Kirby-Bauer Test for Bacterial Resistance to Particular Antibiotics
The Kirby-Bauer Test is a standardized method to establish whether a particular strain of a bacterial species is susceptible or resistant to one or more antibiotics. The test is based on the size of a zone of inhibition, or clearing, in a lawn of bacteria. This zone appears around a disc containing an antibiotic and results from the diffusion of that antibiotic into the surrounding agar combined with the killing or inhibition of bacterial growth. Usually the diameter of the zone is measured, and the larger the diameter, the more likely the bacteria put on the plate are resistant to the antibiotic in the disc in question.
The Kirby-Bauer test does not differentiate between bacteriostatic or bacteriocidal effects; both produce a zone of inhibition. To determine if bacteria are merely in stasis, rather than dead, a sample from the zone inhibition can be inoculated into nutrient broth. If bacteria grow in the broth, the action of the antibiotic was probably bacteriostatic, not bacteriocidal.
Several things must be considered in order to make the zone of inhibition for one antibiotic comparable to another antibiotic. First, the test must be standardized and performed in the same way. Since the zone of inhibition that forms around a disc is directly related to the diffusion rate of the antibiotic, the concentration of the agar, the temperature of incubation, the size of the discs, the concentration of the antibiotic in the disc, and the growth time allowed must all be the same for all antibiotics tested. The Kirby-Bauer test always uses a standard agar recipe, called Mueller-Hinton agar, which provides a constant agar density and nutrients between different plates used in a test.
One diffusion factor cannot be controlled or kept constant in the Kirby-Bauer test: the size and shape of the antibiotics being tested. Because small molecules diffuse faster than large molecules, some antibiotics will form larger zones of inhibition than others. That is, two antibiotics may produce the same zone diameters but the first one, having the smaller molecular size, might create a large circle simply because it diffuses faster. The second antibiotic, having a larger molecular size, might diffuse more slowly but achieve the same diameter. So in fact, the zones of inhibition cannot be directly compared.
To control for molecular size and its affect on diffusion rate, tables showing cut-off diameters are used in interpreting the zones of inhibition. They show if the bacteria being tested are in fact susceptible or resistant. What stands out in such a table is the fact that a substantial zone of inhibition may form around some antibiotic discs and yet the bacteria on the plate are still considered resistant. One would normally think that any clearing around an antibiotic disc means the bacteria around that disc are susceptible. This is not how the Kirby-Bauer test is assessed; you must first look at a table of cut-off diameters to judge susceptibility or resistance.
To use the table, first measure the diameter of the zone of inhibition around a particular antibiotic. If only a partial circle of clearing is available, measure the radius from the disc center to the edge of clearing and multiply by 2 to get the diameter. Using the diameter, look to see whether the zone diameter in the table is sufficiently wide to call the bacteria on the plate "susceptible", or so narrow that the bacteria are called "resistant".
Table I. The susceptibility or resistance of bacteria to particular antibiotics, based on the diameter of the zone of inhibition in the Kirby-Bauer plate test.
Antibiotic Disc Symbol Resistant Intermediate Susceptible
Chloramphenicol C < 12 mm 12-17 mm > 18 mm
Erythromycin E < 13 mm 14-22 mm > 23 mm
Penicillin (for gram+ bacteria) P < 28 mm --- > 29 mm
Penicillin (for gram- bacteria) P < 14 mm --- > 15 mm
Streptomycin S < 11 mm 12-14 mm > 15 mm
Sulfanilamide G, SMZ, SMX < 12 mm 13-16 mm > 17
Tetracycline TE < 14 mm 15-18 mm > 19 mm