The Kirby-Bauer Antibiotic Susceptibility test is used to determine the effectiveness of a collection of antibiotics in preventing bacterial growth through the use of the “disk diffusion method”. By challenging the growth of the bacteria with a variety of antibiotics, it is possible to determine which antibiotics are effective vs. not effective in preventing growth. This informs decisions on which antibiotics are of potential benefit for treating a disease caused by this microbe and those that would not be effective.
Plates of Mueller Hinton agar are seeded with cells from a pure culture of bacteria to create a potential lawn. Next are added in an equidistant pattern paper disks each impregnated with a different antibiotic. The prepared plates are incubated overnight to allow the bacteria to grow. During this time, the antibiotics are diffusing away from their paper disks creating a gradient with higher concentrations near the disk and lower concentrations as the distance from the disk increases. The next day the plates are removed from the incubator and evaluated. The sizes of the zones of inhibition are measured and compared with standardized charts to determine whether the microbe is resistant, intermediate in resistance, or susceptible to each antibiotic.
The antibiotics commonly used differ for Gram – and Gram + bacteria, in part due to the anatomy and physiology of these two major groups of bacteria. Often, additional testing using other alternative antibiotics can provide a more refined and focused examination of the resistance and susceptibility patterns for a specific unknown microbe.
Typical Antibiotics Used for Kirby-Bauer Test vs. Gram – Bacteria:
- Chloramphenicol
- Kanamycin
- Nalidixic Acid
- Nitrofurantoin
- Sulfa Drugs
- Tetracycline
Typical Antibiotics Used for Kirby-Bauer Test vs. Gram + Bacteria:
- Chloramphenicol
- Erythromycin
- Novobiocin
- Penicillin G
- Streptomycin
- Tetracycline
What is the purpose of the test?
The purpose is to see if the microbe is sensitive or resistant to an antibiotic. This information can be used in the identification of the microbe or in decisions on which antibiotic might be used for the treatment of an infection.
How is antibiotic resistance determined?
We use the Kirby-Bauer disk diffusion method. Bacteria are spread across the plate in a thick suspension to support confluent growth across the entire surface – this is called a bacterial “lawn”. Disks containing antibiotics are added immediately after the agar is inoculated and the plates are placed into the incubator so that the lawn can form. Meanwhile, the antibiotic diffuses away from the disk, with the area next to the disk having a higher concentration than is found farther away. In areas where the concentration of antibiotic is high enough to prohibit the bacteria from forming a lawn, a zone of inhibition forms. After overnight incubation, the lawn has formed and any zones of inhibition represent areas where an antibiotic was effective in preventing growth. Zones of inhibition are measured (diameter in mm) and compared to standardized results for each antibiotic to determine whether the bacterium was sensitive to the antibiotic (zones of inhibition were larger than the standard size expected) or resistant (zones of inhibition were smaller than the standard size expected).
What medium is used?
The medium typically used for a Kirby-Bauer Sensitivity Test is Mueller-Hinton agar plates. This medium supports the growth of most organisms and can be enriched with blood when required to test more fastidious organisms.
How is the test performed?
A plate of Mueller Hinton agar is selected and used to create a bacterial lawn. Antibiotic-impregnated disks are added aseptically to the lawn. The plates are inverted and incubated overnight, after which zones of inhibition are measured (diameters are measured in millimeters) and compared to standardized charts for interpretation of results..
What reagents are added?
None.
Performing this test in the VUMIE Online lab
Inoculation of Medium
1. Select Mueller Hinton agar medium.
2. Complete the process of a tube-to-lawn aseptic transfer using a sterile cotton-tipped applicator (swab) to inoculate the medium. Forgotten how to do these things? Watch the “Show Me How To” videos.
3. When you attempt to replace the tube cap and plate lid, you will be prompted to add the antibiotic disks. Do this, and then be sure the cap and lid are replaced.
Incubation of the Inoculated Medium
4. Place the inoculated plate into the 35-37 C incubator.
5. Press the New Day button to move forward 24 hours. Forgotten how to do these things? Watch the “Show Me How To” videos.
Determination of Test Results
6. Incubate this test for 24 hours. Retrieve the incubated culture. If the test protocol was followed as described above, a lawn of the culture will have grown and zones of inhibition will be visible revealing areas where growth was prevented due to presence of the antibiotics.
7. Select “Record Results” option when clicking on the plate. A new image will appear showing the same plate with zones marked and disks numbered.
8. A table will appear that contains the list of antibiotics present on the disks and the size ranges for zones representing the standard values for antibiotic resistance (R), intermediate resistance (I), and susceptibility (S). These are specific for each antibiotic. There is also a column that provides the measurements for the zones on your plate produced by the antibiotics you placed there.
9. Compare the sizes of the zones for each of your antibiotics with its standard values in the table. In the final column, provide your interpretation of the results for whether the microbe tested shows resistance, intermediate resistance, or susceptibility to each of the antibiotics.