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OfflineBlue Helix
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Pouring cooling liquid agar on mycelium - does it kill it?
    #26988761 - 10/16/20 04:09 PM (3 years, 3 months ago)

I was wondering if any of you have experience pouring cooling agar with antibiotics and hydrogen peroxide on mycelium.  I'm trying to streamline the antibiotic agar sandwich method to scour bacteria from cultures that have hard-to-see bacteria running with the mycelium.  I need this because LCs are not as resistant to bacteria than agar plates.  It would just be so much easier to pour warm/hot agar on a wedge than cut a hole and move it over.  That would also make sure there aren't holes in the covering.  I just don't know if mycelium can handle that kind of heat for a minute or two.


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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
    #26988793 - 10/16/20 04:20 PM (3 years, 3 months ago)

You can do it, it's called a hot pour. I've never done one, but I would guess it'd have to be at a cool enough temperature to not kill the mycelium, I don't know the exact minimum temp at which mycelium is killed, someone else probably does

But if you're trying to clean up some bacteria, you could also do the regular hot pour technique where you pour liquid agar over your current culture and then do transfer the mycelium as it pokes up through the layer of agar your poured over it. I'd guess a bacterial colony would have a harder time directionally upwards and that's why it's supposed to work

Like I said, I've never done it, that's just what I've read about it, so maybe wait for the opinion of someone who has done a hot pour


Edited by meowjinx (10/16/20 04:21 PM)


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OfflineBlue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: meowjinx]
    #26989082 - 10/16/20 07:53 PM (3 years, 3 months ago)

Quote:

meowjinx said:
You can do it, it's called a hot pour. I've never done one, but I would guess it'd have to be at a cool enough temperature to not kill the mycelium, I don't know the exact minimum temp at which mycelium is killed, someone else probably does

But if you're trying to clean up some bacteria, you could also do the regular hot pour technique where you pour liquid agar over your current culture and then do transfer the mycelium as it pokes up through the layer of agar your poured over it. I'd guess a bacterial colony would have a harder time directionally upwards and that's why it's supposed to work

Like I said, I've never done it, that's just what I've read about it, so maybe wait for the opinion of someone who has done a hot pour




Yeah, I tried it today.  I monitored the temperature so once it got down to 115F (supposedly it solidifies around 107.6F) I drew it in a syringe and squirted it on a culture that I had chilled.  The mycelium had 1% of standard 3% hydrogen peroxide in it and an antibiotic.  If it works to clean the bacteria out and it pokes through clean, I'll definitely post about it here.  It was pretty easy, so it sure would be great if it worked.


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OfflineBlue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
    #26989102 - 10/16/20 08:06 PM (3 years, 3 months ago)

I'm going to just keep a LC jar with antibiotic agar in it on the shelf.  When I need to clean up a plate that I know has mycelium running with bacteria (meaning I cannot sector it out since it's running on or in the mycelium), I'll microwave the antibiotic agar just enough to liquify (~110F or so), draw it up like an LC, and squirt it over a problem plate around 1/16th inch thick.  In theory, this sounds amazingly easy, but if it's so good, I wonder why I don't hear folks talking more about it, especially those using LCs where it's really important to inoculate with a close to totally-clean starting plate.  Imagine being able to totally clean a plate that you know has bacteria running with the mycelium by a simple injection over it the top of it and waiting a week!  That would be awesome!  I might even do it as a simple kind of preventative if the culture is very old. 

Anyone with experience with this, please let me know in this thread because it almost sounds too good to be true!


Edited by Blue Helix (10/16/20 08:18 PM)


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OfflineYeetusdeetus
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
    #26989225 - 10/16/20 09:47 PM (3 years, 3 months ago)

I’ve done a couple hot pours on half colonized plates without antibiotics and they worked out fine. I just poured at normal pouring temp, scraped the emerging myc with a loop and did a serial dilution on the new plate. Then just transfer to a new plate and it’s ready to go

The serial dilution is probably overkill but I figure if you’re gonna put in the effort to do a warm pour you might as well go the extra step


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OfflineBlue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Yeetusdeetus]
    #26989344 - 10/16/20 11:58 PM (3 years, 3 months ago)

Quote:

Yeetusdeetus said:
I’ve done a couple hot pours on half colonized plates without antibiotics and they worked out fine. I just poured at normal pouring temp, scraped the emerging myc with a loop and did a serial dilution on the new plate. Then just transfer to a new plate and it’s ready to go

The serial dilution is probably overkill but I figure if you’re gonna put in the effort to do a warm pour you might as well go the extra step




In the case of LC serious bacterial issues are rare but if you have them, you won't get a viable LC.  If you have it, even if the culture looks perfect on agar the LC goes cloudy and the mycelium dies (it becomes strings of dead mycelium since the bacteria destroys it).  If you don't have a bacterial problem or if you don't have too much I guess, the LC goes clear and mycelium dominates in small balls (that is if you do constant stirring I mean.  if you don't constant stir you get these kind of crappy LCs that are weak and look like snot but still work).  So in many ways an LC is a very good test of purity.  How did you know you had a bacterial issue and how did you know it was resolved?

If this works, it will really be a godsend since it's far easier than sectoring away from bacteria (which is impossible anyway for bacteria that runs with the mycelium like what I'm dealing with now).  For one thing, I don't even need a still air box to inject hot antibiotic agar through a septum, and I'm a BIG fan of any injection-based steps (my entire growth process is injection-based)!  It'd be amazing if it worked since it's so simple and fast.  I'm skeptical, though.  If it works so well and is super easy like that, why isn't everyone doing it?  It would almost be dumb to do anything else.  It should be as common as sectoring away from bacteria, but I seldom hear of people doing it.  The power to totally scour a culture of any bacteria (hidden or not) without sectoring and only a simple injection!?  I mean come on!  That's kind of like a superpower in my mind!

PS - and if you don't mind telling, about how many days did it take to break through the agar layer?  And to do the transfer, did you just gently scrape the top or what?  I'm trying to imagine what the next steps will be like.


Edited by Blue Helix (10/17/20 12:39 AM)


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OfflineKizzle
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
    #26989500 - 10/17/20 02:44 AM (3 years, 3 months ago)

It's not necessary. If you use a medium that inhibits the growth of bacteria you should have no trouble getting bacteria free mycelium from the edge of the colony.


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OfflineBlue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Kizzle]
    #26989514 - 10/17/20 03:12 AM (3 years, 3 months ago)

Quote:

Kizzle said:
It's not necessary. If you use a medium that inhibits the growth of bacteria you should have no trouble getting bacteria free mycelium from the edge of the colony.




Yes, I know the theory.  What you are talking about didn't work for bacteria that rides with mycelium.  It works fine for the usual type, but that is so easy to get rid of that there is no reason to even mention it.

By the way, I doubt this will work either.  If I know one thing about agar heads it's that they exaggerate how well things work.  Usually the reality of the practice is many times harder than the simple theory or just doesn't work at all.  For example, how I was told I would be able to sector away from bacteria all the time.  That was a flat out lie.  Sure for the simple type of bacteria in the cute little pictures, that works.  But for the type of bacteria I'm talking about in this thread, it doesn't.  And that's the whole point here - getting rid of bacteria that rides with mycelium and cannot be detected before it spoils a LC.


Edited by Blue Helix (10/17/20 03:24 AM)


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OfflineForresterM
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
    #26989555 - 10/17/20 04:33 AM (3 years, 3 months ago)

Blue helix, I've seen some very experienced growers in the edibles forum deal with your issue of difficult bacteria riding along in a culture several times years ago.  It just could not be seen, at any time.  In the end they ended up having to trash the culture.

I will be very interested if you can find a succesful way to get rid of that.


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OfflineYeetusdeetus
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Forrester]
    #26989809 - 10/17/20 09:36 AM (3 years, 3 months ago)

I poured over my t5 ms plates that had some irregular growth around the wedge, looked like bubbles under the myc and uneven compared to the newer growth towards the edge. There were also what appeared to be very small clear metabolites near the wedge, I thought that was weird because every time I’d read about someone experiencing metabolites they were an amber/yellow color. Suppose it could’ve just been condensation but most of my t5 plates from that session had it even though the plates had been sitting in the sab unwrapped for a couple days prior to inoculation.

I believe I read someone say that you should keep an eye on the plates for a day or two because that’s when the myc would poke through. Can’t remember exactly how many days it too to see growth emerge but It was definitely less than a week, more than two days though.

I just scraped it with a loop being very careful not to dig into the agar. I think I read someone say you need to check on them very often because once you can see the myc then the bacteria will not be very far behind. Idk how true that is but I didn’t wanna chance it

No idea if the bacteria was meshed in but the receiving plate appeared clean


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Edited by Yeetusdeetus (10/17/20 09:38 AM)


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OfflineKizzle
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
    #26989845 - 10/17/20 10:06 AM (3 years, 3 months ago)

If you're thinking the bacteria is traveling across the surface of the mycelium so it's not touching the antibiotic I doubt that's the case because the mycelium has to be wet for bacteria to move quickly over it. Antibiotics aren't effective on 100% of the bacteria so if you want an absolutely pure culture there's no way to guarantee that. It might reduce your contamination rate at best.


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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
    #26989902 - 10/17/20 10:53 AM (3 years, 3 months ago)

Dear Blue Helix,

big fan of your work here. I am new to this forum and do not mean to hijack your thread or something and certainly I am not advanced enough to give you advise.

However, I think this tek I wrote about here could of interest to the situation you describe:

https://www.shroomery.org/forums/showflat.php/Number/26981589#26981589

And my follow up from today in that same thread:

https://www.shroomery.org/forums/showflat.php/Number/26989823#26989823

If you would go to the link of that scientific study I linked to in the first post, that would be great. It is very interesting to read very much indeed. They claim they had better success than antibacterial agar and the tek is so simple.

It would be an honor to have a legend grower trying it out !


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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: xspak]
    #26989921 - 10/17/20 11:09 AM (3 years, 3 months ago)

Will re edit this.


Edited by Rumblestrip (10/17/20 11:42 AM)


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OfflineBlue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: xspak]
    #26989998 - 10/17/20 12:00 PM (3 years, 3 months ago)

Quote:

xspak said:
Dear Blue Helix,

big fan of your work here. I am new to this forum and do not mean to hijack your thread or something and certainly I am not advanced enough to give you advise.

However, I think this tek I wrote about here could of interest to the situation you describe:

https://www.shroomery.org/forums/showflat.php/Number/26981589#26981589

And my follow up from today in that same thread:

https://www.shroomery.org/forums/showflat.php/Number/26989823#26989823

If you would go to the link of that scientific study I linked to in the first post, that would be great. It is very interesting to read very much indeed. They claim they had better success than antibacterial agar and the tek is so simple.

It would be an honor to have a legend grower trying it out !




Wow these techniques are really interesting!  They all seem to hinge on this idea that mycelium can borrow through agar but bacteria has trouble doing so.  I might try the cover slide technique next.  It would be easier than cutting a piece of agar, but it might no be simpler than the hour pour actually (kind of hard to beat the simplicity of just squirting a hot agar layer).  Anyway, thank you for chiming in.  This thread is proving very helpful!


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OfflineBlue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Kizzle]
    #26990002 - 10/17/20 12:05 PM (3 years, 3 months ago)

Quote:

Kizzle said:
If you're thinking the bacteria is traveling across the surface of the mycelium so it's not touching the antibiotic I doubt that's the case because the mycelium has to be wet for bacteria to move quickly over it. Antibiotics aren't effective on 100% of the bacteria so if you want an absolutely pure culture there's no way to guarantee that. It might reduce your contamination rate at best.




Kizzle I honestly do not know what is going on, and without proper microscope work, I might never know.  All I can tell you is this: this culture does not look like a wreck by any means.  It was started by a dirty print I'm sure, but it doesn't look any different than many others I've grown out.  The difference is this: when I take an edge piece of growth of this culture and put it in LC, the LC goes bacterial every single time.  I have even made control LCs to make sure it's not something else, and I'm certain it is not.  The LC is becoming infected by the culture, so from that I am saying there is bacteria somewhere in that culture.  I can't know where, though, and I don't know how it maintains itself either.


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OfflineBlue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Forrester]
    #26990019 - 10/17/20 12:21 PM (3 years, 3 months ago)

Quote:

Forrester said:
Blue helix, I've seen some very experienced growers in the edibles forum deal with your issue of difficult bacteria riding along in a culture several times years ago.  It just could not be seen, at any time.  In the end they ended up having to trash the culture.

I will be very interested if you can find a succesful way to get rid of that.




Yeah, I'm sure professional growers do encounter this at times.  I have an interesting story about this culture.  It has been totally bizarre!  Originally, I had a somewhat cloudy LC from it, but it certainly wasn't dead.  So I decided to (a) use it for some bags and (b) give it to a friend who was itching for an LC even though it was cloudy.  My bags only half colonized.  Half of them stalled which is pretty typical for a cloudy LC (you really shouldn't be using cloudy LCs).  The bags that made it did fruit, but it was a weak pin set - overall a failure really.  The friend who took the cloudy LC, expanded it and it spontaneously cleaned up.  But when I tried to restart a second LC with the plate, it suddenly did not only cloud up but would die each time too (I've tried four times now).  So now I have a plate that has been expanded a few times, and it looks okay.  It even has nice rhizomorphs, but it no longer will form a good liquid culture.  This work with the hour pour is my last ditch attempt to clean up the culture.  If this doesn't work, I will probably have to give up and ask for a sample of clear LC from my friend back or just go back to a print.

What trips me out about this is that the growth on agar looks fine.  I don't see anything wrong with it at all.  But the fact that when you use it for an LC, it clouds up and smells off, is proof that something is wrong with it.  Somehow bacteria has set up on that plate, and somehow it's not visible.  I've not seen this before, but then again, I try to get away from plates as soon as I can.  The second I get a viable LC, I often store the LC in a vacutainer and just toss the plates entirely.  I like to store genetics as cold-stored LCs and, as a back up, prints too.


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OfflineeLeSDenes
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
    #26990069 - 10/17/20 01:02 PM (3 years, 3 months ago)

:popcorn:


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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
    #26990112 - 10/17/20 01:41 PM (3 years, 3 months ago)

Quote:

Blue Helix said:
What trips me out about this is that the growth on agar looks fine.  I don't see anything wrong with it at all.  But the fact that when you use it for an LC, it clouds up and smells off, is proof that something is wrong with it.  Somehow bacteria has set up on that plate, and somehow it's not visible.




That was what they were running into as well, although it's even more interesting you found a way to prove it via LC.  I had not thought of that back then when we were discussing but it would have been a really good way to get the bacteria to show up!


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-------------------

Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.


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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Forrester]
    #26990203 - 10/17/20 02:50 PM (3 years, 3 months ago)

what trips me out even more that agar growth looks fine, LC looks fine, but on grain it stalls after shake before almost finishing without any obvious sign of bacterial contamination


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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: eLeSDenes]
    #26990228 - 10/17/20 03:11 PM (3 years, 3 months ago)

Got pics of this culture is mr helix? Have you tried the josex method? Highly recommended:awesomenod:


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