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InvisibleHelltick
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How do I isolate on agar jar?
    #6669932 - 03/14/07 06:29 PM (16 years, 11 months ago)

All my work is done in a still air box using sterile tools...




Do I just take a piece of the most aggressive growth using tweezers and transfer to a fresh agar jar?

Here is a link to my journal to explain my prep...

http://www.shroomery.org/forums/showflat.php/Cat/0/Number/6613630/an/0/page/0

BTW...culture was started from tissue of a fresh stype.
Thanks.


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  My Agar Tek. 
  My Greenhouse Tek. 

"It's not good when it's done, it's done when it's good"


Edited by Helltick (03/14/07 06:41 PM)


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Invisiblemusher_420
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Re: How do I isolate on agar jar? [Re: Helltick]
    #6670091 - 03/14/07 07:15 PM (16 years, 11 months ago)

I'm a newbie to isolations and I'm not 100% on the micro-biology involved, but I believe if you have taking the sample from a stipe of a fruit you already have an isolate. It's been my experience that a cloned fruit will produce fruits with similar morphology and fruiting characteristics.

Transfer a wedge to a grain jar or us the LC method and fruit that baby off. Take another wedge and put it into a sealed jar or sterile distilled water, this will preserve the culture if you find it has favorable fruiting characteristics.

To isolate from spores you must first inoculate the plate with multispore by streaking from a print or a few drops of a syringe. I suggest streaking as using spore solution tends to cause more bacteria problems for me.

Once the spores have germinated you will notice different segments of growth. Transfer a small wedge from the best growing segments to new jars until you no longer see the segmenting. This will now be an isolate. It's pretty self explanatory once you've had a bit of hands on experience. The idea is to isolate > test > store. Good Labeling or record keeping is important.


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InvisibleSwan Song

Registered: 02/08/06
Posts: 559
Re: How do I isolate on agar jar? [Re: musher_420]
    #6670109 - 03/14/07 07:23 PM (16 years, 11 months ago)

Yes you have the Idea. Just isolate the aggressive myc place on the agar, place in incubator for a few days to make sure that no bacteria had gotten onto the agar. then if it is clear of contams, continue to incubate and watch to make sure that the myc is growing the same as the myc you isolated from.
after it has colonized the agar. cut a wedge, and spawn your grain/LC.
from there you can do G2G transfers to keep the clean super strain. and while doing that, you can put another piece in a vacuum tube with agar, let it just start to colonize then refrigerate. Will keep for quite a while.


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InvisibleHelltick
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Re: How do I isolate on agar jar? [Re: musher_420]
    #6670137 - 03/14/07 07:31 PM (16 years, 11 months ago)

^^Thanks for that!
Silly me...I thought it was to isolate from possible contams that may have been passed from the given tissue during the transfer.

One question though...could I ,say add 20 cc of sterile water from sterile syringe to the jar...shake the shit out of it, draw back to sterile syringe, and have a viable inoculate?

All done within a still air box of coarse.

Thanks again!


--------------------
  My Agar Tek. 
  My Greenhouse Tek. 

"It's not good when it's done, it's done when it's good"


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InvisibleBacchus
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Re: How do I isolate on agar jar? [Re: Helltick]
    #6671582 - 03/15/07 04:04 AM (16 years, 11 months ago)

Yes, that approach should work. In the past, I've moved samples from agar to test tubes and agitated with 4cc of sterile water using a syringe. I've used this method when testing the fruiting ability of several substrains and only need to inoculate two jars with each.


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OfflineRogerRabbitM
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Re: How do I isolate on agar jar? [Re: Bacchus]
    #6671990 - 03/15/07 09:34 AM (16 years, 11 months ago)

Just so everyone is clear in their understanding, we do two types of 'isolation'. One is to isolate away from possible contaminants which is what you'll be trying to do with your cloned tissue.

The other type of isolation is when we isolate individual strains away from each other after inoculating with hundreds, or even thousands of spores. That's a completely different process.
RR


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OfflineHobosapien
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Re: How do I isolate on agar jar? [Re: RogerRabbit]
    #6673095 - 03/15/07 02:33 PM (16 years, 11 months ago)

ay,


so roger, your saying one reason to clone (away from contamination) would be to save an otherwise desirable trait; and the other would be to acquire said desirable trait?

hope i didnt just open a whole can of worms here....

(apologies to thread stater.) 'JUS BEEN JACKED!


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Offlinefromthemoon
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Re: How do I isolate on agar jar? [Re: Hobosapien]
    #6673505 - 03/15/07 04:12 PM (16 years, 11 months ago)

I think I finally get it.

When cloning, it's easy to get a contam onto the agar w/ the mushie tissue.  If the tissue is able to grow over part of the agar, even with a contam present, one can cut a small peice free from the contam and transfer to a new agar dish.  I guess this would be repeated until a contam free plate is grown.  I've never tried cloningyet, but now that I get it I'll give it a try :wink:

When isolating a strain (your spore source should be contam free, otherwise the contams most likely would over power the plate before the spores germinate), conditions are sterial to start and one is germinating their spores on the agar.  With repeated transfers, one is picking fast growers, strong myc, etc, until there is one clear mcy strain.  Then we fruit all the different strains we isolated and hope one of them colonizes 100 times faster than the norm and produces 100 times more yeild than the norm.

right?


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Look at the atoms in the air and allow your mind to see the other planes that coexists in the same physical space where you sit. There are vibrational strings in each and every atom containing infinite realities, universes, and dimensions. Multidimensional beings and entities are RIGHT IN FRONT OF YOU!!! Only gravity and mental energy can pass through these different atomic branes and mushrooms can break the mental/spiritual membranes separating one reality from the other. TAKE A LOOK!


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OfflineRogerRabbitM
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Re: How do I isolate on agar jar? [Re: fromthemoon]
    #6673941 - 03/15/07 06:17 PM (16 years, 11 months ago)

Yea, you have it. Sometimes, even with a clean sporeprint, you'll get a bit of contamination on the dish along with the mushroom mycelium. This is easy to transfer away from, by taking a small piece of mushroom mycelium and moving it to a new dish. Always take mycelium from the leading edge of the growth because this is the farthest away from the contaminant. We also transfer individual sectors away from each other so individual strains can grow out to determine the best fruiting ones.

When cloning wild mushrooms, there's almost always molds and bacteria present, so it takes several transfers to get a clean culture.

I do a full strain isolation and cloning tek on my DVD with very clear, closeup pictures(video) so that should make it much easier to understand for visual learners. I'll let everybody know when it's out. Everytime I try to give a release date, I get some new software or graphics, and 'have' to put them in too and it sets the time back. I'm also seriously anal about quality, so have filmed many of the segments again and again until I'm happy with them. It all takes time. It shouldn't be more than a month or two though, but I'm also working on copyrights and trademarks to protect my work, so I have to wait for the lawyers on that one.
RR


--------------------
Download Let's Grow Mushrooms



semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
Thomas Edison


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InvisibleBlutjager
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Re: How do I isolate on agar jar? [Re: RogerRabbit]
    #6674378 - 03/15/07 08:23 PM (16 years, 11 months ago)

I have yet to have any success with agar work,mostly because there is not really all that much information on it to learn from and no real step by step "tek's" demonstrating agar work.

Saying I am looking forward to buying your dvd is an understatement,I am more than happy to wait for it and it is well worth having to wait for while you redo things to improve them,I'm sure there will be more than a few things to be learned from your DECADES of experience and with all you know there is probably always going to be "Just one more thing" that you wanted to put on it:rockon:.I have been trying to grow for yrs on and off with no success buying books I did not understand and crappy ripoff kits that yielded me every color of the rainbow in petri-dishes and 6 page instruction booklets that explained nothing and were obviously ran off on a copy machine with no attention payed to quality whatsoever:thumbdown::slitwrist:

Than I found the Shroomery and when I think about how much progress I have made and all that I have learned in just my short time here it boggles my mind,I can only imagine how much I will know when I have been doing it as long as you have:hehehe:


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InvisibleHelltick
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Re: How do I isolate on agar jar? [Re: Blutjager]
    #6674404 - 03/15/07 08:28 PM (16 years, 11 months ago)

I have a pictorial in the works...check it out.

http://www.shroomery.org/forums/showflat.php/Cat/0/Number/6613630/an/0/page/0

keep in mind its not done yet...still to picture the cloning process and things...but the Agar/jar prep works awesome.

tissues just rip thru that agar...never have a contamed LC again!

gl.


--------------------
  My Agar Tek. 
  My Greenhouse Tek. 

"It's not good when it's done, it's done when it's good"


Edited by Helltick (03/15/07 08:33 PM)


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