AbstractIn this study, we demonstrated the label-free continuous separation and enrichment ofBacillus subtilispopulations based on length using viscoelastic microfluidics.B. subtilis, a gram-positive, rod-shaped bacterium, has been widely used as a model organism and an industrial workhorse.B. subtiliscan be arranged in different morphological forms, such as single rods, chains, and clumps, which reflect differences in cell types, phases of growth, genetic variation, and changing environmental factors. The ability to prepareB. subtilispopulations with a uniform length is important for basic biological studies and efficient industrial applications. Here, we systematically investigated how flow rate ratio, poly(ethylene oxide) (PEO) concentration, and channel length affected the length-based separation ofB. subtiliscells. The lateral positions ofB. subtiliscells with varying morphologies in a straight rectangular microchannel were found to be dependent on cell length under the co-flow of viscoelastic and Newtonian fluids. Finally, we evaluated the ability of the viscoelastic microfluidic device to separate the two groups ofB. subtiliscells by length (i.e., 1–5 μm and >5 μm) in terms of extraction purity (EP), extraction yield (EY), and enrichment factor (EF) and confirmed that the device could separate heterogeneous populations of bacteria using elasto-inertial effects.