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InvisibleSpunkyMonkey88
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Is this plate worthy of being used as an innoculant?
    #26382668 - 12/15/19 05:24 AM (4 years, 1 month ago)

As I understand using agar is for isolating genetics and getting as close to a monoculture as possible and to isolate that culture away from any other contamination.

So if I take a peice of tissue and clone it by cutting a tiny tissue sample out of the center of a stem then put it on agar, shouldnt that be a monoculture?

Or is it not because it has two sets of genes, one from each parent?

Either way if said tissue sample is growing well and looks clean, as far as I can tell, would it be safe to use an agar wedge taken from the outer edge of growth and use it as an innoculant?

Or would there more than likely be some hidden Contamination within the mycelium?

Seems bizarre that I do not see any contamination on my plates with tissue samples...

Edit/ I noticed the 12:00 position looks a little off, is that potential contamination?



Edited by SpunkyMonkey88 (12/15/19 10:01 AM)


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InvisibleBph
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: SpunkyMonkey88]
    #26382842 - 12/15/19 07:47 AM (4 years, 1 month ago)

Looks to be bacterial. I'd transfer it a few more times and see what happens. Although my experience with bacterial infection was that I never out ran it. Make a transfer then grow it out and see. Good luck.


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InvisibleSpunkyMonkey88
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: Bph]
    #26382905 - 12/15/19 08:35 AM (4 years, 1 month ago)

Thanks for the input but where do you see bacteria growing?


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InvisibleSpunkyMonkey88
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: SpunkyMonkey88]
    #26383268 - 12/15/19 12:17 PM (4 years, 1 month ago)

Anyone? Sorry to bump but I need to know whether to transfer to new plates or If I can drop a couple wedges into an LC...


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Re: Question pertaining to cloning on agar EDIT: Pics [Re: SpunkyMonkey88]
    #26383276 - 12/15/19 12:21 PM (4 years, 1 month ago)

I’d do a transfer from about 2 o’clock


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OfflineCybin_man
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: SpunkyMonkey88]
    #26383280 - 12/15/19 12:24 PM (4 years, 1 month ago)

Should transfer just to be safe. 3oclock looks pretty good but use your best judgement only your eyes can see the plate in front of you. You don’t want to make a LC just to find out it’s fuckt later.
Especially because that’s the clone sample plate.


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InvisibleSpunkyMonkey88
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: Cybin_man]
    #26383330 - 12/15/19 01:03 PM (4 years, 1 month ago)

Quote:

Unknown error said:
I’d do a transfer from about 2 o’clock



Quote:

Cybin_man said:
Should transfer just to be safe. 3oclock looks pretty good but use your best judgement only your eyes can see the plate in front of you. You don’t want to make a LC just to find out it’s fuckt later.
Especially because that’s the clone sample plate.




2 & 3 O clock are awfully close to the 12 o'clock section that almost looks like halted growth.. u guys see that? U cant see the very edge because of the glare, I'll have to take a better picture when I get home...

If you imagine a perfect circle of mycelium and then you take your pinky and push down on the 12 o'clock part, that's what it reminds me of.


Edited by SpunkyMonkey88 (12/15/19 01:04 PM)


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Offlinetrubblesome
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: SpunkyMonkey88]
    #26383367 - 12/15/19 01:40 PM (4 years, 1 month ago)

from what I've read, people seem to let the clone grow out a bit, make a transfer, then if the transfer is clean, they let that grow out and make a pin in the dish, then pluck that pin to start a new plate and use that plate to inoculate.


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InvisibleSpunkyMonkey88
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: trubblesome]
    #26383386 - 12/15/19 01:58 PM (4 years, 1 month ago)

Quote:

trubblesome said:
from what I've read, people seem to let the clone grow out a bit, make a transfer, then if the transfer is clean, they let that grow out and make a pin in the dish, then pluck that pin to start a new plate and use that plate to inoculate.




That's a good idea, but is it necessary to wait for a pin? Couldn't I just take a agar wedge out?

Hos long does it take for a plate to pin?


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Offlinetrubblesome
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: SpunkyMonkey88]
    #26383393 - 12/15/19 02:01 PM (4 years, 1 month ago)

I mean, idk, just what i've read in my research. have clone plates brewing right now but haven't transferred yet. I would definitely not inoculate with a first clone plate though.


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Offlinesan pedro guy
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: trubblesome]
    #26383400 - 12/15/19 02:05 PM (4 years, 1 month ago)

not every plate will pin tho

Just take transfers until it looks clean.

Sometimes its on the second transfer sometimes its on the 6th

clean is clean


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Re: Question pertaining to cloning on agar EDIT: Pics [Re: trubblesome]
    #26383410 - 12/15/19 02:11 PM (4 years, 1 month ago)

There’s no need to clone a pin from a plate that is already cloned. But I would make at least one transfer from an original clone plate even if it looks clean.


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OfflineSFS96
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: Unknown error]
    #26383447 - 12/15/19 02:30 PM (4 years, 1 month ago)

:whathesaid:  yeah I’d take a couple of transfers before going to grain to makes sure you have a clean culture


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OfflineA.k.aM
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: SFS96]
    #26383453 - 12/15/19 02:36 PM (4 years, 1 month ago)

I’m at the same stage with you man. I have 4 clone samples with a few T1s from each and just took a couple T2s yesterday.

I figure might as well put the t1s to grain as long as they look like they’ll make it rather than toss them.

This agar stuff is pretty confusing though for me as far as what plates I need to keep around.

Your clone came out nice though I had a bunch just like yours and then some that were crazy bacterial.


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InvisibleSpunkyMonkey88
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: A.k.a]
    #26383476 - 12/15/19 02:54 PM (4 years, 1 month ago)

Quote:

san pedro guy said:
not every plate will pin tho

Just take transfers until it looks clean.

Sometimes its on the second transfer sometimes its on the 6th

clean is clean




But I dunno what clean looks like TBH

I thought this plate looked clean.


Quote:

SFS96 said:
:whathesaid:  yeah I’d take a couple of transfers before going to grain to makes sure you have a clean culture




Ya probably gonna transfer and let it grow out and of looks good ima go with it.


Quote:

A.k.a said:
I’m at the same stage with you man. I have 4 clone samples with a few T1s from each and just took a couple T2s yesterday.

I figure might as well put the t1s to grain as long as they look like they’ll make it rather than toss them.

This agar stuff is pretty confusing though for me as far as what plates I need to keep around.

Your clone came out nice though I had a bunch just like yours and then some that were crazy bacterial.




Ya it really is. I had some t2s that looked fucking awesome, straight rhizos so I took some sectors and transferred to new plates but then they all grew this thin wispy mycelium... I was besides myself. I opened one up and smeared it with my finger and smelt it and it smelt like straight fungus. So I took the other three and three threw the whole fucking plate into 3 different jars, and figured itd be better to focus on clones any way... so well see what happens with them.

Here's one of those plates I'm talking about:


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OfflineA.k.aM
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: SpunkyMonkey88]
    #26383533 - 12/15/19 03:26 PM (4 years, 1 month ago)

Yeah it’s weird stuff. I pulled two samples from the same stem put them both on the same batch of agar and one is a super flat thin circle and the other is crazy rhizo.



Same stem like 1/4 inch apart.


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Edited by A.k.a (12/15/19 03:31 PM)


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InvisibleSpunkyMonkey88
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: A.k.a]
    #26384482 - 12/16/19 04:15 AM (4 years, 1 month ago)

Quote:

A.k.a said:
Yeah it’s weird stuff. I pulled two samples from the same stem put them both on the same batch of agar and one is a super flat thin circle and the other is crazy rhizo.



Same stem like 1/4 inch apart.




That rhizo tray looks bad ass!


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InvisibleInfiniteDreams
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: SpunkyMonkey88]
    #26384864 - 12/16/19 10:40 AM (4 years, 1 month ago)

One thought... another variable is the agar pour itself.  The density of the agar could effect the growth pattern as well as the level of nutrition present.


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InvisibleSpunkyMonkey88
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: InfiniteDreams]
    #26384882 - 12/16/19 10:51 AM (4 years, 1 month ago)

Quote:

InfiniteDreams said:
One thought... another variable is the agar pour itself.  The density of the agar could effect the growth pattern as well as the level of nutrition present.




Ya but I keep my nutes and agar consistently at 2%

Anyways I’m not understanding why I can’t use a agar wedge from this plate to inoculate a LC? Does anyone see any contaminates because I don’t?


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Offlineverum subsequentis
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: SpunkyMonkey88] * 1
    #26384931 - 12/16/19 11:16 AM (4 years, 1 month ago)

That plate MAY be clean but has a few "red flags". Looking at cultures on agar is something that takes a little practice. Important things to look for (other than obviously visible contams" are consistent/ organized growth. On your plate, if you look closely, you'll notice the myc going from "flat" to "puffy" in quite a few places. This would qualify as inconsistent/ unorganized growth.

It's not impossible to get clean cultures on you first plate with cloned tissue but, as a rule of thumb, it's good to do another transfer. Specially when the culture is showing probable signs of underlying contams.

I'd take a transfer or three from the most organized leading edges to new plates. You can put the rest of the plate to grain or LC if you want but i wouldn't promise the results would be good.


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InvisibleSpunkyMonkey88
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: verum subsequentis]
    #26385128 - 12/16/19 01:12 PM (4 years, 1 month ago)

Quote:

verum subsequentis said:
That plate MAY be clean but has a few "red flags". Looking at cultures on agar is something that takes a little practice. Important things to look for (other than obviously visible contams" are consistent/ organized growth. On your plate, if you look closely, you'll notice the myc going from "flat" to "puffy" in quite a few places. This would qualify as inconsistent/ unorganized growth.

It's not impossible to get clean cultures on you first plate with cloned tissue but, as a rule of thumb, it's good to do another transfer. Specially when the culture is showing probable signs of underlying contams.

I'd take a transfer or three from the most organized leading edges to new plates. You can put the rest of the plate to grain or LC if you want but i wouldn't promise the results would be good.




Thanks for the response, I am going to do just that.

This is the plate as of today. I was thinking of taking a couple sectors from between 6:00 & 9:00... what do you think?


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Offlineverum subsequentis
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: SpunkyMonkey88]
    #26385138 - 12/16/19 01:16 PM (4 years, 1 month ago)

I'd go with 5-6 personally but that's just me.


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InvisibleSpunkyMonkey88
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Re: Question pertaining to cloning on agar EDIT: Pics [Re: verum subsequentis]
    #26385141 - 12/16/19 01:17 PM (4 years, 1 month ago)

Will do:thumbup:


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