Question

You perform the disk diffusion test to test the antimicrobial drug resistance characteristics of a strain of Salmonella typhi. The ciprofloxacin disk produces a 15 mm zone of inhibition. Why exactly is the zone 15 mm and not 16 mm or 14 mm? Compare and contrast the components of the agar 14 mm away from the disk and the components present at 16 mm away from the disk.

Answer 1

Disk diffusion testing:

• The antimicrobial agent is incorporated (in different concentrations), into disks, tablets or strips (of specific dimensions).
• The agents then placed on solid medium of standardized, documented formulations. Usually Muller-Hinton Agar.
• The standardized medium amount: 0.5 mm (+/- 4. Aapproximately 25 mL in a 90 mm circular plate, 31 mL in a 100 mm circular plate, 71 mL in a 150 mm circular plate, 40 mL in a 100 mm square plate. The plates should be dry, with no droplets of water.
• Microbial colonies are used in form of suspension. Turbidity determined (Mc Farland standard: 0.5, approximately 1x 10 8 cfu/ml). Usually 15 minutes suspension preferred.
• The plates are inoculated by swab/ streak technique.
• Disks containing the agent are usually placed after 15 minutes of inoculation. Normally 6-8 disks per plate. Control is maintained, with disk without agent.
• Incubation of plates as per specific time as per organism (usually 24-48 hours).
• The plates with confluent lawn growth are considered.
• The zone of inhibition is measured and compared as per AST documents.
• Thus, for a specific set of antibiotic  and organism (in this case ciprofloxacin and Salmonalla typhi combination, a specific inhibitory zone is obtained (15cm) as other conditions are maintained as specific.

• The zone of inhibition measures the area upto which the antibiotic has diffused and inhibited the growth of the organism being tested.
• The larger the zone of inhibition, more sensitive is the organism towards that antimicrobial agent.
• Thus, the media within 14 cm will contain the antibiotic. While, the media within 16 cm will lack the antibiotic.