Droplet-array culture enables single-cell isolation of bacteria directly from blood

Bacteremia and sepsis require rapid diagnosis and treatment, but these are often hampered by slow or inconclusive blood culture results. Here we describe an alternative to traditional blood culture, leveraging common laboratory equipment and a droplet array to isolate and expand bacteria in a single step. The method neutralizes inhibitory whole-blood components through lysis, concentrates sample bacteria, and uses a droplet array to enable single-bacteria isolation and subsequent in-droplet expansion. Seven species of bacteria (four gram negative, three gram positive) were spiked into 1 mL of whole blood at a concentration of approximately 100 colony-forming units (CFU) and subsequently processed. The mean time to colorimetric detection via WST-8 dye was 11.5 h (range 9-13 h), compared with [≥] 28 h typically required for conventional blood culture and subculture. Colony counts per droplet followed a Poisson distribution for both gram-negative ({lambda} = 0.288) and gram-positive ({lambda} = 0.320) organisms, and means of 86.2% and 88.4% of occupied droplets, respectively, contained a single CFU. This method eliminates the need for additional subculture after positive blood culture. By partitioning the sample, the workflow limits competitive exclusion and preserves the composition of polymicrobial samples, while yielding viable colonies substantially faster than conventional methods. Blood processing at low CFU counts (3-5 CFU/mL) with both S. aureus and E. coli consistently recovered viable bacteria (1-3 CFU/mL).