The Kirby-Bauer test, known as the disk-diffusion method, is the most widely used antibiotic susceptibility test in determing what treatment of antibiotics should be used when treating an infection.  This method relies on the inhibition of bacterial growth measured under standard conditions.  For this test, a culture medium, specifically the Mueller-Hinton agar, is uniformly and aseptically inoculated with the test organism and then filter paper discs, which are impregnated with a specific concentration of a particular antibiotic, is placed on the medium.  The organism will grow on the agar plate while the antibiotic “works” to inhibit the growth.  If the organism is susceptible to a specific antibiotic, there will be no growth around the disc containing the antibiotic.  Thus, a “zone of inhibition” can be observed and measured to determine the susceptibility to an antibiotic for that particular organism.  The measurement is compared to the criteria set by the National Committee for Clinical Laboratory Studies (NCCLS).  Based on the criteria, the organism can be classified as being Resistant (R), Intermediate (I) or Susceptible (S).

kirby bauer japan
Equipment used for Kirby-Bauer testing
in Japan.


The culture used in this test has to be the Mueller-Hinton agar because it is an agar that is thoroughly tested for its composition and its pH level. Also, using this agar ensures that zones of inhibitions can be reproduced from the same organism, and this agar does not inhibit sulfonamides.  The agar itself must also only be 4mm deep.  This further ensures standardization and reproducibility.

 The size of the inoculated organism must also be standardized (using barium sulfate standards, McFarland standards).  The reasons are because if the size of the inoculum is too small, the zone of inhibition will be larger than what it is supposed to be (“the antibiotics will have a distinct advantage”) and if the inoculum is too large, the zone of inhibition will be smaller.
kirby bauer us
Equipment used for KB testing
in the U.S.


(Steps omitted include the preparation of McFarland Standard, and the standardization of Bacterial Suspension)

1.     Using a asceptic technique, place a sterile swab into the broth culture of a specific organism and then gently remove excess liquid by gently pressing the swab against the inside of the tube.

2.     Using the swab, streak the Mueller-Hinton agar plate.  To obtain uniform growth, streak the plate with the swab in one direction and then rotate the plate 90° and streak the plate again in that direction.  Repeat the rotation 3 times.  Allow the plate to dry for approximately 5 minutes.

3.     Antibiotic discs can be dispensed onto the agar using an Antibiotic Disc Dispenser. 

4.     Using flame-sterilized forceps, gently press each disc to the agar to ensure that the disc is attached to the agar. 

5.     Plates should be incubated overnight at an incubation temperature of 37°.


  After the plates have been incubated, there should be a noticible “clearing” zone around each of the antibiotic discs.  The diameter of each zone should be measured and recorded in millimeters (mm).

   Each measurement can be compared to a zone-size interpretive chart.  Using the chart, the organism can be characterized as being resistant, intermediate or susceptible to the specific antibiotic.  Intermediate susceptiblity means that some inhibition from the antibiotic occurred but not sufficiently enough to inhibit the growth of the organism in the body.

zone of inhibition link
Click picture for link to the


                                                                                1.     Factors that influence zones of inhibition:

                                                                            Concentration of bacteria spread onto agar plate

                                                                            Pathogen susceptibility

                                                                            Antibiotic diffusion effects

                                                                            Agar depth

                                                                            Growth temperature

                                                                            Nutrient availability

                                                                            Drug antagonists

                                                                                2.     Factors that influence the diffusion of the antibiotic:

                                                                            Concentration of antibiotic

                                                                            Molecular weight of antibiotic

                                                                            Water solubility of antibiotic

                                                                            pH and ionization

                                                                            Binding to agar