The plasmid-located colistin resistance gene confers low-level resistance to colistin, a last-line antibiotic against multidrug-resistant Gram-negative bacteria. Current CLSI-EUCAST recommendations require the use of a broth microdilution (BMD) method with cation-adjusted Mueller-Hinton (CA-MH) medium for colistin susceptibility testing, but approximately 15% of all MCR-1 producers are classified as sensitive in that broth. Here we report on an improved calcium-enhanced Mueller-Hinton (CE-MH) medium that permits simple and reliable determination of -containing Colistin susceptibility testing was performed for 50 -containing and isolates, 7 intrinsically polymyxin-resistant species, and isolates with acquired resistance to polymyxins due to and mutations, respectively, and 32 -negative, colistin-susceptible isolates of , , , , , and serovar Typhimurium. A comparison of the colistin MICs determined in CA-MH medium and those obtained in CE-MH medium was performed using both the BMD and strip-based susceptibility test formats. We validated the data using an isogenic IncX4 plasmid lacking Use of the CE-MH broth provides clear separation between resistant and susceptible isolates in both BMD and gradient diffusion assays; this is true for both -containing isolates and those exhibiting either intrinsic or acquired colistin resistance. CE-MH medium is simple to prepare and overcomes current problems associated with BMD and strip-based colistin susceptibility testing, and use of the medium is easy to implement in routine diagnostic laboratories, even in resource-poor settings.