Over the past 50 years genetic analysis in microbiology has relied predominantly on selections and plate assays using chromogenic enzyme substrates - for example, X-gal assays for the detection of beta-galactosidase activity. Recent advances in fluorescent assays and high throughput screening technologies have paved the way for the rapid isolation of mutants that confer complex phenotypes and for the quantitative analysis of the evolution of new traits in bacterial populations. This Review highlights the power of novel single-cell screening technologies and their applications to genetics, evolution and the biotechnological uses of bacteria.