Phenotypic Testing of Bacterial Antimicrobial Susceptibility:Agar Dilution

Phenotypic testing of bacterial antimicrobial resistance has been widely used in clinical and diagnostic microbiology laboratories. These methods have been well studied and standardized. They have the advantages of being low cost, easy to perform (automated systems), and interpretation criteria readily available for commonly encountered organisms. These assays also are essential for new resistance discovery.
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 clavulanic
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 μL of 1:10 dilution of the suspension with either sterile broth or saline is used to inoculate the agar in order to achieve 10000 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 staphylococci.
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.
For agar Dilution method for Mycobacterium tuberculosis read : Lectures on applied clinical microbiology