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Invisiblecyanara
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Monokarions
    #14505289 - 05/24/11 04:16 PM (12 years, 8 months ago)

I was wondering what would be a good way to get monokariotic mycelium from agar, and how to isolate it? I would like to start exploring the finner aspects of agar work.

edited to remove the apostrophe, which indicates possession, not plurality. Sorry, I'm a grammar and punctuation nerd, especially in advanced mycology.
RR


Edited by RogerRabbit (05/25/11 08:58 AM)


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Offlinetr8orjohn
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Re: Monokarion's [Re: cyanara]
    #14506385 - 05/24/11 07:42 PM (12 years, 8 months ago)

Dilution and streak plating of spore solutions until you get single "colonies"

-jon


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Terry M said:
Looking for rules that other people use successfully. Sterile procedure is just a set of rules that work.


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Invisiblecyanara
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Re: Monokarion's [Re: tr8orjohn]
    #14506423 - 05/24/11 07:49 PM (12 years, 8 months ago)

how much spores on the end of an enoculation loop would you want?
how much liquid to liquid on the dilution rate?
aprox how many plates does it take to get a monokarion?


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OfflineRogerRabbitM
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Re: Monokarion's [Re: cyanara]
    #14506555 - 05/24/11 08:12 PM (12 years, 8 months ago)

You better have thousands of petri dishes if you're going to attempt to breed monokaryons.  A better bet would be to swipe a minimum amount of spores on a dish and give them a few days after germination. Then, take all the growths you find and transfer tiny pieces of them to new dishes.  This way, you're starting with dikaryons, hundreds of them, to do your breeding.

3 section petri dishes cut the amount of plates you buy by two thirds.
RR


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semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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Invisiblecyanara
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Re: Monokarion's [Re: RogerRabbit]
    #14507064 - 05/24/11 09:40 PM (12 years, 8 months ago)

so would you take a very minimal ammount of spores from both types of spores, (i.e. Brazilian and GT) and deposit them on the same plate and as soon as one would notice growth (assuming that the dykarions are from both spore types) transfer and keep transfering everytime new growth occures?
thanks for your patience in this matter as I would really like to learn this aspect of mycology more.


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OfflineFeelers
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Re: Monokarion's [Re: cyanara]
    #14508641 - 05/25/11 07:05 AM (12 years, 8 months ago)

1. I don't know the answer to this but what I would do - take your inoculation loop, get a small amount of spores, then transfer it to a sterile jar with 100cc water and shake.
2. Then with your syringe take out out 10cc's and place into different jar with 90 cc's in it and shake.
3.From this solution take 10cc's put into another different jar with 90cc's water and shake.
4. From this solution take 10cc's put into another different jar with 90cc's water and shake.

Since I don't know how much dilution is required to get reliable monos, you could just take out 1cc and do a few less steps, but the long way will hopefully find a good reliable dilution factor.
At each step (apart from step 1), take out a small amount out of the jar) into your syringe and spread a few drops around onto a petri, so you get a thin spread, kinda like running a little oil around a fry pan.

If I have done my math correctly jar 2 will be 10x dilution, 3 will be 100x, 4 will be 10000x. I'd wager that at step 4 you will have a good chance of finding monos, but you could always try for a 5th jar with a 10 million dilution!

Or you could use the standard z type agar swipe, but that's a bit less exact. :thumbup:


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OfflineRogerRabbitM
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Re: Monokarions [Re: Feelers]
    #14508897 - 05/25/11 08:55 AM (12 years, 8 months ago)

Quote:

1. I don't know the answer to this but what I would do - take your inoculation loop, get a small amount of spores, then transfer it to a sterile jar with 100cc water and shake.
2. Then with your syringe take out out 10cc's and place into different jar with 90 cc's in it and shake.
3.From this solution take 10cc's put into another different jar with 90cc's water and shake.
4. From this solution take 10cc's put into another different jar with 90cc's water and shake.




It sounds like you went to paul's seminar.  However, crossing monokaryons is a long row to hoe.
RR


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semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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Invisiblecyanara
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Re: Monokarions [Re: RogerRabbit]
    #14509022 - 05/25/11 09:42 AM (12 years, 8 months ago)

Hey Roger, thanks for the Grammar lesson, wasn't born here so My spelling is sometimes under par.
Since crossing Dykarions seems a better suit could I germinate on a plate and transfer already growing mycelium? Like what you did with the Red Boy?:congrats:


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OfflineRogerRabbitM
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Re: Monokarions [Re: cyanara]
    #14510179 - 05/25/11 02:35 PM (12 years, 8 months ago)

Sure.  Breeding dikaryons will give somewhat less diversity but you won't need nearly as many petri dishes.

Redboy was a bit different. In that case, a few thousand spores that were degraded and wouldn't germinate were placed on a petri dish and monokaryotic mycelium was allowed to crawl across the plate.  How the monokaryons crossed with the spores without them germinating is a mystery.  I suspect the monokaryotic mycelium 'digested' the spores and obtained the cross that way, but the exact mechanism remains unknown.
RR


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semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
Thomas Edison


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Invisiblecyanara
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Re: Monokarions [Re: RogerRabbit]
    #14510616 - 05/25/11 04:07 PM (12 years, 8 months ago)

thanks i'm still however a little perplexed with the whole crossing dikaryons as I thought, both mycelials will just form a ridge?
should I just introduce two dikaryons or dikaryons mycelia with spores from another type?


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Offlinedr harry bush
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Re: Monokarions [Re: cyanara]
    #14520104 - 05/27/11 10:21 AM (12 years, 8 months ago)

Hi Cyanara

This post might interest you.
http://www.shroomery.org/forums/showflat.php?Cat=0&Number=14520091&page=0&vc=1#14520091

regards
Dr Harry


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Invisiblecyanara
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Re: Monokarions [Re: dr harry bush]
    #14520309 - 05/27/11 11:23 AM (12 years, 8 months ago)

thanks for the link.. the interesting thing is that the Trenskei in te pic and in most pics don't resemble te original genetics from T.H.E.?


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Offlinedr harry bush
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Re: Monokarions [Re: cyanara]
    #14520427 - 05/27/11 11:56 AM (12 years, 8 months ago)

yes that is right. theses are from my own wild print. and i cloned from a very good flush that produced many medium size mushrooms. i cloned for quantity, strength and bruising. i do however have tk other tks im busy with one was cloned for the shape of the caps ( a very nice convex shape cap which reminds me of a fairy mushroom you see in pictures) and got a clone of a tk for its size and the foam like mycielum it gets over the casing. this bigger clone has about 10 subs that i am busy sectoring out and going to trade get a nice full iso.


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Invisiblecyanara
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Re: Monokarions [Re: dr harry bush]
    #14520449 - 05/27/11 12:01 PM (12 years, 8 months ago)

nice. so I take it your from south africa?


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Offlinedr harry bush
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Re: Monokarions [Re: cyanara]
    #14520485 - 05/27/11 12:11 PM (12 years, 8 months ago)

lol i travel alot... :grin:


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