon as I can after setting up the sterile needle and syringe?

After getting LC in the syringe, would you test a couple of drops on agar first (or even a single grain bag), then save the syringe for a few days before using it to inoculate the rest of the bags? Or would you just first test a cloned culture onto a new agar plate, then if clean use that to make LC with a poke, and trust it is a clean culture and simply immediately use the 60cc syringe on your bags?

Just trying to figure out the best procedure for using the LC with 60cc syringes and self-healing ports on spawn bags. Also how many CCs of LC would you use for a 1lb spawn bag?


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Re: The Biopsy Method: A different way of doing LC. [Re: shadyshroomie]
    #27179538 - 01/31/21 04:55 PM (1 month, 3 days ago)

I’m considering one of these to extract LC from a media bottle - the outlet tube had a luer lock port that you hook up to and pull out LC.



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Re: The Biopsy Method: A different way of doing LC. [Re: bongoman]
    #27179548 - 01/31/21 05:04 PM (1 month, 3 days ago)

https://www.shroomery.org/forums/showflat.php/Number/27170204#27170204

https://www.shroomery.org/forums/showflat.php/Number/27171066#27171066

Apparently those things are expensive as hell. I've been doing something similar myself for years on the cheap.


Edited by Josex (01/31/21 05:55 PM)


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27179563 - 01/31/21 05:16 PM (1 month, 3 days ago)

@shadyshroomie, it's fine to inoculate the broth through a SHIP as long as you find a way to keep it sterile until you inoculate, otherwise I think it's risky.

After getting the LC in the syringe you can test it on agar if you want and keep the syringe somewhere clean until you have the results from the test and decide to use the syringe on grains. I rarely test tho, won't lie to you.


Edited by Josex (01/31/21 06:06 PM)


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27179854 - 01/31/21 09:12 PM (1 month, 3 days ago)

Thoughts on dealing with a dripping needle?

Once I've flamed my needle and squirted to cool it down, I find the dripping needle to get in the way of taking the poke and also think I may have lost a biopsy sample as it dripped out before I could squirt it to my broth.


Edited by bongoman (01/31/21 09:12 PM)


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Re: The Biopsy Method: A different way of doing LC. [Re: bongoman]
    #27180378 - 02/01/21 08:20 AM (1 month, 3 days ago)

Do as quick a squirt as you can to avoid dripping. It's going to be enough to cool the needle.


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27180822 - 02/01/21 01:23 PM (1 month, 3 days ago)

Quote:

Josex said:
@shadyshroomie, it's fine to inoculate the broth through a SHIP as long as you find a way to keep it sterile until you inoculate, otherwise I think it's risky.

After getting the LC in the syringe you can test it on agar if you want and keep the syringe somewhere clean until you have the results from the test and decide to use the syringe on grains. I rarely test tho, won't lie to you.





Awesome thank you. So if I were to wrap the jar in foil in my PC (it is an instantpot...) place directly from pot to SAB, spray alcohol on foil, then unwrap right when I plan to use syringe, would that be considered pretty sterile?

I guess if I am making one use LCs there is not much of a reason to not just open the container and aspirate in my SAB...

Speaking of that, say I make 120ml LC. Would you recommend using two separate sterile syringes? Or after using up one, could you re-aspirate it again?


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Re: The Biopsy Method: A different way of doing LC. [Re: shadyshroomie]
    #27182563 - 02/02/21 12:02 PM (1 month, 2 days ago)

Once the syringe is loaded you should be able to use it till you run out multiple time?

So you are saying testing a culture beforehand isnt something you do anymore?

So id look to inoculate 12 jars per 60ml syringe and I could load them all up at the same time and then test them.

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?


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Re: The Biopsy Method: A different way of doing LC. [Re: Edmunter]
    #27182643 - 02/02/21 12:44 PM (1 month, 2 days ago)

Quote:

Awesome thank you. So if I were to wrap the jar in foil in my PC (it is an instantpot...) place directly from pot to SAB, spray alcohol on foil, then unwrap right when I plan to use syringe, would that be considered pretty sterile?




It's a matter of trying things out and see what works for you. I never did what you say, however I know at least one person who does that routinely with success. You can test it if you're unsure.

Quote:

I guess if I am making one use LCs there is not much of a reason to not just open the container and aspirate in my SAB...




Easier said than done. The few times I did that it felt so awkward and I was so clumsy that it all felt like a huge fat vector. If you're going to be opening the lid at all you might as well just pour the LC. I was a proud pourer myself, def x100 easier and safer than opening the lid to aspirate.

Quote:

Speaking of that, say I make 120ml LC. Would you recommend using two separate sterile syringes? Or after using up one, could you re-aspirate it again?





Separate.

Quote:

Once the syringe is loaded you should be able to use it till you run out multiple time?




I prefer to aspirate what I'm going to use. If the syringe still has LC in it after using it I simply toss it and store the empty syringe for later use (I reuse my syringes, they get sterilized empty in the PC). Just ways to skin a cat, I know people who like to keep filled syringes around and they use it as they see fit, just not what I like.

Quote:

So you are saying testing a culture beforehand isnt something you do anymore?




People should do what feels right to them and I would not tell anybody not to test their LC's.

I don't test because I'm confident in my game, do what feels right to you.
The myc sample that you take with the poke is orders of magnitude smaller than the smallest wedge you can take, reducing risks tremendously. The procedure to noc the liquid without opening the lid (that's what I do) is also tight and safe as hell, so the only plausible vector there could be is the culture itself but I'm very confident in my ability to judge the cleanliness of a culture. If you also feel the same way you probably don't need to test.

Also, I've done countless LC's over the years and I was able to tell the 3 or 4 that got contaminated by just looking at them, so I didn't need to test them to know they were bad, although many people say sometimes LC's can look clean and be contaminated so that's something to consider too. Unfortunately, I haven't experienced a contaminated LC that looked clean, so I can't talk about that.


Edited by Josex (02/02/21 12:52 PM)


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27182675 - 02/02/21 12:57 PM (1 month, 2 days ago)

So Ideally if I wanted to inoculate 30 jars with 3 60ml needles I should load them up.  Agar test them and then use to be bullet proof.

Im really liking thi method as a safe way when green hits the fan.


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #27182684 - 02/02/21 01:00 PM (1 month, 2 days ago)

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).


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