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Offlinesillysyban
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Agar to Grain question
    #26654321 - 05/07/20 03:11 AM (4 years, 8 months ago)

Hi Good peoples,

I have a few agar plates that I ran from spores. I had a few contams but a few are clean with no contamination. I want to do some transfers to new agar but also want to run a MS and then clone good looking mushrooms.
My question is, is it OK to take some wedges out and transfer in my SAB and then drop the rest of the plate into a grain jar or will leaving the agar dish open for that long as I transfer to other Agar dishes cause contamination in the grains when I drop the agar wedge in to the jar?

Cheers.

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Offlinemushpunx
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Re: Agar to Grain question [Re: sillysyban]
    #26654325 - 05/07/20 03:20 AM (4 years, 8 months ago)

In a still air box I try to keep things open only as long as I have to - I open the lid, make my transfer, close the recover and then close the donor plate. After you've made your transfers, just put the lid back on while you get your grain jar ready to be inoculated.

Before I inoculate grains I always make *at least one transfer away from germination first.

With a flow good you can keep stuff open longer but a still air box it's best to make smooth, decisive motions as quick as possible.


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Offlinesillysyban
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Re: Agar to Grain question [Re: mushpunx]
    #26654343 - 05/07/20 03:32 AM (4 years, 8 months ago)

Cheers mushpunx,
OK so you suggest to do a transfer anyway before inoculating grains? Will that make it more of an isolate and then cause less diversity in the fruiting tub to clone from or will there be still a few strains in one transfer and to get an isolate you would have to transfer a few times?

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InvisibleLadysKnight
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Re: Agar to Grain question [Re: sillysyban]
    #26654409 - 05/07/20 05:06 AM (4 years, 8 months ago)

As I see it, there's no need for any dish to ever be uncovered. The plate stays on the floor of the sab, lift one side of the lid, cut and remove wedge, lower lid, raise one side of receiving dish/jar lid, insert wedge, lower lid.


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Offlinemushpunx
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Re: Agar to Grain question [Re: LadysKnight] * 1
    #26654416 - 05/07/20 05:14 AM (4 years, 8 months ago)

Quote:

LadysKnight said:
As I see it, there's no need for any dish to ever be uncovered. The plate stays on the floor of the sab, lift one side of the lid, cut and remove wedge, lower lid, raise one side of receiving dish/jar lid, insert wedge, lower lid.




You can uncover it. Sometimes I have to stabilize the dish with my left hand while making a cut with scalpel - I'll set the lit sterile side up on a jar or something- then replace it right away.
The receiving plate, I usually just lift up the lid with my left hand, drop the wedge and put it back.

Just never move anything over top open media in an SAB or between the flow and media with a FH.


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OfflineFrankRhizo
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Re: Agar to Grain question [Re: sillysyban]
    #26654421 - 05/07/20 05:26 AM (4 years, 8 months ago)

Quote:

sillysyban said:
Cheers mushpunx,
OK so you suggest to do a transfer anyway before inoculating grains? Will that make it more of an isolate and then cause less diversity in the fruiting tub to clone from or will there be still a few strains in one transfer and to get an isolate you would have to transfer a few times?




It takes more then a few times to gain a true isolate. Don't worry there will be plenty of genetics after a few transfers.

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Re: Agar to Grain question [Re: sillysyban] * 1
    #26654422 - 05/07/20 05:26 AM (4 years, 8 months ago)

Quote:

sillysyban said:
Cheers mushpunx,
OK so you suggest to do a transfer anyway before inoculating grains? Will that make it more of an isolate and then cause less diversity in the fruiting tub to clone from or will there be still a few strains in one transfer and to get an isolate you would have to transfer a few times?





It's just to make sure the culture is clean - even if the germination plate looks clean oftentimes the myc has germinated over top or next to bacteria or mold colonies. So make a transfer to another plate and use that to inoculate your grains. Often I'll do two transfers to be safe.

Whenever you make a transfer, you are probably cutting out at least some of the genetics, but it doesn't matter. You are just looking for a clean MS culture to inoculate grains with.

In a culture germinated from spores there are hundreds to thousands of strains present. If I am isolating strains it typically takes me 8 to 10 transfers before I see obvious sectoring. Then once I take a transfer from each sector to a new plate, those cultures might still show some sectors and I might still have to make another transfer or two of each of the next plates until they become single sector isolated strains. At that point, I'll have 10 - 20 different strains, which I label, fruit and test for potency + growing factors. There might only be one or two good ones, if I'm lucky.

Strain isolation takes a lot of work and patience!
It makes more sense for us home cultivators to clone. That is still quite a bit of work (and eating a lot of mushrooms if you wanna test potency 😂), taking lots of clones and fruiting them out, searching for potent, high yielding fruiters.


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Offlinesillysyban
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Re: Agar to Grain question [Re: mushpunx] * 1
    #26654481 - 05/07/20 06:23 AM (4 years, 8 months ago)

Quote:

mushpunx said:
Quote:

sillysyban said:
Cheers mushpunx,
OK so you suggest to do a transfer anyway before inoculating grains? Will that make it more of an isolate and then cause less diversity in the fruiting tub to clone from or will there be still a few strains in one transfer and to get an isolate you would have to transfer a few times?





It's just to make sure the culture is clean - even if the germination plate looks clean oftentimes the myc has germinated over top or next to bacteria or mold colonies. So make a transfer to another plate and use that to inoculate your grains. Often I'll do two transfers to be safe.

Whenever you make a transfer, you are probably cutting out at least some of the genetics, but it doesn't matter. You are just looking for a clean MS culture to inoculate grains with.

In a culture germinated from spores there are hundreds to thousands of strains present. If I am isolating strains it typically takes me 8 to 10 transfers before I see obvious sectoring. Then once I take a transfer from each sector to a new plate, those cultures might still show some sectors and I might still have to make another transfer or two of each of the next plates until they become single sector isolated strains. At that point, I'll have 10 - 20 different strains, which I label, fruit and test for potency + growing factors. There might only be one or two good ones, if I'm lucky.

Strain isolation takes a lot of work and patience!
It makes more sense for us home cultivators to clone. That is still quite a bit of work (and eating a lot of mushrooms if you wanna test potency 😂), taking lots of clones and fruiting them out, searching for potent, high yielding fruiters.





Quote:

mushpunx said:
Quote:

sillysyban said:
Cheers mushpunx,
OK so you suggest to do a transfer anyway before inoculating grains? Will that make it more of an isolate and then cause less diversity in the fruiting tub to clone from or will there be still a few strains in one transfer and to get an isolate you would have to transfer a few times?





It's just to make sure the culture is clean - even if the germination plate looks clean oftentimes the myc has germinated over top or next to bacteria or mold colonies. So make a transfer to another plate and use that to inoculate your grains. Often I'll do two transfers to be safe.

Whenever you make a transfer, you are probably cutting out at least some of the genetics, but it doesn't matter. You are just looking for a clean MS culture to inoculate grains with.

In a culture germinated from spores there are hundreds to thousands of strains present. If I am isolating strains it typically takes me 8 to 10 transfers before I see obvious sectoring. Then once I take a transfer from each sector to a new plate, those cultures might still show some sectors and I might still have to make another transfer or two of each of the next plates until they become single sector isolated strains. At that point, I'll have 10 - 20 different strains, which I label, fruit and test for potency + growing factors. There might only be one or two good ones, if I'm lucky.

Strain isolation takes a lot of work and patience!
It makes more sense for us home cultivators to clone. That is still quite a bit of work (and eating a lot of mushrooms if you wanna test potency 😂), taking lots of clones and fruiting them out, searching for potent, high yielding fruiters.




Thanks for the answers. Legends.
One more question. I do have a few plates where germination started in a few spots and I’m sure of genetic variety in those but my agar was very dry and out of 40 odd plates maybe 10 germinated. Some of those started from just one tiny dot and the growth looks very tementose type mycelium.
In your experience do you see it very much where just one spore germinates and creates monokaryotic mycelium or is this really rare and the reason for the tementose mycelium is there are heaps of strains there.
I’ve been reading heaps and it’s only confused me more 😂.

As for having to eat heaps of mushrooms to test for potency I guess we all have to make sacrifices mate 🤪.

Really appreciate it  👍

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Offlinesillysyban
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Re: Agar to Grain question [Re: FrankRhizo]
    #26654497 - 05/07/20 06:41 AM (4 years, 8 months ago)

Quote:

FrankRhizo said:
Quote:

sillysyban said:
Cheers mushpunx,
OK so you suggest to do a transfer anyway before inoculating grains? Will that make it more of an isolate and then cause less diversity in the fruiting tub to clone from or will there be still a few strains in one transfer and to get an isolate you would have to transfer a few times?




It takes more then a few times to gain a true isolate. Don't worry there will be plenty of genetics after a few transfers.




Wicked. Thanks FrankRhizo. Cool name. My dad was a Frank. ✌️

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Offlinemushpunx
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Re: Agar to Grain question [Re: sillysyban]
    #26655055 - 05/07/20 12:27 PM (4 years, 8 months ago)

Rhizomorphic or tomentose don't really mean so much. You'll notice if you take a very tomentose culture and put it to agar mixed with low nutrients it will turn Rhizomorphic and ropey looking - and vice versa.


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Offlinesillysyban
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Re: Agar to Grain question [Re: mushpunx]
    #26655777 - 05/07/20 06:40 PM (4 years, 8 months ago)

OK I didn't know that. Awesome. Cheers.

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