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OfflineBlue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Forrester]
    #27001239 - 10/24/20 01:01 PM (3 years, 3 months ago)

So the scales of bacteria versus mycelium in the LC finally tipped last night toward mycelium.  This morning, the LC has flocculated the malt solids into small mycelium balls.  When this happens, the LC never goes back to bacterial in my experience.  I might expand it out one more time just to give it a totally fresh start, but that's not necessary.  This is what an LC looks like the day it flips:



The size of the balls is so small because there were a lot of malt solids in this LC.  They had been ground up through the ordeal, so this is typical of that.  Although it's kind of hard to tell from the animated GIF, the cloudiness between the mycelium pieces is gone.  That's because the mycelium sequestered all the malt solids and bacteria into the balls.

If I open this, it might still have some bacterial smell, but that's because it was on the edge and fighting just days ago.  You cannot expect all that bacterial smell to just vanish, but the bacteria doesn't have the upper hand here.  A fresh LC inoculated with this will not have a bacterial smell and the balls will be larger.

So this is proof that a culture that I failed to expand 4 times previously due to bacteria could be saved using an agar sandwich technique as outlined elsewhere on the site.  I had to see it for my own eyes to believe it, but this LC makes it undeniable.  The agar sandwich process did clean up the culture enough to tilt an LC toward a mycelium takeover.


Edited by Blue Helix (10/24/20 01:06 PM)


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OfflineForresterM
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
    #27001510 - 10/24/20 04:11 PM (3 years, 3 months ago)

That is awesome.  I think we still have a lot to learn when it comes to LC and this is some great information to have.  Nice work :thumbup:


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OfflineBlue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Forrester]
    #27001630 - 10/24/20 05:40 PM (3 years, 3 months ago)

Quote:

Forrester said:
That is awesome.  I think we still have a lot to learn when it comes to LC and this is some great information to have.  Nice work :thumbup:




As for the question of what makes a "bad LC" versus a "good LC" on a microscopic level, I'm not entirely sure.  Someone in another version of this thread, thinks it has nothing to do with the relative domination of bacteria versus mycelium.  Well, if that is true, then I stand corrected.  However, I could not find bacteria in a clear LC with my microscope.  Maybe I didn't do it right.  I am going to do some more lab work on this in the next week using a very good microscope with a mycologist friend if he has time.  If there is bacteria in there, he should be able to find it.

What I can say for certain is that if the liquid is clear and the mycelium blobs are numerous (for constantly-spun) the LC will work. If you use a LC that is cloudy, smells bad, and has no mycelium blobs in it or just dead strings of mycelium, the bags will probably fail.  Expanding the LC on agar also clearly demonstrates if it is good or not since the layer of liquid over the agar will encourage any bacteria in it to grow exponentially. 

Mold usually manifests in very rapidly growing less blobby and quickly becomes very dense in a LC, and once you've seen it a few times, it's pretty easy to spot because it grows just too fast (at least the types I had did). Generally mold shouldn't be a big problem unless you are being really sloppy with your agar work.  Mold on agar is pretty easy to spot so clean it up before you use the plate to inoculate an LC.

I have had luck with spores direct to LC, but I don't recommend it unless you personally know that the print is exceptionally clean (as in it was taken in a clean room without any of the mushroom flesh touching the print).  Generally you can assume prints are not clean enough to go directly to an LC, though.


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Offlinesmalltalk_canceled
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
    #27001636 - 10/24/20 05:42 PM (3 years, 3 months ago)

Quote:

Blue Helix said:
Quote:

Forrester said:
That is awesome.  I think we still have a lot to learn when it comes to LC and this is some great information to have.  Nice work :thumbup:




As for the question of what makes a "bad LC" versus a "good LC" on a microscopic level, I'm not entirely sure.  Someone in another version of this thread, thinks it has nothing to do with the relative domination of bacteria versus mycelium.  Well, if that is true, then I stand corrected.  However, I could not find bacteria in a clear LC with my microscope.  Maybe I didn't do it right.  I am going to do some more lab work on this in the next week using a very good microscope with a mycologist friend if he has time.  If there is bacteria in there, he should be able to find it.

What I can say for certain is that if the liquid is clear and the mycelium blobs are numerous (for constantly-spun) the LC will work. If you use a LC that is cloudy, smells bad, and has no mycelium blobs in it or just dead strings of mycelium, the bags will probably fail.  Expanding the LC on agar also clearly demonstrates if it is good or not since the layer of liquid over the agar will encourage any bacteria in it to grow exponentially. 

Mold usually manifests in very rapidly growing less blobby and quickly becomes very dense in a LC, and once you've seen it a few times, it's pretty easy to spot because it grows just too fast (at least the types I had did). Generally mold shouldn't be a big problem unless you are being really sloppy with your agar work.  Mold on agar is pretty easy to spot too.





This is some great information for people who are physically inspecting their lcs, trying to make sense out of LCs. Thank you for this post.

But i have a dumb question, and that is how many times you did transfers of this culture to new dishes/something before you decided that either a) the bacteria was riding along

or b) that transfering "away" from the bacterias as described in this thread, doesnt work

im just curious


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Edited by smalltalk_canceled (10/24/20 05:52 PM)


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OfflineBlue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: smalltalk_canceled]
    #27003778 - 10/25/20 10:34 PM (3 years, 3 months ago)

Just to emphasize what a LC that is already going is like, this LC was started about 36 hours ago from the 25ml of the clean one above.  As you can see, it's within a day of being plenty thick to use (actually it could be used now really even while it still needs a good 6 to 12 hours to flocculate the malt solids which will clear it up). 



Also when I open this one, there will be no bacterial smell, and that's because once you expand a good clear LC, bacteria is completely gone.  There is zero bacteria as can easily be established via microscope or agar plate.  It'll never smell bacterial because it's not there.

So I expanded 25 to 500ml within a day and a half, but you could just as easily expand and 500ml LC into several gallons in two days if you wanted.  It's really endless, which is one reason I like LCs; they are fast.


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