Semi-automated colony-forming unit counting for biosafety level 3 laboratories

Biosafety level 3 decontamination precautions motivate measuring microbial colonies using consumable photography instead of expensive automated plate counters or smartphones, and assaying drug treatments-with multiple concentrations per treatment, replicates, and controls-produces hundreds of images. Here, we present a protocol for semi-automated image analysis by hand-tuning three parameters. The parameters control for non-uniform colony growth and artifacts such as lid condensation, reflections, and plating streaks. We describe steps to prepare images, tune parameters, and plot dose-response relationships. For complete details on the use and execution of this protocol, please refer to Larkins-Ford et al.¹.