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OfflineSolipsis
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Monokaryons
    #26393831 - 12/21/19 10:43 AM (4 years, 1 month ago)

I read they are not just weakly growing but also shortlived, how shortlived are we talking? Care to give an example? :smile:

In culture how far can you transfer them and won't they be okay on slant or wedge?


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Re: Monokaryons [Re: Solipsis]
    #26394104 - 12/21/19 02:21 PM (4 years, 1 month ago)

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.


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OfflineElrik
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Re: Monokaryons [Re: Solipsis] * 7
    #26394723 - 12/21/19 10:16 PM (4 years, 1 month ago)

Quote:

Solipsis said:
I read they are not just weakly growing but also shortlived, how shortlived are we talking?



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! :tongue: ]
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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InvisibleSmartattack
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Re: Monokaryons [Re: Elrik]
    #26394803 - 12/21/19 11:28 PM (4 years, 1 month ago)

Quote:

Elrik said:
Quote:

Solipsis said:
I read they are not just weakly growing but also shortlived, how shortlived are we talking?



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! :tongue: ]
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.






:notamused:  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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Invisiblemurderlabz
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Re: Monokaryons [Re: Smartattack]
    #26394821 - 12/21/19 11:56 PM (4 years, 1 month ago)

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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OfflineElrik
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Re: Monokaryons [Re: murderlabz]
    #26394912 - 12/22/19 02:44 AM (4 years, 1 month ago)

Quote:

Smartattack said: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.



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 :smile:
If I recall correctly I got my procedures from Stamets' "Mushroom Cultivator - A Practical Guide to Growing Mushrooms"
Thanks Stamets, your awesome :wink:

Quote:

murderlabz said:
Did you run on low nute agar?



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


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Edited by Elrik (12/22/19 02:56 AM)


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OfflineSolipsis
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Re: Monokaryons [Re: Elrik]
    #26395052 - 12/22/19 06:58 AM (4 years, 1 month ago)

Thanks a lot for the info :laugh:

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?


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Re: Monokaryons [Re: Elrik]
    #26395145 - 12/22/19 08:19 AM (4 years, 1 month ago)

Quote:

Elrik said:
Quote:

Smartattack said: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.



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 :smile:
If I recall correctly I got my procedures from Stamets' "Mushroom Cultivator - A Practical Guide to Growing Mushrooms"
Thanks Stamets, your awesome :wink:

Quote:

murderlabz said:
Did you run on low nute agar?



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







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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Re: Monokaryons [Re: Elrik] * 1
    #26395173 - 12/22/19 08:38 AM (4 years, 1 month ago)

Quote:

Elrik said:
Quote:

Solipsis said:
I read they are not just weakly growing but also shortlived, how shortlived are we talking?



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! :tongue: ]
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.



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.


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OfflineElrik
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Re: Monokaryons [Re: Solipsis] * 1
    #26395352 - 12/22/19 10:26 AM (4 years, 1 month ago)

Quote:

bodhisatta said:
So you think you have mono growth because of one micrograph without a clamp connection in the image? Lol



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:

bodhisatta said:
Show me the growth starting with one isolated spore so i and anyone else can actually be confident.



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:

bodhisatta said:
Heck even show me that you got spores appropriately diluted enough to even attempt your project.



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:

bodhisatta said:
Ive diluted spores streaked and by the time you see growth with your eye they're long dikaryotic.



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:

Smartattack said:I sincerely HOPE it can be that easy.



That's how I felt before I tried. I was quite thrilled when I found out it IS this easy!
Quote:

Solipsis said:It's surprising that you are not at least using spore dilutions...
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.

...did you mean that aside from checking clamp connections you have also seen proof of successfully achieving crosses, from those later generations?



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. :laugh:
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! :smile:


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Re: Monokaryons [Re: Elrik]
    #26395400 - 12/22/19 10:48 AM (4 years, 1 month ago)

Where is Workman, he didn't retire did he?


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Re: Monokaryons [Re: murderlabz]
    #26395433 - 12/22/19 11:18 AM (4 years, 1 month ago)

At the very least stain the slides and show me cells with only one nucleus.


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OfflineElrik
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Re: Monokaryons [Re: bodhisatta]
    #26395546 - 12/22/19 12:26 PM (4 years, 1 month ago)

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 :smile:
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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Re: Monokaryons [Re: Elrik]
    #26395785 - 12/22/19 03:15 PM (4 years, 1 month ago)

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


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Re: Monokaryons [Re: bodhisatta]
    #26395884 - 12/22/19 04:09 PM (4 years, 1 month ago)

In other words, I'm picturing those "respectable distances" to be seperating spores by about the width of a hair?


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Re: Monokaryons [Re: Smartattack]
    #26396012 - 12/22/19 05:29 PM (4 years, 1 month ago)

He was right in saying not all spores are viable.
Quote:

RogerRabbit said:
I've ran several tests with the microscopes to determine spore viability.  A brand new print will give about 1 spore in 100 that will germinate.  After two weeks, that drops to about 1 spore in 500.  At one month since taking the print, less than 1 spore in 1000 will germinate, and after one year, you're doing good to get 1 spore in 10,000 that is still viable and it drops even faster after one year...
RR



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  :shrug:


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Re: Monokaryons [Re: bodhisatta] * 1
    #26396551 - 12/22/19 11:31 PM (4 years, 1 month ago)

Quote:

bodhisatta said:
Look how close those spores are in that second picture its 400x they're within one field of each other.


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.


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Re: Monokaryons [Re: Elrik] * 1
    #26396573 - 12/23/19 12:13 AM (4 years, 1 month ago)

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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Re: Monokaryons [Re: Muad.Dweeb]
    #26396867 - 12/23/19 06:46 AM (4 years, 1 month ago)

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.


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Re: Monokaryons [Re: Solipsis] * 2
    #26396944 - 12/23/19 08:05 AM (4 years, 1 month ago)

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. :hehehe:


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