Dissecting Bacterial Growth Variants by Cellular Phenotypic Profiling and Backtracing

Bacterial cell populations are phenotypically heterogenous, including, e.g., exponentially growing cells, besides nonreplicating persisters that tolerate antibiotics, or so-called "viable-but non-culturable" cells. The lack of biomarkers and the dynamic, reversible nature of these cellular growth phenotypes make it challenging to differentiate them and study their underpinning molecular characteristics. Here, we present step-by-step protocols for our recently developed technique termed "Cellular Phenotypic Profiling and backTracing" which uses index sorting to connect the experimentally determined growth fate of single cells with their phenotypic characterization using fluorescent probes. The method is illustrated using the opportunistic Gram-positive pathogen Staphylococcus aureus grown under dormancy-inducing low pH conditions and a live-dead staining combination. The method is easily adaptable to other species of interest and may accommodate a broad variety of fluorescent probes for cellular phenotypic profiling.