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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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Quote:
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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Quote:
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.
Quote:
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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Quote:
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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Yeetusdeetus


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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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Quote:
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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Forrester
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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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Quote:
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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Forrester
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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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Quote:
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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