7.3 Antimicrobial Susceptibility Test: Disk Diffusion

Define antibiotics

Natural antimicrobial agents produced by microorganisms

Define antimicrobials

Synthetic agents used to treat bacterial infections

Describe the disk diffusion method

Inoculated muller hinton agar plate with a microorganism is exposed to different anti microbic paper disks, each representing a antibiotic. Plates are incubated to allow growth of the bacteria and time for the antibiotics to diffuse into the agar. After incubation, if the organism is susceptible to the antibiotic, a clear zone of inhibition will appear around the disk. ONLY MEASURES BACTERIOSTATIC

Define the zone of inhibition

Describes the area around the antibiotic disk where the concentration is high enough to stop bacterial growth

What area of the zone of inhibition is crucial to interpret and why

Junction of the zone because it tells us where the concentration of the antibiotic has become too low to effectively stop growth

Define the minimum inhibitory concentration (MIC)

The junction of the zone of inhibition, where it describes the lowest concentration needed to stop bacteria growth

Does the disk-diffusion test quantify the MIC?

No

What are important aspects unique to the kirby-Bauer (disk diffusion) method?

Standardization of results requires the following:

1. Mueller-Hinton agar with a pH of 7.2 and 7.4

2. Depth of 4 mm in either 150 or 100 mm petri dishes

3. Incubation period of 16-18 hours

4. Inoculation made with broth culture to match a 0.5 McFarland turbidity standard

5. Measure zone of inhibition in millimeters (mm)

Who determines the disk concentration of each antibiotic?

US food and drug administration

Why do we use the Mueller Hinton agar?

To standarize results and prevent interference with the antibiotics

How does the antibiotic from the disks diffuse into th agar?

The wet environment of the agar allows the antibiotic to diffuse laterally and inward the agar via passive diffusion

What might be the consequence of plating the plates 2 mm deep instead of 4 mm?

A thin Mueller Hinton agar would result in the antibiotic diffusing more laterally, causing a larger zone of inhibition to appear

What might be the consequence of plating the plates 7 mm deep instead of 4 mm?

A thick Mueller Hinton agar would result in the antibiotic diffusing more inward rather than laterally, causing a smaller zone of inhibition to appear

Does the agar have antibiotic beyond the zone of inhibition? How does the answer tie with MIC?

Yes, there is antibiotic beyond the zone of inhibition, but at concentrations below the bacteria’s MIC, so the bacteria can grow there.

Explain the procedure of 7.3 for inoculation of E. coli and the placement of the disks

1. Using a cotten swab inoculate E. coli from broth onto agar plate

2. Streak entire plate with E. coli and rotate to perform another streak (3X) = confluent growth

3. Dispense cotton swab

4. Using flamed forceps or disk dispenser, dispense disk 4 cm away from each other

5. Incubate 16-18 hours