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OfflineBlue Helix
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Relationship of bacteria and mycelium in/on various nutrient media
    #26988563 - 10/16/20 04:22 PM (10 days, 12 hours ago)

Recently it has come to my attention that mycelium and bacteria can easily co-exist on an agar plate yet not impede the mycelium too much.  As such you cannot see it well either.  I wasn't aware this could happen until I found a culture that looks fine on agar but consistently contaminates and stalls with bacteria in LC.  My opinion of this before actually seeing this with my own eyes would have been that it is impossible--and I made all sorts of excuses as to WHY it was happening like immediate stirring of the LC--but I am positive it is possible now because I am seeing it, stirring the LC or not.  After several agar transfers without any luck cleaning the culture of bacteria, I'm now attempting an antibiotic/hydrogen peroxide agar sandwich to do it.  I have no idea if that will work, but even though there is little information about it here, people on this site claim it has worked for them.  So I figured it was worth a shot.

I have a couple questions: first, have others had luck with these agar sandwich techniques and do you have any pointers for doing it?  And, secondly, why would bacteria not overwhelm mycelium on an agar plate yet do so in a liquid culture?  What is it about liquid cultures that tip that balance so much toward bacteria?  Microscopically what is going on?

Incidentally, I've seen the same thing with PF Tek-style jars which are incredibly resistant to bacterial domination, far more so than agar even.  Half the spore syringes you can purchase from vendors are totally bacterially-contaminated as is simple to prove with agar, yet for some reason, they usually work in a PF Tek-style brown rice flour jar.  For this the same questions apply: why is it that the PF Tek jars are so resistant to bacterial domination?  What's going on?


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OfflineBlue Helix
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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: Blue Helix]
    #26988866 - 10/16/20 07:02 PM (10 days, 9 hours ago)

Since agar solidifies around 107.6F, I'm going to allow liquid agar to cool to around 117 and then squirt a layer of it on top of a chilled agar plate with the mycelium fragment on it.  I think that should prevent the temperatures from being very high for long enough to kill the mycelium fragment, and it should make a perfect encasement of the mycelium fragment.  I'll report here if it works.


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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: Blue Helix] * 1
    #26989943 - 10/17/20 01:23 PM (9 days, 15 hours ago)

You're actually supposed to use hotter agar to encapsulate. Its called the hot pour. An old method. The agar layer your pour cools down so quick it doesn't tend to harm mycelium.

I think just transferring is the best way to beat contamination though. Reliable and quick.


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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: Blue Helix]
    #26993035 - 10/19/20 03:51 PM (7 days, 12 hours ago)

Quote:

why would bacteria not overwhelm mycelium on an agar plate yet do so in a liquid culture?




Taking a stab here, but bacterial growth is generally much more localized on a 2D agar plate than in a 3D liquid culture - they tend to grow as relatively isolated colonies instead of a diffuse suspension of single cells.

I'm imagining an agar plate with a lawn of bacterial growth on the left side and a mat of mycelium on the right. If the two species are equally competitive at the center of the plate where they meet, that "front line" doesn't really move in either direction, and both bacteria and mycelium can co-exist just consuming the resources that are present on their respective sides of the agar.

In a liquid culture, there aren't any clear front lines to hold and the mycelium can't effectively "wall off" sections of media for itself. The fast-dividing, free-floating single celled bacteria have a better advantage and might quickly overwhelm the mycelium in suspension culture.

Quote:

I'm now attempting an antibiotic/hydrogen peroxide agar sandwich



Why not add the antibiotic/H2O2 directly to the LC media? Does it just use up more antibiotic that way?


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OfflineRogerRabbitM
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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: Blue Helix]
    #26993175 - 10/19/20 05:09 PM (7 days, 11 hours ago)

Pour the agar when it's still piping hot.  140F to 150F agar isn't going to kill mycelium for several minutes and by then it will have cooled down anyway.  The idea is to 'pasteurize' the bacteria long enough for the mycelium to crawl up through the top layer. 'shave' off tiny pieces of the mycelium as soon as you see it and put on a new antibiotic plate.

I've had the same problem with bacteria manifesting itself in grains but not bacteria, usually with cloned outdoor edibles.  Antibiotic agar helps until you get it cleaned up, then go back to regular agar.
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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: RogerRabbit]
    #26993278 - 10/19/20 06:22 PM (7 days, 10 hours ago)

i had some luck with a hot pour once. except in my case i had mold, not bacteria but the photos might still give you some insight. howevver unlike RR says, my mushroom mycelium never grew up through the top layer but the mold sure did. i was able to take a transfer from the top after it grew outwards between the layers. i poured mine when the agar was warm, not hot (maybe 130F?) and i used a stiff mixture if i remember correctly. maybe that's why it didn't grow up through the top layer.


the dirty plate before the hot pour. no matter how many transfers i took from this plate, clean myc would never reveal itself



this a few days after the hot pour. notice the clean mushroom mycelium at the 2:00 position growing outwards away from the mold



taking a transfer



the new plate looked like this



i was able to clean it up with one more transfer


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OfflineD3monic
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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: FriedEgg]
    #26993315 - 10/19/20 06:50 PM (7 days, 9 hours ago)

Misty on FB also suggested water agar. No nutes just agar and water. The myc rapidly expands out looking for food while the bacteria stalls with no food source. I was actually going to pour some today as part of an experiment thread


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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: D3monic]
    #26993342 - 10/19/20 07:20 PM (7 days, 9 hours ago)

:rockon:
To the last 3 posts


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OfflineBlue Helix
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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: RogerRabbit]
    #26993830 - 10/20/20 01:24 AM (7 days, 3 hours ago)

Quote:

RogerRabbit said:
Pour the agar when it's still piping hot.  140F to 150F agar isn't going to kill mycelium for several minutes and by then it will have cooled down anyway.  The idea is to 'pasteurize' the bacteria long enough for the mycelium to crawl up through the top layer. 'shave' off tiny pieces of the mycelium as soon as you see it and put on a new antibiotic plate.

I've had the same problem with bacteria manifesting itself in grains but not bacteria, usually with cloned outdoor edibles.  Antibiotic agar helps until you get it cleaned up, then go back to regular agar.
RR




Hi, RogerRabbit!!!  Hey nice to see you are still around after all these years!

Okay, so I tried this antibiotic agar encasement in several different plates.  I did it a couple ways.  I poured over a sample of the fruiting body interior in two and used plates that were already growing in the other two.  The issue with pouring it over the fruiting body piece was that I forgot that they float.  If you do that way, you had better allow the mushroom piece to attach to the plate for couple days or it'll float and the layer might be really thin.  For the other two, I poured it on top an existing culture.  Definitely pouring it on top of an existing culture is the easiest.  I mean don't see how something CAN be much easier than microwaving a sterile antibiotic LC agar jar, drawing out 20ml, and squirting it over an agar LC jar or dish (of course it's way easier for a agar jar rather than petri dish since you don't even have to get out the SAB)!

Of those the four, one broke through in only 3 days.  It looked fine.  It almost looked too good - exploding through like that.  It was super easy to scrape the growth with razor-sharp blade that made its way through the agar and flick it directly in the LC.  I'll know in three days which way the LC will go (good growth only or bacteria with the good growth that takes over), and I'll post it here.  As I said before, if this works, I have no idea why I wouldn't use it all the time!  It's ridiculously easy.  Just squirt some hot agar on a plate?  Seriously easy stuff - my kind of technique, but I'll believe it when I see it.

Another thing is that I used chloramphenicol at 35ug/ml.  I later found out that the recommended dosage is about 3X that at 100ug/ml for agar cultures.  Still, though, it sounds like the antibiotic is kind of optional anyway, and I also had 1% H2O2 (of a 3% solution).  Between the two, the bacteria should be having at least a bad hair day, enough to tip the balance.


Edited by Blue Helix (10/20/20 05:41 AM)


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OfflineBlue Helix
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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: Blue Helix]
    #26995067 - 10/20/20 07:56 PM (6 days, 8 hours ago)

So the first scrape does not look promising, but that was of a piece that was not too deep in the agar.  As I expected, it started to grow and has formed a gel cover, which usually means it's bacterially contaminated.  I'll be trying a second scrape today, one that is deeper in the agar, but it was a cool agar cover.

I think the highest chance of success will be the deep (~1/8") hot-covered specimens.  That seems the most likely to have killed or left behind the bacteria.  Again, I still am very skeptical, but I'll keep everyone informed.


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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: D3monic]
    #26995584 - 10/21/20 02:47 AM (6 days, 1 hour ago)

Quote:

D3monic said:
Misty on FB also suggested water agar. No nutes just agar and water. The myc rapidly expands out looking for food while the bacteria stalls with no food source. I was actually going to pour some today as part of an experiment thread




Now that I think about it- agar is made of agaropectin (a mixture of galactans- carbohydrates) and agarose (a polysaccharide- more carbohydrates). I wonder if the fungi can make do, chewing on the agar, while the bacteria usually cannot. It can't be very efficient though, or the medium would liquefy.


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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: Bloo]
    #26996106 - 10/21/20 12:28 PM (5 days, 16 hours ago)

So, I let the first scrape jar settle, and I was wrong.  There is definitely not bacteria in there since the sugar solids settled very well.  I do not see that the mycelium scrape made it yet, but that is not particularly unusual.  It can take several days before that happens.  The second scrape jar also does not have bacteria, but again, I don't see evidence of the mycelium making it yet.  I am starting to think this actually might work after all.  Mycelium can take a few days to take off in a LC, but as long as there is no clouding up (bacterial infection), it usually does.  Even the smallest piece of it does.  I'll keep you informed.


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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: Bloo]
    #26996163 - 10/21/20 12:57 PM (5 days, 15 hours ago)

Quote:

Bloo said:
Quote:

D3monic said:
Misty on FB also suggested water agar. No nutes just agar and water. The myc rapidly expands out looking for food while the bacteria stalls with no food source. I was actually going to pour some today as part of an experiment thread




Now that I think about it- agar is made of agaropectin (a mixture of galactans- carbohydrates) and agarose (a polysaccharide- more carbohydrates). I wonder if the fungi can make do, chewing on the agar, while the bacteria usually cannot. It can't be very efficient though, or the medium would liquefy.




I do not care about identification of mushrooms.  In fact, I find it kind of boring, and I assume some identifiers would find culturing similarly boring.  I think it's a fundamental difference in mental circuitry. I was not descendent from a line of naturalists I assume.  Now, don't get me wrong: I definitely see the tremendous value in their work, but that doesn't mean it's the kind of work I like personally.  I like tromping through the woods with those guys, though; they can tell me what I'm seeing, which is very nice.  But, still, I can't remember Latin mushroom names for shit, but friends of mine like Alan Rockefeller can rattle off thousands and in Latin no less.  That's his gig, though.  No I prefer to buckle down to the nuts and bolts of something, and I don't so much care the names of those nuts and bolts.  I care about their function.

But this growth selectivity issue is interesting because in many ways it stands between the natural world and the cultivation world.  It's a big part of what we aren't understanding.  Think of the thousands of mushroom we can identify yet not fruit in a controlled setting, for example.  The study of the selectivity issue could reveal at least some part of why we need to spend hundreds of hours in the middle of some Peruvian rain forest to find some rare kind of mushroom versus some controlled setting.  That's why this problem in particular is so interesting. 

The natural world is dying; we as a species are going to die with it if we cannot save it because we are wholly dependent on it.  I know that most people are in total denial of these basic facts (particularly the identifiers since it's so easy to ignore the facts when you routinely spend your time in the last vestige of the natural world not practically on its deathbed), but those harsh facts are very simple to demonstrate.  And that makes this subject even more important.  The length of time we have left--be it 50 years or thousands--really depends on how well we can understand and so preserve that which we are mindlessly destroying to accommodate our deep population overshoot.  How can we possibly start self-sustaining ecosystems on other planets when we don't get how the ones we are blowing away work today?  Do you remember Biophere II?  Well, it didn't end well for a reason, and that reason is that we don't understand the full picture of how all the natural world's ecosystems tie together too well yet.

For example, why can't bacteria make it through hot-pour agar but mycelium can?  What gives mycelium such an advantage on agar MEA versus some bacteria?  Why can you inject bacterially-contaminated spore-laden water into a PF Tek cake and still expect mushrooms to emerge from it in weeks?  Under what circumstances can bacteria eat spores and under which can't they?  You see all these questions are so incredibly basic, and yet I can't find the answers to them.  I can find what mushroom grows in some hell hole in God knows where on Mushroom Observer, but I can't find what makes an MEA agar surface selective for mycelium?  I mean come on!  It's insanity to me!

All of this reminds me of one of my personal heroes, the late Richard Feynman (a largely self-taught genius).  I wish I were even a tenth the genius of that man, but he did dabble in biology one time.  He said he was amazed at one aspect of it - how incredibly easy it was to find a topic that wasn't well understood.  I have to agree.  Compared to mathematics or physics, biology is very easy to find and reveal mysteries.


Edited by Blue Helix (10/21/20 01:52 PM)


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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: Blue Helix]
    #26996198 - 10/21/20 01:14 PM (5 days, 15 hours ago)

I made a statement on this site that I thought mycelium would die in LC if the piece it too small and one starts to spin it right away.  I think I was wrong about that.  I'm starting to think I tricked myself into thinking it because of the bacterial issue.  I wish I could remove all those posts now.  I hate to spread misinformation or information not well tested.  Bummer.


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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: Blue Helix] * 1
    #26996355 - 10/21/20 02:58 PM (5 days, 13 hours 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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OfflineD3monic
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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: Blue Helix]
    #26996908 - 10/21/20 09:31 PM (5 days, 7 hours ago)

You’re excitement is beautiful, I love it! I’ll have to give the hot pour a try. I take it it you are pouring as thin as possible or are you pouring average thickness?

Curious if this would have any benefit over molds as well.


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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: D3monic]
    #26996948 - 10/21/20 10:11 PM (5 days, 6 hours ago)

Quote:

D3monic said:
You’re excitement is beautiful, I love it! I’ll have to give the hot pour a try. I take it it you are pouring as thin as possible or are you pouring average thickness?

Curious if this would have any benefit over molds as well.




Not sure if it's really as thin as possible.  I'm pouring between 1/8" and 1/16" thick, which is about as thin as you really can pour.  I'm not so sure about molds, but really, I never seem to have much trouble with those.  Molds are a lot closer to what we are growing that bacteria.


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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: Blue Helix]
    #26996951 - 10/21/20 10:14 PM (5 days, 6 hours ago)

Bacteria Is my bane, I just get the thin halo that hides in my myc, even when it looks clean on agar it rears its ugly head on grain. I’ll give this a try and possibly work it into my routine on a regular basis if it works well.


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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: D3monic]
    #26997147 - 10/22/20 01:05 AM (5 days, 3 hours ago)

Quote:

D3monic said:
Bacteria Is my bane, I just get the thin halo that hides in my myc, even when it looks clean on agar it rears its ugly head on grain. I’ll give this a try and possibly work it into my routine on a regular basis if it works well.




I think you'll find that this works for those cases where you cannot sector the bacteria away because the two are running on the plate together.  Until I wrote this, I had no idea that could even happen.  I thought the reason people used agar was because that did not happen being virtually a 2D grow surface.  I guess I was totally wrong.  It's just one more reason to leave agar behind as soon as you have a viable liquid culture.  To me agar is only a means to obtain a liquid culture, and once I have the liquid culture, the plates are usually just thrown out.


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Re: Relationship of bacteria and mycelium in/on various nutrient media [Re: Blue Helix]
    #26999638 - 10/23/20 02:27 PM (3 days, 14 hours ago)

I want to update this thread: I have started two LC for two different scrapes of antibiotic agar.  One was very thin because I poured the agar hot on a piece of mycelium that floated up.  This LC failed.  It looked okay at first, but the cloudiness developed faster than the mycelium; that is the mycelium lost the battle over bacteria.

The second jar was created off a scrape of mycelium that grew through a 1/16" thick (or so) piece of cold mycelium.  This LC has developed a lot of mycelium, and it appears to be doubling every 12 to 24 hours.  There is a very slight cloudiness still, but given the rate of the mycelium growth, I think it's beyond the point that bacteria could stall it.  I'll get a picture once it clears out in the next 24 to 48 hours.

What does all this mean?  It means this technique of scouring bacteria from a culture using a piece of agar it must grow through is a valid technique that works (I used antibiotic agar but I'm not sure that would even matter given I was using a concentration of antibiotic that was far too low).  I don't know of any other way to achieve this end, although I'm sure there are others.  You cannot just sector away bacteria if it runs with the mycelium, but this technique scours enough of it away that you can start a viable LC, which will remove any remaining bacteria as it completes.  Once you have a viable LC, you can throw away the annoying agar plates.

I'm not sure why there isn't more written about this technique--although there are some nice write ups--but I wanted to chime in as a big proponent of LCs that this does work.


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