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Amazon Shop for: ½ Pint Jars, Agar, Brown Rice Flour, Herb Grinder, Laminar Flow Hood, Rye Grain, Vermiculite

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Offlinefunkyballoon
Pooh-Bah
Registered: 01/19/99
Posts: 311
Last seen: 15 years, 3 months
NEW Tissue Culture Technique - No agar, No flow hood, easy as hell
    #74390 - 02/14/00 03:07 PM (16 years, 9 months ago)






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OfflineLizard King
King Lizard

Registered: 10/04/99
Posts: 1,998
Loc: GA
Last seen: 10 years, 3 months
Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74391 - 02/14/00 03:26 PM (16 years, 9 months ago)

Cool!

I'm gonna give that one a try :smile:

The Lizard,



--------------------


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InvisibleLinkA
Intrinsically No Good

Registered: 07/29/99
Posts: 2,311
Loc: Lost Woods, Hyrule
Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74392 - 02/14/00 03:59 PM (16 years, 9 months ago)

damn, that's awsome, funky. how well does it work for you? i'm gonna have to try that. you need to come around more often. first SOS, now this. you're gonna have to register to become an official mad scientist :wink:

Link

------------------
In the beginning the Universe was created...
This made a lot of people angry and was widely regarded as a bad move.
-- Douglas Adams, The Restaurant at the End of the Universe



--------------------
Wonder had gone away, and he had forgotten that all life is only a set of pictures in the brain, among which there is no difference betwixt those born of real things and those born of inward dreamings, and no cause to value the one above the other.  -- H.P. Lovecraft "The Silver Key"


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Offlinefunkyballoon
Pooh-Bah
Registered: 01/19/99
Posts: 311
Last seen: 15 years, 3 months
Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74393 - 02/14/00 06:45 PM (16 years, 9 months ago)

Something I forgot to mention: It is a very good idea to inoculate the culture jar without removing the tape from the holes. Just stick the needle right through, then get another peice of tape ready to seal the puncture wound.

So far it has worked perfectly for me. Fabulous colonization rates, MASSIVE pinhead formation.



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OfflinePanTrop
enthusiast
Registered: 11/03/99
Posts: 288
Loc: Washington
Last seen: 14 years, 10 months
Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74394 - 02/14/00 08:02 PM (16 years, 9 months ago)

ohhhhhhhh, YEA now thats COOL! Right on Funky! I will have to try that one!!!!


--------------------
" My mind dont work if my spine dont jerk!"


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Offlinelestat
newbie
Registered: 11/18/99
Posts: 34
Last seen: 15 years, 1 month
Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74395 - 02/14/00 09:47 PM (16 years, 9 months ago)

hey, funky b.
cool tek! would there be any problem using a blender instead? i got like no coffee grinder, man.

peace


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Anonymous

Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74396 - 02/15/00 12:43 PM (16 years, 9 months ago)

Sterile Tissue Cloning Procedure

1. Create a past of brown rice flour and water (or your choice of
substrate), spread evenly in the bottom of a canning jar, seal,
pressure cook at 15 psi for 30 minutes.
2. Pour about 1/3 cup of 3% h2o2 into a jar.
3. Obtain a clean coffee grinder; fill with water to the brim of
the metal cup, insert a fresh and cleaned mushroom stem
(no cap) and proceed to grind for 10-15 seconds.
4. Use a clean spoon to transfer some of the resulting mixture into
the h2o2 jar.
5. Let it fizzle for a couple minutes, then draw into a syringe.
6. Inoculate jar created in step#1, or simply inoculate pf-style
jars.

Tips if Culture Contaminates

1. Extract a piece of healthy mycelium.
2. Cut mycelium into tiny pieces with razor blade.
3. Transfer pieces to a small pool of h2o2 and then suck into
a syringe.
4. Inoculate another culture jar with it.



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Anonymous

Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74397 - 02/15/00 01:35 AM (16 years, 9 months ago)

um, you ... rock (if I try this and it works, explitives will be added)

I'm guessing by the way, that organic rye flour/quinoa/etc. could be used as a substitute, I'll try and post my results.

-peace



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Anonymous

Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74398 - 02/15/00 01:53 AM (16 years, 9 months ago)

um, you ... rock (if I try this and it works, explitives will be added)


BTW, for those of us that are anal, what is the amount of BRF/water to use for one 1/2 pt. jar?

-peace[/B][/QUOTE]



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Anonymous

Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74399 - 02/15/00 06:08 AM (16 years, 9 months ago)

Yo LINK,

I think this one deserves the growing archives....

EXCELLENT!!!!!!!!!!



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Anonymous

Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74400 - 02/15/00 11:28 AM (16 years, 9 months ago)

Overall a fantastic Mad Scientific idea!!! I bow to you on this one... but uhh, why not fill coffee grinder to rim with H2O2 not H2O and save a step?

Another idea would be to use this to immedatly transfer to a liquid media like honey water or malt solution... again.. WOW!!!

ThE JafF

[This message has been edited by ThE JafF (edited February 15, 2000).]



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InvisibleLinkA
Intrinsically No Good

Registered: 07/29/99
Posts: 2,311
Loc: Lost Woods, Hyrule
Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74401 - 02/15/00 02:11 PM (16 years, 9 months ago)

hey Burple, good idea, i'll see what ythan and aphex have to say

Link

------------------
In the beginning the Universe was created...
This made a lot of people angry and was widely regarded as a bad move.
-- Douglas Adams, The Restaurant at the End of the Universe



--------------------
Wonder had gone away, and he had forgotten that all life is only a set of pictures in the brain, among which there is no difference betwixt those born of real things and those born of inward dreamings, and no cause to value the one above the other.  -- H.P. Lovecraft "The Silver Key"


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Offlinecamel
old hand

Registered: 04/03/99
Posts: 414
Last seen: 13 days, 11 hours
Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74402 - 02/15/00 05:24 PM (16 years, 9 months ago)

I PERSONALLY would much rather do agar than anything else. It is sooooo incredibly easy, you do not even know til you try it.

peace
camel



--------------------
Don't do drugs.


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Offlinefunkyballoon
Pooh-Bah
Registered: 01/19/99
Posts: 311
Last seen: 15 years, 3 months
Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74403 - 02/15/00 06:53 PM (16 years, 9 months ago)

ThE JafF,

I would have skipped that step, but on my Western Family bottle of h2o2 it says that it should not be shaken. Doesn't give a reason, but I just assumed they know what they're talking about.



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Anonymous

Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74404 - 02/16/00 12:30 PM (16 years, 9 months ago)

I believe that is just becasue of how unstable peroxide is.. That is why you are supposed to store it in a cool place, that is why it is in a brown bottle, etc..

A whole lot of it, maybe most of it will be destroyed as the mushroom comes in contact with it. The way you do it, there is a lot larger peroxide/shroom ratio... So your way is probably a better way to do it after all..

I will say that I just did my first experiments with honey water.. Whoever came up with this deserves an applause... I got growth in just under 24 hours in 4 out of 6 jars (Rye flour/Vermiculite).. I am sure the other 2 are not far behind.

From 1 squirt (1 cc) of spore water I am sure one could innoculate 100 jars just by waiting 10 days for the honeywater to grow (from spores)..

I also reinoculated some more honey jars, and of course I have growth there as well.. Damn this is quick easy and cheap.. The real test is to wait a week and make sure nothing contaminates, but I have faith..

If it works well, I think that the combination of your technique and the honey will greatly improve yeild. Isolated strains perform sOOOOooooo much better.

So guess who's tek I will be trying next?

ThE JafF

[This message has been edited by ThE JafF (edited February 16, 2000).]



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Offlinefunkyballoon
Pooh-Bah
Registered: 01/19/99
Posts: 311
Last seen: 15 years, 3 months
Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74405 - 02/16/00 03:23 AM (16 years, 9 months ago)

Honey jars.... that's something I haven't heard of. Let me guess - honey + water? I'm assuming the ratio is 4/96 by weight, correct?


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Anonymous

Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74406 - 02/16/00 10:03 AM (16 years, 9 months ago)

Well its actually much easier to measure.

1/2 teaspoon per 250ml water... I just cook it up in 1/2 pints with the lids the right way, not inverted, and a hole for the syringe. I cover with foil while cooking, remove to inoculate, and immediatly cover with electrical tape. Works fantastic so far. I will keep everyone updated.. The important thing seems to be not to overcook the honeywater, or you will carmalize it.. So try pressure cooking for 20 min, that seems to work well.

ThE JafF



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Anonymous

Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74407 - 02/16/00 12:31 AM (16 years, 9 months ago)

Ok, it's the next morning and all jars show growth...Did another 6 jars as well. This is fast...

Also I have no idea what I was talking about before. You could still use peroxide in the coffee grinder and then transfer a spoon or two of this to more peroxide.. No reason to introduce any more contamination than necessary.

ThE JafF



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OfflineNCubensis
member
Registered: 07/09/99
Posts: 62
Last seen: 7 years, 9 months
Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74408 - 03/30/00 11:55 PM (16 years, 8 months ago)

Bump. So, anybody have success with this h202 tek?


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Anonymous

Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74409 - 03/31/00 12:12 PM (16 years, 8 months ago)

I tried it and it worked for in all the jars, with ZERO contamination.... I am trying it again with
7 quarts of chantarells, 7q shitake, 7q crimini... Will report in a few days...

ThE JafF



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Amazon Shop for: ½ Pint Jars, Agar, Brown Rice Flour, Herb Grinder, Laminar Flow Hood, Rye Grain, Vermiculite

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