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  1. Bacteria experience substantial physical forces in their natural environment including forces caused by osmotic pressure, growth in constrained spaces, and fluid shear. The cell envelope is the primary load-carrying structure of bacteria, but the mechanical properties of the cell envelope are poorly understood; reports of Young’s modulus of the cell envelope of E. coli are widely range from 2 MPa to 18 MPa. We have developed a microfluidic system to apply mechanical loads to hundreds of bacteria at once and demonstrated the utility of the approach for evaluating whole-cell stiffness. Here we extend this technique to determine Young’s modulus of the cell envelope of E. coli and of the pathogens V. cholerae and S. aureus. An optimization-based inverse finite element analysis was used to determine the cell envelope Young’s modulus from observed deformations. The Young’s modulus of the cell envelope was 2.06±0.04 MPa for E. coli, 0.84±0.02 MPa for E. coli treated with a chemical known to reduce cell stiffness, 0.12±0.03 MPa for V. cholerae, and 1.52±0.06 MPa for S. aureus (mean ± SD). The microfluidic approach allows examining hundreds of cells at once and is readily applied to Gram-negative and Gram-positive organisms as well as rod-shaped and cocci cells, allowing further examination of the structural causes of differences in cell envelope Young's modulus among bacteria species and strains. 
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