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InvisibleThomsen
I'm a teapot


Registered: 09/27/19
Posts: 364
Have i finally found monocultures?
    #26830103 - 07/18/20 02:29 AM (3 years, 6 months ago)

Hey guys.

I've been doing transfers from the original plates 10-12 times now, and i've noticed that all the sectoring seems to be gone. At least i think that it is.

They are B+. Don't mind the numbers and letters on the plate.

Plate 5,1


Plate 5,2


Plate 6,1


Plate 6,2



This last one is a clone from my last grow. Transfer zero.

Plate B+ C_0


What do you think? Monos?


Edited by Thomsen (07/18/20 02:03 PM)


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Offlinew00tmycelium
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Re: Have i finally found monocultures? [Re: Thomsen]
    #26830166 - 07/18/20 04:32 AM (3 years, 6 months ago)

Blurry pictures but I definitely have the same problem, they don't look like strong rhizo growth but that doesn't mean a ton, they still have the radial pattern.  What substrate/agar are you using?

EDIT: Also what do you mean by transfer zero?  From what I learned here, T0 is spore plate, T1 is transfer out of that I think.


Edited by w00tmycelium (07/18/20 04:32 AM)


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Offlineredhandmat
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Re: Have i finally found monocultures? [Re: w00tmycelium]
    #26830192 - 07/18/20 05:11 AM (3 years, 6 months ago)

No thats definitely not a monoculture. ITs just tomentose growth. It doesnt really matter if its rhizo or tomentose. You probably just change the agar recipe enough for the growth to change from rhizo to this. ITs fine. But its not mono.


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InvisibleThomsen
I'm a teapot


Registered: 09/27/19
Posts: 364
Re: Have i finally found monocultures? [Re: redhandmat]
    #26830807 - 07/18/20 01:36 PM (3 years, 6 months ago)

Quote:

w00tmycelium said:
Blurry pictures but I definitely have the same problem, they don't look like strong rhizo growth but that doesn't mean a ton, they still have the radial pattern.  What substrate/agar are you using?

EDIT: Also what do you mean by transfer zero?  From what I learned here, T0 is spore plate, T1 is transfer out of that I think.




It's MEA agar. The B+ T0 is a clone transfer.

Quote:

redhandmat said:
No thats definitely not a monoculture. ITs just tomentose growth. It doesnt really matter if its rhizo or tomentose. You probably just change the agar recipe enough for the growth to change from rhizo to this. ITs fine. But its not mono.




What tells you that it's not mono cultures? You seem very sure.


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Offlinesouthbounpachyderm
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Re: Have i finally found monocultures? [Re: Thomsen]
    #26830817 - 07/18/20 01:39 PM (3 years, 6 months ago)

Quote:

Thomsen said:

What tells you that it's not mono cultures? You seem very sure.




It takes way more work to get there. Everyone would have a monoculture if you could get it within 4 transfers. You really can't.


--------------------


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InvisibleThomsen
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Registered: 09/27/19
Posts: 364
Re: Have i finally found monocultures? [Re: southbounpachyderm]
    #26830825 - 07/18/20 01:43 PM (3 years, 6 months ago)

These plates has been transfered about 10-12 times. A couple of time was a poor choice of words.


Edited by Thomsen (07/18/20 01:44 PM)


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InvisibleDrboomer
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Registered: 09/22/19
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Re: Have i finally found monocultures? [Re: Thomsen]
    #26830935 - 07/18/20 02:44 PM (3 years, 6 months ago)

I don't see rhizo growth just tomentose


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Offlineredhandmat
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Re: Have i finally found monocultures? [Re: Drboomer]
    #26830946 - 07/18/20 02:51 PM (3 years, 6 months ago)

Get them to rhizo so you at least can see that there is no sectoring. You can see nothing with that tomentose growth. I have T3s that look a lot like these, maybe exactly. And I know mine are not mono.

12 transfers do sound like a lot. And it should be possible to get to mono on it, but not if you are not transferring the right piece of growth. here you can read some about it on this thread:

How to identify mono


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Offlineredhandmat
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Re: Have i finally found monocultures? [Re: Drboomer]
    #26830981 - 07/18/20 03:08 PM (3 years, 6 months ago)

Quote:

Drboomer said:
I don't see rhizo growth just tomentose




Monoculture can be tomentose, but it may be hard seeing the sectoring on it.


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InvisibleThomsen
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Registered: 09/27/19
Posts: 364
Re: Have i finally found monocultures? [Re: redhandmat]
    #26831013 - 07/18/20 03:26 PM (3 years, 6 months ago)

Quote:

redhandmat said:
Get them to rhizo so you at least can see that there is no sectoring. You can see nothing with that tomentose growth. I have T3s that look a lot like these, maybe exactly. And I know mine are not mono.

12 transfers do sound like a lot. And it should be possible to get to mono on it, but not if you are not transferring the right piece of growth. here you can read some about it on this thread:

How to identify mono




But you were so sure that it is not monos. Now you're telling me that I can't see if they are monos because of the morphology. So how could you state in your first answer that they definitely not is monos?


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OfflineA.k.aM
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Re: Have i finally found monocultures? [Re: Thomsen]
    #26831027 - 07/18/20 03:38 PM (3 years, 6 months ago)

You can still see sectors in those but it is a little harder than rhizo.

By monoculture do you mean it’s down to one strain? People use monoculture and isolate in different ways, it’s confusing.


There was an old thread where I think verum tried to get PE monocultures and went like 100 transfers before stopping. Sometimes they would look solid and then he would transfer to make sure and they would change again.

Some people think it’s not really even possible to do, that the myc will mutate or whatever and sector out again.


In any case extreme isolation causes extreme attributes, most of which are no good. It seems like the few legit growers I’ve seen mess with it end up deciding doing a few transfers until you’ve got a clean consistent plate ends up performing better.


--------------------
LAGM2020


Edited by A.k.a (07/18/20 03:41 PM)


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Offlineredhandmat
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Re: Have i finally found monocultures? [Re: Thomsen]
    #26831033 - 07/18/20 03:43 PM (3 years, 6 months ago)

Because they look almost exactly like plates I have that are T3. When I moved them to T4 and got rhizo I could see the sectoring. Likewise with yours, you cant really tell they are for sure this way.

But to be fair, in my first comment I should have stated that I do not for sure know. I should have said I for sure don't think so. And hey, Im more or less a noob still, so hopefully someone who knows much more than me can help you out mate.

Edit 1: I see Aka just replied. He is for instance a million times more experienced than I am.

Edit 2: I zoomed into the pics real well and I can tell you what I see (or what I think I see :laugh:):

-plate 5.1: Left pic, you can see between 9'oclock and 9.30 o'clock there is a sector
-plate b+: right pic between 10 and 11 you can see a sector darker than the rest (more pronounced) starting in the middle and going all the way to between 10-11
-Plate 6.1 right pic you can see myc right before 12 to 12.30 o'clock is a bit ahead of the rest, probably a sector there too.
-plate 6.2: right pic, you can see an indention at 11. and around 1 too.

All of these may be indicators of sectors where some have slower or faster growth than others. And I think that if you got better pics and played with contrast etc you'd see more



Edited by redhandmat (07/18/20 03:55 PM)


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InvisibleThomsen
I'm a teapot


Registered: 09/27/19
Posts: 364
Re: Have i finally found monocultures? [Re: redhandmat]
    #26831870 - 07/19/20 03:24 AM (3 years, 6 months ago)

Quote:

A.k.a said:
You can still see sectors in those but it is a little harder than rhizo.

By monoculture do you mean it’s down to one strain? People use monoculture and isolate in different ways, it’s confusing.




A single cell culture is what i mean by monoculture. I guess it's possible for the myc to mutate but it would surprice me if it were from one plate to another. The thought of an substitution reaction and the change of an aminoacid causing a mutation great enough for the cells to sector seems unlikey.  A frameshift mutation maybe could if it changed a vital protein but it shouldn't occur for everyone who does the transfers.


Quote:

redhandmat said:
Because they look almost exactly like plates I have that are T3. When I moved them to T4 and got rhizo I could see the sectoring. Likewise with yours, you cant really tell they are for sure this way.

But to be fair, in my first comment I should have stated that I do not for sure know. I should have said I for sure don't think so. And hey, Im more or less a noob still, so hopefully someone who knows much more than me can help you out mate.




With all due respect I don't think that people should state things in that way if they aren't sure. The reason i'm asking is because i'm not sure my self, so the point is not to have other people not being sure with me :shrug:

The last part i can use. Much better. Thanks. :cheers:


Edited by Thomsen (07/19/20 03:27 AM)


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InvisibleThomsen
I'm a teapot


Registered: 09/27/19
Posts: 364
Re: Have i finally found monocultures? [Re: Thomsen]
    #26831873 - 07/19/20 03:26 AM (3 years, 6 months ago)

Quoted instead if edit :facepalm3:


Edited by Thomsen (07/19/20 03:27 AM)


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Offlineredhandmat
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Re: Have i finally found monocultures? [Re: Thomsen]
    #26831921 - 07/19/20 04:32 AM (3 years, 6 months ago)

Have you seen pictures of mono cultures? There are a couple threads in the forums that have pretty good pictures. One of the threads is about cleaning on agar. And the picture of a mono culture in that thread looks nothing like your plates. So that's what Im basing my answers on. As well as the non-uniformity of your plates. I dont know how much more I need to motivate my answers :frown:


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InvisibleThomsen
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Registered: 09/27/19
Posts: 364
Re: Have i finally found monocultures? [Re: redhandmat]
    #26831941 - 07/19/20 05:04 AM (3 years, 6 months ago)

If you see this post

https://www.shroomery.org/forums/showflat.php/Number/18430998

And looks at this picture you can see that around 11 o'clock and 3 to 5 o'clock you have outer edges that if further out than the rest. So beside for the morphology they look the same to me.

In this picture you have a much more outgoing edge which i do not have in my piture.

So yeah, i have seen pictures of monos and compared them. If you have a agar wedge that is enlongated you obviously won't have a completely circular growth like in this picture.


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Offlinemushpunx
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Re: Have i finally found monocultures? [Re: redhandmat]
    #26831942 - 07/19/20 05:06 AM (3 years, 6 months ago)

IME it can take up to 10 transfers from spore germination before I even start to see real obvious sectoring.

Once I see sectoring, I take a transfer from each individual sector I can identify to new plates. I aim to take from about 10 sectors from one culture. They might still be sectoring some and they each might need more transfers before they are proper isolates.
Once I have those ten isolates it's time to carefully label and test each one out.
The goal is to end up with many isolates from a culture. There might only be one or two keepers. It can take a while to fruit them all out and test the potencies.

It can help if you have a tomentose culture to decrease the nutrients in your agar recipe, the culture will grow out more rhizomorphic.

Honestly strain isolation is a lot of work for our purposes. It makes more sense to me to hunt for really nice clones to test out, you will get a really great performing culture much faster this way.

Once you have a good clone you can always keep making transfers until you end up with a monoculture. It will take a lot less transfers.


--------------------

Amateur Mycologists United
AMU Q&A


Edited by mushpunx (07/19/20 05:13 AM)


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InvisibleThomsen
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Posts: 364
Re: Have i finally found monocultures? [Re: mushpunx]
    #26831951 - 07/19/20 05:15 AM (3 years, 6 months ago)

I did start these cultures from MS and they are transfers from the original culture for some time back now. I did see sectoring back then. They are probably also transfered more than 10 times, but i lost track as i have been working with them for some time. I do know that these cultures have been transfered more than 10 times.

From now and then a plate has grown out the whole plate without showing any (clear sectors, as shown in the thread). Therefore i thought i was there now.

But i will make a low nute agar mix next week and see how it looks when they (hopefully) change morphology.


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Offlinemushpunx
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Re: Have i finally found monocultures? [Re: Thomsen]
    #26831966 - 07/19/20 05:34 AM (3 years, 6 months ago)

Right on. It is definitely a lot easier to see sectors on a ropey culture.

If you're aiming just to get one monoculture, just continue to keep transferring the same culture taking tiny slivers and eventually you'll be left with just one strain


--------------------

Amateur Mycologists United
AMU Q&A


Edited by mushpunx (07/19/20 11:46 AM)


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InvisibleThomsen
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Registered: 09/27/19
Posts: 364
Re: Have i finally found monocultures? [Re: mushpunx]
    #26832048 - 07/19/20 07:07 AM (3 years, 6 months ago)

The plan was to do as you stated earlier, to select the most potent strain ant just grow that so that i know the potency each time.

Thanks for the help and congrats with the TC tag punx!


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