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Anonymous

Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74411 - 03/31/00 10:12 AM (20 years, 5 months ago)

DJCarcass

There are 3 major things you need to know about H2O2:

1. It gets broken down by mushroom/mycelial tissue when the cell walls are breached
2. Most growth medium also breaks it down unless heat treated.
3. It kills spores!

So if you put it in contact with spore prints or spore water then you will just kill the spores.

IMHO the recent enthusiasm about it's use in shroom growing is mostly from people who have not actually tried to use it. It has severe limitations.

I have developed a method which uses it at every stage, including in the actual growth medium. This virtually eliminates all contamination and means that you can colonise your substrate in the same trays that you fruit in. You have to use pressure cooked grain (wheat or rye) to prevent the H202 being degraded or you can use Weetabix (spun, whole grain pillows) as this is unreactive.

Having got it to work I have returned to using straight-forward agar in a ?5.00 home-made glovebox. It works out far simpler and quicker.

Rush Wayne's book on the subject has some methods which look promising for wood loving shrooms. It uses compressed-paper fuel pellets. He also have a recipe for H2O2 agar which is excellent for cloning.

Rad



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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 (20 years, 5 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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InvisibleHippie3
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Registered: 11/07/99
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Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74412 - 03/31/00 07:31 PM (20 years, 5 months ago)

The JafF said
"I will say that I just did my first experiments with honey water.. Whoever came up with this deserves an applause..."

that would be cluB99.


peace
hip


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[This message has been edited by Hippie3 (edited April 11, 2000).]



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OfflineHumidity
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Registered: 04/02/00
Posts: 358
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Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74413 - 04/02/00 02:50 PM (20 years, 5 months ago)

Funkyballoon sounds great!
Just a couple of quick questions.

What do you do with the rice/water mixture after the mycelum grows?

Thanks.



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_____________________________________________________________________________________
"The greatest enemy of knowledge is not ignorance, it is the illusion of knowledge." -Stephen Hawking


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Anonymous

Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74415 - 04/02/00 11:38 PM (20 years, 5 months ago)

Where can I get H2O2 easily? How much does it cost?

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OfflineHumidity
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Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74416 - 04/02/00 11:43 PM (20 years, 5 months ago)

H2O2 is hydrogen peroxide. You can buy it at the grocery store. It is not very expensive.


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_____________________________________________________________________________________
"The greatest enemy of knowledge is not ignorance, it is the illusion of knowledge." -Stephen Hawking


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Anonymous

Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74417 - 04/03/00 09:32 AM (20 years, 5 months ago)

Ohhh. Sorry I didn't know.

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OfflineDan
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Registered: 05/11/01
Posts: 187
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Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74418 - 04/04/00 10:54 PM (20 years, 5 months ago)

So this tek would be called: agar tek without the agar? WTF? Why not go to the trouble and actually go on to agar? All the steps are the same for sterility? Is this some sort of proverbial stepping stone into the arena of agar from PF?

Look, agar is just plain EASY, ok? PF tek only made it harder to teach people how to actually culture sterile mycelium properly.

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Anonymous

Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74419 - 04/09/00 11:41 PM (20 years, 5 months ago)

so...any more success stories concerning these methods. I'm wondering how well the h2o2 does in sterilizing regardless, really, of whether the myc. is agar grown or disected.


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Anonymous

Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #74420 - 04/10/00 11:37 PM (20 years, 5 months ago)

I love how people are so ready to defend agar simply because its the "right" way to do it... Lets see how all the steps for sterility are the same...

First agar..

Choose your agar mixture (there are hundreds of recipes to choose from) and prepare it. You can get premade mixtures, or mix your own.
note: this involves boiling all your ingrediants, sometimes straining out solids (depending on the recipe). You will need containers to do the agar work in, typically petri dishes, but wide mouth jars will work.. If you use plastic petri dishes, you must sterilize your agar mixture in a sperate container and somehow manage to pour it into your dishes, WHILE HOT! You must do this in a sterile location and keep them in a sterile location so they don't suck in dirty air while they cool.

Then once they solidify you just innoculate them, either by isolating a strain from another agar dish, cutting it out (hoping you have selected a single strain) and transfering it (in open air again) into the new fresh petri dish.

Of you can skip right to the culturing of a fresh mushroom to isolate a strain. This involes slicing open a stem and cutting out a section of the inside mycellium, and transfering to the fresh dish (all while praying your scalpel is clean and that nothing gets in your dish while it is open, or lands this clean piece of mycellium while you transfer it, or that you didn't contaminae it by touching the outside of the mushroom with ANYTHING once you sliced open the mushroom)...

And now you still have to wait until the dish grows out until you can use it. If there is contamination you have to keep trying to reisolate your strain from this dish into another dish. Then if you have managed to do this without contaminating it, you still have to take a risk again when you use this stuff to innoculate your substrate... either by usuing wedges (and openeing your substrate jars wide open) or blending it up.. (another open container)...
time... minimum 10 days... practically... 14-21 days.

Ok and now the new peroxide method..

Put piece of stem in coffee grinder with water or h2o2... No sterile conditions necessary...

Blend....No sterile conditions necessary...

Spoon into 1/3 cup peroxide...no steil conditions necessary...

Suck up with syring... No sterile conditions necessary...

Innoculate... time 5 minutes...

Yeah thats the same...


As a side note:

I do have quart jars of birdseed with all three of those species growing,(shitake, crimini, chantarelles) although I did make one stupid mistake... I bothered with the Chantarelles... the mycellium is growing, but I am pretty sure they can't be cultivated... I didn't realize this when I started.. Oh well... I will try anyway for the fun of it..

ThE JafF



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InvisibleMyco-420
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Sound's interesting to me..... [Re: Anonymous]
    #313906 - 05/10/01 01:00 PM (19 years, 4 months ago)

hmmmmmmmmm. What do you guys think?

Someone should email me a bowl.


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InvisibleBrownPastures
old hand

Registered: 03/16/01
Posts: 968
Loc: here
Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: Anonymous]
    #313973 - 05/10/01 02:42 PM (19 years, 4 months ago)

your funny man... edible mushrooms are ... well you know... but the thing is i'm not sure if your just being "clever" and just saying your growing smak-takis and whatever else you were grownin'... :laugh: as for the coffee grinder thing with adding LIQUIDS into a coffee grinder?!?!? what kind of grinder do you have? i'd like to get my grubby hands on a WATERPROOF COFFEE GRINDER.... ne-way ... all that H2o2 makes me kinda nervous...:D.... what do you guy's say ??? peace

"like a Japanese Cowboy or a blind man on skates"Ween


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OfflineoOjonahOo
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Re: Sound's interesting to me..... [Re: Myco-420]
    #313974 - 05/10/01 02:44 PM (19 years, 4 months ago)

Wow, this post was back in the day!
Too bad the pics are gone....;(




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OfflineTaz
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Registered: 03/18/01
Posts: 1,090
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Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: BrownPastures]
    #313982 - 05/10/01 02:52 PM (19 years, 4 months ago)

yo I would like to know the brand name of that coffee maker too...........who sells it? wal-mart, k-mart?

"Out of chaos...comes order...." AND THE FSR..


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OfflineTlaloc
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Registered: 05/03/01
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Last seen: 18 years, 5 months
Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #313988 - 05/10/01 03:00 PM (19 years, 4 months ago)

what happend to the picts!???? there not loading for me,....and im on t1 damn it.

I wanna see too.....WAAAAAAAAAAA!<sob> ;0(



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Bill Hicks (1961-1994) "SQUEEGIE YOUR THIRD EYE! "


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Offlinepsylo330
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Registered: 05/08/01
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Last seen: 18 years, 4 months
Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: Tlaloc]
    #314000 - 05/10/01 03:11 PM (19 years, 4 months ago)

Cool! Dos that tek work with a stem of a mushroom U find outside?

"What da fuck??!!"


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I am completly mad so dont listen to a word I say its the littleblueguyz fault.If I don'tpost anything for a few days, it's that I have suicided myself or I had decided to live on Mars.


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Offlinepsylo330
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Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: psylo330]
    #314654 - 05/11/01 12:03 PM (19 years, 4 months ago)

g

"What da fuck??!!"


--------------------
I am completly mad so dont listen to a word I say its the littleblueguyz fault.If I don'tpost anything for a few days, it's that I have suicided myself or I had decided to live on Mars.


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InvisibleMyco-420
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Registered: 09/29/00
Posts: 66
Loc: BFE
Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: funkyballoon]
    #314679 - 05/11/01 12:36 PM (19 years, 4 months ago)

awwww fuck it man... use suran wrap in the bottom or just go guerilla style and sacrifice the coffee grinder for the hobby



Someone should email me a bowl.


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OfflineMAIA
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Re: NEW Tissue Culture Technique - No agar, No flow hood, easy as hell [Re: Myco-420]
    #314715 - 05/11/01 01:08 PM (19 years, 4 months ago)

Moulinex 123 should also work to mix the h2o2 with tissue.
BTW, how many shroom tissue do you need to inoculate one agar petri dish (I was thinking 0,5cm x 0,5cm)

== :) Spread the Spores :) ==


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Use, do not abuse; neither abstinence nor excess ever renders man happy.
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