In clinical laboratories where a large number of pathogens isolated from various infected body parts are presented daily, such assessment is carried out routinely by the disk diffusion method known as the Kirby-Bauer method. In this method, an agar plate is uniformly inoculated with a carefully adjusted lawn of the pure test organism. Paper disks, each impregnated with a defined concentration of a particular antibiotic, are placed on the agar surface. Antibiotics diffuse radially from the circular disks into the agar medium while bacterial growth proceeds normally in areas of agar where the antibiotic concentration gradient is not inhibitory to bacterial growth. Upon incubation, circular zones, in which no bacterial growth occurs, are revealed around certain antibiotic disks. These are known as zones of inhibition and the diameters indicate whether the test organism is susceptible, intermediately susceptible, or resistant to a particular antibiotic.
The Kirby-Bauer disk diffusion method, or culture susceptibility test, is rigorously standardized as recommended by the Clinical and Laboratory Standards Institute (CLSI), formerly known as The National Committee for Clinical Laboratory Standards. Standardization involves all variables included in the procedure, such as the type of culture medium, the composition of Mueller-Hinton agar, pH, the thickness of the agar, inoculum suspension standardization against standard McFarland turbidity tubes, age of the organism, and the method of inoculation. Also standards are followed for selection, preparation, and storage of antibiotic disks as well as conditions of incubation, measurement, and interpretation of results.
The CLSI issues document M2 for details of performance of disk diffusion tests and document M100 for extensive interpretation of results. Interpretive criteria to assign a zone diameter for a hypothetical antibiotic are derived from scattergrams (also known as scatterplots) that are used to establish mimimum inhibitory concentration (MIC) and disk diffusion interpretive criteria, also called breakpoints. The scattergram represents results from MIC tests and disk diffusion tests of many strains with a hypothetical antimicrobial “X.”
Breakpoints are established by testing several hundred isolates by the standard CLSI disk diffusion and MIC methods. The MIC and corresponding zone diameter are plotted for each isolate to construct a scattergram. Next, MIC breakpoints are established following analysis of:
1. The distribution of MICs
2. Pharmacokinetic and pharmacodynamic properties of the antimicrobial agent
3. Clinical data correlating individual MIC results with patient outcomes
Then the disk diffusion breakpoints are established by:
1. Examining the scattergram to determine the zone measurements that best correlate with the resistant, intermediate, and susceptible MIC breakpoints
2. Calculating the percentage of isolates that demonstrate disagreement between the disk diffusion and the MIC interpretations from scattergrams
The animation demonstrates the steps of the Kirby-Bauer test starting with the selection of isolated colonies of the test organism followed by preparation and standardization of the inoculum suspension. The inoculum is then evenly spread on a Mueller-Hinton agar plate and finally antimicrobial disks are laid on the surface of the agar medium. Separate sections of the animation demonstrate incubation, the development of zones of inhibition, and then measurement of the zones. The final section shows the interpretation of results, especially of problematic zones of inhibition usually encountered in every day routine in the clinical laboratory.
This is an original Flash-based animation that should be useful in demonstrating many fundamental aspects of this important, widely applied, microbiological technique. Realizing the absolute importance of adhering to the recognized standardization references, this animation intentionally and exclusively relied on scientific data provided by CLSI for this test (2).
This animation is intended to be used as an adjunct to clinical laboratory instruction. Students must be reminded that the procedure is highly regulated and controlled and must be accompanied by a rigorous quality assurance program including performance by certified and/or licensed personnel when the results are to be reported in clinical settings.
1. Cavalieri, S. J., et al. 2005. Manual of antimicrobial susceptibility testing. American Society for Microbiology, Washington, DC. http://www.asm.org/ASM/files/ccLibraryFiles/Filename/000000002484/Manual%20of%20Susceptibility%20Susceptibility%20Testing.pdf.
2. Wikler, M. A., et al. 2006. Performance standards for antimicrobial disk susceptibility tests; approved standard, 9th ed. Clinical and Laboratory Standards Institute document M2-A9. Clinical and Laboratory Standards Institute, Wayne, PA.