I need to determine yeast viability upon exposure to H2O2. I need to prepare YPD plates containing 0.4mM H2O2.
I am not certain whether H2O2 should be added before pouring the plate into a cooled agar, or if one can cover the plate directly with H2O2 solution. In the first case I am afraid that H2O2 might be degraded, and in the second case concentration might not be the same over the whole plate.
One more question: how long are such plates stable: over a day?
If anyone has such an expertise that would be very helpful.
Hi there,
You should add H2O2 to cool-down molten agar right before pouring plates to be sure of the concentration. And then the best is to use plates straight away to avoid the inconvenience of storage without exactly knowing about the stability of the reagent in rich media.
I agree with Dominique. Furthermore, hydrogen peroxide is sensitive to light. Therefore you should incubate the plates wrapped with aluminum foil, for example.
Best regards.
If the viability is what you are measuring, why not to treat the yeast first in solution and then plate serial dilutions of surviving yeast onto agar plates?
I would tend to agree with Julijus, why not treat the yeast in solution with the desired concentration of H202, or better yet a range of concentrations, grow for a designated amount of time and measure with optical density and confirm with solid agar plating.
I see the point Julijus is making. Were I to conduct such reseach, I'd probably have a go with serial dilutions. The yeast cells would be treated with H2O2 and after that I'd start a culture in a plate. Good way to check the survival after the H2O2 treatment. However, Barrys' suggestions also make a lot of sense to me. Additionally, you shall be aware that optical density measurment isn't always fully reliable. In my previous studies I was thinking about checking optical density. Unluckily, the method proved itself to be a bit useless in my research. Growing yeast on plates would be crucial anyway.
Best luck!
You're correct to be concerned at stability of H2O2 in agar media. To that point, you;ll need to chemcialy validate that hydrogen peroxide as added to media achieves a stable level as intended. It's not clear - are you investigating inhibition or kill? As Szymon suggested, it would be more valid to simply add yeast to a fresh solution of hydrogen eroxide diluted as needed and plate out, comparing viable count to versus control. .
Be aware that many fungi produce catalse
Does anybody know a paper or protocol how to make these H2O2 agar plates ?
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