CEFTRIAXONE for antimicrobial susceptibility testing of bacterial cultures by disk diffusion method of Bauer-Kirby.
PRINCIPLE: Disc diffusion test is a qualitative test method. This method is based on the principle that antibiotic-impregnated disk, placed on agar previously inoculated with the test bacterium, picks up moisture and the antibiotic diffuse radially outward through the agar medium producing an antibiotic concentration gradient. The concentration of the antibiotic at the edge of the disk is high and gradually diminishes as the distance from the disk increases to a point where it is no longer inhibitory for the organism, which then grows freely. A clear zone or ring is formed around an antibiotic disk after incubation if the agent inhibits bacterial growth. The disk diffusion method is performed using Mueller-Hinton Agar (MHA), which is the best medium for routine susceptibility tests because it has good reproducibility, low in sulfonamide, trimethoprim, and tetracycline inhibitors, and gives satisfactory growth of most bacterial pathogens.
Prepare sterile media plates of MUELLER HINTON AGAR (TM 339/TM 236) for rapidly growing aerobic organisms as per user’s instruction. The agar depth should be 4.0 ± 0.5 mm. Inoculate the plates using inoculum of turbidity comparable to that of standard 0.5 McFarland by lawn technique using a sterile cotton swab. Altertively, the inoculum can be standardized by other appropriate optical method (0.08 - 0.13 OD turbid suspension at 625 nm). Allow the inoculum to dry for 5 - 15 minutes with lid in place. Apply the antibiotic disc(s) aseptically, using sterile applicator or forceps.
Place the antimicrobial discs with centers at least 24 mm apart. For fastidious organisms and for Penicillin and
Cephalosporins, the discs should preferably be placed with centers 30 mm apart.
Incubate immediately at 35 ± 2°C and examine after 16-20 hours or longer, if necessary. For fastidious organisms
incubate at appropriate temperature and time.
Measure the zone of inhibition and record the diameters of the zones to the nearest millimeter using a calibrated
instrument.
Prepare sterile media plates of MUELLER HINTON AGAR (TM 339/TM 236) for rapidly growing aerobic organisms as per user’s instruction. The agar depth should be 4.0 ± 0.5 mm. Inoculate the plates using inoculum of turbidity comparable to that of standard 0.5 McFarland by lawn technique using a sterile cotton swab. Altertively, the inoculum can be standardized by other appropriate optical method (0.08 - 0.13 OD turbid suspension at 625 nm). Allow the inoculum to dry for 5 - 15 minutes with lid in place. Apply the antibiotic disc(s) aseptically, using sterile applicator or forceps.
Place the antimicrobial discs with centers at least 24 mm apart. For fastidious organisms and for Penicillin and
Cephalosporins, the discs should preferably be placed with centers 30 mm apart.
Incubate immediately at 35 ± 2°C and examine after 16-20 hours or longer, if necessary. For fastidious organisms
incubate at appropriate temperature and time.
Measure the zone of inhibition and record the diameters of the zones to the nearest millimeter using a calibrated
instrument.
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