5.4.2: Antimicrobial Sensitivity Test (Kirby-Bauer Method) Activity

Inoculation of Mueller-Hinton Agar Plates

Label three Mueller-Hinton agar plates for each of the bacteria to be assessed.

1. Turn on the Bunsen burner and aseptically obtain a cotton swab.
2. Obtain a bacterial culture. Remove the cap. Pass the mouth of the tube through the flame of the Bunsen burner.
3. Insert the swab into the tube to acquire a sample of the bacterium.
4. Pass the tube through the flame of the Bunsen burner and recap the test tube, and return the tube to the test tube rack.
5. Use the cotton swab applicator to introduce the bacterial sample to the surface of the Mueller-Hinton agar. Swab the surface multiple times rotating in the plate in between swabs. The surface of the agar should be completely covered in bacteria in order to create a lawn of growth.
6. Place the used cotton swab into a biohazard bin or temporarily into a beaker of disinfectant until they can be sterilized.
7. Turn off the burner and let the plates dry for 3-5 minutes.

Application of Antimicrobial Disks

Apply antimicrobial disks using one of the following methods:

Antibiotic Disk Dispenser

1. Load the desired antibiotic disks into the disk dispenser.
2. Remove the lid from the plate and place the dispenser over the plate.
3. Press down on the plunger to dispense the discs onto the surface of the agar.
4. Use sterile forceps to tap each disk gently to secure it to the agar.

Sterile Forceps

1. Sterilize the forceps by dipping them in a beaker of 70% of ethanol and then passing them through the flame of a Bunsen burner.
2. Pick up an antibiotic disk with the sterile forceps and place it on the surface of the agar. Tap it gently to ensure it adheres to the agar.
3. Repeat this for each antimicrobial disk to be placed on the agar plate.

***Note*** Ensure that the antimicrobial disks are at least 15 mm from the edge of the plate and that the disks are spaced at equal distances from one another.

4. Invert the plate and incubate it at 37°C for 16 - 18 hours. Repeat this process for each plate inoculated.

Measuring Zones of Inhibition

Obtain the Mueller-Hinton agar plates that were incubated.

1. Use the provided metric ruler to measure the zone of inhibition for each antimicrobial disk on each plate. Measure the diameter of the zone of inhibition across the center of the antimicrobial disk. Write your answer in the provided table.
2. Compare the measurement with the Antimicrobial Sensitivity Standardized charts on the next page for each antimicrobial disk. Record whether the data indicates susceptible (S), intermediate (I), or resistant (R).

Repeat this for each plate.