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OfflineFractal420
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Some agar noob questions
    #22431535 - 10/25/15 11:45 AM (8 years, 3 months ago)

I just got into using agar and its really cool/fun, so far have some TC TC's (Treasure Coast tissue culture) from my last little project (which was MS). I picked out a potent 2nd flush guy and scraped from the inside. Out of the 4 plates i made with that tissue, 3 of them are doing well. The other one is acually very aggressive but has contams. Still, maybe a transfer could save it? Either way, i have clean ones i can use but the contammed one is the largest piece of stem i used, and ended up growing like 4x faster. Theyre all growing in the cold, like 65 degrees.

Besides that, decided to try PE spores from a syringe onto 4 plates. Once again, 3 look clean (much more spotty than the TIssue though, the tc seems to grow very strongly, MS is developing but all in spots. Here are the nice ones
Tissue TC is growing pretty fast, MS PE def slower. Cold temps def attribute to slower growth but prolly less chance of contam?



And here are the two "bad" plates. One is a Treasure Coast tissue culture, and other one is a PE MS. They look more aggressive than the other plates and im thinking the fact that theyre growing among contams means theyre probably more "evolved?" When it comes to dealing with baddies? Or should i just toss em?


(Left to right: PE MS and TC tissue, PE MS, TC TC)

I actually think these look really interesting, and they are the first agar plates i ever started. I got hooked up with some gourmet plates and spawned some shiitake to get into it, but ive been wanting to work with isolation and stuff. Should i let the (clean) plates colonize 100% or can i just cut a piece and spawn a grain master of PE? The dirty ones i would def transfer first if anything.

Any knowledge greatly appreciated!!

Might make my first LC today too, but i really do like Agar :smile:


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Offlineactive029
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Re: Some agar noob questions [Re: Fractal420]
    #22431556 - 10/25/15 11:49 AM (8 years, 3 months ago)

\i have limited experience with agar... i think you would cut the desireable piece before 100 percent colonization... thats what i did... cool thread cool pics... hopefully i can learn something on this one, also how did you choose your shrooms for cloning?


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OfflineFractal420
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Re: Some agar noob questions [Re: active029]
    #22431570 - 10/25/15 11:54 AM (8 years, 3 months ago)

^ thank you! Had a Treasure Coast MS project. Usually on the second flush, I simply picked out a fruit that looked nice and like it would be really potent (tried it lol, it was!) and so i ripped it open per tek and scraped the inner 4 tissue into 4 plates. 3 remain clean as of now, the 4th, well you see lol.

The PE, i simply squirted like 1.5cc into each plate, took like 5 days as usual, but then started to see some slow growth


--------------------
Dreaming of That face again.
It's bright and blue and shimmering.
Grinning wide
And comforting me with it's three warm and wild eyes.

Prying open MY third eye



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Offlineactive029
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Re: Some agar noob questions [Re: Fractal420]
    #22431621 - 10/25/15 12:04 PM (8 years, 3 months ago)

i think 1.5 cc is to much.... r u talking spore solution?


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OfflinePsilosoulful

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Re: Some agar noob questions [Re: active029]
    #22431645 - 10/25/15 12:07 PM (8 years, 3 months ago)

Yeah 1.5 cc will be flooding your plate with solution. All you need is one drop right in the center of the plate!


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OfflineFractal420
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Re: Some agar noob questions [Re: active029]
    #22431666 - 10/25/15 12:11 PM (8 years, 3 months ago)

it varied. I ended up using like 6cc over 4 plates. I think one of them got more like 2cc (hard to tell in the glovebox) and thats prolly the one that got contammed but is also growing the fastest. I meant more like 1.5cc on average. i think besides that one, the rest got more like 1cc, because there is still 4cc left in the syringe :smile:

I think the ones that are working are more like 1cc each, but i liked how people had sectioned places, so id squirt a bit in diff corners. Just experimenting. I hope to get some awesome PE ::crosses fingers::

I hear people try to get different sections to cross over.

Like i said, its my first real agar project :smile:
Trying to read as much as possible on isolation though and of course watched the RR vid


--------------------
Dreaming of That face again.
It's bright and blue and shimmering.
Grinning wide
And comforting me with it's three warm and wild eyes.

Prying open MY third eye



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InvisiblePastywhyteMDiscord
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Re: Some agar noob questions [Re: Fractal420]
    #22431667 - 10/25/15 12:11 PM (8 years, 3 months ago)

I'm going to be gentle. Those are a mess. If it was me I would throw them all in the trash. Growing in contams does not make it evolved. Just means it has some fight to live. But that is not a rare trait, most cubes will do the same. But you still need a clean culture to work with.

The best plan is to try and keep everything either in the center or in a line. I melt a divot with a hot scalpel in my agar plates and try to drop the spore solution into it. Just one drop. Don't let it roll around the plate. If it gets everywhere its really hard to see whats clean and whats not.

Those mold colonies in your clone plates are indicative of poor procedure. I don't know if you're using an sab or flowhood but you need to tighten up. Molds rarely hitch rides on tissue biopsy,  usually contams at that point are bacteria. Never reach over the media, use flamed or sterilized tools, let air in the SAB settle for 20 seconds or so after bringing in flamed tools before opening a plate or jar.

Its good your trying, this will teach you much. Consider those plates a lesson learned, pitch em, do some more reading, and try again :thumbup:


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OfflineFractal420
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Re: Some agar noob questions [Re: Fractal420]
    #22431688 - 10/25/15 12:16 PM (8 years, 3 months ago)

^Also its like two diff things im trying to do. One is, yeah, get some good PE traits from agar opposed to my usual MS > grain thing, but also thought it would be really cool to clone a nice TC fruit.

Most of the tissue cultures besides the contammy one look like they wouldnt need transplants... Should i wait till 100% colonization or no need for that. I try to keep them pretty well wrapped also. I figure, get it to 100%, use it, and store it in a fridge (once its colonized)

Also: I dont mind ditching the contammy plates. Just wanted to know if i should wait for the clean ones to colonize
:shrug:


--------------------
Dreaming of That face again.
It's bright and blue and shimmering.
Grinning wide
And comforting me with it's three warm and wild eyes.

Prying open MY third eye



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OfflineFractal420
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Re: Some agar noob questions [Re: Fractal420]
    #22431698 - 10/25/15 12:18 PM (8 years, 3 months ago)

As for why those two plates got messed up, i actually know the reason. Its because i accidentally touched those two. Anyway though. Thats fine, tossing the moldy ones isnt really a problem. I guess will just focus on the clean ones

Was always taught that with agar contams arent a big deal if its in diff sections and can be transferred.

Pasty: did you mean youd toss out the bunch of clean ones too? I dont see a problem with those. Although in the pic, you only see one of the tissue cultures (not referring to the moldy ones)


--------------------
Dreaming of That face again.
It's bright and blue and shimmering.
Grinning wide
And comforting me with it's three warm and wild eyes.

Prying open MY third eye



Edited by Fractal420 (10/25/15 12:20 PM)


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Offlineactive029
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Re: Some agar noob questions [Re: Pastywhyte]
    #22431701 - 10/25/15 12:19 PM (8 years, 3 months ago)

Quote:

Pastywhyte said:
I'm going to be gentle. Those are a mess. If it was me I would throw them all in the trash. Growing in contams does not make it evolved. Just means it has some fight to live. But that is not a rare trait, most cubes will do the same. But you still need a clean culture to work with.

The best plan is to try and keep everything either in the center or in a line. I melt a divot with a hot scalpel in my agar plates and try to drop the spore solution into it. Just one drop. Don't let it roll around the plate. If it gets everywhere its really hard to see whats clean and whats not.

Those mold colonies in your clone plates are indicative of poor procedure. I don't know if you're using an sab or flowhood but you need to tighten up. Molds rarely hitch rides on tissue biopsy,  usually contams at that point are bacteria. Never reach over the media, use flamed or sterilized tools, let air in the SAB settle for 20 seconds or so after bringing in flamed tools before opening a plate or jar.

Its good your trying, this will teach you much. Consider those plates a lesson learned, pitch em, do some more reading, and try again :thumbup:





theres the real deal... nice try tho man. i found cloning to pieces of cardboard easier than agar and also i only used tissue for clones... not spores....


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Offlineactive029
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Re: Some agar noob questions [Re: active029]
    #22431710 - 10/25/15 12:20 PM (8 years, 3 months ago)

chuck em all


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InvisiblePastywhyteMDiscord
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Re: Some agar noob questions [Re: Fractal420]
    #22431720 - 10/25/15 12:23 PM (8 years, 3 months ago)

Quote:

Fractal420 said:
As for why those two plates got messed up, i actually know the reason. Its because i accidentally touched those two. Anyway though. Thats fine, tossing the moldy ones isnt really a problem. I guess will just focus on the clean ones

Was always taught that with agar contams arent a big deal if its in diff sections




Once the mold sporulates it gets everywhere.  Best to transfer before crazy colors show up. With the amount of mold there it will be very hard ti transfer away. When we talk about cleaning cultures we are usually only talking about tiny spots, not 2/3 of the plate. Once mold gets meshed in it can be really tough to get clean. Not impossible but difficult.

Agar is killer. Cardboard is a poor substitute especially for cubes. Sure it can colonize but thats about it. It does hide contams nicely tho which show up on agar so they don't end up in your grains.


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OfflineFractal420
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Re: Some agar noob questions [Re: active029]
    #22431726 - 10/25/15 12:25 PM (8 years, 3 months ago)

So Pasty, should i toss the clean ones? Is that what youre saying?


So if i am to just toss all of them, (like i said, you can only see one of the tissue cultures in the clean plates), what would be the reason for that? Do you see bacteria developing? Looks to me like myc growth is happening, these are pretty new and just starting to grow. Why toss clean plates without seeing how theyd develop?

I am not referring to the ones with obvious contams, the ones in the first pictures


--------------------
Dreaming of That face again.
It's bright and blue and shimmering.
Grinning wide
And comforting me with it's three warm and wild eyes.

Prying open MY third eye



Edited by Fractal420 (10/25/15 12:25 PM)


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InvisibleKalistis
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Re: Some agar noob questions [Re: active029]
    #22431734 - 10/25/15 12:26 PM (8 years, 3 months ago)

6 cc between 4 plates is insane. I don't think I used an single cc between 20 plates. Just a drop. There are more than enough spores in a single drop.

Good luck!


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OfflineFractal420
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Re: Some agar noob questions [Re: Kalistis]
    #22431750 - 10/25/15 12:30 PM (8 years, 3 months ago)

^more like 4cc over 4 plates, because like 2cc accidentally went into the contammy plate. As of now, i guess ill toss the contammed 2 plates, let the other 3 MS and 3 TC get a chance to develop. I see nothing but white mycelium. If anyone sees anything else, point it out! :smile: (the TC tissue culture, the thing in the middle is the tiny piece of stem, not a contam

DONT MISUNDERSTAND! The ones in the bottom left, those are TISSUE culture, and the other 3 in the pic, those are MS PE. These are not just all MS. Just the ones with spotty growth those are MS PE that are just starting to germinate. Theyve only been growing like a week (in the cold). The more aggressive ones are Tissue



(And there are a couple more TC cultures that are growing pretty fast like the TIssue agar in the bottom left)

Thanks for the luck :smile: Kalis

Can always make more plates if need be, but id see where the clean ones go first


--------------------
Dreaming of That face again.
It's bright and blue and shimmering.
Grinning wide
And comforting me with it's three warm and wild eyes.

Prying open MY third eye



Edited by Fractal420 (10/25/15 12:36 PM)


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OfflineMoxyOx
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Re: Some agar noob questions [Re: Fractal420]
    #22431782 - 10/25/15 12:37 PM (8 years, 3 months ago)

That's not myc man. There are many species that have white fingers, but myc has a specific patterned growth to it.

Not only that but it is all over the place, other posters have mentioned this. If you can, cut a small divide of ropy rhizomorphic growth and transfer to a clean agar and keep doing transfers until you have something clean.


--------------------
No one behind, no one ahead.
The path the ancients cleared has closed.
And the other path, everyone's path,
easy and wide, goes nowhere.
I am alone and find my way.


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Re: Some agar noob questions [Re: MoxyOx]
    #22431802 - 10/25/15 12:42 PM (8 years, 3 months ago)

There is so much happening in there its impossible to say if any are clean or what parts might be clean. That's why we only use a drop and keep it in the center, so we can see whats going on. Maybe there are some clean areas in those plates but it's impossible to say what or where. Would be less headache later to start over.


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OfflineFractal420
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Re: Some agar noob questions [Re: Pastywhyte]
    #22432006 - 10/25/15 01:44 PM (8 years, 3 months ago)

well, the Tissue cultures seem to be fine then at very least :smile:

As for the MS PE i can make other ones with less solution, but ill keep the MS for now until i see what happens.

The tissue cultures all look like the one in the bottom left. I dont see a problem with those at least, and theyve been knocked up with tissue. The TC for sure is all healthy myc.

Was just wondering what other people thought, my first agar work. So ill continue my TC tissue work and hope for the best with the MS plates. I actually didnt know how much to start with, heard many conflicting things. Will hope for the best and make more :smile:

Probably like 0.25cc in my next MS plates, maybe literally just a drop

I have high hopes for the tissue though. You can only see one of them here (bottom left) but all my other tissue agar plates look like that too: pretty aggressive growth from the middle out, considering its only been a week.

Can post close ups if anyone is interested :shrug:

Im enjoying the agar experimentation. Trying to build up my technique.


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InvisibleKalistis
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Re: Some agar noob questions [Re: Pastywhyte]
    #22432020 - 10/25/15 01:49 PM (8 years, 3 months ago)

It looks like entirely too much growth and awfully milky looking to me. Here's a pic of my first MS to agar attempt. Keep in mind on these plates I used 1 drop or less. On a few I used an inoculation loop which probably had half of the MS of the plates with a single drop.



Then the first transfer:


Second Transfer:


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OfflineFractal420
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Re: Some agar noob questions [Re: Kalistis]
    #22432088 - 10/25/15 02:07 PM (8 years, 3 months ago)

Well, next time i know to just use one drop and then do a quick transfer. I was just experimenting, but anyway, There are several pieces of mushy in each plate which is why there is growth all over it. This mycelium looks to be healthy to me....i have a few of these tissue plates



--------------------
Dreaming of That face again.
It's bright and blue and shimmering.
Grinning wide
And comforting me with it's three warm and wild eyes.

Prying open MY third eye



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