Volume of antibiotics per agar plate - (Feb/26/2009 )

Hi there,

I would like to do a dilutional plating of yeasts on agar plates containing different drug concentrations. I would like to spread the drugs on the already dried plates. Does anyone know which volume I will have to plate to get a certain final concentration?

Cheers, chalet2

chalet2 on Feb 26 2009, 04:02 PM said:

Not easily done. Do you know the volume of agar on the plate? Where the plates made with a measured volume of agar?

Since the experiment appeared to be that test the effects of different drug concentrations on yeast, it would be better to make a new set of agar plates with a set measured volume.

The volume is exactly 25 ml per plate

well in that cause it is quite easy

Volume1 * concentration1 = volume2 * concentration2

Let
Volume1 = volume of agarose
concentration1 = desired antibiotic concentratioin

volume2 = volume of stock antibiotic that you need to add
concentration2 = concentration of stock antibiotic.

This is the equation you use.

Why is the volume of the plate important? I just want to spread it on top of the plate. So, it won't diffuse through the whole plate anyways.
Thanks for your help

actually a good point (what was I thinking )

I think it would be best to remake the agar plate. Get some molten agarose (cool it down to about 50-55C) and pour 25ml into a universal tube. Add the antibiotic to the molten agarose. Then shake and pour the agar into a petri dish.

If one can't be certain that the antibiotic won't diffuse the entire way, how will can we determine the concentration of agarose that the yeast growing on the plate are exposed too.

The antibiotics will diffuse once on the agarose. It won't simply sit on the surface of the plate. From memories of Xgal spreading, diffusion of the antibiotic once on the plate is rarely even. (More concentrated on the center, and probably surface). Thus the concentration of antibiotic won't be even. So it would be better to make the antibiotic treated plates from molten agar.

Most antifungals are poorly soluble. What medium are you using?

To add to pernese's comments, please decide what your controls will be. You should be confident that the antibiotic is present and effective as intended.

GeorgeWolff on Feb 27 2009, 04:29 AM said:

Are antifungals soluble in things like DMSO, ethanol . . . Then you could just make a 1000x stock and add 25 ul to each 25 ml liquid agar, vortex, and pour.

Well, it is a certain drug that I wanna spread and assay for uptake by yeast. The purpose of the exp is to see at which concentration the yeasts won't be able to grow anymore. Since I already poured the plates I was wondering how much of each concentration I would have to spread and what the final concentration would be.

As another said - "spreading" is not going to give you any confidencere. concetration. Don't care the vehicle used to bring an insoluble antifungal to an agar plate assay (ethanol, DMSO, whatever) - poorly soluble in water is poorly soluble in agar.