Exercise 24-B
ANTIMICROBIAL SENSITIVITY TESTING
AGAR DIFFUSION METHOD OR KIRBY-BAUER TEST
Introduction
Antimicrobial drugs are a class of chemotherapeutic agents used in the treatment of infectious
diseases. They can generally be divided into four groups as follows: 1) synthetic antimicrobial drugs,
2) antibiotics (substances produced at least originally by living organisms and used to control bacteria),
3) antiviral drugs, and 4) anti-protozoan and anti-helminthic drugs.
In clinical situations, once the causative agent of a specific disease has been isolated, the physician
needs to know as quickly as possible, which antimicrobial drug will be most effective against it. The
use of filter-paper disks impregnated with antimicrobial agents is one method that can readily provide
this information.
Antimicrobial impregnated disks were introduced in the late 1940’s when penicillin came into
widespread use. Since then, a multitude of new antimicrobial drugs has been discovered, and much
experimentation has been done to develop a method to accurately and reliably test their effectiveness.
In the Agar Diffusion Method, also known as the Kirby-Bauer Test, a Petri plate containing an agar
medium is inoculated, "seeded", uniformly over the entire surface with a standardized amount of a test
culture. The organism type being tested may be mixed with a small amount of liquefied agar and poured
over the agar surface, or may be streaked onto the surface using a cotton swab dipped in broth culture.
Next, filter paper disks impregnated with known concentrations of chemotherapeutic agents are placed
on the solidified agar surface. During incubation, the antimicrobial agents, or drugs, diffuse from the
disks into the agar, and inhibit the growth of sensitive organisms in a zone immediately surrounding
each disk (the zone of inhibition). Since the concentration of each drug decreases progressively the
farther it diffuses from the disk, the concentration at the outside edge of the zone of inhibition represents
the minimum inhibitory concentration (MIC) for that drug. The diameter of the zone of inhibition
may be measured with a ruler; however, a wider zone does not necessarily indicate greater antimicrobial
activity. The diameter of the zone of inhibition is influenced by the rate of diffusion for the
antimicrobial agent being tested, the depth and type of agar medium being used, the incubation
conditions, and many other factors. For this reason, Agar Diffusion tests are always performed under
standardized conditions, and results are evaluated using predetermined tables.
For the Kirby-Bauer method as sanctioned by the FDA and the subcommittee on Antimicrobial
Susceptibility Testing of the National Committee for Clinical Laboratory Standards, the recommended
medium is Mueller-Hinton Agar, pH adjusted to 7.2 - 7.4 and poured to a uniform thickness of 4 mm
in Petri plates. For certain fastidious microorganisms, 5% defibrinated animal blood (sheep, horse, or
other) may be added. Inoculation is made from broth cultures with a specified turbidity. High potency
disks are placed on the agar surface, and the plates are incubated for 16 - 18 hours at 37o C. Zones of
inhibition are measured to the nearest millimeter, and the results are evaluated.
The minimum inhibitory concentration (MIC) as described above is one example of a breakpoint,
i.e., a discriminatory antimicrobial concentration used in the interpretation of the results obtained
during susceptibility testing to define isolates as susceptible (sensitive), intermediate or resistant.
