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OfflineTryptx
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Best agar method
    #20260101 - 07/11/14 09:32 PM (10 years, 6 months ago)

So I have light malt extract and agar.  What's the best ratio.  And what's best for making a mono culture out of; spore, clone,  or lc.  Guessing clone.

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InvisibleMudaFuka
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Re: Best agar method [Re: Tryptx]
    #20260123 - 07/11/14 09:34 PM (10 years, 6 months ago)

I use PDA so I cant really help you with LME ratios but I would say starting with a clone is the fastest way to an isolate.


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Re: Best agar method [Re: Tryptx]
    #20260142 - 07/11/14 09:38 PM (10 years, 6 months ago)

my fave recipe:

22 grams agar
20 grams ME
1000 mL hot water

1. clones
2. T1
3. T2
4. T3
5. T4

You should have a monoculture by T4 or T5

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InvisibleGhatti
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Re: Best agar method [Re: Mykes logos]
    #20260187 - 07/11/14 09:47 PM (10 years, 6 months ago)

That's a very rich recipe. Go 500ml water, 10g agar, 8-10 grams LME.

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OfflinecronicrMFacebook
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Re: Best agar method [Re: Ghatti]
    #20260306 - 07/11/14 10:09 PM (10 years, 6 months ago)

Quote:

Ghatti said:
That's a very rich recipe. Go 500ml water, 10g agar, 8-10 grams LME.



thats like the same recipe cut in half lol


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InvisibleGhatti
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Re: Best agar method [Re: cronicr]
    #20260325 - 07/11/14 10:13 PM (10 years, 6 months ago)

Well damned if it ain't. Whoops.

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Re: Best agar method [Re: Ghatti]
    #20260336 - 07/11/14 10:15 PM (10 years, 6 months ago)

:lmafo:


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OfflineDos Ounce
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Re: Best agar method [Re: MudaFuka]
    #20260393 - 07/11/14 10:26 PM (10 years, 6 months ago)

:trololol:  :threadmonitor:

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OfflineKizzle
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Re: Best agar method [Re: MudaFuka]
    #20260458 - 07/11/14 10:43 PM (10 years, 6 months ago)

Am I the only one who streaks my plates? When you streak the spores across the plate the amount of spores deposited on the agar decreases as you progress down the streak. At the end of streak there are the least spores which means the least amount of strains produced in that area and the least amount of transfers required to get a monoculture. 5 transfers to get an isolate seems kind of excessive.


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Invisiblenucleophile
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Re: Best agar method [Re: Kizzle]
    #20260566 - 07/11/14 11:08 PM (10 years, 6 months ago)

Yessir, I also enjoy a good streaked plate, old microbiology habit. Haven't started enough spores on plates to see how well it works for isolating but it's certainly a fine thing for germinating.

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Re: Best agar method [Re: Kizzle]
    #20260579 - 07/11/14 11:14 PM (10 years, 6 months ago)

Quote:

Kizzle said:
Am I the only one who streaks my plates? When you streak the spores across the plate the amount of spores deposited on the agar decreases as you progress down the streak. At the end of streak there are the least spores which means the least amount of strains produced in that area and the least amount of transfers required to get a monoculture. 5 transfers to get an isolate seems kind of excessive.




I prefer to fruit bulk MS, then take clones from the healthiest, best-looking clumps (1st flush). Starting from MS on plates is fine, too tho, yeah... I agree with you.

I always assume newbies need more transfers away from contamination and tend to transfer larger pieces... 5 transfers is definitely too many, but I think they'll find success by then. Assuming they transfer as soon as new growth appears they shouldn't wear the mycelium out.. right?

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Re: Best agar method [Re: Mykes logos]
    #20260582 - 07/11/14 11:15 PM (10 years, 6 months ago)

i rarely see sectoring before 4 transfers from spores:shrug:


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InvisibleGhatti
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Re: Best agar method [Re: cronicr]
    #20260725 - 07/11/14 11:54 PM (10 years, 6 months ago)

What? I usually see sectoring on 1st transfer from my spore plate.

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Re: Best agar method [Re: Ghatti]
    #20260730 - 07/11/14 11:56 PM (10 years, 6 months ago)

you may see some sorta sectoring but not real strong sectoring that makes isolating easier


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InvisibleGhatti
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Re: Best agar method [Re: cronicr]
    #20260823 - 07/12/14 12:21 AM (10 years, 6 months ago)

I got shit all lucky with my last sleeve and had strong rhizomorphic growth on a spore germination plate. Like at the point of germination and could identify a sector by the time it neared the edge of the plate.(gave me a good place to start anywho)

Made my job a lot shorter.

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OfflineKizzle
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Re: Best agar method [Re: cronicr]
    #20260836 - 07/12/14 12:23 AM (10 years, 6 months ago)

Quote:

I prefer to fruit bulk MS, then take clones from the healthiest, best-looking clumps (1st flush). Starting from MS on plates is fine, too tho, yeah... I agree with you.

I always assume newbies need more transfers away from contamination and tend to transfer larger pieces... 5 transfers is definitely too many, but I think they'll find success by then. Assuming they transfer as soon as new growth appears they shouldn't wear the mycelium out.. right?



I don't even bother with strain isolation normally and streak mainly for the purpose of making it easier to spot and isolate mycelium from any contaminants in the spores. If I was intent on getting a monoculture though I believe that's the fastest way.

Quote:


i rarely see sectoring before 4 transfers from spores:shrug:




That's basically the point I was trying to make. You can see sectoring on the first plate when you streak.


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InvisibleGhatti
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Re: Best agar method [Re: Kizzle]
    #20260866 - 07/12/14 12:31 AM (10 years, 6 months ago)

I've taken to using a bread tie as my inoc loop.

Strip it down to just wire and either touch the very tip to agar, then the outside lightest ring of a print or lay it down and use it that way.

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Re: Best agar method [Re: Ghatti]
    #20260877 - 07/12/14 12:33 AM (10 years, 6 months ago)

i always streak but am yet to get a mono culure that fast mind you i've been doing alot more cloning these days then isolating


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Re: Best agar method [Re: cronicr]
    #20260894 - 07/12/14 12:38 AM (10 years, 6 months ago)

i cool loop/blade in receiving plate, and gently, lightly touch/dab the print (instead of swiping/streaking the print)  and swipe one line back in receiving plate.  ill do three lines, 1/2" apart, 1" long about, for same reason kizzle streaks.  def can see sectoring by plate 4
some times even by plate two, u can begin to see where it would begin to sector if left to grow



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InvisibleGhatti
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Re: Best agar method [Re: blindingleaf]
    #20260908 - 07/12/14 12:41 AM (10 years, 6 months ago)

Next time you go from print, try using the tiniest point you can find. Ie: needle point, teeny tiny paperclip, bread tie, etc. It drastically cuts down on the amount of plates you go through.

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Re: Best agar method [Re: Ghatti]
    #20260919 - 07/12/14 12:44 AM (10 years, 6 months ago)

yeah but the idea from spores is to get  more then one isolate so ya sorta want some good sectoring:shrug:


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Re: Best agar method [Re: cronicr]
    #20260922 - 07/12/14 12:45 AM (10 years, 6 months ago)

true that!!
there will def be enough sectors tho


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Re: Best agar method [Re: blindingleaf]
    #20260925 - 07/12/14 12:46 AM (10 years, 6 months ago)

not as much as normal i guess ur right.  but it would be hard for me to test that many isolates at once!!


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OfflinecronicrMFacebook
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Re: Best agar method [Re: blindingleaf]
    #20260930 - 07/12/14 12:47 AM (10 years, 6 months ago)

oh i was just talking about trying to get to an iso in as little plates as possible..thats not my goal
Quote:

blindingleaf said:
not as much as normal i guess ur right.  but it would be hard for me to test that many isolates at once!!



yeah i rarely test them all lol, normally find a good one fairly fast but i'll admit my last two times turned out crap for me which is y i went back to dfa and agar pins:hehehe:


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InvisibleGhatti
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Re: Best agar method [Re: blindingleaf]
    #20260935 - 07/12/14 12:48 AM (10 years, 6 months ago)

I usually start several spore plates at a time just in case one is covered in funk. I'd rather just use one of the others than to try to spend the time cleaning which might cause me not to grab the pieces I want.

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Re: Best agar method [Re: cronicr]
    #20260936 - 07/12/14 12:48 AM (10 years, 6 months ago)

yea cloning way better for me too!


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InvisibleGhatti
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Re: Best agar method [Re: Ghatti]
    #20260945 - 07/12/14 12:50 AM (10 years, 6 months ago)

What's dfa? And I've never had my agar pin, how long do they normally take to pin and do you have to let them colonize the entire plate?

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Re: Best agar method [Re: Ghatti]
    #20260956 - 07/12/14 12:53 AM (10 years, 6 months ago)

dogfood agar, tends to pin in under a month no problem if you leave the plate to colonize(any recipe will), you can then drop that pin on anopther plate or just toss it right in a grain jar


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Re: Best agar method [Re: cronicr]
    #20260974 - 07/12/14 12:55 AM (10 years, 6 months ago)

Ahh gotcha. I tend to always throw my plates in the fridge before they colonize fully.

Do you let them pin just so you know you have a fruiting strain?

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OfflineKizzle
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Re: Best agar method [Re: cronicr]
    #20260985 - 07/12/14 12:57 AM (10 years, 6 months ago)

Quote:

cronicr said:
i always streak but am yet to get a mono culure that fast mind you i've been doing alot more cloning these days then isolating



Well me too. I actually think you're better off using clones without trying to isolate them any further. Occasionally though I've isolated individual strains for side-by-side experiments.

I was just throwing it out there because Mykes logos mentioned needing 4-5 transfers when you can get one in more like 2-3 transfers. I should probably mention the streaks I make are nearly the circumference of the plate, it has to be long enough.


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Re: Best agar method [Re: Kizzle]
    #20261491 - 07/12/14 04:44 AM (10 years, 6 months ago)

Quote:

Kizzle said:
Quote:

cronicr said:
i always streak but am yet to get a mono culure that fast mind you i've been doing alot more cloning these days then isolating



Well me too. I actually think you're better off using clones without trying to isolate them any further. Occasionally though I've isolated individual strains for side-by-side experiments.

I was just throwing it out there because Mykes logos mentioned needing 4-5 transfers when you can get one in more like 2-3 transfers. I should probably mention the streaks I make are nearly the circumference of the plate, it has to be long enough.




I rarely ever go past T2 from clone but I assume noobs may transfer larger pieces and may have a tougher time with contaminants... and if you make an isolate from MS than you're much more blind than you would be from clone, and may be wasting your time.

But you can do both... many ways to get it done :hatsoff:

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Re: Best agar method [Re: Mykes logos]
    #20261542 - 07/12/14 05:14 AM (10 years, 6 months ago)

I have stones growing on one of my ATL plates. I was wondering if I can transfer it and revive the plate.

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