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InvisibleJoshua
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Agar...what next? *pics*
    #541960 - 02/06/02 12:19 AM (23 years, 18 days ago)

To start off with: for some of you this is a general cultivating question. However, I feel that agar culturing is an advance technique, that is why I am posting in this thread.


I have recently innoculated several agar plates w/ a multispore innoculation. I have fluffy mycelium as shown in the pics.

I am inquiring on my next course of action. I see no rhizomorphic mycelium on my plates. Should I transfer wedges to grain? or should I continue with a possible isolation? This is my first successful attempt at agar culturing and I am in need of advice.

In the future what would be my best course of action for culturing strong mycelium.
I could fruit from BRF and clone from there.
I could repeat my current process.
What other methods work well and will not waste my time?





Joshua


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OfflineAnnoA
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Re: Agar...what next? *pics* [Re: Joshua]
    #542006 - 02/06/02 12:58 AM (23 years, 18 days ago)

Transfer a few small wedges from each dish to new plates. Then the growth should sector.

I allways go the "frut them once from multispore then clone a good looking fruit" path.

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Invisiblealtarego
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Registered: 10/25/01
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Re: Agar...what next? *pics* [Re: Joshua]
    #542302 - 02/06/02 09:10 AM (23 years, 18 days ago)

Early plates are often too dense with strains to see secotring. Transfer very small wedges and the differences should become obvious.

I agree with Anno that cloning makes it much easier to get a pure strain, though it is fun to see strains emerge from the chaos of a plate.

See my new post for a followup question on this one.

- AE

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OfflineSuntzu
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Re: Agar...what next? *pics* [Re: Joshua]
    #542332 - 02/06/02 10:11 AM (23 years, 18 days ago)

Hey Joshua, welcome to 'advanced' :smile:  I've read your other stuff and I like your spirit.

As usual, Anno is right. 

Here's how I think about it. . .in that mottled mat of growth you have many many individual genetic combinations.  Some non-mated/un-mate-able growth, some mated hyphae that lack clamp connections [but still might look like decent growth].  Those boundary zones are a good indication of multiple crossings. 
Visualize hundreds of these potentials on a single multispore agar inoculation.  If you put a chunk to grain or a chunk to agar, either way you've begun the process of weeding out the slower growing ones and if grain, the ones not capable of competing on the 'new' food source. . .whittling down the number to the ?couple/few? combos that are represented in the flush.  With this species though, you'll usually get just fine results--no reason why you can't subculture to grain and agar!  The whole 'rhizomorphic' thing is based on the observation that such growth is usually more aggressive and pins well; and if pure, will tend to pin more uniformly if cased/initiated right. 
I've had senesced mycelium that was still rhizomorphic, though.  Also, might be the generation, but the ecuador print I've used always gives fuzzy mycelium. . .even after repeated transfers, though it fruits admirably.  Along the same lines, clones from fruits may not be as rhizomorphic as you'd think. . .at least on first transfer.

The way to do it, IMO, is to keep track of your cultures.  When you get a nice flush, clone one or two.  The more flushes you see, the more you'll be able to choose from.  If possible, make slants or water stocks [easy] for LT storage; this gives you a library to go back to after observing a given flush.  Just remember that unless it's pure, the mycelium growth will go through the selection process again upon the next expansion.  Which could be a good thing, depending.
It's nice to have multispore growth in LT storage, though; you can basically go years without germinating spores.

One of the nicest strains I ever had was taken off a PF cake.  I'm pretty sure it was pure; Ever notice how sometimes on a cake you'll get patches of very rhizo growth?  Pair of tweezers, a lighter. . .         

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OfflineAnnoA
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Re: Agar...what next? *pics* [Re: Suntzu]
    #542489 - 02/06/02 12:32 PM (23 years, 18 days ago)

>Pair of tweezers, a lighter. . .

Hehe,  that?s exactly how I selected  the culture for these Mazatapec. :wink:

 

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OfflineJuSMe13
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Re: Agar...what next? *pics* [Re: Anno]
    #542582 - 02/06/02 02:01 PM (23 years, 18 days ago)

Wow, I would love a print of those Babies!
very impressive


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OfflineSeussA
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Re: Agar...what next? *pics* [Re: Suntzu]
    #542659 - 02/06/02 03:10 PM (23 years, 18 days ago)

Nice info, thanks! What are the benefits of slants vs petri dishes for long term storage?


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OfflineSuntzu
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Re: Agar...what next? *pics* [Re: Seuss]
    #543720 - 02/07/02 12:58 PM (23 years, 17 days ago)

Don't have a lot of time now, but here's a water culture pic. . .the growth is only a couple days;



I will never go back to slants after using water cultures, they are simpler to make and according to the references will last longer. If you use slant-type tubes, storage space is the same for both. I think the key is getting as little agar as possible in the suspension; less opportunity for contams and the fungi don't seem to mind. I also find it easier to remove a few drops of the water suspension rather than try to pick out some growth off of a slant [or dig through mineral oil. . .ugh].

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OfflineAnnoA
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Re: Agar...what next? *pics* [Re: Suntzu]
    #543861 - 02/07/02 03:16 PM (23 years, 17 days ago)

>according to the references will last longer

The reference:

http://www.mycoculture.org/ftpdir/culture-storage-technique

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