High-throughput protocol for assessing antibiotic resistance using agar dilution, acoustic liquid handling, and semi-automated image analysis.

Conventional minimum inhibitory concentration (MIC) assays used to determine the antibiotic susceptibility of microorganisms are inherently low-throughput and labor intensive. Here, we present a high-throughput agar dilution MIC assay to efficiently assess resistance profiles across large collections of bacterial isolates. We describe steps for agar plate preparation, culture spotting, imaging setup, and semi-automated image-based data analysis of this high-throughput assay. This approach enables simultaneous determination of antibiotic resistance levels for 384 strains across multiple antibiotics within a single workflow. For complete details on the use and execution of this protocol, please refer to Van den Bergh et al.1.