SRP064906 PRJNA298997 Escherichia coli genotypes associated to phenotypes isolated in millifluidic Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypes analysis are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals cells were sampled and individually compartmentalized in 100 nL drops. Growth of their progeny through 15 generations was monitored using a fluorescent protein reporter. The amplification of heritable changes over multiple generations yields clusters of narrowly defined phenotypes reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence heritable phenotypes. Mutations corresponding to each class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods. Escherichia coli