Dedikaryotization With Gregor Jones

We have spent an incredible amount of time thus far tearing into our infamously high yielding commercial dikaryons to develop a gourmet monokaryon culture bank here at Cap N Stem. The goal at the end of the day is to understand these monokaryons on a deeper level and to develop new breeding programs. Last week we launched our expansive episode on the process of mechanical dedi-kary-ot-ization over at mycowizards.com and this week we figured we would release an entire conversation on the topic between Sir Gregor Jones and Erik Lomen to bridge the gap between the audiophiles and the die hard MycoWizards Deep-Dive subscribers and community members. A big thanks must go out to @protoplastpurveyor for simplifying the mechanical dedi process. Keep getting weird with fungi ya'll! See ya next week for "Cloning A Wild" only available at MycoWizards.com 

Transcript

0:20

Ah, nothing like cores in the morning. Just kidding, at 6:40pm anyways, I'm gonna say it like I always do, good morning. Micah, Wizards of the world, how y'all doing today? What's new? What's shakin, what's uh, controversial in the world of mushroom cultivation? Well, I can sure tell you that dedic characterization is full of controversy, which is interesting. I don't understand why, because it's quite straightforward in a practice, in a idea, in a concept, it is esoteric, and it is very much kind of gate kept in a way, but it's also fucking fascinating. And I want everybody who's interested in mycology, or amateur mycologists, or even just cultivators in general, with a flow hood and some plates to try this, because it's cool. And if you're feeling yourself at the end of your ropes and you're just tired of farming mushrooms, you gotta get back to the basics, and you gotta pour some fucking goddamn petri dishes and play with cultures and find life again in this thing called mycology that I encourage you to head over to mycowizards.com and watch the video we just released last Friday on mechanical dedicatedization. I'm not gonna go crazy in this intro, trying to explain mechanical dedic characterization, everybody. If you don't know what it is, look that shit up. It's pretty fascinating. We adopted this particular protocol from a channelman named Julian matucci from Imperial scientific if you don't know who Julian is, we did a Micah wizards episode with him not that long ago. Go listen to it. Talk about this concept, this idea of splitting a diary on a fruit body, forming mushroom strain into its two monokaryotic constituents. And just how you do that, I didn't even know it could really be done. You know, I wrote a whole thing on this for like, a subreddit page and send it out to some folks, and I don't know it's it fascinates me. It shows the plasticity of the organism and its ability to, like, create very interesting sort of connections that are necessary for multinucleatic cell development in order to form fruit bodies. But it doesn't create nuclear fusion until, like, an actual fruit body is formed. It's, I mean, this is fucking crazy shit y'all. I mean, it's so cool. So we spent a lot of time making this video, this mechanical deduct characterization video, a few people came up and, you know, and out of the Instagrams and in the emails and DMS and all that, and I think they're a little protective of it, for some reason I don't know why I, you know, I'm not even gonna hesitate to try and figure out rationalize why? All I know is mechanical, dedicatedization, according to Julia mattucci, who originally got the concept from a publication by the ecologist leolaura out of Mexico, I believe in the early 80s, was doing this. He was calling it monokeratization, or monokeratization, yeah, no, that's what I said. Anyways. Dedicateization is a pretty simple concept, but it requires some finesse with the scope, having some extra plates, having time and being really interested in looking at clamp connections, identifying SEPTA and really just like observing these fungi in a way in which I know that's where most of us originally got stoked. So get back to it. Go check that video button here, in its very own raw form, is the conversation that we recorded to sort of overlay and bring some structure into that mechanical ddec characterization video. I figured it was interesting. It's probably worth putting out there, and I hope y'all get something out of it. So without further ado, here's the conversation between myself and Gregor Jones. Oh, I do the art of taking down the bigger person, gentle art mushrooms. Okay, so what we're here to talk about today is mechanical dedicatedization. Do you want to talk about, specifically, the differences between something like mechanical. Chemical and like just using these blenders, versus Protoplast fusion versus Protoplast isolation. Oh, so right now we're just doing a mechanical dei characterization. So just meaning no chemicals or anything, we're literally just putting some agar into a blender jar, blending it up and then trying to recapture that growth and that lysing of the cells actually opens them up so it can allow for the nuclei to essentially get separated, since they're not they're each in their own nucleus. They're not in one giant nucleolus, right? So when you lyse your cells, you're actually able to capture some of these. It's kind of the idea. It was Lille, Leal Egger and Hubert. I wrote it down. But 1970 Leo, Leal and Edgar Hummer, I think 1974 they were the first ones that kind of came out with protocol, and it's just been used and adapted for all mechanical ddec characterization people. So there's really not much innovation that's gone on since its original invention. And we're using a protocol that the guy went and even he references this original paper, and there's also another group, and they were, I think, 10 years later, but they essentially just did the same, but with Shi Taki. So it's some of these hardier varieties that you can kind of get away with this mass visceration. So right now, we've only tried the blue oysters and shiitakes, and we're still in early stages, but yeah, we blended some. It's a funny art, I think, and it's something that's not quite, as we've pointed out, you have to play a game of like, I just call it SEPTA septic lamp. It's like, duck, duck, goose. Yeah, is that a SEPTA? Is it a clamp? Is my contact dry or Yeah, so, and that's, yeah, the way to go after is you just have to look for clamps. And kind of the idea seems to be you'll just kind of reduce your amount of clamps every time. And so you're essentially trying to capture a hyphae or an extension that has no clamps so far, and then you've just taken that growth from that point onwards. And so that's kind of the mechanical aspect, is you just grow your globular colonies, you blend them, recapture, look under the microscope. It's very kind of monotonous, I guess, like the like the crude, yeah, in terms of lysing cells and, yeah, we're just blending, you know, you're not really like going in and capturing a hyphae one at a time. Or, you know, most people, when they think of this, they still think of spores. But we're doing, we're trying to capture the genetics we already have, instead of only capture new genetics, essentially, right? And then from there, you know, interplay. How can we make things better? Or then with spores after that, how do our mons and the traits we like line up or affect all the new ones? So, I guess we'll go back a little bit. But chemical, dedicate characterization seems to be the other big one. I think it's benomil, which is a fungicide itself, and it'll just help to break down the cell walls and essentially make your protoplasts. Oh, so protoplasts is just with a enzyme to lyse open those cell walls. And as we said, right? If we open up that cell, the nuclei aren't both, like, connected to each other, so you can kind of state, yeah, right. So even if you lyse it and then it goes to repair, you might have both captured. And I'm still a little new, the protoplasts understanding of it. It's mainly used for to recapture the nuclei for like, leafs, plants and all that type of things. So if we were in terms of just like we wanted just genetic material, right? That's where all this technology comes from. But it can be applied under the right settings to capture your mons to grow out from there. So that's the chemical. Is benomal. I don't know the enzyme that lyses the cell, but just some cellulase, I would imagine, sure, just to get through that, or beta gluconase, even if we're trying to get through the rigid xys of the scaffold structure. But yeah. And so that's how chemical way would work, which seems like it's been tested on a lot more other strains, whereas mechanical, it really just seems people have done it for oysters. And I think that's where Julian is really pulling from azalea Laurel stuff, because that in particular seems. Him to be it ended with shizakis, like in the 80s or something, yeah, and, and he's also doing it with salosity species, which seems very, I mean, non problematic. It seems like he can recapture, you know, party, yeah, yeah. I've seen too many, I guess, failures or no troubleshooting, most people are kind of have success with this method, yeah. So, you know, that's also where some weirdness comes in. Of adjusting. We're going off protocol that used philosophy strains, and now we're using Pleurotus or lentinola strains. So, yeah, some, you know, adjusting there and whatnot. But from papers, these are the two that it's good to blend, yeah, good to capture, yeah. And you know it this also does make sense for the philosophy breeding world, because I don't know what there's like 300 reported strains of mushroom now for Yeah, or, I don't even know species. People still call them cyan essence or cubes. So it's like the dog breeds, yeah. Is it a grassland philosophy, or is it a dumb, loving philosophy? Cubensis is, I think, the one that the majority of these people are working with outside of Julian. I don't see too many people working on this stuff, but some of the impetus to do a lot of this monoculture work is due to the fact that nobody is really doing that in the gourmet world and Julian and Imperial scientific and there's some other company in there. I always forget the name of they're sort of working to build this mono library, which I think is really important to distinguish and point out is like the cubens people are sort of paving the way for breeding to become a really normalized thing. That's all they do. It's huge. Yeah, one of the things I mentioned yesterday about, you know, ASCO, my seeds being much more stable in their monokaryotic form is something like you stated, they don't really grab that second nuclei until they really need to. And that could be a major issue when it comes down to, like, expansions through, you know, generations for developing fruits. So I think this sort of idea of keeping a monokaryotic library for dikaryotic production is something that needs to be explored, you know, something that needs to be like, kind of put out there. So that's that's really our goal. And talking about this stuff is to create a mom library, yeah, and it opens the door for, you know, the person at home now, to kind of do their own citizen science. And you know, if they buy them on you're like, Okay, so those are proven genetics that I like, totally my oyster, or my philosophy is not doing too well. What if I cross? Maybe it likes going with this one and you can really, yeah, all right, if I want to test a wide range of spores, yeah, maybe I'll pair them up with something I already know is good, yeah, so at least I'll have a baseline in order to grow off of, yeah, um, but yeah, growing the man bank, you just don't see it anywhere, no, and doesn't exist for I mean, I guess I even kind of understand it. It's because you're not buying a mushroom that would be able to fruit if you got Amman culture, right? So I guess that's where, like, the why would I put investment into that? But now, since the world's kind of caught back up to this breeding, yeah, we're able, actually, to, I don't know, justify it's kind of a cool thing as well, because I could even think about, like, research applications in colleges and stuff like performing this protocol and capturing whatnot. Well, I think it's also very important for this emerging interest in using iNaturalist and DNA data sets, because if you're trying to sequence these things, there is a point at which dikaryotic or strain based or mushroom producing data doesn't really help the grander scheme of things, if it's monokaryotic data that you're extracting through a DNA extraction process, then you're actually getting relatively clean data from a clean you know? Yeah, I know we touched on this on, like, our last talking head, yeah, the mom died. So check it out. If you haven't already check out. But yeah, it was because, you know, if you always have two sets of DNA, yeah, that you're analyzing, you're always going to have like, a shift couple percents that are off, Yeah, most definitely, and most of that is coming from fruit body, like the extractions too. So I feel like there's a lot of excitement around identification. I was talking to Kate from just for a moment. She was the lab manager at North spore forever down at their mycology outside thing, and she was interested in this idea that I just. Randomly blurted out in conversation about these kind of interesting shakeouts of the DNA land, and that folio to adioposa, from a DNA data perspective, is not showing up as folio to adioposa anymore. It's showing up as other species. Does that mean it's actually different species? Not necessarily, yeah, you know, we don't have enough clean data. There's all like, just public, shared, you know, non private sort of amalgamations of information. Yeah. And then, you know, you What's that fun tutoring line of how much different does a genome have to be in order to actually define it as your new species. So we're going off of just anything. All the mushroom farms have different foliotas that they're growing, or oysters, precisely. That's where I think building, you know, longer lasting data, clean phylogenetic trees gets you to understand the phylogeny a little bit better, and gets you to really understand what the evolutionary traits of these mushrooms are. Yeah, these things evolve, and they're still evolving. And there's some that are in a state of flux or evolution as we speak. And, yeah, well, I mean, that's what we're trying to capture, just even going into spawn world, because that is the main idea behind spawn, yeah, yeah. I think it is just interesting, kind of getting away from so much focus on spores as your monos, yeah. And actually just kind of, let's sift through the leads. First see we got, actually see, yeah, see what we got. And then we can compare that to a spore instead of because if we already started Spore, then you go, Hey, but then, if I compare it now to my other one, my dye carry on that that spore came from, yeah, what's different? What's the same, right? Yeah, you're just kind of muddying the waters, right? Still, yeah. I mean, you proved this in regards to the yellow oyster tobacco, right? Of trying to, like, create different little di colonies. Yeah, that are subjects we don't have to go too far into, but the reality works. That's all I need to know. If you're trying to keep a particular type of morphology intact, it's important to understand what both of those model carry ons are doing, and what do they look like, what do they express, and maybe match them up to other ones DNA wise, to really see if they're particular on offs, if you will. Yeah, to really say, like, Oh, you want thick, robust caps, it is like, right here in the DNA, which means let's create a lot of Mons, knowing where a successful mon is, and then try and replicate around that, yeah, to produce similar diary of extremes, yeah. Instead of just being like, yeah, spores four, we'll just see what happens. I mean, that's well, that's also why that appeals so much to the philosophy world, because that's all they're trying to do, is just one species mutate. Find it weird, weird little dog breeds of Yeah, that's true. What does Mike always say? Like, this used to be a wolf, you know, like, if you keep mutating, did you hear that picture, if you keep, yeah, muddling in the same species, you don't allow for there to be other interspecies sort of breeding programs and such. And it also opens the fun door, because there's that one research paper I found where they got just essentially basiliomycetes With that are diary ons and just, yeah, the Ganoderma and the Pleurotus. What want to see that fruiting by, I know they never went that far. They just see that's fine. Like, why have we explored this? Yeah, well, and you know, Shima, furry black oyster, right? That's the whole story of nevermind with this, getting those spores and then mixing it with the king trumpet or blue. And I love how clean that patent looks. But, you know, that took, like, a decade of consistent attempts at crossing different things. I'm noticing. I'm starting to realize slow not working all the time. That's research. So that's okay. It's also why, like, I think helping everybody involved in the micro wizards community to, like, really engage with this research is really important, especially because, you know, like, I love that Julian has set the precedent for, like, trying to do it in the Cube World. Nobody else has done that. You know, trying to, like, come up with these, like modern monokaryotic banks, has just been kind of left to the void. But well, now we have a giant cryo Yeah, that's how. So let's, I mean, this is kind of interesting and mildly off topic, but important to. Keeping them on library is most of these mons will be kept cryogenically stored? Is there any safety practice protocol or otherwise important things to note when storing model, carry ons next to dye, carry ons next to again, have plastic bags, I don't know. Is that really? I mean, yeah, if everything's sealed and away, yeah? But if you are worried about that, and you're just having, you know, parafilm Petri plates instead of bagging them all up, sure, you'd probably want different fridges, right? I've seen my son push its way through like parafilm, yeah, exactly, yeah, right. Or keep a monoculture if you were gonna bring it to a fruiting room, yeah, you think you need your own mono fruiting room, right? To try to test that out, just in case there are other spores from anything else, and if they're monos. And if we now have seen from this one paper that monos of different species can get together, right? Or you're gonna so if you were gonna go to fruiting trials from your Mons, I think you would definitely want your own setup. And then, yeah, if you're not bagging up everything, and you're just existing in the world of parafilm, you probably want, like, a solid, yeah, divider wall, separate fridge, yeah? But I think once we're in the terms of cryo, we're each in our individual rye buried cells. So you wouldn't have to worry about it there, but they're just sort of stationed, yeah? I mean, it's just good lab practice. Kind of keep everything separate. Don't let them all touch. You know, if you are a pair of filming, don't let your mons reach your edge of your plate, because then the two mons are on top of each other. Sneaky little fingers might come up. Nature finds a way Exactly. I think it's really important too to just sort of like talk about these weird crosses, like putting a Ganoderma and a Pleurotus into a single, what two nucleitic cell, you know, whatever kind of side cell, yeah. Is Like, where did you see something like that in my brain? It was only because Damon just gave me, like the shrimp of the woods. But you take that mushroom variety, and I think it's honey. Mushrooms are the ones that create this, and in nature, they kind of find one another. And they're usually that's when this, like weird mutation or abort comes in. Yeah, I can't remember. Can't remember the mushroom. Yeah, I get it, except with the lobster as well, yeah. And it's not really a periodization. I think if you actually, like, took those cells apart and did a which might be interesting with if we just cloned that, you know, and through a dedicatedization, what would we get in terms of cellular growth in there, would there be a honey looking variety, or a honey, you know, mon and a, I think it's Ecto, Ecto, whatever. Forget, it's blanking whatever the shrimp of the woods actually is in its binomial state. Would you get a complication of cells there? Because I think that's what's happening, there's an interaction, and possibly a mate that creates, like on board, it doesn't have any spore basidium producing structure whatsoever. It's just these like weird globular formations. And it's, as far as I know, the only papers or studies that have come out is so that there's some interaction there, where it only occurs, and you'll see all three of the mushrooms, so to speak. Oh, yeah, different stages. Yeah, that one's shrimp, yeah. So it's got something to do with the convergence, and that is kind of what we're getting at. We're talking about these things like interspecies breeding programs. Can aganoderma cell or mon mate successfully with a pleurode syringe on. And if they do, will that produce some sort of fruit body or mutation or abort, or is it just an evolutionary step? So, yeah, I don't know too much about that. I mean, it's weird to say a pure parasitization, yeah, because I'm sure it's more of like a Oh, thanks for breaking down all those nutrients. I'm gonna just take, because they don't write at the mycelial level. It doesn't seem like the shrimp is taking over the mycelial mat. No, of its reaction our other one, yeah. So it's like it's coming in contact with it, and then just going, Oh, fruiting body, and just following that. So it's, yeah, I mean, it's probably not pure parasitic. I mean, lobsters kind of look like because you get much eaten and stuff. There's like, an outer skin, yeah, I've never seen like, like a normal and then a lobster, right? Together together, it's always just all lobstered. Yeah, I mean, this is just making me think, because we're trying to do the tramella fukiformis stuff as well, and that's that interaction where, right people say parasitizes, but it does seem it's just more of the symbiosis when they meet each other, then it's almost like the tremella employs the high Fox lawn and even makes it into feathery mycelium. And so it's otherwise, yeah, they suck. So it's like it almost feels like they're employing it. And then the tremella is just kind of behind, like, Okay, go that way. Like, going that way, yeah. And it just kind of follows. So you just need the turtle thing. You just need that one little like wake up call to the tremella, essentially. And then, yeah, do they work together? Or does it make the tremella now active, right? Did what? What's in that DNA? Does it even have all the enzymes to break this stuff down? But it just doesn't use them all the time until it's go time for high Fox salon. And these are all questions we're trying to put out there, because they all inform Why monokaryotic work is important. It's not just like finding the best, most amazing strain combinations. Yeah, it really is like creating interesting observations and data sets to inform more research. You know, I guess I'm trying to think most of my cilium in nature would be mono, yeah, right. It's only we only see the fruiting bodies. But Well, this is where that effect comes in, because if a diary on forms, a mon comes along. It pulls it out, and it says, Oh, we've got another diary on over here. Like, I think this is very obvious in chanterelles. That's one of the questions I asked this dude, Thomas, or Thomas, I don't know how he pronounces his name, sorry, but this is the Clark University guy studying the tiger saw Gill. Oh, yeah, right. But it's, I wonder if these two forms form on the same log, that one has this sort of membrane over the gills and one doesn't. Are they actually just two sort of dikaryotic strands side by side, even though you would assume they would be the same, it's not like everything is the honey mushroom and, yeah, the massive, you know, whale that it is out there in Oregon or Washington or whatever. It's not all that, you know, that's a weird, weird instance. So walk us through the basic steps really simplistically, in terms of how we're going about dedicaratization. We're starting with a diary on and then we go from there, yep. So yeah, have our dye carry on, and ideally want to capture it on a typho end. So it's not just this giant mat that we're trying to take from because kind of the whole theme of this is little amounts seem to go a long way. So you have our dye carry on, then your however many Erlenmeyer flasks or mason jars that you want to inoculate and test, and then just your blender ball jar with your DI water or regular water. You run everything through the sterilizer, except the culture, of course, take it all out, cool it all down, and then you're just going to take, yeah, about a half centimeter cube from the edge of your your leading edge, the apical zone of growth, and you're going to take just, yeah, like a little cube from there, toss it into your now sterilized blender jar, blend it all up. And I've been having success at about 20 and 25 seconds, and that's just for plerotis right now. I've been seeing that success. Shishaki is still waiting on them to see. This hasn't been long enough. They take about a week to start globulizing. Anyways, blend it all up and make sure you can actually see it getting blended, instead of just saying staying as a chunk. So you'll see, like, the little flakes of mycelium and cell tissue just kind of existing. And so then you're gonna take your pipette, and I guess missed a step early. Meyer flask, just have 1% to whatever volume you're using, of glucose and peptone, yes. So small percent of nutrient because into this, like mon blob forming, yeah, yeah. So we're just doing a ratio of 1% by weight, or, I guess, volume to weight. So 100 mils, one gram of each. 150 mils, one and a half grams of each, right? That's just kind of what we're doing now, and that has been working, so not gonna, why mess with it? Yeah. And so that's what's inside of your flask that you're going to be inoculating. So blended, get your micropipette. And I've been doing five micro. Liters, and that's been giving me a good amount of colonies. You're welcome to do, like five ones if you want to try to grab from like, different spots of your ball jar. But I've just had success if you kind of, you see some chunks just kind of aim right in there, grab out your five microliters, toss it in the Arlo Meyer, yeah, give it a little swirl, and then just let it hang out. And yes, that's just how we kind of get it all started. The sterilize, the blend, the small inoculation, little shake, so that it's not just all together. Yes, they did capture two Mons, and if they're just stuck together, then you've now you're back to square one again. That's where I'm at, yeah, so that's I've noticed about after three days for the oyster mushrooms, you do start seeing like little balls, essentially, I guess they call them pellets when it's a submerged culture, is kind of the term people use, because you will see, once they get a little bigger, you will see like pellet of white in the middle, and then just kind of like the glob that forms around it, and that's all mycelium, but it's that part in the middle stops its growth once It gets surrounded, and then everything else is just kind of sending nutrients. And yeah, that's the one thing that's weird to think about in liquid cultures, is because everything, even fungi in nature, it's all osmosis, right? So if you're just a literal net, just hanging out, yes, sucking up the peptone and the glucose, it's just funny to think about, oh, I think that's why we're playing around with aeration in the liquid culture component, because you're trying to, like, relieve some of that stagnant state, yeah. But this is also an important consideration for when you're doing this dedicateization, you're keeping that Erlenmeyer flask very still, so that these colonies are just they're literally growing in an orb, blob fashion, because they all kind of grow out at the same time three dimensionally, and that's kind of this fascinating thing to try and recover. You know? It's like, when is enough enough? Pull it, you know, yeah. So not get foreign DNA on it, yeah. And so right now, after the three days, you kind of see, I've been seeing about like seven to 10 colonies per Erlenmeyer flask. And yeah, with the pipette, you can just go in, suck up one colony at a time, and then just place them around a plate. Obviously give enough space so that you can let them grow and not touch each other. Yeah. And so this is kind of the first, like trial step. So once you get your sorry, she's she's derailed me. She's like, once you get your work, once you get your image in my chest, scratch skin flakes all over you. So this part is just like, kind of like a testing to see. Is it just all dikaryons, or do we have a reduced in the amount of clamps? Because it doesn't seem like the clamps go away right away. No, it seems like it kind of takes it a sec, and then it's like, Oh, why am I even making clamps anymore, right? So there's only three of me, yeah, I do wonder if that's the, like, a weird interaction. If you've always been making clamps, that part of your genome is just turned on, right, but then you take away one so it kind of just goes less, Oh, that's interesting to know. Yeah. Well, I think you're right, because, you know, Julian talks about this in his protocol protocol, protocol, just a little bit and saying, like, you do this a few times, because you're always going to have some of the dikaryotic, you know, nuclei. There's too many. It's this is where that dominance thing comes in, where, like, there is a dominant recovery. But when you're trying to get a symmetrical recovery, therefore two bones from the die, you're really kind of playing with fire, trying to, like, isolate two things and get lucky, yeah, and see, that's the one part I don't like, yeah, that's sciences. But, I mean, that's a lot of science. It's unproven. So, yeah, it's kind of like finding those or and that makes sense as well, if there's just kind of one hanging out. But since we've just macerated it, it's probably not thinking about doubling up all its genetic material in the moment. Probably just wants to exist. So anyways, once you isolate a couple of the colonies, you don't want to grab all of them right now. Just grab a few, bring them under the microscope, and we're going to look for clamps, and we should see less, so like 30% and less was his, our Julian guy's goal, um, and, yeah, I mean, you'll, you know, if you look at a dye carry on plate, even at the edges, it's like, oh, plant, clamp, clamp, clamp, clamp. Here you are, kind of like looking. You're like, oh. That's clamp, yeah? And there's one so you will find them and then so, and you just kind of hope it's less as if it is just clamps everywhere. You got to go back to square one, yeah. And I think this is also where you take them, and you put them onto multiple dishes too, to really try and see how that colony keeps growing, yeah, if you want it, yeah, we'll form a diary on if there's still enough of the opposite nuclei, yeah. So yeah, that's kind of like you can do that from there, as in, oh, I don't know if it's a mon yet, but you're welcome just to keep kind of culturing those out. I haven't had success with that in terms of reduction in amount of clamps, but you do start seeing different growth, yeah, and it's not, but it's never like, Oh, that was that growth. That was that growth. Yeah, it is this weird spectrum that you kind of end up with, yeah, even though you're like, I only had two here, but yeah, so that's kind of like fun to figure out, and you can kind of keep an eye on it, because definitely some you look in, you go, Oh, those are the same. Yeah, yeah. Anyways, so once we've kind of confirmed that there's less clamps, we're gonna sterilize blender jars all again, one for each strain or vessel, of course, dump in that deed I characterization solution with your globs and blend it all again for another 2030, seconds, whatever you feel comfortable with. That's just kind of the spot I've found to work. Toss them back in the flask, let them sit again, and then kind of rinse and repeat from there. Yeah? And so on his protocol, he just does the two, and then he goes to isolate and monitor growth. But I'm sure you know, if you kept doing it, I think it's just he don't want to get over blendy, because then you're just causing so much cell damage. Yeah. Let's see the other question I have, and something we'll learn over time, I guess, as we do more and more of this dedicatedization is like, how much is too much, and how much cell death actually impacts the Mon? So then, when it's re bred with the other mon to create the original diary on how different is it? That's the biggest question I have. Is like recombination and then fall through a spawn, production, substrate, production, fruiting. What does it look like? Are the same traits still, there are original traits that were originally, say, off. You know how oysters, like lose their flavors? You sort of like take them from nature. And it's got to be just like a system of recovery for it's, oh yeah. Well, are you eating a wild oyster, right? Have you? Yes. So the smell and the flavor is very, like fragrant, right? At least to me. Every time I've I've harvested a wild oyster, it's like almost a minty aromatic, like we've only had the late oysters, okay, caps, yeah, yeah. So there's, there's a few different ones you'll find around up here in particular, like getting closer and closer, there's a DIA more, is there? Yeah, it's not anymore, but it's not there. Yeah, you ate it didn't Yeah. Well, not telling you why things are even when you were this close with somebody you know last year and it got taken from me, wasn't me? You're one of one of the three. It was you Ethan or Sam. Most likely. Sam is Mike. Sam's quite the taker. He likes the he likes eating mushrooms. He likes just taking things, take and ask questions later, exactly. That's why we need them around. Yeah, exactly. He builds in the forts. Where'd this wood come from? Don't worry about it. All right. So that sort of makes sense. That tracks in regards to, like, what the protocol is and how far you can keep going with it, yeah. And so I'm just doing the second blend and then monitoring after that, or reculturing after that. Cool for these kind of first runs, because just stick with the protocol, mess with stuff once we've done it. And yes, that's just kind of where we're at right now. My real curiosity, and something I really want to play with in this whole thing, is putting these mons on liquid culture, sort of program, similar to how we make liquid culture. For dikaryotic strains, and see what it does, how it grows, if there's differences, if there's adaptations to the recipe, or to the aeration, or non aeration. And I mean, we aren't growing them originally in a very like, yeah, a broth, so you might reduce some lag time even, yeah, you might be kind of interesting, yeah, yeah. It could be kind of a fascinating endeavor into, like, quick recovery almost, or it's almost like pre or post ddec characterization, mono. Know, capturing conditioning or something, where you recondition those monocarians Before storing them or something. But I also think blending two monocarians in a liquid apparatus or liquid culture to return into a diary on could be an interesting way to see how they re interact. Yeah. I mean, I think that would be a little better than plate Yeah, all the time, yeah, still just kind of be locked in and stay in there and take some generational moves. Yes, if you double, let them meet each other, hopefully they like each other, cut the middle. I remember you, yeah, oh, it's been so long. Yeah, it's so true. Yeah. What else this D decaratization stuff? People are really protective of this. I've seen in the community, and it's probably worth mentioning even some people like in the I don't think there's too many of you in the micro Wizard World, but a few people mentioned it when we did that, maundai Come on a sighting episode, there's a few people who are just like, almost gatekeeper, like weird where they're like, you know, if you're gonna do this episode on D Day characterization, you better fucking do it right? Oh yeah. Weird messages where I felt like I was, like, putting a fire to then have a conversation like a normal human. I mean, I guess I can kind of unders. If you're gonna sell a mono, you gotta have a lot of trust. You did it's a mono. Yeah, yeah. But also the problem, like, I think this person, an individual I'm thinking of, like, thought that there's a lot of people just talking about it, but nobody really knows what they're doing. And it's like, not a very complicated thing, but it is. It does have a morphology that's questionable, you know, in terms of its actual like effect, like you're saying. It's not hard science. You need to, like, very slowly tease this organic compound apart. And there's only two research papers that people reference, which is fucking crazy. I think you don't really get that these days. Yeah, I think that's what makes this whole like thing so exciting. Every time it's re brought up. I'm like, right? This is fucking new, yeah? Because yeah. I mean, the tons of videos on spores and monocarians are approaching into, like, the modern language that people are using, yeah? And they're talking about fungi and breeding and all that. But again, it's all back to the spore realm, yeah? So, right. I mean, I brought up, it's funny. I imagine everybody knows what I know. I try not to think anybody's, like, unintelligent or beyond whatever. It's just like, Yeah, can I talk to a monetary we're at this, this talk for this guy, Thomas, talking about these Tiger tooth, soggy, whatever's lentinus to Grannis, and I asked him that I was just like, are you DNA sequencing? You said you're gonna sequence this stuff. Are you gonna sequence the monocarians or dicarans? And he had a very quick explanation. But this kid out of nowhere is like, What in the fuck are you talking about? And like, all of a sudden he took hold and started explaining man die sort of concepts in a very different way, to which I was like, Oh, this guy, I don't think even knows about dedicaratization. He's coming at it from like, oh, any organism that is a multi nucleic sort of organism can have these cells. You can say, Go on inside it. Didn't say, mon, ma and die. Nothing of that. He was just coming almost at it from like a school script. And I was like, Well, that was cool. So he explained it to this guy in a way I would not explain it. And and then I talked to this kid afterwards, and I was like, you know, the man die thing is kind of interesting. And I think it just kind of like fucked his mind up a little bit. I was like, Oh, this is harder to grasp for something. Well, most people, you tell it's got two nuclei, yeah, they're like, What? Right? Yeah, it kind of throws you off anyways. And also, yeah, I don't know where the term carry on came from, but I've only heard it really mushroom so far, I didn't hear throughout college or anything interesting, yeah, so it also then makes sense, if, like, What are you talking about, right? It's just a foreign term. Yeah, terms are nice, but it can be too many. Yeah, if you're trying to talk to a general public. It's like, do I need to give a vocab list before? Yeah, and now you can talk. I love these glossary terms that are kind of antiquated now because they're on, like, dead end HTML websites, but there are a lot of term. Technology recaptures and then, like almost mutations, where you're like, What is this word? Even the word dedicaratization is always going to be a spell chunk. It's not a real word. You can call it a real word, but Shakespeare would be proud nobody else. So it's kind of cool. It's fringe information, snickering. Peanut gallery. Anyways, shit, that's a relatively clear and concise way in which I think this stuff works. Is there anything you don't think we covered that we should talk about in particular? Man, oh, man, what did you think of that episode? If you want more of this Micah wizardry, stay tuned, because we are turning up the heat. We are shooting for a release a week on Michael wizards.com and a release a week on Spotify, Apple, iTunes, slinger, Stitcher, Witcher, really, you know, wherever the fuck you get your podcast. So be on the lookout for that ear candy, because it's coming your way. This podcast is produced and sponsored by Maine cap and stem. Need cultures, spawn, substrate, equipment, raw materials, consultations. Reach on out to the amazing team of cap and STEM at WWW dot C, A, P, N, S, T, E, m.com, if you want to support this podcast, swing on over to Michael wizards.com and become a member of the largest culture bank on the fucking planet. Seriously, our team is focused on bringing you commercial, gourmet and experimental cultures from around the globe. So become a member of our culture club at Michael wizards.com today. Don't dig the flow hood work. That's okay. Come on in for the videos, the deep dives, the exclusive interviews and phenoflics. Yeah, that's what we're calling them, Motion Picture flicks focused on the phenotypes we are working with every day. Don't want videos. Don't dig the fungus on dishes. Just want to support this podcast. Well, shit, you can do that too for five bucks a month to keep this show on the road. Speaking of the road, I have to send a special thanks to the new and improved and magical and mysterious Micah wizards team, consisting of Mr. Michael Vance, Dr Daniel Ashley, as well as Tyler Crawford and the flow hood man Sir. Gregor Jones, okay, I'm Erik lomen. Stay weird, my mica wizard family, and hopefully we'll see you out there in hyperspace. Space, space, space. I like that effect. I.