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

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

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

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

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

Student Workstation Materials

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

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

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