|Posted on Friday, October 12, 2001 - 09:41 pm:||
Sterile Tissue Cloning Procedure
1. Pour about 1/3 cup of 3% h2o2 into a jar.
2. Obtain a 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. note: a blender can be substituted for a grinder.
3. Use a clean spoon to transfer some of the resulting mixture into the h2o2 jar.
4. Let it fizzle for a couple minutes, then draw into a syringe.
optional: the syringe can be partially filled with sterile honey water beforehand to provide nutrients to sustain the tissue.
5. use immediately or refridgerate for a few days only.
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.
plagarized and modified by hip from funkyballoon's post at the shroomery
* Notes from Nan: A Blender is much better than a coffee grinder.
Also Dirty Prints can be sometimes be cleaned by germinating a small quantity of the contaminated spores in clear liquid culture, then go fishing with a big needle. Gently suck up some clean mycelium into a syringe pre-loaded with 2.5 cc Sterile Water and 2.5 cc H202 3%, suck in a cc or two of air and shake the hell out of the syringe. Wipe the needle, then let sit 10 minutes with the needle up and covered (it will squirt if the needle is not up). Shoot clean Dextrose jars and some Agar Plates at the rate of 1/4 cc per culture.
|Posted on Monday, December 31, 2001 - 08:49 pm:||
Does the h2o2 cloning tek give good results? The one where you grind up a stem in water and pour the mix in peroxide. Does a clean piece of stem need to be taken from the inside of the stem or will the whole stem work?
Is a glove box important or can this be done in open air?
|Posted on Monday, December 31, 2001 - 10:28 pm:||
if you are using a glovebox and cutting tissue from the inside of the mushie... i don't think the H2O2 would be necessary... i think that the H2O2 cloning tek is so you wouldn't have to be as clean...
|Posted on Tuesday, January 01, 2002 - 01:03 am:||
Is it as effective? If it is, it sounds much easier than cutting the stem and using a glovebox.
When you use peroxide is a glovebox and cuting tissue from the inside necessary.
|Posted on Tuesday, January 01, 2002 - 01:06 am:||
i think that that is the whole philosophy of the h2o2 tek... but peroxide does do some harm to mycellum (sp?), but if it didn't work it wouldn't be in the archives... i have never personally tried it so i really can't say much more about it
|Posted on Tuesday, January 01, 2002 - 03:03 pm:||
it isn't a very good way to chose, there are many organisms unaffected by peroxide.
you'll prolly get better results in a glovebox, and get your tissue from inside the shroom.
Mr. Tambourine Man (Tambourine_Man)
|Posted on Friday, January 04, 2002 - 08:20 pm:||
After the blended mushroom and water mixture is "spooned" into the H2O2, should the fizzy part on top be drawn into a syringe or the liquid part below the top fizziness? The liquid part below looks clear except for bubbles. Does it contain some of the mushroom material or just the fizzy part on top?
Dr. Cubesis III (Newbieshroomer)
|Posted on Friday, January 04, 2002 - 09:30 pm:||
Hey Tamborine man, gonna piggyback your thread, hope ya don't mind
Interesting... My friend never uses h202 with his cultures, or honey/karo teks rather...
He usually uses them about 7 days after they are innoculated... Should he be using h202???
|Posted on Friday, January 04, 2002 - 10:52 pm:||
he is referring to a peroxide tek for cloning... you should only use H2O2 when you are having problems... like if you think you have a contamed liquid culture
|Posted on Saturday, January 05, 2002 - 12:07 am:||
I have not run this tek, but seems logical to me. Give it a good stir after the bubbles subside some and then suck it up. The liquid should appear cloudy and there should be plenty of tissue disbursed, suck it up.
I see some obvious problems with this tek, I would like to get some feedback.
|Posted on Saturday, January 05, 2002 - 01:08 am:||
yeah, i'd like to hear from someone who's made it work.
Mr. Tambourine Man (Tambourine_Man)
|Posted on Saturday, January 05, 2002 - 01:17 am:||
Nan and Hippie-
Has it failed for you guys? What do you think its problems are?
|Posted on Saturday, January 05, 2002 - 01:23 am:||
i only tried it twice, contam'd both times.
i just don't think peroxide is effective enough.