Microbiology andCell Culture

Activity 3.2: Disk Diffusion Test

(Modified Kirby-Bauer Test)

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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

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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

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Skills to Master

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

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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

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Student Workstation Materials

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Performing a Modified Kirby-Bauer Test

Play video: Modified Kirby-Bauer Test

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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

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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