Abstract
PySpore is a Python program that tracks the germination of individual bacterial endospores. Here, we present a protocol for segmenting spores and quantifying the germination properties of individual bacterial endospores using PySpore. We describe steps for using GUI-based tools to optimize image processing, annotating data, setting gates, and joining datasets for downstream analyses. We then describe procedures for plotting functionality tools without the user needing to modify the underlying code. For complete details on the use and execution of this protocol, please refer to Ribis et al. (2023).1.