|By Nan (Nanook) on Tuesday, October 23, 2001 - 06:56 pm:|
a recipe for one liter of "No pressure" agar medium:
12 gms agar
12 gms light malt powder
0.5 gm processed nitrogen source (rotate between gelatin, soy milk powder, milk powder, low sodium soy sauce, etc.)
5-7 wood fuel pellets
small chip (0.1 gm? enough to turn the solution slightly yellow) from synthetic B vitamin complex pill
1 liter clear water, free of particulates
1) Let your jar containing this medium (and your petri dishes, if reusable) sit in boiling water in a covered pot with a heavy lid for about forty-five minutes, which allows time to melt the agar and kill any live organisms in the medium (it may be advisable to steam reusable petri dishes even longer).
2) Then remove the jar and let it cool, adding the peroxide as in the first recipe. The peroxide will kill any spores remaining in the medium. I add slightly more peroxide to non-autoclaved Plates, about 8 mls per liter of medium. In general I find that non-autoclaved peroxide plates contaminate more often than autoclaved peroxide plates, but they still do considerably better than plates made without peroxide.
Watch out for drips of agar medium that land on the outside of your petri dishes. If these are not cleaned off, they will grow mold within a few days, and the spores will diffuse into the plates and start germinating at the outer edge of the agar.
If you are working with reusable petri dishes as I am, clean them carefully after you take out the old agar. Even the smallest amount of old medium left in a plate, if it is not in contact with the peroxide in the new agar the next time you use the plate, can grow mold and become a jumping-off point for contamination.
A benefit of pouring plates when the agar is so cool, is that there is considerably less condensation on the inside of the lids, than if you pour hot. This obviously means that you won’t have to take special measures to get rid of condensation, such as shaking out the lids, or warming the plates to evaporate the droplets. And you’ll be better able to see what is happening in your plates. However, the agar surface still needs some drying, so I let the plates sit at room temperature for a day, lightly covered with a sheet of wax paper to keep dust off, before I use them. Plates with agar medium that has been steamed will be wetter than plates with medium that has been pressure cooked, because of the lower cooking temperatures and shorter cooking time, so they will need to be dried for a longer time.
If you have extra agar medium after your plates are poured, the excess will remain sterile stored in the refrigerator. When you get around to using it, you can melt the agar again, but note that you will need to add peroxide again, because the heat of melting will have destroyed what you added the first time
See: Peroxide Manual Vol. I