How to prepare antibiotics disk - (Dec/26/2010 )

Hi guys, i need some help here.

i wanted to perform disk diffusion test and i'm having difficulties finding the protocol for preparation of antibiotics disk.

the disk i wanted to prepare is ampicillin, 10ug per disk.

regards

I don't know how to make it "properly" but when I was testing some substances for antibiotic activity I simply used disks made from thick filter paper with office hole punch, placed those disks on Petri dish and add the substance with pipette (1-5 uL).

And -- remarkably -- you can do this while leaving out the disk. The main reason for the disk is so you can make up many ahead of time, dry them, and use them conveniently. Punch out disks of filter paper, load 10 ug of ampicillin onto the disks, dry, and store (probably at 4C dry).

i read from some where that Grade 1 Whatman paper is used for the disk. is it too thin to be used as antibiotics disk?

how long am i going to sterilize the disk if i going to use UV sterilization method? 15 minutes on each side enough?

I would punch them, wrap in aluminum foil, and then autoclave them. The type of filter paper won't matter, as long as the liquid is absorbed without excess. Whatman No. 1 will work fine.

how should i prepare the 10ug ampicillin disk? add 1ul of 10ug/ul ampicillin to the disk or add 5ul of 10ug/ul?

sorry for my bad english

If you want 10 ug, then you add 10 ug. So, that means 1 ul of a 10 ug/ul solution. The disks will easily absorb up to about 5 ul I think, so you might be better off making a 5x dilution down to 2 ug/ul, add then adding 5 ul. This would cut down on variation due to pipetting errors.

now I'm facing a problem. My control which is E.coli ATCC 25922 doesn't form inhibition zone around the ampicillin disk, my my other test subjects are.

Could the strain that I'm using is not E.coli ATCC 25922? It doesn't grow on 100ug/ml ampicillin LB agar though.