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Phony Phone

Registered: 09/19/20
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Agar transfer, avoiding isolation
#27001555 - 10/24/20 04:44 PM (3 years, 3 months ago) |
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Hello shroomery, sorry if this has been asked already. I wanted to ask, if you wanted to transfer a clone mycelium from plate #1 to plate #2 but you wanted to avoid further isolating the strains, is the solution to this to just grab a big enough wedge compared to a tiny one?
Thanks for your time.
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Lenz
Misunderestimated


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Re: Agar transfer, avoiding isolation [Re: Phony Phone]
#27001633 - 10/24/20 05:41 PM (3 years, 3 months ago) |
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I was wondering about this myself recently cause I've been taking a bunch of transfers from clone plates.
Here's a good thread about it: https://www.shroomery.org/forums/showflat.php/Number/23660093#23660093
Quote:
Homesteader said: Hey morty great post and you are definitely on the right track brother! This topic is one I struggled with most when starting on Agar. What I've noticed is that a clone is usually comprised of multiple isolates fused together through anastamosis. They will hold together as one uniform 'type' of mycelium for several transfers. However, 3-5 transfers (Increase in P value) later I notice sectoring of once stable strains. Transferring these sectors will leave you with an isolate that no longer contains all the genetic material that once made up your cloned fruit and may no longer even have the capacity to fruit. To combat this I keep as low P value as possible and avoid unnecessary transfers. I also change up the nutrient concentrations of my Agar blend and sometimes alternate the actual source of carbohydrates (one time PDA, next time MEA).
The advantage to mycelium of a cloned fruit is the fact that it is an exact replica of the parent. The disadvantage here (so I'm told) is that some or many of the isolates that make up the cloned fruit may in fact only have the ability to grow vegetatively and have no ability to fruit. Thus, robbing nutrients from the isolates that do have the ability to fruit. This should make no difference whatsoever as the strains that make up a clone are anastomosed and can share their nutrients across the entire network. In theory they work together and not against one another.
To answer your question though: yes you are isolating by trying to clean up your plates and yes you could most certainly end up with an isolate that has no ability to fruit. Is this likely to happen? Not really and it depends on how many times you are transferring. Keep in mind, if you are grabbing rhizomorphic sectors of fast and healthy growing mycelium than you will almost always have a fruiting strain.
Sorry if some of this info is redundant but I felt this is an important topic to cover for people getting started on Agar.
I can't vouch personally for this but a TC in that thread seems to be saying essentially the same thing, that it's not really something to worry about.
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Brian Jones
Club 27



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Re: Agar transfer, avoiding isolation [Re: Phony Phone]
#27001821 - 10/24/20 07:54 PM (3 years, 3 months ago) |
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It's hard to get isolations even if your trying, taking multiple sequences of transfers from clearly delineated sectors. I would keep transferring small pieces of agar to lower the odds of contamination.
I think your theory was good, but it's not really a problem.
-------------------- "The Rolling Stones will break up over Brian Jones' dead body" John Lennon I don't want no commies in my car. No Christians either. The worst thing about corruption is that it works so well,
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Phony Phone

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Re: Agar transfer, avoiding isolation [Re: Brian Jones]
#27002894 - 10/25/20 02:41 PM (3 years, 3 months ago) |
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thank you both! So it appears that if you were to isolate the strain cluster synergy of the "isolate" clone, u have to go 4-5+ tranfers to start having problems, correct?
some plates might have contam issues so they will need at least 1 more transfer. i am having trouble recognizing the sectors early on as they are so joined up together (the non obvious ones) that I cannot tell if im grabbing a single sectors or multiple "invisible" ones.
How do you guys tell your sectors apart?
Another small question, what does the alternation between agar recipes do for the health of the myc? why is it beneficial to it?
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Big_Dub
I'm just some guy



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Re: Agar transfer, avoiding isolation [Re: Phony Phone]
#27002958 - 10/25/20 03:07 PM (3 years, 3 months ago) |
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you always want to make tiny transfers. the point of transfering is to get clean healthy mycelium. less is more
look for nice spots, and take those. dont get caught up worrying up sectors. just grow some mushrooms
-------------------- split_by_nine said: click me you fuck do the right thing
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Phony Phone

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Re: Agar transfer, avoiding isolation [Re: Big_Dub]
#27002978 - 10/25/20 03:22 PM (3 years, 3 months ago) |
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with the above information mushrooms might not grow if clones keep getting transferred though 
Quote:
Keep in mind, if you are grabbing rhizomorphic sectors of fast and healthy growing mycelium than you will almost always have a fruiting strain.
why are rhizomorphs certain fruiters?
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Big_Dub
I'm just some guy



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Re: Agar transfer, avoiding isolation [Re: Phony Phone]
#27002988 - 10/25/20 03:31 PM (3 years, 3 months ago) |
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rhizomorphic mycelium does not equal fruiting. that is a myth. if you want a fruiting culture, wait for pins to show upp an agar, then transfer the pins
-------------------- split_by_nine said: click me you fuck do the right thing
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Phony Phone

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Re: Agar transfer, avoiding isolation [Re: Big_Dub]
#27002997 - 10/25/20 03:37 PM (3 years, 3 months ago) |
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great idea. thanks how do you recognize sectors?
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Big_Dub
I'm just some guy



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Re: Agar transfer, avoiding isolation [Re: Phony Phone]
#27003002 - 10/25/20 03:45 PM (3 years, 3 months ago) |
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not necessary ime.
just take something from the leading edge. dont overthink this
-------------------- split_by_nine said: click me you fuck do the right thing
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verum subsequentis
seeker of truth



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Re: Agar transfer, avoiding isolation [Re: Phony Phone]
#27003010 - 10/25/20 03:51 PM (3 years, 3 months ago) |
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OP, this is a good question and one that many have thought and asked. As far as I know, no one has a concrete answer. Most of us that have been at it a while were worried about it (as you are) at first but then learned that it doesn't seem to be much of a concern.
I've done lots of tests to try and figure this out and still don't have a perfect answer. I used to always try to take a piece from each obvious sector anytime i was transferring an obviously sectoring clone. I've done a lot of trying one sector vs trying another vs trying all of them together and never came to a solid conclusion. Best i can say is that it's probably not worth tripping on but in theory, you could end up snatching a less optimal set of genes.
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Phony Phone

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Alright, thanks for the tip. Guess keeping the master plates is a fail-safe method to fall back onto if everything else comes out to be bunk.
I have 2 more questions, 1 is, in the refrigerator, how long will cube mycelium live at say around 44 f (6-8 Celsius)?
2, I've read Stro's thread on sectors but I am still kind of confused about it, for example when you have a colony half tomentose - half rhizo on agar, is this sectoring? And on a rhizo growth, are different growth speed patterns obvious sectoring? And within that sector, how do you tell if it's a monoculture sector or just dense strains? is the only way to distinguish that even more transfers until you get the obvious "lines of separation"? Sorry if i got any terminology wrong, feel free to correct me.
Thanks for your time.
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verum subsequentis
seeker of truth



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Re: Agar transfer, avoiding isolation [Re: Phony Phone]
#27003038 - 10/25/20 04:12 PM (3 years, 3 months ago) |
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Properly kept cultures will last a good long while in the fridge. I haven't done exact tests to give an exact answer but I've pulled them out a year later. Thinner plates dry out faster than thicker plates and wrapping them really really well helps. They don't need to breathe in cold storage so you can seal them up nicely.
Slants are what you want for real long term (years) storage.
Sectoring is just what it sounds like. If you can see a difference between this and that it's a "sector". It's not really worth tripping on. The confusion comes when noobs start to think that sectoring = genetic difference absolutely. This is not the case. I have done quite a bit of fooling around with sectors and seen everything and anything possible happen.
I'd love to give you a simple yes or no but.... The best advice is just to transfer from the fastest growing/ most organized growth, do so until the whole culture looks nice and clean, put to grain... to bulk... to fruit. If it kicks ass, keep growing it.
Check my main threads. I did a little write up for some one that i called "masters and slaves...." and i documented a little isolation that shows shitloads of cultures going plate to plate to plate to plate to......
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Phony Phone

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yup, will do. thanks a ton. I got 1 more little thing, what is the theory of alternating between recipes when you transfer myc? What is in that method that help myc from accumulating senescence errors?
Really helpful info
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verum subsequentis
seeker of truth



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Re: Agar transfer, avoiding isolation [Re: Phony Phone]
#27003093 - 10/25/20 04:37 PM (3 years, 3 months ago) |
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The UNPROVEN RUMOR is based on the assumption that the genetics will get so used to surviving off of that type of food that it will get lazy and become a slower growing culture. I have seen no evidence of this in my own life. As a noob i took the rumor seriously and tried to fuck around and find out. I found out that I found no advantage to switching things up and have only and always used Malt extract agar since then.
Others may disagree, I'm just stating what I've seen in my own rainbow farming life.
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Phony Phone

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Say one had only 1 slant and that slant started running out of myc from transfers, would one then take the final bit of myc left in the slant and make a new master slant or a petri? I am afraid this is kind of a silly question but I really wonder what techniques people utilize to keep their favorite cultures running.
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verum subsequentis
seeker of truth



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Re: Agar transfer, avoiding isolation [Re: Phony Phone]
#27003235 - 10/25/20 05:49 PM (3 years, 3 months ago) |
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I'd have a master plate going already and make a new slant from the master plate. If you don't have one, make one so you can verify it's still good and then make another slant.
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NecroMyce
Mostly Ghostly



Registered: 05/12/13
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Quote:
verum subsequentis said: I'd have a master plate going already and make a new slant from the master plate. If you don't have one, make one so you can verify it's still good and then make another slant.
I have been tagging along and I see you post everywhere, Verum. Always helpful bro! Thanks for helping us both out. I have a quick question to chime in, now do You deal with a plate that has a contam in it? Just move slowly and try not to touch the mold or knock the Petri dish hard to avoid disbursing spores everywhere? Here is a few plates with mold or some contam, to give a example
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verum subsequentis
seeker of truth



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Re: Agar transfer, avoiding isolation [Re: NecroMyce]
#27003400 - 10/25/20 06:54 PM (3 years, 3 months ago) |
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You got it. In a still air box in a still room, you gently unwrap (we should talk about your wrapping) the plate. Then flame sterilize the scalpel until red hot outside the box and gently move it into the box. Be sure not to disturb the air in the box in any way. Let you're scalpel cool in the air or cool it quickly in a fresh plate. Every move you make should be gentle and smooth. Once cool reach in and gently take a transfer from as far away from the away from the contam and from the best looking myc around.
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NecroMyce
Mostly Ghostly



Registered: 05/12/13
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I know, I didn’t have any parafilm at the time so I used plastic wrap! Lol and so basically we are looking to just clean up our cultures by doing transfers over time and try to only keep it around 2-3 transfers. What is usually colonization time with agar inoculation?
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verum subsequentis
seeker of truth



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Re: Agar transfer, avoiding isolation [Re: NecroMyce]
#27003624 - 10/25/20 08:40 PM (3 years, 3 months ago) |
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I use glad cling wrap cut into rolls that are just wider than a petri.
2-3 transfers is just a guideline we give people to make sure they know to make sure they've actually achieved a clean plate. It's generally recommended that noobs make one transfer even after they think it's clean. Just because it's better safe than sorry and noobs don't have well trained eyes yet.
Colonization times depend on many factors. Grain prep/ ambient room temp/ how much agar you put in/ how much grain it's trying to colonize/ Vigor of the culture. Two to three weeks is about right though.
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