Effects of Microplate Type and Broth Additives on Microdilution MIC Susceptibility Assays [Analytical Procedures]

The determination of antibiotic potency against bacterial strains by assessment of their minimum inhibitory concentration normally uses a standardized broth microdilution assay procedure developed more than 50 years ago. However, certain antibiotics require modified assay conditions in order to observe optimal activity. For example, daptomycin requires media supplemented with Ca²⁺ and the lipoglycopeptides dalbavancin and oritavancin require Tween-80 to be added to the growth media to prevent depletion of free drug via adsorption to the plastic microplate. In this report we examine systematically the effects of several different plate types on microdilution broth MIC values for a set of antibiotics against Gram-positive and Gram-negative bacteria, both in media alone and in media supplemented with commonly used additives Tween-80, lysed horse blood, and 50% human serum. We observe very significant differences in measured MICs (up to 100-fold) for some lipophilic antibiotics, such as the Gram-positive lipoglycopeptide dalbavancin and the Gram-negative lipopeptide polymyxins, and find that non-specific binding plates can replace the need for surfactant additives. Microtitre plate types and any additives should be specified when reporting broth dilution MIC values as results can vary dramatically for some classes of antibiotics.

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