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OfflineLogicaL ChaosM
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Spore Printing Directly on Agar?
    #26612165 - 04/19/20 06:50 AM (3 years, 9 months ago)

Anyone do it? Whats the growth look like after you print?

I bet it looks pretty cool :cool:


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InvisiblebodhisattaMDiscordReddit
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Re: Spore Printing Directly on Agar? [Re: LogicaL Chaos] * 1
    #26612267 - 04/19/20 07:50 AM (3 years, 9 months ago)

Someone probably has. But that's way too many spores and a dealing with contamination guarantee


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InvisibleJawn876
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Re: Spore Printing Directly on Agar? [Re: bodhisatta] * 1
    #26614019 - 04/19/20 10:30 PM (3 years, 9 months ago)

Its quite common with Cordyceps using petroleum jelly to stick a mature Cordyceps fruiting body to the lid of the plate


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Offlinepoisoned
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Re: Spore Printing Directly on Agar? [Re: Jawn876] * 1
    #26614539 - 04/20/20 07:32 AM (3 years, 9 months ago)

Sounds like a fun, but quite useless experiment. Do it and show us the pics.


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OfflineOldManRiver
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Re: Spore Printing Directly on Agar? [Re: LogicaL Chaos] * 1
    #26618947 - 04/21/20 10:50 PM (3 years, 9 months ago)

You'd get at best a one use spore culture, because the plate is going to be viciously contaminated, you'd have to clean, and then the remains would be useless.

The beauty of a spore print is that it is inert.  You can store it, and sample from it several times.  It keeps well, doesn't require refrigeration.  None of these things would be true of your suggestion.


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InvisibleAndyHinton


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Re: Spore Printing Directly on Agar? [Re: OldManRiver] * 1
    #26667208 - 05/12/20 06:33 PM (3 years, 9 months ago)

Interesting! I wonder if taking a sterile print from a fruit in vitro, and pressing that to the plate, would be better?

One of my sporadic projects is stabilizing a donko shiitake strain. 10 generations of spore-to-shroom cracked caps and I'm good.

But the main problem I run into is the capacity to accommodate enough genetic diversity. I don't use disposable plates, and even if I did, I couldn't afford to run 50 spore germination plates.

Could be a nice side project if you have a few choice fruits to print. Spore mating seems to be all about selecting the fittest growth from the most diverse possible progeny.

Germinating and transferring from a whole spore print would select the best genetics of the cell line. Please try this and report back.


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Edited by AndyHinton (05/12/20 06:36 PM)


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OfflineSolipsis
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Re: Spore Printing Directly on Agar? [Re: AndyHinton]
    #26677305 - 05/17/20 05:15 PM (3 years, 8 months ago)

Wow that could be a pretty interesting timelapse :laugh:

Yeah i was also thinking of an in vitro fruit otherwise i wouldnt like to try. maybe with distilled water agar only...

My prediction based on even just applying a bit too much spore matter to agar is it will make a messy warzone of different strains, so many that they inhibit and gridlock each other... maybe sectors too small to see with the naked eye? but perhaps very cool with microscope photography!

I'm skeptical about putting so many strains on agar together, Andy... i just have doubts that the competitive process makes any sense anymore, wouldn't you get too much of a chaos theory effect to rely more on the results than with a 'reasonable' amount of spores?

That said.. i have wondered about hypothetically doing a MS grow and sterile mixing everything up at various points during the grow, so much that maybe you approximate like 1 strain per spawn grain (lets really hope its not less here haha)... it might seem crazy but my idea if grown out from a rather flat spreading of the spawn and an overly generous spawn rate is you might get such fierce competition between all those strains that while "fair" because homogenized... you get limited results per strain.
But the point would not be the result in absolute terms but putting the strains through such intense competition that *relatively, the winner is really badass.


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OfflineRainbowmycelium
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Re: Spore Printing Directly on Agar? [Re: Solipsis]
    #26679430 - 05/18/20 05:28 PM (3 years, 8 months ago)

Tried this but no growth yet .. it's been about 6 days.. could be the agar I made which turned some strong rhizomophic myc into fluffy tomentose myc.


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InvisibleSannyTIES
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Re: Spore Printing Directly on Agar? [Re: LogicaL Chaos]
    #26681264 - 05/19/20 02:23 PM (3 years, 8 months ago)



Edited by SannyTIES (05/19/20 02:46 PM)


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Invisiblepanne cyanne
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Re: Spore Printing Directly on Agar? [Re: SannyTIES] * 1
    #26681311 - 05/19/20 02:47 PM (3 years, 8 months ago)

ive done this by placing invitro caps on agar plates.
it does work colonizing agar fast.

personally?
i never saw any advantage or exceptional use for the practice.


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OfflineGreenHorns
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Re: Spore Printing Directly on Agar? [Re: Jawn876]
    #26681320 - 05/19/20 02:49 PM (3 years, 8 months ago)

Quote:

Jawn876 said:
Its quite common with Cordyceps using petroleum jelly to stick a mature Cordyceps fruiting body to the lid of the plate



Are you kidding me!? Is this true for all cordyceps? I went through so much to get my culture to grow. I'd love further reading on this topic if you can suggest some.


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InvisibleJawn876
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Re: Spore Printing Directly on Agar? [Re: GreenHorns]
    #26681856 - 05/19/20 08:25 PM (3 years, 8 months ago)

It is a preferred method for collecting/culturing Cordyceps spores as far as I know. I can't really speak for "all" Cordyceps but it works well for C. militaris. Not sure which other Cordyceps are grown, I've personally only seen people grow C. militaris. I know they use (or at least used to use) Tolypocladium inflatum for Ciclosporin production but I don't know what methods they used for culturing.
As for further information on printing C. militaris directly on agar, William Padilla-Brown talks a bit about it in the 'Cordyceps Cultivation Handbook'. Info on Cordyceps cultivation in general can be found in that book, some threads here on these forums, Ryan Paul Gates at Terrestrial Fungi has a good video on youtube, William Padilla-Brown at Mycosymbiotics also has some videos on youtube including using PCR for mating type IDs for strain breeding, and I think Alex Dorr at Mushroom Revival has some youtube videos too


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OfflineSolipsis
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Re: Spore Printing Directly on Agar? [Re: Jawn876]
    #26682733 - 05/20/20 07:27 AM (3 years, 8 months ago)

ok.. i found it easy to just print wild Cordyceps militaris first and then germinate on agar :shrug: , although not much can be seen of the spore fragments. possibly i 'double printed' to various extent to avoid contam, and got zero contam.

but yea i have heard about sticking a teleomorph to lid of a plate. for in vitro fruits it seems practical enough, without the risk of contam it could simply save time for breeders i guess.


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Offlinevinnie boombotz
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Re: Spore Printing Directly on Agar? [Re: Solipsis] * 1
    #26682808 - 05/20/20 08:20 AM (3 years, 8 months ago)

Quote:

Solipsis said:
Wow that could be a pretty interesting timelapse :laugh:

Yeah i was also thinking of an in vitro fruit otherwise i wouldnt like to try. maybe with distilled water agar only...

My prediction based on even just applying a bit too much spore matter to agar is it will make a messy warzone of different strains, so many that they inhibit and gridlock each other... maybe sectors too small to see with the naked eye? but perhaps very cool with microscope photography!

I'm skeptical about putting so many strains on agar together, Andy... i just have doubts that the competitive process makes any sense anymore, wouldn't you get too much of a chaos theory effect to rely more on the results than with a 'reasonable' amount of spores?

That said.. i have wondered about hypothetically doing a MS grow and sterile mixing everything up at various points during the grow, so much that maybe you approximate like 1 strain per spawn grain (lets really hope its not less here haha)... it might seem crazy but my idea if grown out from a rather flat spreading of the spawn and an overly generous spawn rate is you might get such fierce competition between all those strains that while "fair" because homogenized... you get limited results per strain.
But the point would not be the result in absolute terms but putting the strains through such intense competition that *relatively, the winner is really badass.




Would a ms from the same print differ in ways from a ms from several different Print?


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OfflineSolipsis
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Re: Spore Printing Directly on Agar? [Re: vinnie boombotz] * 1
    #26683117 - 05/20/20 11:25 AM (3 years, 8 months ago)

Depends. if you grow an isolate and it fruits and you make several different prints, the spore (fragment)s will contain the shuffled genetics of the isolate no matter what print you use. Or if you grow MS multiple times from 1 and the same print.
Cause if the parents are identical you might as well pile up the spores the genetically identical fruits produce. (Ignoring epigenetics for a second to keep things manageable)

But if you compare spores coming from entirely separate strains say from across the world, then yeah there are just different genetics involved.

The fact that spores reshuffle genes partially obscures the direct connection to the parent and therefore the difference between geographically/genetically different parents. But nevertheless, they're "simply" different strains in that case.

correct me if i'm wrong of course


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InvisibleAndyHinton


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Re: Spore Printing Directly on Agar? [Re: Solipsis] * 1
    #26689121 - 05/22/20 08:54 PM (3 years, 8 months ago)

Quote:

Solipsis said:
I'm skeptical about putting so many strains on agar together, Andy... i just have doubts that the competitive process makes any sense anymore, wouldn't you get too much of a chaos theory effect to rely more on the results than with a 'reasonable' amount of spores?



Dunno. I haven't tried this and it's not a priority right now. But going by the numbers, a strain that outcompetes billions or trillions of others would be more virulent than one that outcompetes only thousands or millions. Like how the current plague became more virulent to outcompete every other disease breeding in the medieval hygiene conditions of the Chinese market.

My thinking goes, "Germinate the whole print, scoop the first polyculture to appear, and develop it on clean plates." But the layout of the spores could undermine the chaos and competition. Chaos is good here. Maybe a stock solution, like a whole print in 100 μL saline, would randomly distribute the spores. Then you could deposit 10 μL each on 10 plates and get roughly the same effect as an agar print.


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Edited by AndyHinton (05/22/20 08:55 PM)


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OfflineSolipsis
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Re: Spore Printing Directly on Agar? [Re: AndyHinton]
    #26690042 - 05/23/20 09:40 AM (3 years, 8 months ago)

I guess it shouldn't be bad - am just wondering if a sort of ceiling effect occurs for 'fair competition' and yeah homogenized inoculant sounds like it could deal with some of the issues.

I understand if it would select for early germinating spores, in massive quantities, but it may preclude selection of strong fast growing strains if the get gridlocked and don't end up being on the edge of the polyculture? I def like your idea, but it would be cool to explore if it is possible that circumstances can prevent proper selection in this way.

Again, homogenization would be one part of the solution but even then if you wanna go overkill with the number of spores you might just get too much of a gridlock - but homogenous gridlock.
Similar story with the 'dominance' of certain strains doing di-mon matings. Maybe if you have too many spores you get so many small dikaryons that you limit the beneficial di-mon combinations that can occur to amplify the dominance of a strain without having to unravel crazy polycultures.

Oh maybe im being dumb and the inoculation of a super diverse polyculture of yours into grain spawn could still offer enough opportunity for amplifying vigor and dominance?

Hey if we hypothetically use in vitro fruits anyway for this, why not put an entire print into LC if it's sterile? now that seems like a lottery with even more potential? xD

yeah if you will be growing a superstrain, keep away your bats and minks.. D:


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InvisibleAndyHinton


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Re: Spore Printing Directly on Agar? [Re: Solipsis]
    #26690310 - 05/23/20 12:35 PM (3 years, 8 months ago)

Quote:

Solipsis said:
Hey if we hypothetically use in vitro fruits anyway for this, why not put an entire print into LC if it's sterile? now that seems like a lottery with even more potential? xD



That sounds more like Russian Roulette, putting spores to broth no matter how clean it is. :smile:

Whenever I personally work with spores, I try to keep it as wild as possible and do minimal strain development. Basically once the polyculture stops visibly fighting itself, I'm done.

Say you have a shiitake block and get 100 pins on it. You select the desired fruit and discard 99% of the flush's genetic potential. You put 10 μL spore suspension on 10 plates, discarding 99.999% or more of each plate's potential. You run the polycultures on agar until it becomes apparent that 3 are worth fruiting, discarding 70% of the genetic potential.

By that point you've got more than enough to work with even if it's like 0.00003% of the flush's total potential (math probably wrong). This thought experiment of mine assumes that spore mating is a black box, and that discarding the most genetic potential increases the relative fitness of what you do select for. It's all about verifying that most potential strains just aren't worth it.

But the gridlock problem is probably quite real, like how you can hop on Tinder and see thousands of hot girls in bikinis, not knowing which ones are worth flirting with. I have to use an analogy because the genetics of spore mating is beyond my understanding. :tongue:

Anyway, I'm still working out how to make friends with entropy in interesting ways. One of my friends has a plasmid that you can mix with a DNA template and transform into a bacteria. It makes the bacteria spit out random proteins. I wonder if there's any crypto application if you sequence the proteins on the plate, for example.


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Edited by AndyHinton (05/23/20 12:39 PM)


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OfflineGreenHorns
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Re: Spore Printing Directly on Agar? [Re: AndyHinton]
    #26690497 - 05/23/20 02:10 PM (3 years, 8 months ago)

A thought occurred. What if you find something like those plastic pizza box tables but one that will withstand being in the pc and place a cap that's been uv treated onto the table sitting in the agar? Enough uv should kill most of the contams leaving just the deepest youngest spores to drop. You would need to hollow the table out in some way but you get the idea.


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OfflinePTreeDish
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Re: Spore Printing Directly on Agar? [Re: GreenHorns]
    #26690887 - 05/23/20 05:45 PM (3 years, 8 months ago)

On the topic of breeding, I've been really enjoying Gary's YouTube channel: Fresh from the Farm Fungi LLC. He has a video series on Breeding Mushrooms and in Part 1: Serial Dilutions and Isolating Haploids he covers some of the techniques for harnessing the genetic lottery of spore prints to generate unique and desirable prototypes that can give cultivators advantages in the market. Highly recommend checking it out.


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