Answer

The most effective way to use these discs is as follows: Transfer some of the bacterial culture (using a loop) into some Nutrient Broth and incubate overnight. The broth should turn cloudy once the bacteria have multiplied sufficiently. Then add the inoculated broth to the (liquid) agar to be used for plating (the amount used depends on how much agar is being used). The agar should be at around 45°C - i.e. not too hot or it will kill the bacteria. Hand hot is about the right temperature, although the temperature should not be allowed to drop too low or the agar will set. The agar-broth mixture can now be poured into the plates (petri-dishes). It is best to apply discs within 15 minutes of the inoculated agar setting. This prevents a pre-incubation of organisms before the antimicrobial discs are applied. Similarly once the discs have been applied, plates should be incubated within 15 minutes or so, to prevent pre-diffusion of the antimicrobial at room temperature. It is essential that discs are in close contact with a moist agar surface. Therefore either use dispensers which have a tamping action on the discs or press them individually after application. Do not allow the agar surface to become dry before applying the discs.
Incubation period: ensure uniform times of incubation for the plates, (e.g. 24-48 hours) at the recommended temperature. A clear zone will form around each disc if the antibiotic has worked.
Antibiotic susceptibility discs can also be applied to streaked plates but the results will not be as good, as streaking will not give as uniform a spread of bacteria as adding inoculated broth to the agar does.