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Edmunter
Mr



Registered: 05/01/13
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Re: Cultivation General Discussion [Re: junk_f00d]
#26172371 - 09/06/19 11:20 AM (4 years, 4 months ago) |
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Quote:
junk_f00d said: So when gearing up to do hundreds of isolates for testing, what's the best way to test them? It's infeasible for me to run hundreds of monotubs and thousands of mason jars (and wasteful even if it was feasible). Is spawning a quart jar to 8x4 disposable foil trays a good idea? IS it worth downsizing to pint jars or smaller just for isolate testing purposes? I couldn't find teks on this so if someone wants to link me that'd be great! Nothing like great, well written advice from the veterans 
I was told looking for isolates is a fools mission unless you want to cross it of the tick list. Finding a decent clone for most people is easier.
Saying that, If you look at my photo below I have 4 shoeboxes in 1 mono so you could test 4 per mono which I use 2 quarts per box. Test 20 a month.....ect
Edited by Edmunter (09/06/19 11:22 AM)
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verum subsequentis
seeker of truth



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Re: Cultivation General Discussion [Re: Edmunter]
#26172380 - 09/06/19 11:24 AM (4 years, 4 months ago) |
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i test in shoes. 1 q spawn to roughly 2-2.5 q sub. i also throw them into tubs and treat just like any other tub
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junk_f00d


Registered: 12/04/15
Posts: 933
Last seen: 1 year, 2 months
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I don't have hundreds of isolates but I'm on the hunt. I like the idea of doing shoeboxes in a mono, it's nice if my fruiting environment is as similar as possible to a monotub so I know I'm getting stuff that flourishes in there. I thought about just getting many tiny monotubs (20+) and stacking them away when not in use.. Is that dumb? I think 10qts is a good size for a single quart but idk. Maybe I'll look into using pint size jars for testing and go even smaller.
Also, who said hunting for isolates is foolish? I mean I also have clone cultures, but I'm always looking to improve and have time to kill so
Edited by junk_f00d (09/06/19 02:45 PM)
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LtLurker
Lost Sailor



Registered: 01/03/18
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Re: Cultivation General Discussion [Re: junk_f00d]
#26172734 - 09/06/19 02:49 PM (4 years, 4 months ago) |
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An isolate is a single set of genetics, a single strain, 2 spores mated only. It takes a really long time, a shit ton of transfers, and a keen eye for vectoring to accomplish a true isolate. Verum's PE isolate project took him over a year to actually get some isolates to test.
It's very common for people to throw isolate around but meaning what we call a clone, or just an even nice culture.
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junk_f00d


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Re: Cultivation General Discussion [Re: LtLurker]
#26172758 - 09/06/19 02:54 PM (4 years, 4 months ago) |
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Quote:
keen eye for vectoring
wdym?
I'm aware it will take time, but the process is fun. I just want some perfectly circular agar plates to call my own and slants I can be proud of 
And from what I've heard it can be accomplished in a much shorter time span than a year..
Edited by junk_f00d (09/06/19 03:00 PM)
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LtLurker
Lost Sailor



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Re: Cultivation General Discussion [Re: junk_f00d]
#26172771 - 09/06/19 02:59 PM (4 years, 4 months ago) |
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ok, i misunderstood thinking you said you had a bunch to test.
If you wanna do it for the love of the game and see what you can do, then fuck yea go for it and share your progress with us.
e/ I suppose the more diluted your starting syringe is the less time it could take. Apparently with the right tools you could even start with an isolate by isolating 2 spores from the get-go.
Edited by LtLurker (09/06/19 03:14 PM)
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junk_f00d


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Re: Cultivation General Discussion [Re: LtLurker]
#26172781 - 09/06/19 03:04 PM (4 years, 4 months ago) |
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junk_f00d


Registered: 12/04/15
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Re: Cultivation General Discussion [Re: junk_f00d]
#26172892 - 09/06/19 04:21 PM (4 years, 4 months ago) |
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I don't understand how accurately propogating non-isolated clone cultures works.
When you clone a non-isolated shroomie and grow it on agar it starts to sector cuz many strains reside in that clone. Got it. Where I'm confused is how to accurately grow this culture - since the beautiful shroom I picked was a genetic hodge podge, simply grabbing any leading edge would, I assume, omit other strains to some extent (similar to how strain isolation works by repeated isolation), and this could change the results similar to how repeated isolation attemps would.. Though AFAIK strains do share genetic information as they merge or whatever, so any random pick from the clone culture plate should still have genetic info from the clone. But on the other end, if you propogate leading edge sectors enough you end up with an isolate, not a clone culture.. Does that make sense?
I guess what I'm asking is if you can perfectly propogate a non-isolated clone culture? Or do you just end up with a closley related new culture? Hope that makes sense I'm just confused on what's going on here.
Edited by junk_f00d (09/06/19 05:58 PM)
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FiatFirmamentum
Stranger


Registered: 04/15/19
Posts: 59
Loc: Central EU
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Re: Cultivation General Discussion [Re: LtLurker]
#26172952 - 09/06/19 05:00 PM (4 years, 4 months ago) |
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Quote:
LtLurker said: An isolate is a single set of genetics, a single strain, 2 spores mated only. It takes a really long time, a shit ton of transfers, and a keen eye for vectoring to accomplish a true isolate. Verum's PE isolate project took him over a year to actually get some isolates to test.
It's very common for people to throw isolate around but meaning what we call a clone, or just an even nice culture.
I believe that fastest way to get isolate would be by using methods established in microbiology as effective.
I have read somewhere figure that fully colonized plate contains ~1g of wet oyster mycelium. Let's assume it's the same for cubensis. Average mass of yeast cell is around 60 picograms. Let's assume it's the same for cubensis. Therefore, we can expect that in fully colonized plate there are ~1.5 x 1010 (!) cubensis cells.
Typical wedge can be easily put in 1.5ml tube, it's around 6% of media volume in plate (108.95 cells). We blend it in 500ml of sterile water, therefore each ml contains around 1750705 cells. That's way too much. Therefore, we throw out 509ml of liquid inoculant (by putting 1ml in sterile syringe), and refill to 500ml. Now it's only 3501 cells per ml. Therefore our 10μL inoculation loop will contain around 35 cells. Assuming standard 90mm Petri plate and 45cm streaking path, we can expect less than 1 cell per cm.
Unfortunately, this reasoning assumes that cells don't clump together, but it still should yield much better results than manual transfers. Other good results can probably come from adapting the biopsy method.
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LtLurker
Lost Sailor



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Sounds like dilution but for mycellium instead of spores. I suppose it could work, but with clumping and anastosmosis, your streaks my still have a similar "strain count", just starting from a smaller piece. Don't take that as definite, just how it's sounding at first glance. Blending's gonna add some unpredictability i'd think.
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tryptkaloids
Learner



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Re: Cultivation General Discussion [Re: junk_f00d]
#26173156 - 09/06/19 07:18 PM (4 years, 4 months ago) |
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Quote:
junk_f00d said: I don't understand how accurately propogating non-isolated clone cultures works.
When you clone a non-isolated shroomie and grow it on agar it starts to sector cuz many strains reside in that clone. Got it. Where I'm confused is how to accurately grow this culture - since the beautiful shroom I picked was a genetic hodge podge, simply grabbing any leading edge would, I assume, omit other strains to some extent (similar to how strain isolation works by repeated isolation), and this could change the results similar to how repeated isolation attemps would.. Though AFAIK strains do share genetic information as they merge or whatever, so any random pick from the clone culture plate should still have genetic info from the clone. But on the other end, if you propogate leading edge sectors enough you end up with an isolate, not a clone culture.. Does that make sense?
I guess what I'm asking is if you can perfectly propogate a non-isolated clone culture? Or do you just end up with a closley related new culture? Hope that makes sense I'm just confused on what's going on here.
The short answer is we don't know. This site is the leading source and none of us have genetic testing equipment. There's a few members here who understand how genes merge so youll have to track them down, theres a lot of info but we don't knkw where the line is
-------------------- "Remember, kids, the difference between science and screwing around is writing it down" -adam savage Flowchart for Recommended plan of action. Learn the tried and true way to grow mushrooms Use the Damn search engine After you know what you're doing, take a break Pick a book, Make some chips! Josex said:Don't take the site seriously bro, ain't worth it.
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Caps McGee
Grandaddy Smurfshack



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Re: Cultivation General Discussion [Re: tryptkaloids]
#26173170 - 09/06/19 07:31 PM (4 years, 4 months ago) |
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Taking tissue and transferring until it's clean; like anyone else... Not trying to sound like an ass, but not sure what you asking exactly? Lol nimrod...
Quote:
rickyswamps said:
Quote:
Caps McGee said:
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tryptkaloids said: My bet is it's a conditions problem
I guarantee it... 10 different clones?
 No way your luck is that bad... I'd bet on excess moisture ... classic newb move
How would you come to that conclusion? The clones are grown in the same conditions as MS tubs. The clones yield, just not as good as the MS tubs. Not my first go at this.
Was looking for constructive advice instead of "classic newb move."
Conclusion drawn from the evidence my friend... suppose the same way quirk deduced conditions the issue... Constructive advice is there: lay off the water... NEWB!
 Also, get your panties out of a wad: i dont look at whos posting what, couldve sworn it was a new registry? I just call em booboo... not that field capacity has changed, just a growing realization that it's slightly wet for mushrooms to have optimal set and forget conditions... But I'll not waste any more of your time with my nonsense... We're a group of scientists sharing information, I didn't mean to hurt you...
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junk_f00d


Registered: 12/04/15
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Re: Cultivation General Discussion [Re: tryptkaloids]
#26173179 - 09/06/19 07:40 PM (4 years, 4 months ago) |
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OK, I had a feeling this was the case. So it seems like the best way then to propogate clone cultures accurately is to take enough tissue samples so that you can afford to drop entire plates into jars (tic tac toe tiger drop style like a baller), so that all sectors/strains are present? I imagine then one sector may become more dominant, but I guess another method would be taking wedges from each sector, putting into a grain jar and just hoping that anastamosis hooks a brutha up.
Just hypothetically. I don't do this personally but I might start just for fun and compare it to what I normally do (just take a wedge from a good looking sector into a new master jar).
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Caps McGee
Grandaddy Smurfshack



Registered: 10/28/17
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Re: Cultivation General Discussion [Re: junk_f00d]
#26173205 - 09/06/19 08:01 PM (4 years, 4 months ago) |
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No... take 2-5 tissue samples per intended clone in case any fail, transfer away from the rotting tissue before introducing the culture to sterile any thing... I've used a few tissue plates and gotten away with it, but generally ends poorly... transfer at least once, until the culture grows out uniform in shape and form before use... spend the extra time and plates to save yourself LOADS more on the back end... short cut and get cut short, ya digs?
If there are sectors displaying (unlikely with an MS clone culture, as it will still contain too many strains to unpack) then transfer those that look the most aggressive and clean...
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Yesum
Furry as Fuc



Registered: 11/05/12
Posts: 13,124
Loc: Central Part of Town
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Re: Cultivation General Discussion [Re: Caps McGee]
#26173242 - 09/06/19 08:24 PM (4 years, 4 months ago) |
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I think there talking about trying to keep original characteristics from a clone. How everytime you transfer from a leading sector you run the risk of loosing characteristics from the original clone
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Caps McGee
Grandaddy Smurfshack



Registered: 10/28/17
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Re: Cultivation General Discussion [Re: Yesum]
#26173254 - 09/06/19 08:33 PM (4 years, 4 months ago) |
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Absolutely... but not transfering runs the risk of never fruiting and losing ALL, not just some characteristics... keep the original tissue plate in cold storage if this is the concern... this will slow or stop the development of contamination, and preserve all available genetics .. thing about that is, even the tissue sample will be of more isolated genetics than the fruit as a whole, and realistically, the characteristics being sought after, very well transferred away from with the chosen tissue, and dried/discarded with the remnant donor
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junk_f00d


Registered: 12/04/15
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Re: Cultivation General Discussion [Re: Caps McGee]
#26173270 - 09/06/19 08:47 PM (4 years, 4 months ago) |
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Quote:
Caps McGee said: thing about that is, even the tissue sample will be of more isolated genetics than the fruit as a whole, and realistically, the characteristics being sought after, very well transferred away from with the chosen tissue, and dried/discarded with the remnant donor
This is what I'm getting at (now you're getting it ). I'm wondering if there's a way, even just hypothetically, to mitigate the slight isolation that occurs when doing tissue transfers from clone cultures.
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Caps McGee
Grandaddy Smurfshack



Registered: 10/28/17
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Re: Cultivation General Discussion [Re: junk_f00d]
#26173278 - 09/06/19 08:51 PM (4 years, 4 months ago) |
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No... clone the whole fruit, hope the rotting tissue doesn't cause bacteria I suppose... I doubt the difference discernable, and suspect this exercise to likely be one of futility
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Yesum
Furry as Fuc



Registered: 11/05/12
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Re: Cultivation General Discussion [Re: Caps McGee]
#26173286 - 09/06/19 08:55 PM (4 years, 4 months ago) |
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Also by not taking from the sector when it displays itself should help to right. Or no. You know. Take a clean piece just not from the leading sector when it shows.
I've actually been thinking about this lately myself.
Of course you have to keep transferring.
I clone whole fruits all the time. Because I'm a gangster. There just not fruits taken from a tub but rather a fruiting petri
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Edited by Yesum (09/06/19 09:05 PM)
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mushboy
modboy



Registered: 04/24/05
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Re: Cultivation General Discussion [Re: Yesum]
#26173298 - 09/06/19 09:08 PM (4 years, 4 months ago) |
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Quote:
Yesum said: Because I'm a gangster.

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