progression of biofilm formation and how it impacts antibiotic resistance 42. This concept could be extended to test various antimicrobial coatings and their properties.

Standard digital assays need a large number of compartments for precise quantification of a sample over a broad dynamic range. We address this issue with an optimized droplet digital approach that uses a drastically reduced number of compartments for quantification. We generate serial logarithmic dilutions of an initial bacterial sample as an array of microliter-sized droplet plugs. In a subsequent step, these droplets are split into libraries of nanoliter droplets and pooled together for incubation and analysis. We show that our technology is at par with traditional dilution plate count for quantification of bacteria, but has the advantage of simplifying the experimental setup and reducing the manual workload. The method also has the potential to reduce the assay time significantly.

Droplet microfluidics disrupted analytical biology with the introduction of digital polymerase chain reaction and single-cell sequencing. The same technology may also bring important innovation in the analysis of bacteria, including antibiotic susceptibility testing at the single-cell level. Still, despite promising demonstrations, the lack of a highthroughput label-free method of detecting bacteria in nanoliter droplets prohibits analysis of the most interesting strains and widespread use of droplet technologies in analytical microbiology. We use a sensitive and fast measurement of scattered light from nanoliter droplets to demonstrate reliable detection of the proliferation of encapsulated bacteria. We verify the sensitivity of the method by simultaneous readout of fluorescent signals from bacteria expressing fluorescent proteins and demonstrate label-free readout on unlabeled Gram-negative and Gram-positive species. Our approach requires neither genetic modification of the cells nor the addition of chemical markers of metabolism. It is compatible with a wide range of bacterial species of clinical, research, and industrial interest, opening the microfluidic droplet technologies for adaptation in these fields.