REQUIREMENTS :
St. MH agar butt (20 ml), St. Petri dish, St. 1 ml pipette, standard antibiotic solutions of known concentration, St. cork borer (external diameter 10 mm), test culture, absolute alcohol, etc.
PROCEDURE :
1. Inoculate 0.2 ml of S. aureus ATCC 6538P culture to 20 ml of cooled, melted MH agar butt; mix and pour into a dry, sterile Petri dish.
2. Allow the agar to solidify and set.
3. Make four wells in each plate using a sterile cork borer.
4. Transfer 0.1 ml of 4 different antibiotic solutions in each well of a plate aseptically using a micropipette.
5. Place the plate in the refrigerator for 30 minutes for prediffusion of the antibiotic solutions.
6. Transfer the plates to an incubator and incubate the plates upright at 37°C for 24 hours.
7. Measure the diameter of zones of inhibition given by each antibiotic solution and interpret.
Notes :
1. Presence of a haze of growth within the zone around the disc indicates drug resistance in the population of the isolate.
2. Quality control should be performed each time a new lot of discs or agar is used. NCCLS has established limits of zone sizes that are acceptable for quality control strains.
Reference strains for quality control : Enterobacter faecalis ATCC 29212, Escherichia coli ATCC 25922, Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923.
For comparative methods, the cultures used are : Escherichia coli NCTC 10418, Pseudomonas aeruginosa NCTC 10662, Staphylococcus aureus NCTC 6571.
