1. Microbiology and Cell Culture
Activity 3.2: Disk Diffusion Test
(Modified Kirby-Bauer Test)

2. Activity 3.2: Disk Diffusion Test
Research Question
Which antimicrobial agent is most effective against E. coli HB101 bacteria?
Objectives
Make one 100 mm LB agar plate
Set up a disk diffusion assay using three chemicals and one negative control
Evaluate the antimicrobial properties of three chemicals

3. Safety Reminders Follow all laboratory safety procedures
If you are allergic to antibiotics, tell your instructor and do not handle the ampicillin antibiotic
Never use the autoclave unless under the direct supervision of the instructor
Use aseptic technique when using bacteria
Wear appropriate PPE

4. Skills to Master Perform a modified Kirby-Bauer test
Use aseptic technique (Activity 3.1)
Inoculate an agar plate for a bacterial lawn

5. Tips
When placing the disks on the agar surface, remember to hold lid directly above the plate as a shield
Use sterile forceps to handle the disks
When placing the disks onto the surface of the agar, press them gently with forceps to ensure that they stick when the plate is inverted

6. Student Workstation Materials

7. Performing a Modified Kirby-Bauer Test
Play video: Modified Kirby-Bauer Test

8. Protocol Highlights/Tips
Remember to use appropriate safety equipment
Follow the aseptic technique at all times
Use sterile forceps to place disks onto the agar surface
When streaking the plate for a bacterial lawn, ensure that the bacteria are evenly spread across the entire surface
Place agar plate atop a piece of paper with an X drawn on it as a guide to draw the X on the bottom of the agar plate
Label all quadrants with the proper labels

9. Summary Make sure to: Record all steps in your notebook
Be careful and wear heat-resistant gloves when handling hot agar or broth
Follow all tips
Incubate with agar side up in an incubator at 37ºC
Measure the zone of inhibition around each disk
Bleach or autoclave plates upon the completion of the experiment