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Peroxide Agar Tek

Explains the use of peroxide in preparation of agar medium

The use of peroxide is unnecessary and counterproductive in agar:

"The whole idea of using petri dishes is to encourage rapid growth of mycelium in order to isolate it away from contaminants or other strains.  Using peroxide in any amount degrades the growth of the culture, defeating the purpose.  It's better to make a series of transfers to isolate your culture, rather then to depend on peroxide, which is as toxic to your mycelium as it is to other species of mycelium or bacteria.  Without the peroxide, you'd have already made two or three transfers by now and be finished.  We have to be careful relying on 20 year old growing advice when we've advanced the hobby beyond that point.  I'm sure your culture will begin to colonize the agar once the peroxide breaks down, but then what's the point of having used it in the first place?
RR"  RogerRabbit

Just thought more people might need to be reminded of this tek. I works wonders! This was a post written by placebo, and commented on by me (mycofile).
First of all, let me begin by saying that this is not my tek, and I'm not taking credit for it. As far as I know, the credit belongs to Dr Rush Wayne. Check out his website www.mycomasters.com. If you find this information useful, and/or would like to find out more about the uses of peroxide in mushroom cultivation, please consider buying his manual.

I've noticed a few people lately asking about agar. I find it very useful and recommend that everyone give it a try. Most people are turned off of agar because of fears of unavoidable contamination problems. I've found that with the use of hydrogen peroxide in my agar mix, I can do transfers in the open, unfiltered air with little worry of contamination.

Many people seem a bit confused as to what peroxide can and can not do. Hydrogen peroxide will kill bacteria and fungal spores and inhibit the growth of yeasts. Unfortunately, mushroom spores fall under the category of fungal spores, so they will not germinate on peroxidated agar because they are killed on contact. The good part about this is that mold spores, such as spores from the dreaded green mold, fall under this category as well and are also killed upon contact with the agar surface. What will grow on peroxidated agar is existing fungal colonies, such as your living mushroom mycelium, because their cells can produce enzymes that will cause the peroxide to be broken down into water and oxygen. The downside, however, is that existing colonies of some molds, such as green mold, are also capable of producing these enzymes, so peroxide will not guard against mold spores that have already germinated. But perhaps the best attribute of peroxide is that it will kill all bacteria, so you will never have a problem with bacterial contamination on peroxide enriched agar.

Enough with the background information already... Tell me how to do it!

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 150 ml of water.
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. Sine 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 ?F so fill a syringe with peroxide and when the temperature falls to 130?F, 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.

A good idea would be to order some antibiotic agar from FP to use for germinating spores. This stuff, while resistant to bacteria, is still susceptible to contamination by mold and yeast, so don't forget to use the proper sterile techniques (like a glove box or bleached down room). Once the spores germinate, the mycelium can be transferred to peroxidated agar with less of a stress on sterility. It is important to remember to keep your scalpels clean and to thoroughly clean and sterilize your plates prior to pouring! If there are small mold colonies left in the plates when you pour them, they will be resistant to the peroxide and will grow.

A range of H2O2 concentrations is acceptable. Psilocybe cubensis seems to tolerate amounts ranging from 1 ml to 7 ml per liter for this mix.(Wayne recommends from 6-10 ml of the 3%uFFFDeroxide per liter of agar medium).
The more you use, the longer "adjustment" period for mycelium to adapt to the peroxidated agar. As a general rule, the least amount necessary is best. But since your mileage may vary, you may modify as to what works for you.
There tends to be a "recovery period" that the mycelium goes through when transferred to agar containing peroxide, especially after the first transfer. The mycelium is slow during this recovery period, as it is building up the enzymes needed to break down the peroxide so it can digest the malt (in MEA). After that, the mycelium quickly picks up speed and grows just as quickly as it would if peroxide were not present. I've found this recovery period to usually last only a day or two.

You are not restricted to the use of Malt Extract Agar, you can use any agar formulation. Just add the appropriate amount of peroxide after the medium has cooled to around 120?F(~50?C).

I have personally used the original formula to make plates that were left unsealed and unfiltered in a nasty NASTY kitchen for up to 2 weeks until they dried out. The agar never contaminated!

~by mycofile
revised by Anno

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