Transcript Activity 3.2: Disk Diffusion Test

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
2
Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com
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
3
Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com
Skills to Master
 Perform a modified Kirby-Bauer test
 Use aseptic technique (Activity 3.1)
 Inoculate an agar plate for a bacterial lawn
4
Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com
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
5
Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com
Student Workstation Materials
6
Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com
Performing a Modified Kirby-Bauer Test
 Play video: Modified Kirby-Bauer Test
7
Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com
Protocol Highlights/Tips






8
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
Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com
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
9
Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com