A robust method for generating, quantifying and testing large amounts ofEscherichia colipersisters

Bacteria can exhibit phenotypes, which makes them tolerant against antibiotics. However, often only a few cells of a bacterial population show such so-called persister phenotype, which makes it difficult to study this health-threatening phenotype. We recently found that certain abrupt nutrient-shifts generate E. coli populations that consist of almost only antibiotic tolerant persister cells. Such nearly homogeneous persister cell populations enable assessment with population-averaging experimental methods, such as high-throughput methods. In this paper, we provide a detailed protocol of how to generate such large fraction of tolerant cells using the nutrient-switch approach. Furthermore, we describe how to determine the fraction of cells that enter the tolerant state upon a sudden nutrient shift and describe a new way to assess antibiotic tolerance with flow cytometry. We envision that these methods facilitate research into the important and exciting phenotype of bacterial cells.