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 [Display omitted] •Detection of colony-forming units under restrictive image capture environments•Images prepared with Fiji, CFU counted with CellProfiler, and quantified with R•Minimal tuning of three parameters to capture CFUs and reject artifacts•Performance on par with manual counting Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.