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Antibiotic susceptibility testing can be done in many ways, including new genomic tests, the MIC broth dilution, and the Kirby-Bauer (K-B) disk diffusion method that you will perform in lab today. Identifying the correct antibiotic to use is critically important, especially in today’s situation with the increasing number of antibiotic resistant bacteria emerging in all populations. Delay of using the appropriate antibiotic, one that the bacterial disease agent is sensitive to, can greatly reduce successful patient outcomes at an alarming rate and can be the difference between life and death. All antibiotic susceptibility tests have limitations. One of the biggest drawbacks of the K-B test is that it is an in vitro test. What happens in a petri dish is not always the same thing that will happen in an actual patient. Why do you think there could be differences (think about this, you will be asked again!)?
In order for the K-B test to be valid across institutions and be accurate for each patient, it must be standardized. This includes the media used, Muellar-Hinton agar, the depth the media is poured, age of the media, etc. As the media ages, or if the depth or moisture content are not accurate, the antibiotics in the disks will not diffuse through the media the same from batch to batch. So consistency in media is essential. The antibiotics are prepared specifically for this test by infusing known amounts into standard paper disks. During the test the antibiotics will diffuse out into the media, based on the molecular size of the antibiotic. The amount of bacteria placed on the plate must also be standardized. This can be done in a variety of ways—usually by prepping a broth culture via a spectrophotometer (how does this work? Think about what you did when you measured the absorbance of your dilutions in the SPC procedure), or by using a McFarland standard. The MacFarland standard method employs a prepared test tube of aspecific turbidity with which one can prepare a broth of the bacterium to be tested to the same turbidity. This helps ensure that a consistent amount of bacteria is plated. Incubation temperature and time are also critical, as well as accurate measurement and interpretation of the zone of inhibition. As bacteria begin to grow on a plate, the antibiotics are diffusing out into the agar. If the bacteria are sensitive to the antibiotic, at some point their growth will be inhibited and a zone of no growth (zone of inhibition) will be apparent around the disk. The diameter of the zone is measured and the size of the zone determines whether a bacterium is determined Sensitive (S), Resistant (R), or Intermediate (I) to the antibiotic. The size and interpretation of the zone is specific to each bacteria-antibiotic pair and is referenced in a chart.
Contributors and Attributions
Kelly C. Burke (College of the Canyons)