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Typically when we're testing the antimicrobial effects of some substance on bacteria growing on an agar plate, we get the area or diameter/radius of the zone of inhibition. But what do we do in cases where the zone of inhibition is not uniformly circular around the antimicrobial agent? How would diameter and radius measurements be of use in these cases and/or how would one adapt these measurements to more accurately reflect the area of the zone of inhibition?

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  • $\begingroup$ I think a picture or two would be worth a thousand words here $\endgroup$
    – MikeyC
    Sep 11, 2022 at 22:13

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We actually did something like this in one of my periods in biology class, all you need to do is take your measurement in millimeters. This measures the radius of the zone of inhibition. Multiply that by two in order to get the diameter.

If it's not uniformly circular, try getting the diameter of the circle you do know, and find the diameter/length/width of the unusual part. Just like in maths when you calculate total volume of a combination of shapes.

By unusual part, I mean the part that makes the circle around the microbial agent non-uniform.

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If your inhibition zones are not uniformly circular (or at least close too it) you may want to revisit your methods and ask why this might be the case.

Are you applying your inoculum in a consistent and standardized manner? Is the inoculum too light or too heavy in any place? Are your disks too close together, or too close to the edge of the plate? Could your disk have moved after application (possibly due to excess moisture or condensation on plates)? Is the uneven zone close enough to another disk for the two agents to interact, either synergistically or antagonistically? (see image)

Illustration of synergisms and antagonism between antimicrobial drugs

Have you looked to see if any special cases apply, like Fosfomycin, in the EUCAST breakpoints table.

If all of those potential causes are ruled out, I'd say you should measure to the nearest (inner most) circular or semi-circular edge of the zone. Since diffusion tests are often used for antibiotic susceptibility testing of clinical isolates, users (and clinicians) are often more interested in the qualitative classification (susceptible, resistant, or intermediate) than the quantitative result. For example, in cases where a double zone in present, the EUCAST method recommends measuring to the inner-most zone, unless otherwise specified, to avoid falsely categorizing a resistant isolate as susceptible.

If it's irregular because it's overlapping with the edge of the plate, with another inhibition zone, or if synergism/antagonism(aka induction) are suspected, you could just measure to the circular part and ignore the overlap/interaction zone, but retesting with more space around the disk would be preferred.

References:

  1. EUCAST clinical breakpoints table, version 12.0
  2. Antibiotics in laboratory medicine, 6th edition. (image credit)
  3. Development of the EUCAST disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories
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