Abstract
Given the low fraction of antibiotic-tolerant persisters and the transient nature of the persister phenotype, identifying molecular mechanisms underlying persister state exit, also called "awakening," is challenging. Here, we describe how persister awakening kinetics can be quantified at the single-cell level, enabling the identification of genes that are important for persister survival following antibiotic treatment. We report step-by-step sample preparation, dynamic recording, and data analysis. Although the setup is flexible, time-lapse microscopy requires a minimal number of persisters being present. For complete details on the use and execution of this protocol, please refer to Wilmaerts et al. (2022).