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Offlinebyo_shroom
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Isolating a mono culture on agar * 1
    #24923712 - 01/18/18 06:53 PM (3 years, 3 months ago)

I'm a total newbie and having some fun with agar.

I transferred my first culture to grains left them to colonise (they appear to be doing okay). I know that was gun ho, but realise I'm like a kid at Christmas with all this new stuff and I just want to play!

The grain transfer was my long shot project. Second to that I've been isolating genetics on agar (at least I think I have).

Aside from the obvious contaminations, I'm also noticing the differences in the mycelium growth. There's the culture that grows like what I refer to spokes on a bicycle wheel. Then there's the more candy floss (cotton candy) culture growth. And then I see some that doesn't seem to have any density/volume to it... it only appears to blur the agar dish.

I watched an online video tek where he said you want to aim for the bicycle spoke mono culture (my words not his).

My question is, what are the advantages of isolating this type of mono culture? I assume it results in faster grain colonisation, and bigger flushes. Is that right?

Also, here's a dish I'm working on. The first image was taken a few days ago, the others were taken this morning.

1)  It was looking really nice from the agar in the centre. But I'm not sure about the growth from the outside of the dish in. Should I transfer this to another dish?



2) Same culture shot this morning



3)



4)



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Offlinewushroow
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Re: Isolating a mono culture on agar [Re: byo_shroom]
    #24923829 - 01/18/18 07:41 PM (3 years, 3 months ago)

:takingnotes:


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OfflineCracked out
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Re: Isolating a mono culture on agar [Re: wushroow]
    #24923864 - 01/18/18 07:51 PM (3 years, 3 months ago)

The bicycle spokes are rhizomorphic growth, which is desired. Rhizo growth=more pins. I'd isolate the rhizo growth. But you can still cut some of the cottony stuff and transfer it and over time it may show rhizo growth. At least in my experience. I dont have much exp with agar, i just started using 2 months ago. Good Luck


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OfflineCracked out
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Re: Isolating a mono culture on agar [Re: Cracked out]
    #24923869 - 01/18/18 07:53 PM (3 years, 3 months ago)

Just saw the heart shape on your agar. lol. pretty cool


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Offlinebyo_shroom
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Re: Isolating a mono culture on agar [Re: Cracked out]
    #24924768 - 01/19/18 02:47 AM (3 years, 3 months ago)

Quote:

Cracked out said:
The bicycle spokes are rhizomorphic growth, which is desired. Rhizo growth=more pins. I'd isolate the rhizo growth. But you can still cut some of the cottony stuff and transfer it and over time it may show rhizo growth. At least in my experience. I dont have much exp with agar, i just started using 2 months ago. Good Luck




Good to know, thanks for the clarification.

The jars that are colonising were the first culture off the spoor print. Maybe they won't fruit as much then.

Lol, yes whatever it is it's formed a perfect heart shape. It makes it easy to compare to it's earlier shape a few days ago and it looks like it hasn't changed.


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Onlinemushpunx
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Re: Isolating a mono culture on agar [Re: byo_shroom]
    #24924874 - 01/19/18 06:37 AM (3 years, 3 months ago)

You can't tell how a culture will perform by if it is ropey and rhizomorphic or fluffy. Doesn't mean much. And a rhizo culture will grow fluffy if you raise the nutrients in your agar enough.
There is a difference however between vigorous myc and weak myc, you should be able to tell that from watching it.

But you shouldn't be trying to isolate  "A" monoculture you should be aiming to get at least 10 out of a culture. The majority will suck when you grow them out, there might only be one worth keeping.

You won't see real obvious sectoring until you've transfered the culture a number of times. Can be 8-10 transfers IME.
Next you take a transfer from each sector to a new plate. If they no longer show any sectoring, these are monocultures, your isolated strains.
Many times what you thought was a single sector might have been a few , and you will need to make another transfer or two before you get an isolate.

Then each of those 10-15 isolates need to be labeled , and fruited out. The best producers are kept. 



Photos of sectoring. Note the one with marker has many more sectors than I marked, its an old photo and I was learning.



Photos of isolates ^


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Offlinebyo_shroom
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Re: Isolating a mono culture on agar [Re: mushpunx]
    #24927193 - 01/20/18 04:07 AM (3 years, 3 months ago)

Awesome run down mushpunx, thanks!


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Offlinecatnip40
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Re: Isolating a mono culture on agar [Re: byo_shroom]
    #24927271 - 01/20/18 06:24 AM (3 years, 3 months ago)

:takingnotes::popcorn:


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OfflineCracked out
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Re: Isolating a mono culture on agar [Re: mushpunx]
    #24927449 - 01/20/18 09:50 AM (3 years, 3 months ago)

I read somewhere that rhizo meant more pins. I cant remember. Either way good to know. I've had some i thought were isolates, then a week later sectors started showing themselves. Lots of patience involved.


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Offlinebyo_shroom
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Re: Isolating a mono culture on agar [Re: mushpunx]
    #24929604 - 01/20/18 06:14 PM (3 years, 3 months ago)

Quote:

mushpunx said:
Then each of those 10-15 isolates need to be labeled , and fruited out. The best producers are kept.




Would you preserve the strain after fruiting, from a spore print? Or store from the agar culture?


Edited by byo_shroom (01/21/18 01:29 AM)


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OfflineCracked out
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Re: Isolating a mono culture on agar [Re: byo_shroom]
    #24930775 - 01/21/18 09:58 AM (3 years, 3 months ago)

I would keep the agar culture of whichever strains fruited the best. If you take spore print, that's just going back to base 1, multispore. Based off of what ive read.


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Offlinewushroow
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Re: Isolating a mono culture on agar [Re: Cracked out]
    #24931729 - 01/21/18 05:41 PM (3 years, 3 months ago)

That's true; you would want to keep the isolated one. Spore printing would give more random genetic scrambles of the isolated fruit.


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Offlineverum subsequentis
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Re: Isolating a mono culture on agar [Re: wushroow]
    #24931914 - 01/21/18 07:15 PM (3 years, 3 months ago)

I've been documenting my isolation process here. i didn't even post any pictures before the fourteenth plate because my hope was to demonstrate isolation and it clearly wasn't happening (sectoring) yet. I'm on my 28th plate (from ms) now and only one is an isolate (i think).



mushpunx, did you isolate from spores? if so, how many transfers did it take you?


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Offlinebyo_shroom
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Re: Isolating a mono culture on agar [Re: Cracked out]
    #24933008 - 01/22/18 07:07 AM (3 years, 3 months ago)

Quote:

Cracked out said:
I would keep the agar culture of whichever strains fruited the best. If you take spore print, that's just going back to base 1, multispore. Based off of what ive read.




I'm total new to cultivation... are you saying that you store the agar plate for each culture that makes it to fruiting? If so, do you keep the agar culture in a preserved condition, or just let it to continue to grow in the petri dish?


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Offlinebyo_shroom
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Re: Isolating a mono culture on agar [Re: verum subsequentis]
    #24933010 - 01/22/18 07:09 AM (3 years, 3 months ago)

Quote:

verum subsequentis said:
I've been documenting my isolation process here. i didn't even post any pictures before the fourteenth plate because my hope was to demonstrate isolation and it clearly wasn't happening (sectoring) yet. I'm on my 28th plate (from ms) now and only one is an isolate (i think).





I think I'll need a few more petri dishes!

Will you segment mycelium from each petri dish culture, or just the select ones that show promising growth?


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Offlineverum subsequentis
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Re: Isolating a mono culture on agar [Re: byo_shroom]
    #24934032 - 01/22/18 04:46 PM (3 years, 3 months ago)

I throw them out only if they look like utter garbage. Otherwise I transfer the best looking part to a new plate. Then I either put the old plates to grain of wrap em and stack em and wait for PINS.


      I like to transfer the rhizo shit but you can't judge them to quickly though. As mushpunx said, they can switch it up all on there own. The one thing people seem to believe is that you can judge how aggressive the culture is by it's rate of growth. So it's a good idea to take a transfer from whatever myc has made it the farthest away from your inoculation point. I only break this general rule if some other (slightly slower) myc is particularly beautiful and calling to me.

Here are a couple of my last plates after I transfered out of them.


Edited by verum subsequentis (01/22/18 05:39 PM)


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OfflineCracked out
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Re: Isolating a mono culture on agar [Re: byo_shroom]
    #24952922 - 01/29/18 07:46 PM (3 years, 3 months ago)

I isolate down until i have about 5 separate substrains of the same "strain" isolated then cut agar and drop in grain jars. Then, let colonize and fruit out. Keep a grain jar of each substrain to the side while they fruit out. Once you see which one does the best, go ahead and do grain to grain transfers of that strain and your in the Game! Also let that strain colonize 2/3 of an agar plate then store in test tube or, anything you can manage to scrape up around the house,and refrigerate it. Then, you'll have it forever


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Mushrooms, Mycology and Psychedelics >> Mushroom Cultivation

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