Accessible, robust ultrahigh-throughput microbiome analysis via integration of uniform droplet-templated...

Microbial phenotypes vary at the single-cell level, shaping key community traits like resilience and adaptability. Yet, current methods either lack resolution (e.g., culturing, sequencing), are too costly, or technically complex, limiting widespread use. To address this gap, we introduce and validate a workflow called DE-SWIRL (Double Emulsion-Sorting Workflow with sImple, Rapid emuLsification), an accessible, low-cost workflow enabling ultrahigh-throughput (~107 microcultures/experiment) screening of individual microbial cells. DE-SWIRL integrates a published droplet-templated emulsification protocol producing uniform double emulsions from monodisperse single emulsions with Fluorescence-Activated Cell Sorting (FACS). We validated that double emulsions of 6 and 24 pL can be reliably formed, with ~45% droplet survival. To address persistent large-particle contaminants, we validated a gating strategy and show it enables accurate screening and sorting. When starting from a monodisperse single emulsion population, oil layer variability is higher for droplet-templated emulsification than for on-chip microfluidics, but maintains a comparably uniform core emulsion while offering substantial time savings. We demonstrate DE-SWIRL's utility by isolating viable strains from a synthetic community with up to 99% sorting purity and isolating droplet cocultures from a mixed community. This workflow provides a fast, accessible, and affordable workflow for screening entire microbiomes at a single-cell level using a fluorescent assay of interest.