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Blue 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) |
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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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meowjinx
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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) |
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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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Blue 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) |
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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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Blue 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) |
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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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Yeetusdeetus


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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) |
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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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Blue 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) |
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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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Kizzle
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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) |
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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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Blue 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) |
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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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Forrester
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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) |
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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.
-------------------- Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here. ------------------- Have some medicinal mushrooms and want to get the most out of them? Try this double extraction method.
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Yeetusdeetus


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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) |
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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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Kizzle
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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) |
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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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xspak
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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) |
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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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Rumblestrip


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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) |
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Will re edit this.
Edited by Rumblestrip (10/17/20 11:42 AM)
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Blue 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) |
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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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Blue 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) |
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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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Blue 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) |
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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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eLeSDenes
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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) |
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Forrester
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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) |
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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!
-------------------- Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here. ------------------- Have some medicinal mushrooms and want to get the most out of them? Try this double extraction method.
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eLeSDenes
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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) |
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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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mushboy
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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) |
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Got pics of this culture is mr helix? Have you tried the josex method? Highly recommended
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Blue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: mushboy]
#26990297 - 10/17/20 04:11 PM (3 years, 3 months ago) |
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One of the culture jars was just covered with antibiotic agar (35ug/ml chloramphenicol + 1% standrad-3%-H2O2). Even though this is a bit deeper than 1/8", I probably have between 1/16" and 1/8" coverage over the rhizomorphs that were reached off the plate, which was the depth I was shooting for:

As you can imagine, it's ridiculously easy to do this. You just have a sterilized antibiotic/H2O2 agar jar ready, pop it in the microwave for 30 seconds, draw out 15 to 20ml of agar from the septum, and inject it into another culture jar's septum. No annoying still air box for this technique - at least not at first. Nothing could be easier, which is why I fear it won't work. If something this simple works, then I'll use it all he time. In fact I might start using it just as standard practice if it works just to make sure there is no bacteria.
I would show you a picture of a culture, but getting pictures of my cultures requires me to actually get my SAB out since I try to not mess with petri dishes often and you can't get a good side picture really. Plus I don't keep cultures for long. Like I said, I run from agar because I hate fucking with it. I'm the LC guy. Petri dishes, culture jars, etc. are part of the world of culturing that requires still air boxes, steady hands, clean technique, etc. - everything I really don't enjoy. In fact, I typically destroy them all as soon as I have a viable LC that is proven for high yield. Once you have a good LC, everything else, except maybe a print or two, is just a waste of time and redundant. A good LC is magic, and you never have to worry about all this bacteria stuff again since LCs defend themselves against bacteria once they are strong.
Edited by Blue Helix (10/17/20 04:22 PM)
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Forrester
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
#26990427 - 10/17/20 06:02 PM (3 years, 3 months ago) |
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Just a thought, but with a little knowledge of antibiotics and bacteria - you might have to run through more than a couple antibiotics in the agar to find one that works against the specific type of bacteria that's in there.
I'm sure you're aware that some antibiotics are not at all helpful against some types of bacteria. Just thinking out loud as it hasn't been mentioned...
-------------------- Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here. ------------------- Have some medicinal mushrooms and want to get the most out of them? Try this double extraction method.
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Blue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Forrester]
#26990462 - 10/17/20 06:23 PM (3 years, 3 months ago) |
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Quote:
Forrester said: Just a thought, but with a little knowledge of antibiotics and bacteria - you might have to run through more than a couple antibiotics in the agar to find one that works against the specific type of bacteria that's in there.
I'm sure you're aware that some antibiotics are not at all helpful against some types of bacteria. Just thinking out loud as it hasn't been mentioned...
Yeah. I'm more or less doing this for fun, so I probably won't go through a bunch of antibiotics. I am also cloning some of the specimens from the tray, so hopefully that will work too. I've thrown enough shit out there that _something_ should stick at least. If this was some master genetic linage, then sure I'd go crazy trying to make it work, but it's not. It's just some random genetics of the Z strain - nothing special.
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Forrester
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
#26990475 - 10/17/20 06:31 PM (3 years, 3 months ago) |
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Yeah I didn't think it was that important for the culture, just more interested in the process
-------------------- Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here. ------------------- Have some medicinal mushrooms and want to get the most out of them? Try this double extraction method.
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Blue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Forrester]
#26996360 - 10/21/20 12:59 PM (3 years, 3 months ago) |
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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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Forrester
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
#26996434 - 10/21/20 01:39 PM (3 years, 3 months ago) |
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That is awesome!
-------------------- Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here. ------------------- Have some medicinal mushrooms and want to get the most out of them? Try this double extraction method.
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sporecap
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
#26996436 - 10/21/20 01:39 PM (3 years, 3 months ago) |
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Great to hear it seemed to have worked for you! I tried a similar version basically at the same time, first results seem promising https://www.shroomery.org/forums/showflat.php/Number/26996310#26996310
How does the myc look like? Mine is very whispy, like the translucent growth one sometimes sees with germinating spores. Do you actually think it could be a good idea to germinate spores like this? They should be much more resistant to a hot-pour, but then they are perfectly protected against drying out, and one should have a bacteria-free culture right from the start.
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Yeetusdeetus


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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: sporecap]
#26996535 - 10/21/20 02:40 PM (3 years, 3 months ago) |
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Hey blue helix, have you heard of josex’s poke method? Seems to work pretty well for a lot of people trying to clean up bacterial cultures
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Blue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Yeetusdeetus]
#26996670 - 10/21/20 04:16 PM (3 years, 3 months ago) |
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Quote:
Yeetusdeetus said: Hey blue helix, have you heard of josex’s poke method? Seems to work pretty well for a lot of people trying to clean up bacterial cultures
If you mean just taking a tiny poke biopsy, yes I tried that. It did not work. This liquid culture would contaminate with bacteria even when I transferred only a dot just several times the size of a period over. It looked great on agar, but once in the LC, it would grow maybe two days then go cloudy soon after with the mycelium being consumed.
I don't know what else to say other than it really seems like the idea of hot pouring over the plate or cutting a piece of agar and laying it on top of the sample cold, both are working. I cannot know 100% yet, but they seems to be working. You can tell when something is working because the LC is not cloudy if you allow it to settle (when spun it will be cloudy due to the malt sugar solids powder). I won't know for sure for a couple days probably, but when I do get a nice liquid culture, I'll post an animation of it here. And if it fails, I'll report here too (I always like to report my failures for something like this too).
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Blue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: sporecap]
#26996695 - 10/21/20 04:32 PM (3 years, 3 months ago) |
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sporecap said: Great to hear it seemed to have worked for you! I tried a similar version basically at the same time, first results seem promising https://www.shroomery.org/forums/showflat.php/Number/26996310#26996310
How does the myc look like? Mine is very whispy, like the translucent growth one sometimes sees with germinating spores. Do you actually think it could be a good idea to germinate spores like this? They should be much more resistant to a hot-pour, but then they are perfectly protected against drying out, and one should have a bacteria-free culture right from the start.
The break-through mycelium looked wispy when I scraped it and put it in the LC. The LC has not gone cloudy in 2 days, which is a good sign. In fact, when I let it settle, it settles pretty fast into a crystal clear solution, which mean the mycelium has probably taken over and flocculating the malt sugar solids even if I cannot see it yet (which makes them settle faster). If I stare at some of the solids with a high-powered flashlight behind the jar, I _think_ I am starting to see a halo around some of the pieces, which is the start of the mycelium taking over. I'll know which way it'll go very soon, but I think it's working.
I'm not so sure about spores germinating on a hot pour. You are talking about a spore which is, if I'm not mistaken, a single cell. Even if it is more than one cell, it's super tiny, and that matters. If you try to germinate spores on 1% (of standard 3% solution) of H2O2 they will die. If you use that same concentration and transfer over a very tiny fragment of mycelium (say a biopsy from a needle), it'll live. It's just that even the tiniest pieces are larger than a single hyphae leaving a spore. Plus the growth before the two single hyphae merge is slow and weak. It's only once they've had sex that they start to act like the mycelium we think of. So I am skeptical that spores would germinate inside of agar, but it sure is worth a try!
Asking around: has anyone tried to germinate spores under a hot agar pour of say 1/16"? Also do spores float? If they do, the hot pour might be out of the question. You'd need to take a sterile agar wedge and put it over them to sandwich the spores between two agar layers if they float (I think they sink, though).
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Yeetusdeetus


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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
#26997520 - 10/22/20 08:43 AM (3 years, 3 months ago) |
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When you took the biopsy did you go straight to lc with it?
I know brf tends to have lower initial endospore counts than conventional grain which is part of why so many people can be successful just steaming cakes. I feel like I read something about Pf cakes being a good medium for keeping bacteria in place while allowing myc to expand, can’t remember why though. Might just be pulling that out of my ass
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Yeetusdeetus


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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Yeetusdeetus]
#26997523 - 10/22/20 08:46 AM (3 years, 3 months ago) |
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bodhisatta said: I would say its harder for bacteria to move in brf cakes so contamination stays localized. But to say bacteria doesn't have an easy time on brf is totally wrong, and the verm being nutritional or non nutritional has nothing to do with it.
Bacteria doesn't move like mold. Even motile bacteria need an aquious medium to get around. Grain jars get shaken and dispersed so that's the problem there. On a cake a small bacterial contamination can usually be washed off.
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bodhisatta said: Even on a petri dish bacteria usually doesn't get too far. Motioe bacteria make bigger patches usually odd shaped too but still don't colonize an entire dish that ive seen. Unless you give the bacteria a helping hand by shaking or whatnot. But its generally a small threat to cakes by nature of how sponge like they are
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Blue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Yeetusdeetus]
#26997643 - 10/22/20 10:08 AM (3 years, 3 months ago) |
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Yeetusdeetus said: When you took the biopsy did you go straight to lc with it?
I know brf tends to have lower initial endospore counts than conventional grain which is part of why so many people can be successful just steaming cakes. I feel like I read something about Pf cakes being a good medium for keeping bacteria in place while allowing myc to expand, can’t remember why though. Might just be pulling that out of my ass
The time I tried this, I used a colonized spawn bag, not the cake. I did go direct to the LC since none of this causes trouble on MEA agar. The same basic thing keeps happening to the LC regardless of how small the piece is or where I took it from. There are signs of life, but sooner or later, the LC goes cloudy. That might be happening with one of the two scrape LCs too, but it's still a bit early for either of them (I've started two so far). Here's a bit more detail.
This cloudy process takes several days to mature, and in the beginning, the cloudiness simply seems to be flouring of the malt sugar solids (whatever they are) through magnet spinning. I don't know how those solids could really be expanding, so I always assumed they are just breaking down to finer and finer powder, giving the illusion of expanding. Eventually, one of two things happen: either (a) I see the mycelium balls start up which sequester all the malt solids and leave a crystal clear solution behind or (b) the cloudiness stops settling when the spinning is turned off even for a full day. In the latter case, I think it means bacterial contamination, and there is no further expansion of mycelium material in a jar like that. The mycelium just disappears or turns into dead strings. I have not verified bacteria on my microscope, but I really don't think I could without a better scope and dye. I have very strong sense of smell, though, and do verify it via that.
I'd like to think that the antibiotic agar scrape jars are working, but I'm still not sure. One of them isn't clouding up now and has formed tight larger pieces of malt sugar solids rather than powder. But neither jar shows the telltale signs of a healthy culture, which are the solids fuzzing up or mycelium balls rapidly expanding and sequestering the malt solid fragments while jar is spinning. Until I see those balls and a clear solution, I cannot claim victory here.
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
#26997843 - 10/22/20 12:10 PM (3 years, 3 months ago) |
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So weird that it doesn’t show up on agar. I wonder how it follows the myc so closely without any kind of indication that it’s there. Maybe if we knew the specific species It’d be easier to figure out how it hides so well
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Blue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Yeetusdeetus]
#26998346 - 10/22/20 05:11 PM (3 years, 3 months ago) |
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Yeetusdeetus said: So weird that it doesn’t show up on agar. I wonder how it follows the myc so closely without any kind of indication that it’s there. Maybe if we knew the specific species It’d be easier to figure out how it hides so well
It hides, but this is not uncommon. I see a battle between bacteria and mycelium most of the time when I start LCs from agar. The only time that doesn't happen is if I inoculate an LC with a few ml of clean LC or a perfect agar culture.
The first scrape jar that was from a very thin layer (the piece of mycelium floated up) finally went bacterial today. The jar smelled pretty bacterial too. Now I have the LC from the deeper cold agar wedge scrape looking good. I definitely saw many mycelium balls in the LC but they are smooth, which is a sign they are still struggling in the bacteria. I do think, though, the mycelium has the upper hand in there. There just isn't enough cloudiness in there compared to the mycelium. I'll know for sure in 24 hours and get a picture.
Just to make sure it's strong, I'll probably take some of that LC and inoculate another jar to make sure the balls are not too smooth like that.
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Blue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
#26999635 - 10/23/20 12:26 PM (3 years, 3 months ago) |
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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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Blue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
#26999992 - 10/23/20 03:57 PM (3 years, 3 months ago) |
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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: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
#27000208 - 10/23/20 06:17 PM (3 years, 3 months ago) |
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Bacteria's ability to hide from us can be quite amazing... whether in the body or right in front of us in culture!
I wonder if you could see it under microscope.
-------------------- Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here. ------------------- Have some medicinal mushrooms and want to get the most out of them? Try this double extraction method.
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Blue Helix
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Forrester]
#27000319 - 10/23/20 07:23 PM (3 years, 3 months ago) |
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Forrester said: Bacteria's ability to hide from us can be quite amazing... whether in the body or right in front of us in culture!
I wonder if you could see it under microscope.
I'm sure with the right dyes I could see the bacteria - no doubt. My scope is not that great, though, and I can smell it anyway. For me, smell is the biggest give away for bacteria and yeast. Even myceium I can tell the difference of say cubensis, pan cyan, and oysters. They just smell different.
The LC from the scrape still has a good 48 hours before I'll know. I think by Sunday evening, though, it'll be clear if the mycelium will pull through enough to take over or if the mycelium will collapse into strings of dead cells and the LC go even more cloudy.
Someone wrote me today telling me that an LC is not a battle. Sorry to disagree, but sometimes (MOST of the time) that is exactly what it is like in an LC. This is one of those cases too. I've few cloudy LCs that were stable, and those had significantly lower yields too. None of the pictures above were from a cloudy LC, and I can count the number of times I've used such an LC on one hand. Usually it IS a battle.
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Re: Pouring cooling liquid agar on mycelium - does it kill it? [Re: Blue Helix]
#27000777 - 10/24/20 03:38 AM (3 years, 3 months ago) |
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Blue Helix said: Someone wrote me today telling me that an LC is not a battle. Sorry to disagree, but sometimes (MOST of the time) that is exactly what it is like in an LC.
Agree 100%
-------------------- Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here. ------------------- 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]
#27001239 - 10/24/20 01:01 PM (3 years, 3 months ago) |
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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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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) |
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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
-------------------- Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here. ------------------- 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]
#27001630 - 10/24/20 05:40 PM (3 years, 3 months ago) |
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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 
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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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) |
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Blue Helix said:
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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 
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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Blue 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) |
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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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