The testing of the antibiotic sensitivity of bacteria on an agar medium: The problem of a double zone of inhibition

When the sensitivity of Micrococcus luteus ATTC9341 to streptomycin, erythromycin, oleandomycin and spiramycin was tested by an agar diffusion method using antibiotic impregnated filter paper disks on unbuffered Penassay Seed Agar two zones of inhibition were observed around the disks after an incubation period of 24 hours at 30 degrees C. The pH of the M. luteus seeded Penassay Seed Agar was measured before and after 24 hours incubation at 30 degrees C and found to be 6.6 and 8.7, respectively. When the Penassay Seed Agar was buffered to pH 6.1 and the sensitivity of the microbe to the antibiotics was tested as before no double zones of inhibition could be observed. The phenomenon of the double zones of inhibition may possibly be due to the pH increase of the medium from a relatively low level to the optimum range of activities of the antibiotics during the incubation period.