Antibiotic susceptibility testing

The isolate to be tested should be obtained in pure culture and 3-5 identical colonies are picked up using a sterile straight wire and inoculated into nutient broth (or Mueller Hinton broth) and incubated at 37^oC for 2-4 hours or until sufficient turbidity is achieved. Alternatively, for testing the fastidious organisms such as Haemophilus spp., N. gonorrhoeae, and streptococci, and for testing staphylococci for methicillin resistance, a direct broth or saline suspension of isolated colonies on 18- to 24-hour agar plate can be made. Either way, the turbidity must be matched to that of 0.5 McFarland turbidity prior to testing. This is done by visually comparing the inoculum tube and the 0.5 McFarland standard against a card with a white background and contrasting black lines. The turbidity of the broth culture is adjusted with sterile broth to obtain a turbidity comparable to that of the 0.5 McFarland standard.This results in a suspension containing approximately 1 to 2 x 10⁸ CFU/ml for E.coli ATCC 25922.

Within fifteen minutes of standardization of inoculum, a sterile cotton swab is dipped inside the tube and squeezed against the sides of test tube to remove excess of fluid. The moist swab is rubbed on the entire surface of a dry Mueller Hinton agar. This procedure is repeated by streaking two more times, rotating the plate approximately 60^o each time so as to obtain an uniform lawn growth. Finally the circular rim of the plate must be swabbed.

The surface of the medium is allowed to dry and filter paper disks containing known antibiotic is placed on the surface and pressed gently to ensure proper contact. Once placed, the discs should not me moved or relocated since diffusion of antibiotic starts instantly. The interdisc distence should be 24 mm to prevent zone overlapping. Ordinarily, no more than 12 discs should be placed on one 150 mm plate or more than 5 discs on a 100 mm plate. Within fifteen minutes of application of discs, the plate is inverted and kept in incubator. The plates are incubated in ambient air or CO2, as required.



After 18-24 hours of incubation, the plates are removed from the incubator and observed for good growth and zones of inhibition around the discs. The diameter of zone of inhibition is measured to the nearest mm and read from the Kirby-Bauer Chart and is reported accordingly as susceptible, moderately susceptible or resistant.

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