With agar dilution the antimicrobial concentrations and organisms to be tested are brought together on an agar based medium rather than in liquid broth. Each doubling dilution of an antimicrobial agent is incorporated into a single agar plate; therefore, testing of a series of six dilutions of one drug requires the use of six plates, plus one positive growth control plate without antibiotic. The standard conditions and media for agar dilution testing are shown in Table 1. The surface of each plate is inoculated with 1 × 10⁴ CFU (Figure 1). This method allows examination of one or more bacterial isolates per plate. After incubation the plates are examined for growth; the MIC is the lowest concentration of an antimicrobial agent in agar that completely inhibits visible growth. The same MIC breakpoints and interpretive categories used for broth dilution are applied for interpretation of agar dilution methods. Similarly, test results may be reported as the MICs only, the category only, or both.

Table1. Summary of Agar Dilution Susceptibility Testing Conditions

Fig1. Growth pattern on an agar dilution plate. Each plate  contains a single concentration of antibiotic. Growth is indicated by  a spot on the agar surface. No spot is seen for isolates inhibited by  the concentration of antibiotic incorporated into the agar of that  particular plate. 

The preparation of agar dilution plates (see CLSI M07-A6 series document, “Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically”) is sufficiently labor intensive to preclude the use of this method in most clinical laboratories in which multiple antimicrobial agents must be tested, even though several isolates may be tested per plate. As with broth dilution, the standard medium is the Mueller Hinton preparation, but supplements and substitutions are made as needed to facilitate growth of more fastidious organisms. In fact, one advantage of this method is that it provides a means for determining MICs for N. gonorrhoeae, which does not grow sufficiently in broth to be tested by broth dilution methods.

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