Agar Dilution

Agar dilution is one of the standardized antimicrobial testing methods. Mueller-Hinton agar (MHA) is used for testing nonfastidious aerobic and facultatively anaerobic bacteria that require no special supplement for growth. To prevent the interference to drug activity, any calcium and magnesium containing supplement should not be added (NCCLS, 1996). Culture medium mentioned above in dehydrated form is commercially available. Preparation of the agar plates should follow the manufacturer's recommendations. Drugs are tested at serials of twofold dilutions with each plate containing one concentration. The range of concentration tested for each drug should cover the CLSI break points and the expected MICs for quality control reference strains. Studies show that the oxacillin MIC for Staphylococcus spp. carrying the mecA gene are detected with increased sensitivity by the agar containing NaCl (Huang et al., 1993). Therefore, MHA with 2% NaCl is recommended for the testing of staphylococci against methicillin, oxacillin, and nafcillin. Plates containing certain agents such as imipenem, cefaclor, and clavu-lanic acid combination have short shelf-lives and should be prepared freshly each time used (Murray, 2003).

Inoculation size is critical in obtaining an accurate MIC value. For standardized agar dilution method, a final inoculum of 104 CFU (colony-forming units) per spot is recommended. A simple way to quantify bacteria numbers in the inoculum is to measure the turbidity of the bacterial suspension used in preparing the inoculum. By either growing several colonies from an overnight culture in a liquid broth or directly suspending colonies from an overnight culture on nonselective agar medium, the bacterial suspension with a turbidity equivalent to an 0.5 McFarland turbidity standard is made to reach a concentration of 108 CFU/mL. The former method is required for testing staphylococci (CLSI, 2005). Approximately 1 to 2 ^L of 1:10 dilution of the suspension with either sterile broth or saline is used to inoculate the agar in order to achieve 104 CFU per spot. To maintain the consistent inoculation size, bacterial suspensions have to be plated onto the plates within 30 min of preparation. By using an inoculum device, multiple samples can be plated on the same plate simultaneously. Whenever susceptibility tests are performed, tested organisms from the prepared suspension have to be grown on the plates without antimicrobial agents to ensure the viability and purity. After the inoculation, testing plates are incubated in ambient air at 35°C for 16 to 18 h. When testing staphylococci, incubation temperature between 33°C and ~35°C should be maintained to ensure reliable results. Extended incubation time (24 h) is required to detect the vancomycin-resistant enterococci and oxacillin-resistant staphylo-cocci. MIC values are determined by examining growth of the bacteria on plates containing various concentrations of antimicrobial agents. The drug dilution in the first plate showing no bacteria growth is recorded as the MIC. For bacteriostatic agents, the drug dilution that inhibits 80% of growth is regarded as the MIC.

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