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OfflineBlue Helix
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Bacterial agar plates that look like mycelium? Is this a thing?
    #26984374 - 10/14/20 12:49 AM (3 years, 4 months ago)

I always knew agar plates sucked big time, but with this latest problem, I'm started to wonder if they are even viable way to culture at all.  I have a plate that I swear looks totally fine to me and is growing great, but I've tried now four times to start my LC from it.  I've expanded it three times, including one time on H2O2 agar.  And each time, the new plate looks just find but the LC is contaminating with bacteria when I use a tiny bit of it from the plate.  I've never had this problem before in my life, but I'm starting to wonder if there is bacteria on the plates and I just can't see it.  Does anyone have experience with agar plates where the mycelium "rides with" bacteria?  Is there any indication when that happens?  And if it does happen, how do you sector away from something you can't even see?  I'm totally stumped on this one.


Edited by Blue Helix (10/14/20 01:36 AM)


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OfflineBlue Helix
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Re: Agar pictures wanted [Re: Blue Helix]
    #26984383 - 10/14/20 01:00 AM (3 years, 4 months ago)

One more thing: I just noticed that the second the mycelium got to the bare glass of my jar that it transitioned into rhizomorphic growth.  It's about a half inch long now.  I have not tried to start an LC using it.  Is it possible that the rhizomorphs would be less likely to have this mysterious bacteria in it?  How often do you guys see nice long rhizomorphic with bacteria?  Basically I'm seeking a way to know if there is bacteria other than using more and more liquid cultures.  I'm getting fed up with wasting time if the agar can't even show bacterial growth on it.  LC do that easily, but I can't sector bacteria out of an LC.  My understanding was that agar's only real purpose is that it allowed one to sector away from contamination, right?  So if that's what it's for, how do you do it if you can't even see the effect of the bacteria on the mycelium?  Or is there something else going on here?


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Re: Agar pictures wanted [Re: Blue Helix]
    #26984401 - 10/14/20 01:33 AM (3 years, 4 months ago)

Just one more note: I just cut some of these 1/2" rhizomorphs riding up the side of the half-pint jar and transferred them to both and LC and a plate.  This kind of growth seemed to have been triggered at the edge of the jar where the mycelium leaves the agar surface (i.e. left its food source).  My theory is that if the bacteria I'm seeing is somehow riding the mycelium, it would only ride it close to the agar, not on the bare glass rhizomorphs.  I don't know if it's right since I've never had this problem before.  In the past, I've taken a little mycelium off a plate, and it was fine in an LC.  This is the first time I've seen a plate of mycelium that is not obviously bacterially contaminated but goes bacteria in an LC repeatedly after several attempts.


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Re: Agar pictures wanted [Re: Blue Helix]
    #26984411 - 10/14/20 01:53 AM (3 years, 4 months ago)

Blue Helix said:
My theory is that if the bacteria I'm seeing is somehow riding the mycelium, it would only ride it close to the agar, not on the bare
glass rhizomorphs.  I don't know if it's right since I've never had this problem before.




Kinda makes sense so I guess it's worth a shot to use the rhizo-growth.


If that doesn't solve it you might want to try T-gel? I use this recipe with agar instead of gelatine
when ever I think something is riding along and it usually sorts it out or I do it from the start
1st tranfer from spore plate onto T-agar following two transfers on normal plates.

Also Josex method to clean things up works ok.

Don't have any photos myself but have a look here in case you haven't seen this already
https://www.shroomery.org/forums/showflat.php/Number/22020260


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OfflineBlue Helix
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Re: Agar pictures wanted [Re: Baba Yaga]
    #26984484 - 10/14/20 04:24 AM (3 years, 4 months ago)

Quote:

Baba Yaga said:
Blue Helix said:
My theory is that if the bacteria I'm seeing is somehow riding the mycelium, it would only ride it close to the agar, not on the bare
glass rhizomorphs.  I don't know if it's right since I've never had this problem before.




Kinda makes sense so I guess it's worth a shot to use the rhizo-growth.


If that doesn't solve it you might want to try T-gel? I use this recipe with agar instead of gelatine
when ever I think something is riding along and it usually sorts it out or I do it from the start
1st tranfer from spore plate onto T-agar following two transfers on normal plates.

Also Josex method to clean things up works ok.

Don't have any photos myself but have a look here in case you haven't seen this already
https://www.shroomery.org/forums/showflat.php/Number/22020260</font>



Quote:

Baba Yaga said:
Blue Helix said:
My theory is that if the bacteria I'm seeing is somehow riding the mycelium, it would only ride it close to the agar, not on the bare
glass rhizomorphs.  I don't know if it's right since I've never had this problem before.




Kinda makes sense so I guess it's worth a shot to use the rhizo-growth.


If that doesn't solve it you might want to try T-gel? I use this recipe with agar instead of gelatine
when ever I think something is riding along and it usually sorts it out or I do it from the start
1st tranfer from spore plate onto T-agar following two transfers on normal plates.

Also Josex method to clean things up works ok.

Don't have any photos myself but have a look here in case you haven't seen this already
https://www.shroomery.org/forums/showflat.php/Number/22020260</font>





This is exactly the kind of info I wanted to see, so thanks.  Of particular importance, I saw a picture of a plate that has bacteria.  I see now why I didn't catch this.  I didn't know that perfectly fine looking plates can be hiding bacteria.  I sort of thought that bacteria held back mycelium growth on plates, but now I see that they can't even do that simple function.  I'll try both of these if the rhizomorphic grow doesn't work.

My feeling now is that probably plates are a lot like LCs.  Just like LCs they OFTEN contain bacteria at first, and you are trying to allow the mycelium a head start to grow past it.  In LCs, they are cloudy sometimes (bacterial issue) and you are trying to get the mycelium to grow fast enough so it resolves (i.e. goes clear).  I am just amazed at how far off I was about agar, though.  I really thought it was easier to see bacterial growth on it.  I thought that was the entire point of using it.  I guess not.  I just thought bacteria would make it so mycelium wouldn't grow at all, but now I can see agar is much less useful than I gave it credit.  You still deal with bacteria, but unlike an LC, it's even harder to spot on an agar plate.


Edited by Blue Helix (10/14/20 05:08 AM)


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OfflineBlue Helix
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Re: Agar pictures wanted [Re: Blue Helix]
    #26984510 - 10/14/20 05:19 AM (3 years, 4 months ago)

After reading this thread I think I finally understand why LCs to bulk yield so much better than agar to grain spawn to bulk (at least in many of the cases I've seen here).  It's because that agar doesn't show many types of bacteria very well whereas LCs always do.  If you have an LC that goes clear on you, it's resolved its bacterial issue--that is you can be certain it has--but if you have a plate that simply colonizes the whole surface, you have zero guarantee that its really clean.  And the subtle clues on agar that tell you if it's clean or not aren't totally obvious (no one even had told me about this issue).  And you might even fruit out with a bacteria-heavy spawn, but that hidden bacteria can reduce the yield and pin set a lot.  I guess I am still learning in this hobby because up until now I'd never heard about this.  I had always been able to resolve it in LCs, but this time I've had trouble.


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Re: Agar pictures wanted [Re: Blue Helix]
    #26985003 - 10/14/20 12:07 PM (3 years, 4 months ago)

how can you know if the bacteria came from agar or popped up when you introduced it to lc? i can only think of using a microscope.  It seems the bacterial plates i notice always have a shade of gray to it. 


I left a semi opened liquid culture jar outside for several days, the tiny agar wedge i used appeared to have cultured in the open jar fine -it looked similar to my clean cultures.


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OfflineBlue Helix
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Re: Agar pictures wanted [Re: trippleblack]
    #26988924 - 10/16/20 05:43 PM (3 years, 3 months ago)

Quote:

trippleblack said:
how can you know if the bacteria came from agar or popped up when you introduced it to lc? i can only think of using a microscope.  It seems the bacterial plates i notice always have a shade of gray to it. 


I left a semi opened liquid culture jar outside for several days, the tiny agar wedge i used appeared to have cultured in the open jar fine -it looked similar to my clean cultures.





Well, right now I'm guessing that the bacteria is from the culture since I've never had trouble cleanly inoculating an LC before, and I don't know why I suddenly would now. 

To clean up the culture, I just decided to encase a mycelium fragment from this plate in two layers of antibiotic hydrogen peroxidated agar.  If the heat doesn't kill the mycelium first, any growth out of the encasement should be clear of bacteria.  Then if I introduce that into an LC and do not see bacteria, I think I can conclude definitely that the bacteria was dormant or otherwise on the agar with the mycelium.  I'll post about it if it works.

I'm allowing the molten agar to cool down to 115F (solidifies at 107.6F) before pouring a thin layer of it over another frozen agar plate with non-frozen mycelium fragment.  I think it should cool rapidly, solidify, and encase the mycelium fragment with antibiotic agar with hydrogen peroxide.  The question is if the mycelium will be exposed to excessively high of temperatures so die as the molten agar solidifies over it.  I guess I'll see.  If this works, it would be an ideal way to do a very old agar sandwich technique that is used to clean up hard-to-see bacteria on mycelium cultures.


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Re: Agar pictures wanted [Re: Blue Helix] * 1
    #26992180 - 10/19/20 12:23 AM (3 years, 3 months ago)

I do a lot of insect cell culture, and yes- it's possible you have unseen bacteria contaminating your culture. For whatever reason, they're not doing well on fungal media but do fine in your liquid culture substrate. Sometimes it's the formulation, sometimes it's the environment (liquid vs. gelled nutrient substrate). The "eyeball test" in this context doesn't mean much: just because your plates look like you have a single organism doesn't mean it's free of others.

There are several tests and they can get fairly complex but the easiest thing to try would be to put your fungal isolates on a non-selective bacterial screening medium, preferably one that suppresses fungal growth, perhaps by having no complex carbohydrates. That may not suppress fungal growth 100% but could be enough to allow the bacteria to multiply.


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Re: Agar pictures wanted [Re: Bloo]
    #26996354 - 10/21/20 12:58 PM (3 years, 3 months ago)

I THINK IT IS WORKING!!!!  BOTH scrapes of the mycelium going through two depths of antibiotic agar, one hot-poured and one cold-sliced, are showing undeniable signs of recovery WITHOUT BACTERIA!  There is a halo of very tiny mycelium fuzz on the malt solids and they settle - meaning that there is no bacteria clouding!  OMG!  This is amazing!  Unless something weird happens now, it looks like it works!  This is truly an amazing discovery!  I can't believe this actually works!  I had tried to sector out of this bacteria on antibiotic agar and normal several times without success, but this technique of covering the piece with agar and scraping off the first little bit that pokes through, IS WORKING!!!


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OfflineBlue Helix
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Re: Agar pictures wanted [Re: Blue Helix]
    #26999993 - 10/23/20 03:58 PM (3 years, 3 months ago)

Below is a picture of the antibiotic agar encasing technique scrape jar.  As you can see, it's still pretty cloudy.  More importantly, though, the balls look tight with no ragged edges.  Some of them look like a gel is over them.  These are all signs that the bacteria might be less, but maybe I'm not out of the woods yet.  LCs should develop rapidly when spun, and the cloudiness should go away within a few days.  Also if the balls are very smooth like that, it means there is a battle going on between the inside of them and outside.  I would say there is a 50% chance this jar will make it, so maybe the technique didn't work after all.



If this doesn't work, I'm just going to start over and move on to a clone.  I'm tired of this.  It's not working as well as I had hoped it would.


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Re: Agar pictures wanted [Re: Blue Helix]
    #27000011 - 10/23/20 04:12 PM (3 years, 3 months ago)

I'm riveted now. How did the agar sammy work out?

Hopefully I'm not too late..


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Re: Agar pictures wanted [Re: tanuki]
    #27000057 - 10/23/20 04:42 PM (3 years, 3 months ago)

Quote:

tanuki said:
I'm riveted now. How did the agar sammy work out?

Hopefully I'm not too late..




Yeah, the jar in the last message was started from a scape off an agar sandwich top.  The mycelium grew through the agar cover just fine, but it doesn't look like from the LC that it worked to get rid of enough of the bacteria to make a viable LC.  I really didn't think it would work either, but for a minute there, yesterday, it looked like it was going to actually work (the solution was not as cloudy and the balls not so smooth).  Then this morning I woke up to more cloudiness and the mycelium balls tightening into little smooth spheres.  LCs can have smooth shapes, but the spherical nature is the giveaway that there are problems still.

Look at this healthy LC with smooth shapes:



They are smooth irregular shapes, though, not tight spheres.  Tight spheres are always bad.  And, of course, the most classic looking constantly-stirred LCs are like this:



See how the balls are spiky like those depictions of a Coronavirus?  Those spikes are a 3D version of the 2D linear and/or rhizomorphic nature of the growth on a healthy plate.  It's what you want to see.

This all applies to constantly-stirred LCs.  If you don't stir them at all, they look like snot and you can't tell much about them.  If you stir them too fast (over about 200 RPM like the cheap stir plates or if you use a PC fan to make one) you can't draw much any conclusions from the mycelium pieces because you are just blending it.  It's no different than taking an agar plate and putting it through a food processor then asking if it's contaminated.


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Re: Agar pictures wanted [Re: Blue Helix]
    #27000209 - 10/23/20 06:17 PM (3 years, 3 months ago)

:ooo::thumbup:
Thanks for that education, kind sir.

I've definitely brewed up some of those little spheres.


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Re: Agar pictures wanted [Re: tanuki]
    #27000323 - 10/23/20 07:26 PM (3 years, 3 months ago)

Quote:

tanuki said:
:ooo::thumbup:
Thanks for that education, kind sir.

I've definitely brewed up some of those little spheres.




Was the LC cloudy too?  If so I bet it smelled off.  Unless you are a smoker, elderly, or have some other sinus issues, your nose is probably your best guide to the health of an LC next to your eyes.


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Re: Agar pictures wanted [Re: Blue Helix]
    #27001244 - 10/24/20 01:02 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.


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Re: Agar pictures wanted [Re: Blue Helix]
    #27003799 - 10/25/20 10:42 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.

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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Re: Agar pictures wanted [Re: Blue Helix]
    #27004373 - 10/26/20 10:43 AM (3 years, 3 months ago)

It never turned cloudy but probably didn't give the mycelium enough time to fight back. I just knew that it wasn't looking right, so I threw it out.

I was under the assumption that ones you get any sort of contamination, it's a lost cause.

I actually haven't ever smelled my LC.. Do you have a filter than you can sniff through or do you open the lid?

I'm still learning to look at the world through my contam goggles and I'm a bit paranoid yet..


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Re: Agar pictures wanted [Re: tanuki]
    #27004384 - 10/26/20 10:50 AM (3 years, 3 months ago)

If you are not using malt sugar,
Quote:

tanuki said:
It never turned cloudy but probably didn't give the mycelium enough time to fight back. I just knew that it wasn't looking right, so I threw it out.

I was under the assumption that ones you get any sort of contamination, it's a lost cause.

I actually haven't ever smelled my LC.. Do you have a filter than you can sniff through or do you open the lid?

I'm still learning to look at the world through my contam goggles and I'm a bit paranoid yet..




If you are not using malt sugar or some other sugar that has solids that precipitate from it, then you might not see any cloudiness if the LC is fresh.  Cloudiness can just be a side effect of those solids or it can be bacteria.  In either case, it can clean up.

There is no contamination that you will get that will harm you through smelling alone.  That is, unless you've actually let it go so bad that it's formed spores or endospores or something on the surface.  Don't sniff things that are obviously bad and scary looking, but yeah, your nose can tell you a lot provided you have a decent sense of smell still.  I don't take off the lid until I'm done with the LC, so I don't get a chance to smell it until I'm done.  But really, for me, the way it looks is plenty of information.  The smell just confirms it, which is nice but not necessary or that important.


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Re: Agar pictures wanted [Re: Blue Helix]
    #27006211 - 10/27/20 12:03 PM (3 years, 3 months ago)

Quote:

There is no contamination that you will get that will harm you through smelling alone.




I should have been more clear, I'm not worried about myself.. just my cultures. I thought that opening the lid would guarantee contam.

I'll have to start giving them more time to resolve the cloudiness.


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