Antibiotic Sensitivity - The Kirby-Bauer Disk Diffusion Test                            Atlas page 93

(See photos at the bottom of this page)

The Kirby-Bauer disk diffusion test is a standardized protocol for determining antibiotic susceptibility. A pure culture of the test organism is inoculated on Mueller-Hinton agar. You must create a "lawn or confluent growth area" on the poured cooled agar using a sterile Q-tip dipped into your unknown organism.  Quickly swab the surface of the agar plate with the Q-tip, then discard the Q-tip in the Biohazard trash.  "Discs" are then placed on this lawn BEFORE incubation.  Place the discs no closer than than 2 cm apart using flamed forceps or tweezers and press the discs into the agar gently. Then incubate the plates upside-down at the optimum temperature for 18 hours. Results are read and interpreted after of incubation.

If the growth of a test organism is inhibited by the antibiotic on a disc, a zone of inhibition will appear. The zone of inhibition is a circular area surrounding an antibiotic disk in which the test organism does not grow. The diameter of the zone of inhibition is calibrated in millimeters and compared with the Interpretive Zone Standards published by the National Committee for Clinical Laboratory Standards. Results are reported as S (sensitive), I (intermediate), or R (resistant).  Note, that many "dots" within a Zone of Inhibition are "mutants" are thus make the agent useless...

Results are affected by factors such as the antibiotic concentration, rate of diffusion of the antibiotic, pH and depth of the culture medium, inoculum density, incubation time and temperature. Thus it is essential that the Kirby-Bauer test is performed under standardized conditions: on Mueller-Hinton agar of pH 7.2 to 7.4, 4 mm. deep, inoculated with a pure culture of microbes matching the McFarland 0.5 standard for turbidity. Under these conditions, the results provide a guide for the potential effectiveness of a particular antibiotic. The plates shown here have been produced following the standardized procedure.

Please ask your instructor for a table which lists the standard S, I and R mm Zones of Inhibition established by the National Committee for Clinical Standards... Also, you must standardize your unknown in a broth using a Spectrophotometer AND have the Mueller-Hinton agar prepared for the plates... 

For the purposes of this class we use the following "rule of thumb" in our evaluations:  S = 21+mm, I = 14-20.99mm, R = 13.99mm or less...

See the Atlas page 93 for additional information

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