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Crispykoot
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expanding mycelium on agar
#23927932 - 12/14/16 10:40 PM (7 years, 1 month ago) |
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Hey folks, I have a quick question about how many transfers to take from a P1 plate and just a general question about how to move forward.
I have a bunch of plates now in the fridge that I need to start putting to grain. Lets say I have 10 MS plates at P1 and want to make P2's. How many transfers from each P1 is reasonable? Do I take 3 transfers or 10? Should I always use a whole plate when transfering up? If you had 10 MS plates at P1 how many would you transfer up and how many would you store? I don't have clones tested yet, so this is what I am working with for now. Always appreciated...
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Josex
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Re: expanding mycelium on agar [Re: Crispykoot]
#23928016 - 12/14/16 11:08 PM (7 years, 1 month ago) |
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It's really up to you man, it's as reasonable to take one sample as it is to take 10. Although if myc is looking nice and healthy in those MS plates I'd feel it'd be kinda wasteful to take one sample only.
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LocN9ne
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Re: expanding mycelium on agar [Re: Josex]
#23928062 - 12/14/16 11:35 PM (7 years, 1 month ago) |
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As many as you are going to need to noc up all of your grain jars man... Go nuttz.
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Intelligentxfruit2



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Re: expanding mycelium on agar [Re: LocN9ne]
#23928080 - 12/14/16 11:51 PM (7 years, 1 month ago) |
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Usually If i'm starting a from scratch and I have the room ill transfer out a few and use the rest of the agar>grain to get some fruits going to test out, maybe get a nice clone if one presents itself
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Crispykoot
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I guess what I'm wondering is this: Is it better to seperate the procedures? Do the agar to agar from one set of plates and then do the agar to grain with a different set of plates. I want to limit contamination.
Do people take a few transfers off of a P1 and then parafilm it back up and throw it in the lunchbox in the fridge to transfer from again later, or do people generally transfer a whole plate at a time? Sorry if this sounds obvious to some...My mind doesn't always figure things out right away...
Edited by Crispykoot (12/15/16 12:29 PM)
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Josex
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Re: expanding mycelium on agar [Re: Crispykoot]
#23929195 - 12/15/16 12:20 PM (7 years, 1 month ago) |
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Normally, the reason to keep a culture in the fridge is to stall its growth because it's a clone or an isolate that you want to test out. No point in storing MS cultures in the fridge, since there are a fuckton of strains still in there and the transfers you took from it have all different genetics.
If you transferred let's say 4 samples from that donor MS plate you can keep the donor plate (at room tempeturature) just in case the receiving plates contam to shit, so that you can go back to the donor plate and snatch new samples.
But after the samples you took from the donor take off and look healthy you can perfectly trash the donor, or if you wish you can use the donor to inoculate some stuff for shit and giggles, taking that the plate still looks healthy, because you can introduce contams in it from your sterile technique.
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Muskoka
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Re: expanding mycelium on agar [Re: Crispykoot]
#23932605 - 12/16/16 02:17 PM (7 years, 1 month ago) |
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The main goal and purpose of transferring agar to agar is to isolate the desired strain of mycelium . It is possible to get ropy mycelium from p1 but more often then not you will need to to isolate and transfer it. Don't take multiple samples for the sake of making Bulk culture. isolate the desired mc and when you have a culture reach its peak then isolate and transfer again and again. Remember the more you isolate and transfer your culture with each generation of careful selection your technically developing your own strain and you want your stain to be robust , so take as many as possible if it's good quality .
Edited by Muskoka (12/16/16 02:25 PM)
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Mycolorado
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Re: expanding mycelium on agar [Re: Muskoka]
#23932631 - 12/16/16 02:27 PM (7 years, 1 month ago) |
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Quote:
Muskoka said: The main goal and purpose of transferring agar to agar is to isolate the desired strain of mycelium . It is possible to get ropy mycelium from p1 but more often then not you will need to to isolate and transfer it. Don't take multiple samples for the sake of making Bulk culture. isolate the desired mc and when you have a culture reach its peak then isolate and transfer again and again. Remember the more you isolate and transfer your culture with each generation of careful selection your technically developing your own strain and you want your stain to be robust , so take as many as possible if it's good quality .
Dude, this is the second post of bad info you've made today. What gives?
Edit: And it's only your second post!
Edited by Mycolorado (12/16/16 02:28 PM)
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Sirtalis


Registered: 10/05/16
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Re: expanding mycelium on agar [Re: Mycolorado]
#23932678 - 12/16/16 02:41 PM (7 years, 1 month ago) |
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I use agar to clean up clones for the wild, not isolate strains. Agar has many purposes. Stop spreading garbage information.
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Muskoka
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Re: expanding mycelium on agar [Re: Sirtalis]
#23932858 - 12/16/16 04:02 PM (7 years, 1 month ago) |
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What's wrong about it ? I'm trying to help here so be my guest and correct any misinformation. I'm not above learning myself. I'm getting my info from my own experiences and a Dalhousie Unisversity professor who's written books and is considered the most distinguished mycologists and scholar on this matter in the world. Any misinformation is a misinterpretation on my part and I apologize
Edited by Muskoka (12/16/16 04:19 PM)
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Mycolorado
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Re: expanding mycelium on agar [Re: Muskoka]
#23932953 - 12/16/16 04:33 PM (7 years, 1 month ago) |
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Transferring has nothing to do with rhizomorphism, nor is rhizomorphism indicative one way or the other of vigor or fruiting ability. I've had plates that have both rhizo and tomentose growth on them and it's the tomentose that is pinning and not the rhizo...I've also had the exact opposite. You'll find nutritional content of the medium/substrate is more influential on the type of growth you have. Also, if you keep isolating down without testing, you might simply be refining a mycelium that doesn't want to fruit or doesn't fruit well.
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Muskoka
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Re: expanding mycelium on agar [Re: Mycolorado]
#23933035 - 12/16/16 05:03 PM (7 years, 1 month ago) |
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that explains a lot thank you. I've used laboratory grade agar and health food store agar but always isolated and transferred a few times before beginning any other process . Threw trail and error And testing in between using what I thought was less desirable mycelium sometimes I found no difference in pinning and yield with transferring x amount of times
Edited by Muskoka (12/16/16 05:06 PM)
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Crispykoot
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Re: expanding mycelium on agar [Re: Muskoka]
#23934655 - 12/17/16 06:57 AM (7 years, 1 month ago) |
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Thank you for the thoughts Much appreciated. I went with the decision to seperate the procedures ie either agar to agar or agar to grain, but not both from the same plate. The reason why I'm keeping MS plates in reserve in storage is that they seem good to have. Faster than going back to a print for varied genetics when needed. I took 10 transfers from each agar plate and made 20 P2 MS plates. Doing agar to grain today. You guys rock. Some of us get stuck on indecision and feedback pushes us through. Thank you.
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Th3Issu3
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Re: expanding mycelium on agar [Re: Crispykoot]
#23934789 - 12/17/16 08:39 AM (7 years, 1 month ago) |
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When working with agar, my goal is clean healthy growth. I'm not trying to isolate anything. So the amount of transfers I do solely depends on that. Each time I transfer from my M.S. plate I also label it "T" then the number, for example; T1,T2,T3,ect... Usually 'T-2' Is where I find myself before cutting the plate up and using it for inoculation.
My process in this, is in steps like this......
MS to petri, labeled 'ms p' 'ms p' to 3 new plates labeled 'T 1' 3, 'T 1' plates to 3, 'T 2 ' plates
*all plates are saved for a week or so in case i need to use them again. But after clean growth on the donor plat is achieved I toss them.
Now depending how the plates look 't-2' is where I start using wedges. Not always... But like i said, depending if they are clean on not.
So I now have 3, 't-2' petri's with clean growth.
I make 16; 1 pint master.
I now take plate 1 and 2 labeled "T-2" and cut it up and use them to innoc the 16 1 pint grain masters. I do this b/c I take the 1 pint grain master after 100 % colonization to inoculate 2, 2 quart grain bags. In which I just use half of the jar in each bag. That's what I use for a tub. About 4 quarts of spawn...
OK now you have one plate left...
First thing i do is make 3 slants. I do this so i dont have to start from scratch...
So I take a small pieces from my t2 agar plate and drop them in the slant and label them T-3' then the strain and date. Those get set aside to fully colonize. Once they are, they get put in a lunch box in the fridge. ( I will will use this in the up coming months)
You still have the 't-2' pate just a few chunks missing...So what do we do...More transfers... Now following what I do here you can keep going down the line as far as you want.
I take the "T-2" petri and make 3 plates. Labeled "T-3"...Then I just do the same thing I did previously. Take two plates for masters then use one to go further down the line.
I try not to go farther than 't-6'. I'm not trying to isolate genetics. Just want clean growth.
When you transfer out of t-6 bring your slant out and do the same thing, work up to t-6.
IDK how others do it, but this has worked well for me. Wish you the best!!
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Crispykoot
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Re: expanding mycelium on agar [Re: Th3Issu3]
#23934822 - 12/17/16 08:59 AM (7 years, 1 month ago) |
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Thanks for that...That sounds like the path I'm on as well. I have to buy some test tubes and start putting slants away. My plates are still pretty young, but I'm going to order the tubes today. Thank you for the thoughtful outline of your process. It really helps.
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Th3Issu3
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Re: expanding mycelium on agar [Re: Crispykoot]
#23934839 - 12/17/16 09:03 AM (7 years, 1 month ago) |
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That's what we're here for my friend.
Also idk how many tubs you plan on doing but that sets me right for doing 16 tubs ever 3 weeks or so...
edit: you can also just use a plate instead of a slant to save in the fridge. Just make sure you have it all wraped up and in a clean lunch pale.
Edited by Th3Issu3 (12/17/16 09:05 AM)
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