PEROXIDE AND BROWN RICE CLONING TEK
Posted 25 April 2005 - 09:43 AM
PF PEROXIDE AND BROWN RICE CLONING TEK
by Professor Fanaticus
The original idea comes from Rush Wayne's cultivation manual "GROWING MUSHROOMS WITH PEROXIDE". The following is a simplification of the idea down to the smallest degree, using brown rice powder, peroxidated water dilutions and small jars.
1. Small jars with lids
2. Brown rice powder
3. Regular store bought Hydrogen Peroxide (3%) antiseptic solution (usually in a brown bottle)
4. Dissection knife and long needle (exacto etc)
5. Living mushroom or fungus, or culture of fungus
As a preliminary, start reasonable clean, but the following is to be done in the open air, without sterile implements or containers.
Mix the peroxide antiseptic and water at a 20% ratio. Example - 2cc peroxide and 8cc water. Place an amount of brown rice powder into the jar and add some of the peroxidated water to get a slurry, or very wet condition. The diluted peroxidated water sterilizes the medium and the jar. For extra experiments, you should try more potent peroxide contents in the brown rice peroxide slurry medium (30% - 40% - 50%). It is so easy, it can't hurt to experiment and you can benefit from it.
Tear the shroom apart and with an exacto knife, excise a small fragment about the size of a match head. With a long needle, knock or scrape the shroom fragment off the exacto blade and place the fragment on the surface of the peroxidated brown rice slurry. If you are working with previously made plate or slurry cultures that have mold growing along with the pure white good fungi, follow this tek using a small fragment of the desired fungi excised out of the culture. If the culture grows back clean, that doesn't mean that it really is clean, because there will be contaminant spores in the mycelium (which is there anyway because of the open nature of this tek). When the culture is to be used PF style, when it is reslurried with fresh peroxide water, the contaminant mold spores and bacteria endospores will be killed while the growing fungi mycelium will survive to grow clean - PF style or any style you like.
Screw the lid tightly onto the peroxide slurry jar and put the jar in a warm place for growth. The culture can be opened and exposed for observation or experimentation without danger to the growing medium and culture.
Under magnification (10x), the fragment of mycelium appears to "melt". It also turns blue, as if it were killed. Within a few days, tiny white hair like tendrils (hyphae) will appear on the "melted blue" fungus. It will grow and fill the the culture jar.
To use the culture for further inoculations, add 20% peroxidated water to the culture, mix the brown rice and fungus, and with a syringe, draw up water and inoculate PF jars, with the standard PF spore syringe technique. This is done nonsterily also because the peroxidated water will kill contaminant spores. But flame sterilize the needle before injecting into the sterile PF substrate. Instead of flaming the needle, an even better way to sterilize the needle (outside of the needle) is to wipe the needle with a tissue soaked with rubbing alcohol. Let the needle briefly dry of the alcohol before injecting. Use the culture well before it grows in. That way, it is much easier to get a usable slurry for the syringe without clogging.
As an addition, try more potent peroxidated water ratios. For instance, a 50% ratio works also. That would be half peroxide antiseptic and half water. Increase peroxide content until the mycelium doesn't survive. Then back off the amount of peroxide and use a near death peroxide load for guaranteed clean results. But a 20% peroxide to water ratio seems to be perfect.
If you are working with previously made plate or slurry cultures that have mold or bacteria slime growing along with the pure white good fungi, follow this tek using a small fragment of the desired fungi excised out of the culture. Make sure you don't have any growing mold or bacteria in the good mycelium because the peroxide won't kill it (as it doesn't kill the good fungi). If the culture grows back clean, that doesn't mean that it really is clean, because there will be contaminant spores in the mycelium (which is there anyway because of the open nature of this tek). When the culture is to be used PF style, when it is reslurried with fresh peroxide water, the contaminant mold spores and bacteria endospores will be killed while the growing fungi mycelium will survive to grow clean - PF style or any style you like.
The only problem is syringe needle clogging. As a remedy, do not allow the culture to fully grow in or get thick. Keeping the culture "thin", allows a good breakdown of the mycelial fragments for use in a syringe. At first, doing the tek can be messy, but learning is quick and easy. Finding and using needles as big as possible is important.
But another route is very possible and actually preferable. Using long glass bulb pipettes are very good to use, but here, one must customize and "tweak" the teks a bit (one must be careful when injecting PF style jars with the bigger pipetts and not to breach the top vermiculite contaminant barrier). Also, flaming the glass pipetts can sometimes break the glass. In this case, always sterilize the outside of the pipette with the rubbing alcohol. Bulb pipettes are actually better to use than needles and syringes because they don't have much problem with clogging as compared to needles and syringes with mycelial slurries. But if the Professor's tek is followed closely and the cultures are not allowed to grow in and get thick, the 18 gauge needles with 10cc syringes work OK.
PRINCIPLES OF THE PEROXIDE TEKS
Hydrogen Peroxide is a powerful antiseptic. The solution of Hydrogen Peroxide bought in a drug store is 3% Hydrogen Peroxide and 97% water. Even at this low concentration, and with further dilutions, the germ killing is potent. But that germ killing power only works for micro fungi spores and bacteria endospores. A micro-organism that has germinated into its secondary form (mycelium), is safe from the antiseptic power of diluted Hydrogen Peroxide. But the ungerminated spores, bacteria endospores, and microbes are all susceptible. If there is bacteria that is growing (germinated), it will not succumb to the peroxidated water (just as the fungi mycelium is not succumbing). Also, any mold that is growing will survive. A clean fragment of shroom flesh or mycelium from a mold contaminated culture has germs all over it, but only in the spore or endospore form. They won't germinate in the peroxidated medium and water or on the recovering mycelia.
The tek is like a tightrope act. The mycelium that is cultured in the peroxide enriched medium can survive and grow, but it is not clean. Any spores or bacteria that are "piggy-backing" on the mycelium and not in contact with the peroxidated medium can come to life if given the opportunity.
After the culture of mycelium grows, it can be rehydrated with more peroxidated water. The spores and bacteria endospores that are "piggy-backing" on the mycelium will die. The mycelium in its fully secondary form, will survive the new peroxidated solution, "cleaned".
BY PROFESSOR FANATICUS
Posted 25 April 2005 - 12:57 PM
It's been a few years since I last grew cakes.
Got the urge while jar shopping.
Definitely trying this TEK next time I clone.
Must the jars be sealed due to the unstable nature of the peroxide?
Could colonized Karo water with possible contamination be fully or partiallly substituted for water?
Can a clean liquid culture be used to noc up H2O2/BRF jars?
If so, should there be any change in the water/karo water/peroxide ratio,
assuming the karo:water concentration is 1tsp karo:100ml distilled water?
Thanks for this awesome cloning TEK...
So many possibilities.
Can't wait to try it!
Posted 25 April 2005 - 06:28 PM
Posted 27 April 2005 - 06:05 AM
Posted 27 April 2005 - 06:15 AM
Posted 27 April 2005 - 11:05 AM
Posted 27 April 2005 - 04:02 PM
Posted 27 April 2005 - 04:16 PM
Posted 29 April 2005 - 08:46 AM
I'm guessing yes, just not sure.
Posted 29 April 2005 - 08:59 AM
Posted 29 April 2005 - 09:05 AM
My last cloning attempt (blender cloning)
made 30 jars of green.
Posted 29 April 2005 - 09:35 AM
Posted 29 April 2005 - 09:51 AM
Posted 29 April 2005 - 10:44 AM
Posted 29 April 2005 - 11:20 AM
Brown rice powder = brown rice flour?
I'm guessing yes, just not sure.
Posted 29 April 2005 - 12:37 PM
My reasoning is that I suspect the tissue may contain surface contams that were picked up on its way out of the dunk.
This gives me a better, although not great, success rate.
I keep some fluorescent light diffuser grid on the bottom of my glovebox so
my work isn't sitting in the various liquids that I dump/spill in there.
Whenever I clone, I also use plain distilled water and/or karo liquid. (PC'd 20-30min)
These usually work out better.
Posted 05 May 2006 - 02:59 AM
The tek is below:
Posted 05 May 2006 - 07:39 AM
Go with agar or proven lc teks for success imo. There are also folks who use peroxidated agar.
I may try experimenting with this again, though not anytime soon.