Cation components of Mueller-Hinton agar affecting testing of Pseudomonas aeruginosa susceptibility to gentamicin

Seven lots of Mueller-Hinton agar were examined for calcium and magnesium contents and their distribution in pools or compartments. Gel disruption and centrifugation yielded the soluble cations, which varied from 9 to 113% of the total calcium and from 76 to 102% of the total magnesium. Throughout the experiments, a standardized disk diffusion test, using Pseudomonas aeruginosa (ATCC 27852) and a 10-mug gentamicin disk, served as an indicator for medium performance. Zone diameters correlated well with the sums of the soluble calcium and magnesium values in the different lots (r = -0.85). Ionized calcium, presumably the biologically active ion, was measured with a calcium-specific electrode. It represented only a fraction of the soluble calcium pool in three lots. Autoclaving resulted in shifts of the cations between the different pools. Addition of magnesium to one medium lot resulted in shifts of soluble and ionized calcium, indicating an interdependence of calcium and magnesium, and zone diameters correlated with soluble magnesium (r = -0.98), soluble calcium (r = -0.96), and ionized calcium (r = -0.96) in this experiment. Manipulation of one medium to match the performance of another showed that excess amounts of both ions were required to obtain similar performance. Satisfactory performance of an individual medium can be obtained by cation supplementation, but simple adjustment will not suffice for all media. The interaction of the other cation pool components must also be evaluated.