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m̶a̶d̶ disappointed scientist Registered: 12/28/09 Posts: 3,398 Loc: the Neitherlands Last seen: 5 months, 18 days |
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I read they are not just weakly growing but also shortlived, how shortlived are we talking? Care to give an example?
![]() In culture how far can you transfer them and won't they be okay on slant or wedge? -------------------- My trade lists:
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Smurf real estate agent Registered: 04/30/13 Posts: 61,889 Loc: Milky way |
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No one probably knows the only people on the entire site who have claimed to have grown monokaryotic mycelium have just that only claimed to have done it.
-------------------- Bod's Library Article behind a paywall? I can try to get it for you
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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Quote: This jar of a monokaryotic PE6 isolate has been consolidating at about 21°C for nearly a month and its still perfectly happy. It amuses me, some of the myths surrounding monokaryons. They are not weak, microscopic, short lived, or hard to isolate or cultivate. In my experience they tend to be even more vigorous! Think about it, a monokaryon needs to find a breeding partner. What would be the best way to do that? Stay 20 micrometres and die after 3 days, or grow like mad all over the place and stay alive for months if needed? Look at these plates, these are Ps. mexicana var. Chicon Nindo from the bottom and the crossed out colonies are the dikaryotic ones: You'll note they are smaller. That's perfectly ordinary in my experience. Monokaryotic strains tend to be beasts. [You may also notice that some of the monokaryotic strains are forming stones, this thrilled me quite a bit and I'm now breeding the stone forming monokaryons with, well, everything! ]People always talk about how they must be difficult to capture. They say you need a very expensive microscope and some other, always unspecified, special equipment. Possibly involving robots. And they like to talk about manipulating spores one by one under magnification in a laminar flow hood. All you need to isolate and manipulate monokaryons is the very simplest, most basic equipment for agar work plus a microscope that gives clear magnification at 400X. Although I would strongly recommend petri dishes instead of jam jars. Take this plate, for instance: Two days ago I harvested a crop of 12 monokaryon candidates from this plate. All I did was streak it very lightly with spores and as soon as I saw an abundance of growth I transferred twelve relatively isolated ~1mm specks of very simple and uncomplicated growth to fresh plates using sterile hobby knife blades. Screw flaming things, I just have tubes of 10 sterile blades. When I use this method I have a 75-100% isolation success rate. The last plate was Atl#7 and 12 of 12 were proved to be monokaryotic under magnification. On this plate the spores were freakishly high in their viability, I only put a speck of spores on that plate and its covered, but I still expect to get at least 6 monokaryons. Proving that an isolate is monokaryotic is where the microscope comes in. You mount some mycelium to a slide and look at the cell-cell junctions for clamp connections. Dikaryotic strains have clamp connections, monos dont. The easiest way to get mycelium from an agar plate to a microscope slide that I've found is to set up a slide with a coverslip on top, put a small drop of dye off center on the slip, take a piece of clear plastic tape and just tap the sticky side against the mycelium so the mycelium leaves a 'thumb print' on the tape. Then stick the tape to the coverslip so the dye spreads out under it as you lay it down. Flip the slip+tape over and firmly press out any bubbles and excess dye. Don't let the tape slide across the slip or it'll damage the cells. Its not hard. I use crystal violet and methylene blue as stains. Methylene blue is dirt cheap. I've also heard of people using blue or black food dye. When monokaryons mate and form a dikaryon they start forming clamp connections and when a cell divides it forms a new clamp connection to the new cell, its how they make sure every cell has two nuclei. Heres a plate with two PE6 strains: The one on the left is dikaryotic, here's how the cell-cell junctions look under 400X: See the half loop connecting the cells? That's a clamp connection. The colony on top is monokaryotic, here's how the cell-cell junctions look under 400X: No loops. When checking, you'll want to look at a bunch of cell-cell junctions. If a clamp is out of plane then it can be invisible, and cells at the leading edge of a colony can be transiently monokaryotic. If I see no clamps I look around at other parts of the slide just to be absolutely sure. Also, if cells are damaged they can split and those splits can look like cell-cell junctions. If you have a microscope and have agar, please dont be afraid of monokaryon isolation and manipulation. Its fun! When you get one just grow it on agar, then use some mycelium water to inoculate a jar of grain. At 100% colonization shake it and inject some spores for it to mate with. Give them 10 days to have their fun before casing the grain or spawning to a minimal amount of substrate. The first generation will be a nice mix of the dominant traits of the two varieties as homogeneous in character as the variety that the spores came from. If you then grow spores from those hybrids, that next generation will be an explosion of diverse traits for you to select from. With a short list of traits your selecting for, and a good number of small grows in each generation, you can breed a new stable variety in as few as 8 generations. Better tripping through science.
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C'mon man Registered: 12/21/18 Posts: 3,775 Loc: A thought |
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Quote: If this were true, care to explain how exactly you are isolating jars full of monokaryon? I have a feeling im not the last to ask your process, and probably the last to do it politely.
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RIP Stoneman Registered: 05/18/19 Posts: 551 Loc: The Multiverse |
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Did you run on low nute agar?
Canadian Journal of Botany - Volume 54 - Page 72 Found inside - Page 72 ... (5) found incidence of clamp connections in dikaryons (a phenotypic character) to be adversely affected by low humidity and nutrition which could result in a dikaryon being mistaken for a monokaryon of Psilocybe cubensis (Agaricales). Edited by murderlabz (12/22/19 12:02 AM)
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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Quote: Haha, I understand your skepticism. I isolated and confirmed the monokaryotic strains exactly as posted above. To colonize a jar of grain I grew the monokaryote out on agar and then I dumped some sterile water into the dish and scrubbed the mycelia off into the water with a wire loop. I made that out of thick steel wire and heated to bright red hot and dropped in water to give it a rough texture. This mycelial suspension was injected into sterilized jars of grain with a wide bore needle. The above posted jar of one month consolidated monokaryotic mycelia came about because I made three jars of that strain, "PE6 MK4.1", and when I shook one prior to injection with PESH spores it turned deep blue, the next jar did the same before I injected KSSS, the third jar I kept so I can make it into tea to see if I can use a myceliated grain assay to test the potency of monokaryotic isolates prior to breeding them ![]() If I recall correctly I got my procedures from Stamets' "Mushroom Cultivator - A Practical Guide to Growing Mushrooms" Thanks Stamets, your awesome ![]() Quote: I've used from 1/8th strength MEA to the standard 2% MEA formulation. Low nute seemed to make it easier to see which tiny colonies were structurally simpler. Go after the cute little squiggles, not the spider webs.* * Edit: to be exact, thats my current practice. I have not confirmed if structural simplicity is an accurate guide to which are monokaryotic Edited by Elrik (12/22/19 02:56 AM)
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m̶a̶d̶ disappointed scientist Registered: 12/28/09 Posts: 3,398 Loc: the Neitherlands Last seen: 5 months, 18 days |
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Thanks a lot for the info
![]() I'm happy to see an open-minded approach. Most of the basics about monokaryons I knew already and luckily I do have the necessary equipment but it's cool to see how you do it. It's surprising that you are not at least using spore dilutions, I would think that just 'visually' picking up even a minimal speck of spores would give you dikaryons because they are so tiny and plentiful and also tend to clump together. Wasn't the combination of spores with a monokaryon the "shortcut" way Workman used to make APE? Afaik you need to add the spores to fully colonized substrate so that they dont get a chance to germinate, mate with each other and joining with other such fresh formed mycelia, before those reach your other strain. I'm confident you know what you're doing but did you mean that aside from checking clamp connections you have also seen proof of successfully achieving crosses, from those later generations? -------------------- My trade lists:
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C'mon man Registered: 12/21/18 Posts: 3,775 Loc: A thought |
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Quote: Sorry, glossed over the entire paragraph in a late night daze. For the reasons that I have read repeatedly I remain skeptical due to process as everything i have read suggests it much harder than that but ill wait for more experienced to chime in. I sincerely HOPE it can be that easy.
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Smurf real estate agent Registered: 04/30/13 Posts: 61,889 Loc: Milky way |
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Quote: So you think you have mono growth because of one micrograph without a clamp connection in the image? Lol Show me the growth starting with one isolated spore so i and anyone else can actually be confident. Heck even show me that you got spores appropriately diluted enough to even attempt your project. Spores absolutely love to clump up and im my experience and also having been a lab technician in a yeast lab pretty impossible to get mono growth without something like a micromanipulator Ive diluted spores streaked and by the time you see growth with your eye they're long dikaryotic. -------------------- Bod's Library Article behind a paywall? I can try to get it for you
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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Quote: No. Re-read my first post in this thread, I specifically say that it is important to look at many clamp connections and hunt around across the mount for multiple reasons. At times when I've made a less than ideal mount and seen 20 cell-cell junctions without clamps I've still made another fresh slide from a different section of the same colony just to be absolutely certain. So far its always been redundant, but I stand by my cautious approach. Quote: Okay, now your just joking right? For a strain to be monokaryotic it doesn't need witnesses. All dikaryotic Psilocybe and Panaeolus species form clamp connections. With the exceptions of the cells at the very leading edge of a colony and neohaplonts made by brutally mashing up cells, no clamp connections in your colony mean its monokaryotic. Those four pictures I took of those two colonies were not the only four cell-cell junctions I observed for those strains. They were more like the sixty-fourth ones I checked, as I had already verified them. Heck, I skimmed over at least 20 more on each strain when taking the picture because my cam tends to be grainy so I had to hunt for photogenic examples. Quote: Simple. Look up, no clamp connections! This isnt a fluke, I've got at least 40 petri dishes of strains of Psi. cubensis, Psi. tampanensis, and Psi. mexicana that are totally lacking in clamp connections. I've even mounted some mycelia from a colonized grain jar to a slide to make sure some lone spore didnt germinate very late and mate with it. Nope, still monokaryotic. I dont know what kind of proof you're looking for. No clamp connections is the definitive proof of monokaryotic status in these species. Quote: I guess the answer is 'practice more'? People here tend to take RRs advice quite seriously. He has repeatedly said that you can get monokaryotic isolates simply by swiping spores to agar very thinly and transferring growth before it starts to expand. This is mycology 101 Quote: That's how I felt before I tried. I was quite thrilled when I found out it IS this easy! Quote: I tried spore dilutions. I don't bother with it now. What I do is to use these yellow plastic 'disposable' loops I have and pick up just a speck of spores. Actually I get a speck on both sides so I can do two petri dishes with one loop. Anyway I kind of tap and streak that speck into a line down the center and then just turn 90° and scrub really thoroughly to expand out from that line. I don't even bother to do the 4 or 5 sector dilution-swiping method bacteriologists use. Look at the above posted plate of Psi. cyan. that I took 12 transfers from, you can see the thick band of colonies where I rubbed the speck of spores and then you can see the perpendicular lines of colonies coming out from it. I try to use less spores, that print was just exceptionally fresh and viable. Injecting spores into a colonized jar could be viewed as a shortcut, I suppose. Its simply practical, unless you really want a specific cross of two monokaryons. Then you either breed them on a plate or inject both together into a grain jar. And you are correct, its important to let the jar fully colonize with monokaryote mycelia before injecting spores. Clamp connections are what prove it as monokaryotic. Beyond that, I've never seen or heard of a rhizomorphic monokaryotic strain of a Psilocybe species so I take rhizomorphic mycelia as a warning that its almost certainly dikaryotic [but I still check] and I am thrilled when jars of fluffy monokaryote show their first little ropes of rhizomorphic growth 10 or 12 days after injecting spores. But the microscope wins the battle. No clamps = monokaryotic. Thanks for pointing out that workman also uses this technique. I don't know why people are having a hard time believing me when I'm in the company of Stamets, RR, Workman, and every mycology textbook in print. ![]() But I really was thrilled when I saw first hand how easy it is, so I guess any doubters will feel the same if they try. And then you can breed a new variety of your own!
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RIP Stoneman Registered: 05/18/19 Posts: 551 Loc: The Multiverse |
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Where is Workman, he didn't retire did he?
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Smurf real estate agent Registered: 04/30/13 Posts: 61,889 Loc: Milky way |
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At the very least stain the slides and show me cells with only one nucleus.
-------------------- Bod's Library Article behind a paywall? I can try to get it for you
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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No offense, it really is hard for me to tell sometimes, but I swear your just messing with me. As such I can see what would happen. I'd take a whole bunch of pictures at 400X, you'd look at my grainy cam pics and call two mitochondria leaning against each other a nucleus. Then I'd use the 100X oil objective to really look inside a cell and you'd say 'well ok, theres one nucleus but I can only see 1/100th of the cell.
All while your smirking because you're most likely just messing with me. Sorry, I have mild aspergers and I really cant tell some times, especially on forums. So instead of wasting an hour photographing one cubensis cell I opted to address another one of your valid concerns. Spore clumping. I recently swiped a plate with some cube spores that were desiccated hard or something, they didn't come off the print easily which often makes lone strains harder to locate. A prime candidate for troublesome clumping. These pics were taken at 40X through a petri dish. I love my microscope ![]() In this pic we are adjacent to a main swipe line, as you can see at the bottom, but even right next to it you can already see spores thinning out into pairs and singles. Then off away from that main track on three of the cross-swipe lines we can see individual spores and spore pairs lined up at respectable distance to eachother. Now, again, this is not with a bacteriologists five sector dilution-swiping technique. I just scrubbed a speck of spores into a stripe down the middle of the dish, turned 90°, and scrubbed in the other direction all over the dish. Now, keep in mind, that even with fresh prints it is entirely normal for only 1% of spores to be viable. Moreover, spores from the same print tend to have a fair bit of self incompatibility. Something of a minor incest taboo where its common for 50% of monokaryote strains to be incompatible with one another because of lack of diversity in their gender alleles. So sometimes two or even three spores can germinate together and still not be able to mate. Bodhi, your an influential dude with lots of grow experience. If you have the gear to do agar work, a fair microscope, and the inclination I really think you should try again to develop a good monokaryote isolation technique. You don't have to believe me. Make your own, test them, and breed them and then see for yourself!
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Smurf real estate agent Registered: 04/30/13 Posts: 61,889 Loc: Milky way |
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Look how close those spores are in that second picture its 400x they're within one field of each other. how in the world did you grab that before it was naked eye visible. You would need microscope aided scalpel work. So yes now im even more sceptical than I was originally given those pictures.
I gave this same project a shot for a long time. Those monokaryons send out chemical Messengers to meet each other. You can't grab threads of mono growth soon enough without the aid of a scope. By the time you see growth they've long linked up to dikartotic growth The tip of your scalpel takes up multiple fields of view at 400x -------------------- Bod's Library Article behind a paywall? I can try to get it for you
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C'mon man Registered: 12/21/18 Posts: 3,775 Loc: A thought |
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In other words, I'm picturing those "respectable distances" to be seperating spores by about the width of a hair?
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Stranger Registered: 09/03/09 Posts: 433 Loc: USA |
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He was right in saying not all spores are viable.
Quote: If spores were streaked thinly enough that in sections just 500 are in a square centimeter and less than 1 in 500 spores are viable I can see the plausibility of being able to locate a few just as they become large enough to see. I dont see the problem with his argument
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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Quote:Oh how I wish I could get 400X through a sealed petri dish! I'm sorry, I may have been unclear, probably because I made mention of my oil objective. When I said the plate pictures showing the spores were at 40X magnification I meant 40X total, 4X objective lens and the 10X eyepieces. Although technically my camera gets a little closer when I stick it into the scope. Those pictures of spores are 1mm tall. The image showing 5 possibly viable spores in a 1mm x 1mm area was not even the most sparsely populated area, it was just a representation of a thinner region of the cross-swipes. I can find areas with just 1 spore, or none. But even at 5 full size spores per 1mm2, that would average to 500 spores per 1cm2. Of which one or a few may be viable, at best, according to Auxins quote of RR. I try to take transfers that are 1.5x1.5mm, try, usually its 2x2mm. I get them when they are just a barely visible squiggle and cut a square around them with the tip of the hobby blade and then cut the agar just under the surface to get a micro-wedge. If there's a 2x2mm squiggle in 1cm2 that really isn't such tight tolerances. I don't know, maybe doing precision mill work just got me used to such things but its only a skill of patience and calm. Edited by Elrik (12/22/19 11:34 PM)
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Seer Registered: 04/13/17 Posts: 148 Last seen: 5 months, 8 days |
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I have regularly been getting Dikaryon isolates in one transfer using swabs and cross steaking. The mosaic of inhibitory zones makes it very easy. Colonies are isolated enough that with a bit more work I don't see why monokaryon isolation would be problematic.
Also, regarding petris on a scope, my friend uses agarose poured very thin so it's almost clear, and views plates inverted on their scope. A sucrose based recipe helps to keep the agarose clear.
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m̶a̶d̶ disappointed scientist Registered: 12/28/09 Posts: 3,398 Loc: the Neitherlands Last seen: 5 months, 18 days |
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Doing the "stripe" and then turning the dish and spreading spores again is basically a known streaking technique only they usually do a little more than one swipe before turning and turn by less than 90 degrees but repeat the step another time.
It's basically doing dilution right on the agar. I still think it could be useful to make a spore suspension first and volumetrically dilute a bit to get closer to your ballpark spore count range before switching to streaking to narrow in. Spore clumping can be helped with a small quantity of a surfactant such as tween. I don't think i would use just any detergent but suspect some classic soaps can work fine. Lube iirc was stamets' old suggestion and glycerol should also help. Glycerol plays biological roles tho so i try to avoid using it too directly. Using a micromanipulator should definitely not be necessary, maybe it was routine for bodh where and when he did it and it could offer certain reliability of routine work or expedite certain parts but if there are beliefs that you need things so fancy it must be an old one. You are of course respectable and know a whole lot but its best to keep an open mind about what we don't know and recognize that science is provisional and the developments in mycology can be great as well as changes in widely held beliefs. Hold your beliefs lightly. At the very least it is common sense that unclumped spore suspension dilutions can give you a spore count/concentration in the order of magnitude to easily give you separated spore germinations on agar and monokaryons. Just the power of volumetric measurement. A little off topic but abuot that mosaic pattern: I have seen it before with I believe monokaryons / irradiated spore cultures and more recently in a thread about mycoviruses. Do you know what jagged edges indicate? Anyway i understand enough of this but just am trying to find the easiest way for that first step to get your ballpark without having to do a lot of work to check different dilutions. I figure it could be easiest to just make a suspension of a very small amount of spores and then just in case a 100-250x dilution of it and use streaking to do the rest of the 'diluting' and zoom in. Having tried that as a 'calibration' or reference i figure all you need to be able to do the next time is like eyeball an amount of spores with about such a 100-250x error margin. Viability of the spores etc is not factored in but some of this will inevitably come down to routine / experience. -------------------- My trade lists:
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Zapologist Registered: 11/27/19 Posts: 166 |
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Amazing what gets forgotten over a decade lol. Both Workman and the_chosen_one discovered their first mono's on agar by shear accident. Not two weeks apart. Both were identified as a strange looking random germination off to the side of the plate. Both were able to isolate them and use for breeding projects. In order to produce different mono's Workman pursued streaking methods while TCO practiced serial dilution. Both had positive results more than once. It was easy enough for them both to get bored with it.. TCO had a few grain jars populated with monokaryon mycelium.. so Elrik, I believe you. I hold a little skepticism simply because it's the internet, and even those guys admitted mistakes from time to time. But your confidence is a good thing. We also need that skepticism from the others. Debate makes good science. I'm looking forward to seeing your crosses.
Solipsis, avoid the detergents for serial dilution. Although you really grabbed my attention with the Tween lol. Never thought of that one and I use the stuff all the time in plant tissue culture. A teaspoon of agar per gallon in your dilution water will work well and safely. This is how PF (RIP) made all those beautiful non-clumping syringes back in the day.
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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My first one was accidental too, when I was fighting to isolate cube mycelium away from mould. I didn't even recognize what it was at first, just that it was fluffy, aggressive, 100% non-rhizo, not sectoring, but clearly cubensis. In the end 60% of the isolates I got from that print were monokaryotic simply because there was so much mould! To actually grow the mushrooms I just put all cleaned dis and monos on a plate together so I could mix them for injecting into jars.
My Psi. tamp. 'Pollock' was similar, the print was very bacterial. I wasnt trying to isolate monos, just to get clean tamp transfers. I put all my isolates under the scope hours before I took 9 grams of cubes, seeing that I had managed to rescue quite a few monos made that one seriously enjoyable trip! It wouldn't surprise me if unrecognized accidental monokaryotic isolates are not uncommon. Some of those non-fruiting strains people find on agar may simply be monokaryotes. It could even add to the explanation of why rhizomorphic growth is a sought after trait, because sometimes those fluffy non-rhizo isolates just wont fruit. Because some are monokaryotes
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Shedding... Registered: 07/30/18 Posts: 413 Loc: Europe Last seen: 1 year, 10 months |
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Wow, this is really interesting. Right now I also have a culture of cubes on Petri dishes from a 1.5years old spore print, which I streaked very diluted (many nonviable spores+dilution). It stayed tomentose even after 5 transfers but looks healthy and very homogeneous. Still, a dish which by now is about 2 months old didn't pin yet. I have two jars of it which are completely colonized, now I'm very interested to see whether they are going to produce some fruits or not... Too bad I don't own a microscope
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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Quote:I know what I would do. Spawn one jar out and try to fruit it. If it doesn't fruit in a month shake the second jar and inject some spores from something else, give it 10-12 days, and then spawn it 1:2 to coir. Colonized jars can sit there consolidating for quite a while and still perform when spawned out [Link]
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Seer Registered: 04/13/17 Posts: 148 Last seen: 5 months, 8 days |
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My streak method is to load a swab with minimal spores from a print. Around the center of the plate, I make a circle of looping motions in the center. Then I rotate the swab to the clean side, and zig zag the full width of the plate, starting at the center and going all the way to to top of the plate. Then from center to the bottom. Then I rotate the dish 90° and zigzag from top to bottom. If I were more on the ball about transfers, I could probably catch monos on demand. I have several plates I suspect are, but they sit in my fridge because I have more pressing projects (dedikaryotization to back-engineer clones).
I do want to see if the mitochondria have any significant influence on fruiting though. I'll be generating monokaryotic cultures from spore to make sure the mitochondria is intact, then test compatibility and cross. After nuclear migration, cultures will be separated and confirmed for clamps and run side by side. Edited by Muad.Dweeb (12/23/19 11:25 PM)
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Mycelium Expander Registered: 05/25/11 Posts: 955 Last seen: 1 year, 11 months |
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I just use the scalpel handle, flame it, let it cool down then just barely touch the spores and swipe. Managed to get lots of monokaryons that way.
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m̶a̶d̶ disappointed scientist Registered: 12/28/09 Posts: 3,398 Loc: the Neitherlands Last seen: 5 months, 18 days |
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Hey guys,
thanks that is very encouraging! I was on a little hiatus :} idk if its really that things were forgotten in a decade or however long it was. I am just quite on my own and not really that in touch and i wasn't finding the Workman stuff i was looking for.Also just interested in engaging in discussion about current thoughts etc about the matter. What about the surfactants such as tween? does it have a bioactive effect on germination? Thanks for the tip about agar. clever! Just today i was hypothesizing to a friend about possibly germination may very well or likely involve various chemical-binding receptors since isnt this how it always goes basically? with ratios of usually mostly polysaccharides but importantly proteins working togethers. So my idea was: isnt the reason for activated carbon to help in germination generally (aside from mycorrhizal benefits) that spores adsorb to the AC and this messes with these proteins on the exospore structure which may improperly trigger germination? Anyway that's another thread. Still i'm curious if tween is bad because it more negatively interferes with those proteins or shields them from detecting anything at all? Just a little bit too much clingy clingy enthousiasm. The dedikaryotization seems very interesting by the way and something i wanna work on with a friend, but it seems mostly useful for sporeless cultivars and rather long term advanced breeding projects. But blood, idk your method vs the textbook streaking method of swiping, turning 60 degrees, streaking out the end bit of the previous pattern and then repeat one more time. Or another 2 more times perhaps, not textbook but i wouldn't arrest you for trying that. Your way definitely spreads stuff around but not in careful steps of order of magnitude. I'll consider all of these mentioned methods and implement what i will after i am done with maintenance. Also i have to do a quick grow of a species to get its spores. -------------------- My trade lists:
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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Concern was raised over the size of mycelial colonies needed for visualization without a scope and practical transfer of colonies to fresh plates.
I have some plates germinating right now so I took this picture of a colony I would transfer if doing it today [I'll wait until tomorrow so more spores can sprout first, hopefully in less spore-dense areas]. This is the size of colonies I can see with my naked eye, the image is of a region 1,13mm wide and 0,85mm tall and encompasses the whole colony. Sorry about it being blurry and not dyed, its still sealed in glass. My transfers are kept as close to 1mm X 1mm as possible, but in practice they tend to be more like 1,5mm X 1,5mm. Tiny, tiny wedges are key for high success in monokaryon isolation in most cases. Though not all. If you have a scope you should check any isolates that stay tomentose, even if you weren't seeking monokaryons. You might be surprised.
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Stranger Registered: 11/09/19 Posts: 406 Last seen: 10 months, 17 days |
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so I'm just barely getting started in this game, but my sense is that there is something about yeah, groups of spores aiding in each other's germination. an entourage effect of sorts, whether it's the actual physical aspect of them being next to each other causing capillary action between them to draw water out of the agar/whatever surface they're on, already germinated spores waking up other spores, or what. I'm tracking several "isolated" spores (within millimeters of each other) daily on a dish, though I haven't seen a peep from any of them yet (it's only been 5 days). have any of you working with scopes and searching for monokaryons seen a single spore under the scope and come back later to actually see mycelium sprouting forth? It seems possible something could be added to stimulate them in the same way being around a clump does, like Solipsis says, some proteins or polysaccharides to tell them to wake up and get to mating, there's water, AND genetics available nearby!
these are the 2 spots I'm tracking right now, the single spore has been isolated to it's own dish, the other 5 are on their own as well, though having read RR's statement on spore viability in the months post print I'm growing doubtful any of these random 6 out of thousands will germinate:
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Smurf real estate agent Registered: 04/30/13 Posts: 61,889 Loc: Milky way |
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RRs statement about spore vitality seems to be completely wrong in practice. I've witnessed nothing remotely close to what he said for spores.
-------------------- Bod's Library Article behind a paywall? I can try to get it for you
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Stranger Registered: 11/09/19 Posts: 406 Last seen: 10 months, 17 days |
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gotcha, good to know. guess I'll keep checking back on them and a few others then.
finally realized I can get the 10x objective down comfortably by...flipping the plate upside down ![]() better pics to come
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m̶a̶d̶ disappointed scientist Registered: 12/28/09 Posts: 3,398 Loc: the Neitherlands Last seen: 5 months, 18 days |
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Actually Elrik.. while i have been generally enthousiastic and believe in a broad sense that one can get monokaryons since there are also just too many people working on this stuff who do have training and meeans of verifications... that post earlier about the PE6 strains did prove nothing i believe, on the contrary.
It would be good to actually see two mating monokaryons or at least a mono and dikaryon mating, and not the sort of absence of evidence is proof kind of thing. TBH on a hunch i did try putting two strains of Cordyceps militaris together on plate.. and while i am tired and lazy now i dont mind showing the plate. I have been back and forth about it and now realize it has not mated despite one spore culture looking a lot like a mono - and with the weirdness of Asco's i admit i dont understand i thought fuck it i wanna see what happens. Well a bit less inhibition between the culture happened but still no real action, they just kinda alpha blend into each other but i see now its nothing. That has nothing to do with dilutions and streaking however i was just playing out a hunch/curiosity. If nothing else, mating a mon-mon or di-mon decently is something i suggest is a challenge for us all, with reasonable means so not micromanipulation or anything like that. I realize not all mon-mon or di-mon may even be compatible per se but i think that would be whats to focus on and not just indirect evidence of having a monokaryon, regardless. I am still waiting on some fruiting to gather the spores i need to start on real crossing experiments. Not cubes btw. Just trying to keep this in the middle here, all sides get skepticism. This is about the science and not about conviction. So let's science each other better. -------------------- My trade lists:
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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I truly don't get why people think this is prohibitively advanced.
I will freely agree that, for hobby mushroom growing, isolating and breeding with monokaryons is advanced work and I will not fault anyone who doesn't choose to go that far. But in true mycology, as a scientific discipline; isolating, verifying, and breeding with monokaryons is beginner level fundamental work. Its whats taught in the first year of college. Literally. I sadly never took the wild-eyed lunatics classes but I knew my colleges professor of mycology and this is exactly what he taught to first and second year college students in the lab course of his beginner mycology classes. Now, I do at least partially agree with the spirit of your point. I dont know why you have the impression I have only indirect evidence I was even working with a monokaryon. As I said before. Be cautious, be suspicious, be attentive and aware, re-confirm everything. But breeding different varieties within a species is as easy as you can get. When I made two crosses with PE6 I wasn't trying to scientifically prove that monokaryons exist or that Psi. cubensis can cross with Psi. cubensis. I was just making a cross. It worked too, every shred of evidence indicates they were valid crosses. There are times for careful mon-mon crosses on petris. I'll give some of my own examples: •I recently isolated and thoroughly verified three Psi. mexicana 'Chicon Nindo' monokaryotic isolates numbered 1.2, 2.1, and 3.4. I bred these to each other on petris. The first two formed clamp connections with 3.4 but 1.2 and 2.1 would not cross with eachother indicating sexual incompatibility, so now I know that. And now I have 1.2 X 3.4 F1 and 2.1 X 3.4 F1 that I can grow out. If 1.2 X 3.4 yields better or is more potent than the other than I'll know that the monokaryotic strain 1.2 is better for breeding than 2.1. Stuff like that is a good use for mon-mon crosses. •I recently [before verifying the Chicon Nindo 3.4 isolate] also isolated three Atl #7 monokaryons. Unlike a cube X cube cross, a Psi. mexicana 'Chicon Nindo' X Psi. tampanensis 'Atl#7' cross would be news! I was also very dubious about it even being possible. I crossed my two Chicon Nindo monos with my three Atl#7 monos by every combination. Sadly, even after a good amount of time, no clamp connections formed at the interfaces of any of the six colony pairs. No cross. If I had got clamp connections on a pair I would have redone that test after intensive re-verification of the monokaryotic lines and I would have grown out both resulting crosses scrutinizing them for intermediate or mixed morphology with as much suspicion as I could muster. To be fair, another recent incident shows why I am emphatic about being cautious and reconfirming monokaryon status and why I put more work into verifying them than many would. It also shows why I am sympathetic to skepticism: I was reviving an old Transkei print with the hope of hunting monos as well as starting up a few jars. I streaked 4 plates thinly. Spore germination was horribly low and I wasn't home in the right window to catch any of the germinated spores just as they become visible, as I like to when hunting monos. Fresh spores really work best for finding monos. So I just let them grow. I soon had 1cm colonies that looked like potential monokaryons on plates ready to be turned into inocula for my grain jars. I mounted some mycelium from the colonies and saw that they were monokaryotic, I couldnt find clamps anywhere. Bonus! I took two transfers each from the healthiest looking three seemingly monokaryotic colonies. But as I said, be suspicious, re-verify. I waited. Both the transfers from one colony soon turned dikaryotic, then one transfer from the second colony did, both transfers from the third colony stayed monokaryotic. I'll never know if a few spores germinated late on the spore-laden transfers or if the colonies were just beginning to undergo dikaryotization and I just wasn't lucky enough to see one of the sparse clamp connections as that occurred. I'm still going to re-re-confirm them before putting them to slants and grain. When I say hunting and taming monokaryons isn't hard I don't mean it can be done with frivolous or poorly developed technique. Don't let your guard down, monokaryons are as sneaky about sex as teenagers. But it's not hard. It just requires skill, precision, and lots of attention.
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Stranger Registered: 11/09/19 Posts: 406 Last seen: 10 months, 17 days |
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so it is possible:
I have 4, maybe 5? single spores on their own plates now. The one above (Plate #4) is the only one to have germinated, but they were printed and streaked Sunday 1/12. lets see what happens
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Stranger Registered: 11/09/19 Posts: 406 Last seen: 10 months, 17 days |
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...or is it?
not satisfied with being unable to see the spore/growth, I dicked around a bit looking for it this morning and found it - still ungerminated, which is clear at 100x: so I guess we wait.
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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You're braver than I.
When over 10% of spores are viable I'm shocked. When 0.1% of spores are viable I'm not surprised. When 0.01% of spores are viable it must be monday. I wouldn't put one spore per plate. I might put 10 spores on a plate in a grid if I had a laminar flow hood, fresh spores, and nothing to do. Mycelium doesn't need to have a witness to be monokaryotic, it just needs to consistently have a total absence of clamp connections through multiple transfers. The smaller the wedges, the better. To find things on petris it helps to write on the bottom of the petri with a super-fine point permanent marker. Just use 90% isopropanol to clean the markings off if you reuse. On a crowded plate that's a great way to point out which colonies you intend to transfer. When starting stone formers I did MS plates, waited until baby stones were forming, and put elongated arrows pointing at my targets like: ---->-----
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Stranger Registered: 11/09/19 Posts: 406 Last seen: 10 months, 17 days |
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i still feel compelled to witness it though, for my own probably insane reasons. im just going to leave these plates alone for a while and if i come back in a week and there is mycelium, great, if not, no big deal. maybe I’ll try again next time I take prints of something. either way it took like ten minutes total between streaking and making transfers. seems easier than properly looking for clamp connections or obsessively checking plates. without sampling and staining and 400x magnification ive never been able to see them well enough anyway.
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C'mon man Registered: 12/21/18 Posts: 3,775 Loc: A thought |
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I was having a thought about this the other day, concerning isolating spores with reasonable distance between them in single form.
I'm wondering if there's a better way to do this right from the start rather than using prints. Here's a thought I had. Rather than taking a cap that is sporulating to make a print, why not place it Gill down on an elevated small grill of some type maybe 3" above your work surface. Given the amount of spores dropped by a cap in short time, maybe taking an agar plate and sliding it under the elevated grill for a short period would catch a few in their natural Cascade out of the cap, I assume they fall out much less clumpy than when in a print and possibly solo much of the time? Some experimenting may need to be done as far as timing is concerned, like with a steadily dropping cap, maybe you would only want something like 1-2 seconds of exposure under the cap with your plate. I feel like this would be a fairly easy way to keep things clean as well since there is no direct contact other than the spores themselves.
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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That's a very good idea to try. If someones technique is good enough that taking several transfers from a plate generally wont contam it, I don't imagine just holding a cap over it for 10 seconds would be a problem and, theoretically, it should lay spores down quite well if it was sufficiently active in sporulation.
My main problem is I usually start from a newly acquired print that I haven't grown out yet, so I can develop the monos while doing my first grow of the variety. Sometimes I'm lucky and the print will be new and essentially undried, the spores act like moist flour. Often the print will be desiccated dry. Great for long term storage, but its like trying to scrape spray paint from foil. Today I'm looking at a few very promising PE colonies. PE genetics will be improved! ![]() ...I just had a wacky thought. Isolate several monokaryons from a given variety, say PE, get them colonized to grain, and then inject with spores from that same variety. I haven't actually seen anyone doing that. It wouldn't be a cross, but an isolation of one half of the genetics. You could then investigate the properties of each group and if one was better than the others you could 1) clone and isolate a great strain, 2) print caps and go from there, and 3) know which mono was better for outcrossing to different varieties. All at once. Dude. Shit, yes. I'm trying this!
Edited by Elrik (01/19/20 11:50 AM)
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Stranger Registered: 11/09/19 Posts: 406 Last seen: 10 months, 17 days |
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im confusing myself with how much ive included in this thread vs my private journal where ive been keeping track of this process but i did something similar in theory to what you posted, smartattack -
“ The less successful plates I lightly drew the loop on the spore print in the same manner, but then I held the loop from above the plate, and used the tip of a flame sterilized scalpel to pull back the loop and then allow it to snap, the idea being a scatter of spores would drift on to the plates. I don't think I let the air hang long enough though for any spores to land on the surface of the agar, and scanning the surface of the plates for spores that could be literally anywhere ended up not being very fun so I'll be focusing on the light streaking method for now” the method i use instead is: “ I lightly drew my loop across some of the areas of my print with a finer spore load, then carefully turned the loop so that those spores were facing "up" on the loop. Then I streaked the plate with the bottom side of the loop, so that the movement would more lightly draw spores off of the loop and increase my chances of finding single spore locations to cut from later. These were very sucessful, I found several locations with individual spores.” this last time i streaked back and forth across the whole plate, then again perpendicular to that streak. it left grids of streak lines that helped me extract the spores that much easier without magnification once they were identified and marked with sharpie live print dropping is something I’ll probably try at some point but i’d be nervous about all the other shit that would fall on to the plate with the spores that cant be seen at 40x on the plate under the scope. with the light streak method I’m gonna have several spores i can transfer p much right away, plus I can just scan along the streak line when looking for spores. the spores arent always visible in the same plane so without the streak lines to give you a sense of your depth of field its difficult to find anything but the largest clumps under the scope. my hypothesis about all of this is that because spores rely on hydrophilic sugars to attract water to the basidium and collect enough weight that they can fall off the gills, they will more often fall in clumps and the mechanical action of drawing the loop over them will break them up. moving them back and forth rapidly allows the dryer spores to flitter off on their own on to the plate. I have absolutely zero scientific documentation of this but my intuition says that there is chemical signaling going on that tells spores that are in contact with one another that there are pairs to be made in short distance from them, as well as capillary action and the hydrophilic sugars to provide moisture and an energy source to the growing spores. im kind of thinking that having to plant that one upside down may help in the long run of tricking the spore in to thinking it will get the necessary genes to make fruit with right away
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Stranger Registered: 11/09/19 Posts: 406 Last seen: 10 months, 17 days |
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soooo I think I'm going to put my project on this on pause - I just don't see myself having the time to really dedicate myself to it at the moment. It also seems like just selecting fruits for the traits I'm looking for will be way easier to get the ones I want.
Something I was thinking about though, if anybody else feels ambitious, is to make a boat load of prints, and then put the spores in to an agar recipe. They'll be deactivated during the PC cycle but the sugars and chemicals should be preserved and hopefully help initiate germination for spores put to the plate, IF that really is a germination mechanism. Anyway.
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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Yeast extract is often used in agar to give the nutrients needed by fungi. Mushrooms eating dead yeast is like people eating monkeys.
It is conceivable that mushroom spores could convey some growth factor that yeast might not. It has been demonstrated in genus Agaricus that growing mycelium can emit volatile hormones that promote spore germination on the other side of the agar plate. Its possible these hormones might be produced better by monokaryons. When clearing plates I've noticed that cubensis monokaryons often have a more fruity or almondy odor than standard dikaryotic mycelium. But I haven't tracked it to see if that really is a monokaryotic thing. Edited by Elrik (01/29/20 12:01 PM)
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Stranger Registered: 11/09/19 Posts: 406 Last seen: 10 months, 17 days |
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oh that's interesting! I've got all these split plates hanging around, maybe when I pick the project back up I'll use some of those with that purpose in mind - cube myc on one side, single spore on the other
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Shedding... Registered: 07/30/18 Posts: 413 Loc: Europe Last seen: 1 year, 10 months |
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My experiment did not turn out very conclusive unfortunately.
What I did was take two cultures which I expected to be monokaryotic since they grew from ~1.5 years old sporeprints and they stayed very tomentose even after ~6 transfers and never showed any kind of rhizo growth. Even after changing for example from MEA to grain water agar and increasing nutrient content. Here is what I started out with, two different cultures but from the same spore print Right before they grow into each other They meet! I was excited whether this change in structure would actually now indicate the "mating" of mycelium and it would start to show some rhizo growth A couple of days later it's not very conclusive There's definitely some rhizo structures forming at the boundary, which these cultures never showed before. But I cannot exclude that this might be caused because they became stressed when "mating" or reaching the edge of the agar plate? Do you think it would be interesting to take a small wedge from the center where it's still tomentose and one from the rhizomorphic edge and try to grow them out? Or has the full culture now become dikaryotic in case this really happened? Just for comparison this is how other cultures from this spore print can look like, from a pinning agar dish which showed almost the same rhizo growth
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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Without a microscope you'll likely never really be sure the tomentose isolates really are monokaryotic.
If I were to attempt to tell if they were I might colonize two grain jars with each and hybridize one of each using spores, then try to fruit everything. True monos would never fruit, but crosses with spores should have no trouble. Dikaryotization should proceed through the colonies if two sexually compatible monokaryons meet. This isn't instant, it takes time. This plate comes from crossability tests I did with confirmed monokaryons I derived from a PE6 bloodline that isn't very rhizomorphic: After they grew together and had their fun for some time I confirmed that clamp connections were present at the union between the two colonies. I then re-wraped the plate just to see what they would do. As you can see, pins formed along the outer border of both colonies indicating that the dikaryotization spread all the way around. Neither ever did go rhizomorphic, damnit ![]() Some day I might refine this PE6 bloodline.
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Logs, wood chips & sawdust Registered: 01/28/20 Posts: 26 Loc: Kijimi |
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Elrik: Thank you for posting this and responding with so much detail. I am really enjoying it and learning a lot!
-------------------- If you are interested in trading mycology club tshirts, or want to help me buy one from your club, send a DM my way
Edited by BabuFrik (07/14/20 04:10 PM)
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m̶a̶d̶ disappointed scientist Registered: 12/28/09 Posts: 3,398 Loc: the Neitherlands Last seen: 5 months, 18 days |
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Yeah your contributions are much appreciated!
I recently streaked Pleurotus citrinopileatus to try and get monokaryons but not sure if i did it quite right. If you do 3 streakings and turn the plate, arent you supposed to get rid of the spores on your loop and only cross your previous path a little to achieve the dilution? Otherwise if you accidentally had too many spores on your loop idk if you lose enough of them when you drag.. I did my best with that and should just review the protocols and methods well.Will read these posts better too - just really busy. -------------------- My trade lists:
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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What you describe is the method used for bacteria.
I've heard of it being used for mushroom spores. Numerous times I tried it, swiping a line on one side, turning the plate and swiping a more or less over-lapping path, and again, etc. until I had either a square or pentagonal layout. Always I had lots of germinations on the first path, a couple on the second, and nothing on the rest. For me spores just don't act like bacteria and all I have to do is spread out a line I rubbed down the center. Try out different methods and see what works for you. My last hunt was another success, I now have six tentatively verified Penis Envy T2 monokaryon colonies and a tub of PE6 ready to go into fruiting to give me clean spores. Back-cross is officially in the works!
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Stranger Registered: 11/09/19 Posts: 406 Last seen: 10 months, 17 days |
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yeah, I could see that method working for spore syringe solution maybe but mostly is for bacteria in suspension.
The method that worked for me: "I lightly drew my loop across some of the areas of my print with a finer spore load, then carefully turned the loop so that those spores were facing "up" on the loop. Then I streaked the plate with the bottom side of the loop touching the agar, so that the movement would more lightly draw spores off of the loop and increase my chances of finding single spore locations to cut from later. These were very sucessful, I found several locations with individual spores. this last time i streaked back and forth across the whole plate, then again perpendicular to that streak. it left grids of streak lines that helped me extract the spores that much easier without magnification once they were identified and marked with sharpie" I verified the individual spores with a scope, but if you don't have a scope, basically if you cant see any groups of spores with the naked eye it's probably worked well.
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m̶a̶d̶ disappointed scientist Registered: 12/28/09 Posts: 3,398 Loc: the Neitherlands Last seen: 5 months, 18 days |
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So in the meanwhile thanks to info from this thread, I have been streaking spores and with P. caerulescens have gotten many monokaryons from 2 dishes.
9 in fact although I only checked 3 under the microscope and couldn't find clamp connections for the life of me, whereas with some other dikaryon culture it was easy to find them. I transfered 9 monokaryons and put them in sets of 3 on plates. On a plate i got them from though it does not appear like there have been any successful matings among them yet. Does anyone know if ALL Psilocybes are bifactorial (so 25% chance of matching mating types)? And can someone show me a plate of successful mating and what that looks like with the resulting sector and the lack of a dead demarcation zone? -------------------- My trade lists:
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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These were confirmed cubensis monokaryons made to mate
Somehow I still have that plate over 2 months later and that line of separation between the two is still visible. Oh, Alan has said nearly all Psilocybes are bifactorial. I never found a list of exceptions, though. Edited by Elrik (04/04/20 10:34 AM)
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m̶a̶d̶ disappointed scientist Registered: 12/28/09 Posts: 3,398 Loc: the Neitherlands Last seen: 5 months, 18 days |
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Thnx good to know about the bifactorial thing.
About the plate: appreciate you showing it. I didn't know there would still be such a demarcation line. I wonder if it is also present with di-mon matings. How come no extra sectors are visible on your plate? -------------------- My trade lists:
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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You noticed that. I did too, several times.
I think the reason mon X mon crosses do not sector is because there is no self-other incompatibility. Assuming no mutation, every strain that appears is either half identical or identical to every cell it meets. Say you try to cross MK4 and MK 5. Here are the options: • They don't cross, each stays as it is. No new strain to sector, though in some almost-crosses the cells can 'fight' where they meet. • MK4 sees MK5 and mates with it like a drunken college student, and vice versa. • MK4 sees other MK4 cells and sees itself, same for MK5. • MK4 X MK5 sees MK4 and sees a 100% genetically compatible sub-component of itself, same if it sees MK5. Outside of mutations, those are the only options and none would lead to recognition of a foreign and/or incompatible strain. So no sectoring. Sectoring will happen with di X di crosses and, I think, mon X di crosses. In both of those scenarios there would be strains that dont recognize each other and cant mate by the standard reproductive mechanism. I've never seen demarcation lines completely vanish. Every time they appeared to it was just one colony growing over the other, rather than a fusion [inter-species cross attempts tend to do that]. I have seen demarcation lines much more severe than the above example, those tend to be incompatible cross attempts. Edited by Elrik (04/05/20 02:30 PM)
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m̶a̶d̶ disappointed scientist Registered: 12/28/09 Posts: 3,398 Loc: the Neitherlands Last seen: 5 months, 18 days |
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Increasingly interesting, increasingly confusing..
(on par for mycology I guess)...I follow most of what you describe about the different scenarios or phenomena taking place. A question about that first: afaik from di-mon interactions you get a nucleus donated by the dikaryon to the monokaryon which is not exactly random. If MK4 x MK5 forms and it sees either mono MK4 or MK5, that would mean the dikaryon MK4 x MK5 will donate the complementary nucleus to whichever of the two it encounters, and it seems like necessarily both are (separately so to speak) encountered pretty much right away. That would amount to a sort of wave backwards of nucleus donations so that the rest of both monokaryons get dikaryotized. On a sidenote, I assume these donated nuclei are just being copied where it is convenient. Earlier I was imagining them migrating.. from... where exactly? Storage unit? Anyway I digress, seems to make sense if that is true / if you agree. Also interesting, i didn't know di-di fusion was possible. Never seen a mechanism explained really. What kind of exchange happens? (Yes yes off-topic! lol) Don't you find it a little weird if through mating it all basically can become the same dikaryon, but it will still refuse to fill in that demarcation line? I guess mycelium dies and perhaps even dies back in the demarcation zone and apparently this leaves some sort of signal for the culture to permanently quarantine that shit. Growing right *over* it ah ok i guess that is a workaround for that. I would like to asap show 2 dishes, one is a dish from a friend with what seems like 2 cubes that i personally thought were monokaryons that mated, and a third sector formed. And another is my own dish right now, caerulescens with a whole bunch of monokaryons present pretty discretely it looks like. Most encounters do not look like they resulted in mating but indeed, some have much more subtle demarcation lines and are even being readily overgrown. I have a slightly more recent pic which is less than a day later, but I think it can all be seen a bit clearer here. BTW I appreciate that i just gotta take a microscope to verify a dikaryon, but ofc thats not what this is about. I have looked around in articles and theses and you name it, and indeed I'm not seeing much about third or even fourth sectors, not unless it's indeed as you say di-di then you can get a new sector. Not sure about di-mon, maybe that is happening on the plate from AFOAF after all. -------------------- My trade lists: Edited by Solipsis (04/05/20 09:55 PM)
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Soulflyer Registered: 08/29/19 Posts: 79 Last seen: 3 years, 5 months |
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Quote:That is my understanding as well. A mon X mon cross is a mon X mon cross where they first touch and then it moves backwards as a mon X di cross. Quote:I really have no idea how nuclei are signaled to migrate and then do the actual migration They also must be signaled to duplicate the needed nucleus so one is available to donate.Quote:My understanding is this [and take it with a grain of salt, it may only be partly correct] When a dikaryotic cell divides the new cell receives just one nucleus, apparently often with a ~90% preference for one nucleus type over the other, the new daughter cell then gets the second nucleus donated to it by the parent cell via a clamp connection. Thus explaining why all dikaryotic cells have clamp connections. This results in the single cell leading edge of a dikaryotic culture being transiently monokaryotic. If this transiently monokaryotic cell runs into another sexually compatible cell, be it actually monokaryotic or transiently monokaryotic, then there is a chance it will pick up its second nucleus from the 'foreign' strain rather than its parent strain. In this way, in a manner of speaking, all mon X di and di X di crosses are technically mon X mon crosses. That's what I've pieced together from various bits of information from books, mycology papers, and Alan Rockefellers posts.
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m̶a̶d̶ disappointed scientist Registered: 12/28/09 Posts: 3,398 Loc: the Neitherlands Last seen: 5 months, 18 days |
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Wow
I guess there must be other genetic effects going on between them as I read about merely some genes being exchanged between di-di, but not sure if this has anything to do with horizontal gene transfer or anything like that. It would be fascinating to learn about what kind of things can be done to increase the chance of di-di fusion. I found this: https://www.differencebetween.co I thought from your explanation it would be the hyphal tip to be the temporarily monokaryotic one but maybe that would create problems far too often. Spitballing here. Can't find much on di-di pairing so far. Anyway... the nice thing about my mess caerulescens plate above full of mono's is being able to see something (probably) about the mating and that sooner or later a dikaryon virtually must be formed which then should cause extensive migrations of nuclei dikaryotizing a lot, most or all of the plate I guess. Unfortunately it may get difficult for me to see which monokaryons that I transferred tiny pieces of and put together on plates, should actually be compatible with one another as a sort of preview. I will be sure to save wedges of the monokaryons plated in triangular patterns and def hope that at least there i should be able to see which mated. I consider this practice. The idea of making mating testers intrigues me: figuring out by putting different combinations together which have complementary mating types. Call the monokaryons something like a, A, b, and B (if that is not confusing). But I don't think that if a x A would form a complementary pair and b x B do too.. that there is a meaningful or testable relationship between say a and b. I guess that's arbitrary but i could be wrong there. Mating testers as reference in case of caerulescens has very limited use (its practice) but it seems pretty cool for cubensis or certain gourmet species. Its all good fun but probably all has limited potential and after that i should probably look into mechanical dedikaryotization and much later protoplasts which if nothing else seems like an expensive occasion. -------------------- My trade lists:
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C'mon man Registered: 12/21/18 Posts: 3,775 Loc: A thought |
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Thread is 👍. Carry on.
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untitled Registered: 04/17/13 Posts: 1,738 Loc: Yurop Last seen: 1 year, 3 months |
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m̶a̶d̶ disappointed scientist Registered: 12/28/09 Posts: 3,398 Loc: the Neitherlands Last seen: 5 months, 18 days |
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Hey all, and Elrik
![]() I was talking with a friend and we were wondering about di-mon matings and what happens when there is only half compatibility, idk about the differences in that given between unifactorial and bifactorial but lets just focus on bifactorial cause at least for me that is what the fungi i work most on are... A prof was not able to give a straight answer (yet)... Aside from that there is another thing which is my mating experiments. So far I have made monokaryons and I have confirmed that caerulescens MS dikaryon does have clamp connections but my monokaryons do not. I put bunches of monokaryons together on agar and it gave both a bunch of sectoring at least apparently, but also it just seemed like the myc was possibly very brittle and i just lost pieces of it during transfer (which did not happen during initial transfers), so i have a bunch of satellite colonies. My method which i don't know if is been done before coincidentally but i (also) came up with this: to stack the agar of one or more dikaryons upside down on agar with MS of another variety or species. The top one should only contain monokaryon, no matter how many. Plates fully grown as possible. Hopefully gravity will give me plenty of contact, and di-mon mating should take place. The verification would be that dikaryon surfaces and it should not be the bottom dikaryon(s) as it shouldn't pass through. I imagine there are tons of patterns imaginable for both the layers, MS will be a bit messy usually... and the monokaryon layer either just one multiple ones clearly visible as marked off island cultures. I started out with the idea of just using one monokaryon and MS plate. The upside to this method would be to be able to give multiple strains the opportunity to mate di-mon, giving a multi-strain top layer - all crossed. I do have plates with 3 monokaryons on a single plate too so i figure this should give even more diversity as it is not limited to 1 blindly chosen monokaryon as parent. What i am curious about is: is there 'someone out there for everybody' and can you breed something ok with a parent that wasnt properly selected? Anyway i am almost ready to sandwich. ![]() It is possible to mechanically turn a dikaryon (say an isolate for simplicity and usefulness sake)... but i wonder if you even get nice genes from only one nucleus of a cool isolate. My guess is that you should get "healthy" genes because some wellbred isolate should generally have healthy genes, but other than that it seems to me it guarantees very little if you make a cross with it. Yes it can carry recessive traits but just going through spores would do that too? I guess my point is i don't really see the advantage of having a nucleus from an wellbred isolate rather than just the meiosis. -------------------- My trade lists:
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m̶a̶d̶ disappointed scientist Registered: 12/28/09 Posts: 3,398 Loc: the Neitherlands Last seen: 5 months, 18 days |
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So, I have since learned what some drawbacks are of that sandwich method ^
Very simply put: MS cannot really be tracked, so depending on what the breeding plan is, the disadvantage is you don't have the parent isolated. In any case... I wanted to report that all signs point towards having made my first successful cross (di-mon), and i wanted to thank elrik for the helpful contributions. I put a monokaryon and dikaryon together kind of on the edge of a plate, not that far apart.. the idea being that there would be a lot further to go for them to interact. Having reached the other side it appears like on the monokaryon side there are sort of radial sectors, a bit like on the edge of a roulette spinner. I allowed some time to pass and those sectors now clearly show clamp connections. I figure there will only be two strains created with the cross since the dikaryon could donate two different nuclei. And i figure as they grew together, they isolated from each other so I would expect them to be alternating strains (like ABABAB etc). The varieties I crossed (RustyWhyte x Orissa) while intentionally having noticeably different phenotypes, were a bit of a random choice of what became available. It will be interesting to see at least, if the offspring F1 looks normal since the leucism and rusty spore color are probably recessive at least. I would need to go to F2. Of course I will need to grow it out to really verify what happened but I think so far my checks such as microscopically checking for clamps have given results 100% as expected. Encouraging to proceed to more interesting crosses and attempt hybridization in gourmets. <3 -------------------- My trade lists:
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Stranger Registered: 12/01/19 Posts: 355 Last seen: 1 day, 3 minutes |
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thanks, so much good informations.
Is anyone counting spores? the route i'm taking, instead of streaking plates and searching for monospores i'll consider dilution. Dilute down to 10 spores per plate.. you can count the spore load under a slide then squirt onto agar when happy. you can very quickly do 10 plates like this, you'll have a lot more monos to choose from and they will be easy to grab.
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Smurf real estate agent Registered: 04/30/13 Posts: 61,889 Loc: Milky way |
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Spores love to clump together. Even with surfactants
-------------------- Bod's Library Article behind a paywall? I can try to get it for you
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If this were true, care to explain how exactly you are isolating jars full of monokaryon? I have a feeling im not the last to ask your process, and probably the last to do it politely.




