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. This article focuses on the use of disk-diffusion antibiotic susceptibility as a test for bacterial identification.
How is antibiotic susceptibility 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 grow. 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 toxic and 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 is a test used to determine general antibiotic susceptibility by challenging a microbe with six general antibiotics. Look for the article on Kirby-Bauer Antibiotic Susceptibility Test to find more information.
In this article, the focus is on using antibiotic susceptibility as a diagnostic test for identification. A single disk of the antibiotic of interest is seeded on the plate and results are either + or -. In VUMIE Online, this is done using nutrient agar (rather than Mueller-Hinton) and follows the general process described for the Kirby-Bauer test. Antibiotics that use this version of a disk-diffusion test for diagnostic purposes are Optochin, Bacitracin, and Novobiocin.
How is the test performed?
A plate of nutrient agar is selected and used to create a bacterial lawn using a sterile cotton-tipped applicator (swab). A single disk of the antibiotic for the test is added aseptically to the lawn. The plate is inverted and incubated overnight, after which the lawn is inspected to determine whether a zone of inhibition is present around the disk (a positive result). No zone is a negative result.
What reagents are added?
Performing this test in the VUMIE Online lab
Inoculation of Medium
1. Select Nutrient 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 disk. 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.
Determination of Test Results
6. Incubate this test for 24 hours. Retrieve the incubated culture. Look for the presence or absence of a zone of inhibition around the disk. Interpret these results.
7. Select “Record Results” option when clicking on the plate. Report the results and dispose of the medium.