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Invisibleweetsie
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23460366 - 07/20/16 06:26 PM (7 years, 6 months ago)

For clarity I doubt you would see rhizomorphs degrade into tomentose mycelium in 20 transfers, at least not with cubensis, I should of used something other than the ":lol:" emoji as I can see it's not clear. That said, 20 transfers would be 6-9 months or so of continuous growth? That seems incredibly wasteful of young cells.

RR lets grow mushrooms - strain isolation:

"Chances are once you transfer from this dish (referencing transfer #3) your next transfer you'll have a mono culture"

The complete guide to cultivating mushrooms - Strain Isolation:

"When starting from spores you may have to transfer your strain to a new dish three or four times before you're able to isolate a single strain"

I really don't agree it takes more than 5 transfers or is in any way that difficult. Dilute your spores, take tiny wedges :shrug:


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InvisibleSupalemonhaze
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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23460425 - 07/20/16 07:29 PM (7 years, 6 months ago)

I'm pretty sure OP didn't dilute his spores, and neither did I(neither do others). It takes an awful lot of time and trasfers  to get senescene strictly on agar. 1 single G2G is probably equivalent to hundreds of agar transfers, especially since we do transfers as soon as possible. Bear in mind that you will be slanting your isolates once you get them, effectively halting senescene. Every single grow from there on out will be from that slant.

AFAIK, p values are taken only after an isolate is achieved.


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Offlinespacechildo
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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23460432 - 07/20/16 07:31 PM (7 years, 6 months ago)

you can quote all you want it aint doing much difference to OP :shrug: when you say cultures "start sectoring again" is the actual sectoring.
you also seem to confuse sectoring with isolates.


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InvisibleMad Season
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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23460437 - 07/20/16 07:32 PM (7 years, 6 months ago)

Uniform doesn't necessarily mean isolated. It can be un-uniform on a different ratio of a different type of nutrients. Uniform is what I aim for. Isolating is a hassle.


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InvisibleSupalemonhaze
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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23460445 - 07/20/16 07:34 PM (7 years, 6 months ago)

Quote:

Mad Season said:
Uniform doesn't necessarily mean isolated. It can be un-uniform on a different ratio of a different type of nutrients. Uniform is what I aim for. Isolating is a hassle.




Yeah, sure is. I kinda enjoy it though. I'm kind of excited to see how uniform the flushes are.


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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23460462 - 07/20/16 07:38 PM (7 years, 6 months ago)

lmao SupaLemonHaze you are the bomb, somehow manage to be extremely helpful without even reading my my post :thumbup: thats quite a talent my friend , thanks again

but again, this is definitely NOT my first grow, not even my 10th grow, just my first time playing with isolates and had some specific questions, questions that might seem less stupid if they are actually read

And i never said i could do it in 3, i asked a question, he asked for links. and i dont think forgetting what i read is the way to learn anything :/ however knowing the context helps (ie, experience, technique)

i am doing my best to keep things in context and clear, for the benefit of anyone who actually wonders about these things

Quote:

weetsie said:


I really don't agree it takes more than 5 transfers or is in any way that difficult. Dilute your spores, take tiny wedges :shrug:



much obliged my friend, i really appreciate the input :smile: that is what i thought, and what my notes say, and what i keep reading. although it could of course take more than that if you use a ton of spores, take huge wedges, etc. thanks for adding some context

Quote:

Mad Season said:
Uniform doesn't necessarily mean isolated. It can be un-uniform on a different ratio of a different type of nutrients. Uniform is what I aim for. Isolating is a hassle.



that makes lots of sense, thanks Mad, honored to have you in my thread :smile: a few of the first MS plates i did looked very even to begin with, even though they were obviously a jungle of strains. they became very uneven after a few days though. What do you do when you start seeing even growth a few transfers into an isolation, but when you suspect multiple strains are present?


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InvisibleSupalemonhaze
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23460480 - 07/20/16 07:42 PM (7 years, 6 months ago)

What he means by uneven is disorganized (I think at least). Different strains will grow at different speeds but if they are all organized (clean), they can be used as a MS inoculation.


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InvisibleMad Season
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23460490 - 07/20/16 07:45 PM (7 years, 6 months ago)

Play with the nutrients and ratios. Lower nutrients to make it more rizmorphic and spread out. Try it on a different media type. All are legit options. If it truly is isolated, it doesn't matter what you do, it'll always be the same growth, and rate.

And by organized I mean this:


It clearly isn't isoalted on any of those, since it's about 3 transfers in, but you can see that the combination of genetics all are growing at the same rates, the cube myc has thick and thin areas that are hard to notice, but still shows it isn't isolated. The panaeolus is almost impossible to tell if it's truly isolated due to the tomentose nature.. My best guess is it isn't isolated. Still extremely organized and ready for inoculating grains tho.


This is disorganized, and still not even something I'd trust until transfered and "isolated" a bit more.


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Invisibleweetsie
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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23460578 - 07/20/16 08:11 PM (7 years, 6 months ago)

Quote:

spacechildo said:
you can quote all you want it aint doing much difference to OP :shrug: when you say cultures "start sectoring again" is the actual sectoring.
you also seem to confuse sectoring with isolates.




I don't know how else to put it.. Ive already explained when I brought up senescens it was a joke, sarcasm call it what you want. I was just trying to suggest how absurd it seems to me that it will take 20+ transfers to get isolates.

Single strain isolates take 3-5 transfers, multiple sources have been given to back that up. Even when you don't dilute spores and start with a big black dot  your first plate you can still get isolates in less than 5 transfers.

Starting with less spores is way less important than taking small transfers. Regardless you can achieve a similar effect as streaking by taking some spores on your loop and stabbing your plate multiple times, I like to go in a spiral pattern starting at the outside. I believe I have seen homokaryotic cultures using this methord with old spores.


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Offlinespacechildo
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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23460596 - 07/20/16 08:16 PM (7 years, 6 months ago)

are you even reading the stuff mad season is posting?

How should I know when you're joking/being sarcastic and when you're serious? :shrug: what does "start sectoring again" mean to you then? or was that another joke I didnt get?


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Invisibleweetsie
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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23460669 - 07/20/16 08:36 PM (7 years, 6 months ago)

Rhizomorphic mycelium can become tomentose as the DNA becomes damaged from too many divisions. I was suggesting that by 20 transfers you would of had an isolated culture for so long that it would start to die of old age and you would start seeing genetically different sectors appearing as it fails.

The main take away for me in this thread is that I need to work on my jokes. :sad:


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Offlinespacechildo
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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23460755 - 07/20/16 09:01 PM (7 years, 6 months ago)

Quote:

weetsie said:
Rhizomorphic mycelium can become tomentose as the DNA becomes damaged from too many divisions.




another joke? or sarcasm? :lol:


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Invisibleweetsie
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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23460776 - 07/20/16 09:06 PM (7 years, 6 months ago)

Nope.


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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23461018 - 07/20/16 10:12 PM (7 years, 6 months ago)

Mad, thanks a bunch, that is super helpful info :smile: brilliant idea to try it on different media and compare characteristics , those are wonderful suggestions and i am going to implement them all :thumbup: i really appreciate your examples

lol i thought your joke was quite funny weetsie, and got it the first time. everything you have said makes lots of sense to me :smile:

btw all the plates pictured started from diluted and somewhat old spores (the first plates in the series). I am about to do another set of transfers using Mckenna's MYPA recipe instead of stamet's which i had been using (7g malt, 1g peptone,.5g yeast, rather than 20g malt, 1g yeast, 1g peptone), hopefully that will encourage less vertical growth and more across (will go back to just MEA if not)


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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23461025 - 07/20/16 10:14 PM (7 years, 6 months ago)

Quote:

spacechildo said:
Quote:

weetsie said:
Rhizomorphic mycelium can become tomentose as the DNA becomes damaged from too many divisions.




another joke? or sarcasm? :lol:



always thought nute content was what played a major role in tomentose v rhizo growth :wink:
maybe i was wrong....learn something new every day :awehigh:


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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: MysticMoteToter]
    #23461060 - 07/20/16 10:26 PM (7 years, 6 months ago)

The two arent mutually exclusive by any means, it could very well be both.

I have never heard of Rhizomorphic mycelium becoming tomentose as a result of too many divisions before, but it is certainly interesting and something i want to look into, seems plausible :smile:

and it seems relatively well accepted that nutrient concentration plays a role in the formation of rhizo vs tomentose growth (and probably in the super dense vertical growth, as in some of my plates which i suspect is a result of too much yeast+peptone+ME)


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Invisibleweetsie
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23461191 - 07/20/16 11:25 PM (7 years, 6 months ago)

Quote:

c10h12n2o said:
The two arent mutually exclusive by any means, it could very well be both.

I have never heard of Rhizomorphic mycelium becoming tomentose as a result of too many divisions before, but it is certainly interesting and something i want to look into, seems plausible :smile:




Quote from TMC

"When a mycelium grows old it is said to be senescing. Senescent mycelium, like any aged plant or animal, is far less vigorous and fertile than its counterpart. In general, a change from rhizomorphic to cottony looking mycelium should be a warning that strain degeneration has begun."


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23461205 - 07/20/16 11:31 PM (7 years, 6 months ago)

What about when it goes from cottony to rhizo? Cause most of my plates when I drop spores is tomentose


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23461208 - 07/20/16 11:33 PM (7 years, 6 months ago)

Quote:

weetsie said:
Quote:

c10h12n2o said:
The two arent mutually exclusive by any means, it could very well be both.

I have never heard of Rhizomorphic mycelium becoming tomentose as a result of too many divisions before, but it is certainly interesting and something i want to look into, seems plausible :smile:




Quote from TMC

"When a mycelium grows old it is said to be senescing. Senescent mycelium, like any aged plant or animal, is far less vigorous and fertile than its counterpart. In general, a change from rhizomorphic to cottony looking mycelium should be a warning that strain degeneration has begun."



weird, i've got some cubensis Redboy culture that will go tomentose on just about anything with nutrition but can't get it to go rhizo to save my life...however, it's also put out one of my most potent clones....i don't think tomentose growth is indicative of anything other than colonization speed as long as it's not wispy or weak looking :shrug:


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InvisibleMad Season
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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie] * 1
    #23461214 - 07/20/16 11:34 PM (7 years, 6 months ago)

Senescence IME gets wispier, not necessarily more tomentose.. Tomentose is still aggressive and thick, it just doesn't need to stretch as much looking for nutrients, so it gets all thick and bunched up.


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