About this test
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. Since this is a simulation, we simply use nutrient agar plates.
How is the test performed?
A plate of nutrient agar is selected and used to create a bacterial lawn. Antibiotic-impregnated disks are added aseptically to the lawn. Th plates are inverted and incubated for 24 hours, after which zones of inhibition are measured and compared to standardized charts for interpretation of results..
What reagents are added?
Performing this test in the VUMIE Online lab
Inoculation of Medium
1. Select the nutrient agar medium.
2. Complete the process of a tube-to-lawn aseptic transfer to inoculate the medium. Forgotten how to do these things? Watch the “Show Me How To” videos.
Incubation of the Inoculated Medium
3. Place the inoculated tube into the 35-37 C incubator.
4. 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
5. Incubate this test for only 24 hours.
6. Retrieve the incubated culture from the incubator.
7. Observe test result. If the test was followed as described above, a lawn of the culture will have grown and zones of inhibition will signify whether the microbe shows sensitivity to the drug. If you a completing a diagnostic test for ID (bacitracin, novobiocin, optochin), any zone of inhibition is indicative of a positive test. If you are completing the sensitivity testing of a range of antibiotics for a Gram + or Gram – microbe, you will need to measure the zones of inhibition of each antibiotic and compare them to a standardized chart to determine whether the microbe is sensitive or resistant to the drug.
8. Record test result.