Subtopic	Posts		Updated
	Cloning a Pure Culture	7		07/01 01:55pm

By Cultivater420 (Cultivater420) on Thursday, April 26, 2001 - 03:15 pm: Edit

I have read, but, am still confused by the process of cloning a mushroom .. I'd like to try an idea given to me by hippie, and clone my next biggest Equador fruit, and try to grow myself a flush of those.. If anyone can help me out, it would be MUCH appreciated

By Hippie3 (Hippie3) on Thursday, April 26, 2001 - 03:27 pm: Edit

first, prepare some peroxidated agar as per -

---

Quote:

"First you need to get a hold of some light malt extract powder and some agar agar. Malt extract can be found at your local wine and beer brewing store, and agar agar can be bought from Fungi Perfecti, among other places. You also need some petri dishes, also available from FP, or you can pour your agar into empty jars instead of plates if you prefer. Next, mix three grams of malt extract and three grams of agar (or, if you bought them pre-mixed, six grams of the mix) with 149 ml of water (you should use 149 ml instead of 150 because you want to account for an extra ml of peroxide that you will be adding later). I measure out my water with an empty syringe, keeping in mind that one cc is the same as one ml. I mix and sterilize this solution in a half pint jar, because the amount of agar solution is right for that size jar and because the size of the jar fits well in the pot that I use for sterilizing it. It works for me, but it is not ideal for pouring and I'm sure a better substitute could be found. Anyway, close up your container of agar and sterilize for 45 minutes. I sterilize mine by letting it sit in boiling water in a covered pot, but you could use a pressure cooker if you wanted to. While it is sterilizing, thoroughly clean your plates if you haven't already done so, and stack them on a tabletop. As soon as is possible without burning yourself, move the jar of molten agar to the tabletop, open the lid, and insert a clean thermometer into the agar. Since hydrogen peroxide decomposes at 140 degrees fahrenheit, you need to let the temperature of the agar drop below that before you add the peroxide. Otherwise it will decompose into water and oxygen and be effectively useless. This is why the peroxide is added after sterilization. I have found that my agar begins to solidify at around 120 degrees fahrenheit, so fill a syringe with peroxide and when the temperature falls to 130 degrees, add one ml to the molten agar and stir it in with the thermometer. Then remove the thermometer and pour your plates, stacking them as you go. I can usually pour nine or ten plates with this amount of agar. If you want to make a larger or smaller amount, simply adjust the amounts of water, agar, malt, and peroxide, keeping all the ratios the same."

---

By Hippie3 (Hippie3) on Thursday, April 26, 2001 - 03:29 pm: Edit

prepare glovebox for sterile transfers.
if you lack a glovebox, try this-

---

Quote:

Build the Cheapest "Glove" Box Possible

Items Required

Cardboard box (13"x15"x10")
Plastic wrap
Duct tape
Dish cloth
Disinfectant spray
Sharp knife or razor blade for cutting cardboard
Rubbing alcohol
No gloves

To raise the odds against contamination, a sterile work area is vital. This is especially true if you want to become independent of outside sources of sterile spore syringes. In a rush I threw together this glove box out of scrounged materials. It has worked so well that I haven't built my dream box yet. First find a clean cardboard box with the above dimensions as a minimum. It doesn't have to be new or pristine, just fairly new with no visible dust or dirt. Cut off the top opening flaps. Cut away the entire front of the box, leaving a 2 inch wide strip of cardboard at the top edge to attach the dish cloth to. Cover the top with plastic wrap and tape down the edges. Using duct tape, attach a dish cloth or towel across the front opening onto that 2 inch strip. The cloth should cover the entire opening of the front of the box like a curtain and you should be able to see into the box through the clear plastic wrap on top. That's it. To use the box, spray down your work surface with the disinfectant spray. Close off all drafts (windows, heater fans, etc). Spray outside of box lightly and saturate the dish cloth. Place bulky items into the box such as grain jars and agar plates. Spray disinfectant into the box until all of the air inside is displaced by the mist. Wash hands with rubbing alcohol and you are ready to work

---

By Hippie3 (Hippie3) on Thursday, April 26, 2001 - 03:32 pm: Edit

when you have prepared everything, and selected the shroom you want to clone,
load the petris, the shroom, and some tincture of iodine, cotton swabs, and a sharp into the box & disinfect everything.
take the shroom and clean it with the iodine, then slice it open and remove a chunk of tissue from the inside, then quickly tranfer it to the agar.

By Hippie3 (Hippie3) on Thursday, April 26, 2001 - 03:35 pm: Edit

grow on the agar as per normal cubensis parameters until it gets big then you can cut out sections of colonized agar for use in preparing syringes, or transfers to jars of grain, other petris, etc.

By Cultivater420 (Cultivater420) on Thursday, April 26, 2001 - 03:52 pm: Edit

Ok... thanks a LOT hippie. Now, for the peroxide, would you happen to know what percentage peroxide is needed/used would you?

As it is now, i am in possesion of a bottle of 3% peroxide. Should it be pure peroxide? and if so, any ideas on where to procure 90% - 100% peroxide?

By Hippie3 (Hippie3) on Thursday, April 26, 2001 - 04:08 pm: Edit

the recipe is figured for the 3% stuff common in the us.
you would not like 100% peroxide, it's a very unstable substance.

By Cultivater420 (Cultivater420) on Thursday, April 26, 2001 - 04:18 pm: Edit

hehe.... Dumb me huh?? hehe.. I've got absolutely NO experience using peroxide in cultivations, except to water casing

By Hippie3 (Hippie3) on Thursday, April 26, 2001 - 04:21 pm: Edit

a trivial, 100% peroxide is used as rocket fuel [oxidizer]

By Alley (Alley) on Friday, May 04, 2001 - 12:40 am: Edit

hey, hippie. can you make honey water and drop in some of the sample tissue from the shroom you would want to clone to make a syringe for cloning?
thanks - alley

By Hippie3 (Hippie3) on Friday, May 04, 2001 - 02:52 am: Edit

yup.
just be sterile, get the tissue from inside the stem, work in glovebox, you can use a little peroxide in the honeywater, since it's live mycellia , not spores.

By Fungusflipper (Fungusflipper) on Friday, May 04, 2001 - 04:32 am: Edit

Hey Hippie,

Will you archive this thread?

By Hippie3 (Hippie3) on Friday, May 04, 2001 - 11:11 am: Edit

sure.