I am conducting agar well diffusion assay using 200 µLof my test extracts.

However, I am using 9mm petri plate bored with wells of 6mm in diameter, in which it will not be possible to accommodate 200µL of the extract. Do I have to pipette 100 µL first, then let it diffuse through the agar, before pipetting the remaining 100 µL? Is this a common method? How long will I have to wait before pipetting the next 100 µL?

If it is not a common method, I calculated that wells with 10mm in diameter will be able to accommodate 200 µL of extract. However, is 10mm too wide for diffusion to occur? How will this affect my results?

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