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teesionbear


Registered: 12/13/09
Posts: 387
Last seen: 4 years, 9 days
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: oh_you_know]
#12125409 - 03/02/10 01:21 AM (13 years, 11 months ago) |
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Quote:
oh_you_know said:
cakes may not be the best. technically you could crumble and spawn to bulk but i believe PE does a lot better as trays or bulk. cakes just dont do it. one of those mysteries. and def keep a few cc's just in case! i think youll enjoy PE. idk why someone wouldnt. i lost me PE spores and stuff, im gonna have to order some new syringes once i get some cash.
he's just doing 2 cakes in order to get a tissue sample for a plate.
he had 10 cc's and doesn't like paying for spores, so he is covering his bases as far as preserving them 
as soon as he has cultures going, he's gonna do grains and monotubs.
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shroombab
Visionary



Registered: 01/15/09
Posts: 78
Loc: Canada
Last seen: 10 years, 10 months
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: teesionbear]
#12127666 - 03/02/10 01:11 PM (13 years, 11 months ago) |
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I think because of my time constraints im going to go spore solution to agar at the start as well, so that I can do sectoring while I wait for my MS grain jars to finish so I can take clones.
SO, in that case. If I do a set of 10 and keep sectoring and sectoring, am I just gambling what each strain will be? Surely the ones that colonize fast and strong on agar will be a good gamble to fruit?
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PrimalSoup
hyperspatial illuminations



Registered: 11/17/09
Posts: 13,568
Loc: PNW
Last seen: 1 year, 5 months
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: shroombab] 1
#12138928 - 03/03/10 11:20 PM (13 years, 11 months ago) |
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What you're saying is, go spores > jars > bulk > fruit and then take a fruit and then take a flesh sample from one good fruit and go straight to agar, which immediately makes a strong isolate?
There is a difference though and it's in the # of cell divisions required to get to the fruit. The longer the culture grows the more cell generations it goes through, so the fruit will have more opportunity for strain divergence/senescence to occur over the isolation on agar, which is earlier in the life cycle.
It will take longer to go to a fruit=>agar=>grain etc than to go MS=>grain=>bulk, and the result may not be enough different to matter.
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if I take a slant from a strong agar isolate and save it, how many times can I use that over and over (by doing agar to agar to LC to grain etc.) surely there is a point at which it stops? or doesn't work anymore? What is the best way to sustain an isolate for use over and over again, negating the need to buy more spores of that strain.
You just keep the strain culture in a couple of slants (for redundancy) in the fridge. When you need to start fresh you plate it out, and put a sample of that in another slant to replace the one you used (you don't put the same slant back in as it needs to grow some, and there's a risk of contamination). Ideally you do this every 6 months or so, but viability varies. Everytime you go back to it the strain has only grown for a few more days, relatively speaking, so it stays very viable.
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I think because of my time constraints im going to go spore solution to agar at the start as well, so that I can do sectoring while I wait for my MS grain jars to finish so I can take clones.

Quote:
SO, in that case. If I do a set of 10 and keep sectoring and sectoring, am I just gambling what each strain will be? Surely the ones that colonize fast and strong on agar will be a good gamble to fruit?
Your MS fruits will not necessarily be isolates and so they may not be true clones. You can isolate from the fruits on agar but it'll take a while.
The "sectoring" is better called isolating, and it will depend on your luck, the genetics of the spores, and so on. You keep isolating multiple cultures (all at the same time) until the plates grow uniformly - different areas of growth are the sectors.
Usually you'd select the most vigorous or most rhyzomorphic areas to transfer further. I'm doing this right now with a PESH strain and some of the plates are mad wild (see pic of a plate on the 2nd transfer after spore germination). In this case I'm doing about 15 samples of different rhyzo styles and will take them all through fruiting on bulk medium, just to select the best fruits/fruiters/potency. Meaning it ain't necessarily that simple...
But I also took this plate and inoculated six grain jars from it because I just liked its looks. 

Peace -PS
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if you stand too close to the machine it'll start to eat youPrimal's simple tested teks and projects: Wheat Prep 2.0 Acidic Tea Tek Potency Project!
Edited by PrimalSoup (03/03/10 11:26 PM)
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