What is the standard for measuring the zone of inhibitions in antibiotic overlay assay (i.e. isolates spot-inoculated on agar then overlaid with soft agar with the test strain)?
There could be two ways:
1. Measure the diameter of the whole zone of inhibition like that of kirby-bauer disc diffusion assay, or
2. Measure the diameter of the inoculum, the diameter of the total zone of inhibition and get their respective areas. The area of inhibition is computed by subtracting the area of the inoculum from the area of the total zone of inhibition.
I have doubts on method one since the total zone of inhibition cannot be fully attributed to the activity because of the inherent bias implied by the varying growth and size of the spot-inoculated isolates.
Any suggestions on this?
Hey Dan. =)
Checking for antimicrobial activity using agar plates can provide some different results depending on few things like the "height" of the media on the plate, if the surface was dry enough when you spot innoculated your samples, the way your isolates grow on solid media, etc... So I would rather use this assay as a qualitative screening, just to determine whether antibiotic production is present or absent, and then go for microdilution techniques for further quantification.
But, in case you want to perform the agar overlay assay, I think you should at least make sure your sample spots have the same size so that you can make comparisons between your strains. You could try using filter disks with a specific volume so that it would "hold" your isolates in place, or even agar plugs. Having the same starting point, you could now measure diameters like in kirby-bauer method. You could also try the cup plate method, let your isolates grow and then overlay with the test-microorganisms...
As for the second method, I haven't seen it used for antibiotic (so far), but it is known to enzymatic production, except that instead of area, you also use diameters. Check for "enzymatic index" or "zone of precipitation" regarding enzyme production and see if the concept serves you. They consist of the result of a quotient between colony diameter and total zone of activity (or vice-versa) that would you allow you to place your samples in low, intermediate or high level of production. But you would have to find a paper with index values specific to antibiotics... Sorry I can't be of much help on this. =(
Good luck!
Hi, Liany. I already did the agar overlay assay yesterday and I am just waiting for the results. I tried to make the spots uniform by using a spot mark as guide. Most of my non-motile isolates were uniform in diameter but I had problems with my motile isolates, they spread quickly on the agar after 24 hours prior to soft agar overlay. It seems that the agar plug method is more appropriate especially for motile bacteria. I'll try to find articles on the concept of enzymatic index for antibiotic screening. Thank you very much, Liany! :)
Hi Dan, How did the agar-overlay approach go? I am wondering if I could use a similar approach for my bio-control assay. Thank you!!!
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