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root9
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Registered: 09/07/13
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Various questions regarding agar, isolates, genetics..
#19173943 - 11/22/13 07:52 AM (10 years, 2 months ago) |
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I'm about to start some agar plates once some supplies are delivered. However, I have a few questions with the process and theory..
I plan on taking a tissue sample from a recent fruit and placing it on agar. The two notable samples I have could come from the largest fruit which wasn't part of a cluster or I could take a sample from a cluster of like 5 mushrooms that were all medium sized. I'm guessing that a tissue sample from a large mushroom would tend to pass on those size traits, so would a sample from one mushroom that was in a big cluster tend to pass on "cluster" traits? I'm kind of leaning towards using the large fruit anyway because some of the mushrooms in the cluster couldn't reach maturity before the bigger ones' veils broke.
I've read something about genetics weakening every G2G transfer. Say I have an isolate on agar.. I take a wedge of that agar and drop it into a jar of WBS and let it colonize. I then use that as a master jar and inoculate 15 other jars. Would one of those 15 jars also work as a master jar identical to the first? How about a third generation master jar created from one of the initial 15? If I remember correctly, the thread about genetics weakening indicated that 3 or 4 G2G transfers is about the most you can squeeze out of an isolate. Does this mean you'd go back to your initial petri dish and make a new master jar?
Storage: Referencing this thread HERE. It seems that I'd be okay putting a petri dish with my isolate in the fridge for a few months and taking it out here and there to make new master jars; any longer and I should definitely go with culture slants.
When my dishes and agar arrive (today hopefully), I may have some questions regarding pouring, parafilm, etc..
One last thing: We all know that LME agar does better with a yellow amethyst crystal sitting on top of the petri dish. I'll be using potato agar. Would I be better off using brown amethyst because the skin of a potato is brown and the frequencies of their chi would harmonize better?
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Midnight Cyclone
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Re: Various questions regarding agar, isolates, genetics.. [Re: root9]
#19174014 - 11/22/13 08:21 AM (10 years, 2 months ago) |
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Some good questions here that are also relevant to me.
I'll bookmark this in the hopes someone more experienced in the area can help you/me out.
One question,
Quote:
I plan on taking a tissue sample from a recent fruit and placing it on agar.
From a recent fruit, meaning one you picked a while ago and now that sample is older and likely dry/contaminated?
I'm confused by this.
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root9
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Registered: 09/07/13
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Re: Various questions regarding agar, isolates, genetics.. [Re: Midnight Cyclone]
#19174062 - 11/22/13 08:34 AM (10 years, 2 months ago) |
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Quote:
Midnight Cyclone said:
From a recent fruit, meaning one you picked a while ago and now that sample is older and likely dry/contaminated?
Picked yesterday and put in a desiccant drying chamber. Hopefully it will still work, otherwise I'll have to pick a fresh one which is no big deal.
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bodhisatta 
Smurf real estate agent



Registered: 04/30/13
Posts: 61,889
Loc: Milky way
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Re: Various questions regarding agar, isolates, genetics.. [Re: root9]
#19174267 - 11/22/13 09:42 AM (10 years, 2 months ago) |
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Quote:
root9 said:
Quote:
Midnight Cyclone said:
From a recent fruit, meaning one you picked a while ago and now that sample is older and likely dry/contaminated?
Picked yesterday and put in a desiccant drying chamber. Hopefully it will still work, otherwise I'll have to pick a fresh one which is no big deal.
it will it's just not as easy or optimal as a fresh one. You'll likely have to battle a bit more with contamination on your dishes is all.
You can clone from any fruit you like. If it's a MS fruit it's got a lot of strains in it still so chances are you'll isolate a strain that's going to perform differently than the host fruit looked like anyway, but it is more likely to find strong genetics from fruits already growing than spores so you're on the right track.
Quote:
I've read something about genetics weakening every G2G transfer. Say I have an isolate on agar.. I take a wedge of that agar and drop it into a jar of WBS and let it colonize. I then use that as a master jar and inoculate 15 other jars. Would one of those 15 jars also work as a master jar identical to the first? How about a third generation master jar created from one of the initial 15? If I remember correctly, the thread about genetics weakening indicated that 3 or 4 G2G transfers is about the most you can squeeze out of an isolate. Does this mean you'd go back to your initial petri dish and make a new master jar?
You should take a read through Frankhorrigan's threads you'll find the answer there and it's a good read There's a g2g thread and some agar ones.
Also +5 here's your first rating!
Edited by Trusted cuItivator (11/22/13 09:45 AM)
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root9
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Re: Various questions regarding agar, isolates, genetics.. [Re: bodhisatta]
#19180782 - 11/23/13 10:53 PM (10 years, 2 months ago) |
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I have a glass of agar solution in the PC right now and a fruit body, scalpel, petri dishes, alcohol, and parafilm sitting in my SAB. I watched the video sample here: http://www.mushroomvideos.com/Agar-Petri-Dishes
My plan was to PC for 30 min, depressurize and put the jar in the SAB, wait like 10 minutes or however long it takes to cool down to "thin honey" consistency, then pour the plates. I have a few concerns about the pouring order of operations though.. After pouring, do I let those plates sit in stacks until fully cooled, then come back to the SAB and put the tissue samples on them, and then parafilm?
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PussyFart
Retired Cultivation Extrodinaire



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Re: Various questions regarding agar, isolates, genetics.. [Re: root9]
#19180862 - 11/23/13 11:30 PM (10 years, 2 months ago) |
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Quote:
root9 said: I have a glass of agar solution in the PC right now and a fruit body, scalpel, petri dishes, alcohol, and parafilm sitting in my SAB. I watched the video sample here: http://www.mushroomvideos.com/Agar-Petri-Dishes
My plan was to PC for 30 min, depressurize and put the jar in the SAB, wait like 10 minutes or however long it takes to cool down to "thin honey" consistency, then pour the plates. I have a few concerns about the pouring order of operations though.. After pouring, do I let those plates sit in stacks until fully cooled, then come back to the SAB and put the tissue samples on them, and then parafilm?
Follow the tek....it does not say to depressurize anything....he says after time is up to turn off the stove and walk away for 2 hours...I would wait 4....depending on the size of your PC and the amount of water you used....the more water volume, the longer it will take to cool.
Pour the agar when you can hold the bottle without needing heat protection for your hands....this is also mentioned in the tek.
I like to make sure I can hold it for 10 seconds while swirling it, just to make sure.
For me I wait 4 hours and then it takes about 30 minutes for it to drop to the right temp for me to hold the bottle.
After pouring let them sit there for a couple hours stacked up and get to work.
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THIS HOBBY IS NOT FOR THE IMPATIENT! PLEASE BE PATIENT, DON'T BE A PATIENT! A Tale of 10 Isolates, GT Cluster Clone Monotubs, RR's Let's Grow Mushrooms DVD, SGFC(Shotgun Fruiting Chamber), Monotub Tek, Damion5050's Coir Tek, TL's Tek List, Frank's Tek List, EvilMushroom666's Pasteurization Tek, How It Should & Shouldn't Look - NEW CULTIVATORS GUIDE *** *** AFGHAN KUSH GROW LOG *** ***
Edited by PussyFart (11/23/13 11:56 PM)
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root9
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Re: Various questions regarding agar, isolates, genetics.. [Re: PussyFart]
#19180903 - 11/23/13 11:42 PM (10 years, 2 months ago) |
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Quote:
Notahacker420 said: Follow the tek....it does not say to depressurize anything....
Follow which tek, the RR video? I could only watch the ~3 minute sample of the agar pouring segment. I hoped to have his video series purchased and watched prior to pouring this agar but the bank xfer's are taking forever. I bought 2 PS4's on launch night and sold one on ebay for huge profit (lol capitalism), but the money transfer to my paypal account is still pending. Once it clears I'll use that for the video series.
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cronicr



Registered: 08/07/11
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Re: Various questions regarding agar, isolates, genetics.. [Re: root9]
#19180932 - 11/23/13 11:53 PM (10 years, 2 months ago) |
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Its ok he just explained it for ya, no need to wait for a vid...you got hacker
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  It doesn't matter what i think of you...all that matters is clean spawn I'm tired do me a favor
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bodhisatta 
Smurf real estate agent



Registered: 04/30/13
Posts: 61,889
Loc: Milky way
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Re: Various questions regarding agar, isolates, genetics.. [Re: cronicr]
#19182129 - 11/24/13 11:01 AM (10 years, 2 months ago) |
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Don't try to make your PC cool down any faster or depressurize it early. Especially when you have agar in it.
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