On the theory of the disk diffusion test. Evidence for a non-linear relationship between critical concentration and MIC, and its practical implications for susceptibility testing of Haemophilus influenzae.

The procedure for disk diffusion susceptibility testing has been worked out for rapidly growing non-fastidious bacteria. Using the general zone diameter breakpoints for interpretation of susceptibility, it was found that clinical isolates of H. influenzae were assigned to the wrong SIR category in fifty per cent of the strains for erythromycin, and ten per cent for doxycycline. New species- and laboratory-specific interpretive zone diameter breakpoints corresponding to the recommended MIC limits were therefore worked out. The Standard Curve regression Analysis (SCA) method used for this purpose is based on the correlation between zone size and disk content, using two reference strains with different MICs. In its original version (the Single strain Regression Analysis, SRA) only one reference strain was used. This equation was found not to be generally valid since the relationship between MIC and the critical concentration is not constant, as was originally assumed. The slope and intercept of the regression line obtained by SCA is species related, while a general regression line based on results from many different species assumes that there is the same relation between the zone size and MIC for all species. Breakpoints for erythromycin and doxycycline calculated by the SCA equation gave more accurate results in routine susceptibility testing of H. influenzae and reduced the error rate from fifty three and ten per cent to four and three per cent for the two antibiotics.