Determination of bacterial resistance to antimicrobials is an important part of the management of infections in patients. The disk diffusion method of Kirby-Bauer has been standardized and is a viable alternative to broth dilution methods for laboratories without the resources to utilize the newer automated methods for micro dilution testing. The agar used is Mueller-Hinton (MH) agar plate which is a microbiological growth medium that is commonly used for antibiotic susceptibility testing. When a 6 mm filter paper disk is impregnated with a known concentration of an antimicrobial compound is placed on a Mueller-Hinton (MH) agar plate, immediately agar is absorbed into the disk form the agar.
The antimicrobial diffuses into the surrounding agar. The rate of diffusion of the antimicrobial is highest closest to the disk and a logarithmic reduction is observed as the distance increases. The rate of diffusion depends on the diffusion and solubility properties of the drug in MH agar and the molecular weight of the antimicrobial compounds.
• Required Chemicals:
o Mueller-Hinton agar
o Antibiotic disks
o Pure culture organism to be tested
o Nutrient Broth
o A sterile swab was dipped into the suspension culture.
o The surface of the MH agar plate was inoculated by streaking the swab over the entire agar surface.
o The swab was discarded.
o The appropriate antimicrobial-impregnated disks were placed on the surface of the agar, using forceps. The plate was divided into four sections, with each section containing one disk.
o The plates were inverted and placed in a 37ºc air incubator for 18 to 24 hours.
o Following incubation, the zone sizes were measured to the nearest millimeter using a ruler or caliper.
o The forceps and other equipment, and the hands must be properly sterilized before use.
o The bud of the cotton swabs must not touch any surface to avoid contamination.
o The disks must be far apart enough to avoid overlapping of the antibiotics.
Research Assistant, ASRBC, ACI LTD