Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

1. Grow E. coli cells in Luria-Beltrani broth (LB) medium to exponential phase (OD = 0.2-0.4).
2. Grow the culture in LB supplemented with 50 μg/mL of cephalexin for 15 min to induce filamentous growth, and then concentrate the culture by 5 times thru centrifugation.
3. Make PEI-coated coverslip by flowing 1% polyethylenimine diluted in water into a flow chamber, and wash with water after a 5-minute incubation.
4. Flow the concentrated cell culture into the chamber, and washed with a mixture of LB and cephalexin(50μg/mL) after 3 min to remove unattached cells.
5. Incubate the chamber at 37°C for 30 min-1 hour to let the attached cells grow before placement on the optical trapping instrument.
6. Make polylysine-coated beads by incubating 0.5-μm-diameter polystyrene beads (Bangs Labs) in 0.1% polylysine diluted in water for 30 min. Then wash the beads 3 times and resuspend in water.
7. Dilute the bead solution by a factor of two into LB with cephalexin (50μg/mL), and add it into the flow chamber.
8. Optically trap a floating bead and touch it to the free tip of a cell. (We use a Mad City Labs piezo stage to control of the motion of the sample.) When a suitable bead/cell combination is found, wash the chamber with cephalexin in LB (50μg/mL) to remove unattached beads.
9. Run custom-written LabView program to apply bending forces to cell and record force-displacement data. Program Details
   1. Calibrate detector response by raster scanning the attached bead within the detection laser beam and recording 3D PSD voltage signals.
   2. Identify cell long axis in microscope image.
   3. Move cell in steps in a direction perpendicular to the cell long axis.
   4. Record the distance moved and the displacement from the optical trap.
   5. Convert displacement to applied force using the previously measured trap stiffness and save tip displacement and force to file.

Secrets to Success: In Step 8), one needs to find a cell with a well-defined stuck end. Some cells are just stuck at one tip, and the bending force at the other tip leads to a whole-cell pivoting rather than bending. A suitable pair is found by bending each cell quickly by hand using the joystick-controlled stage motion.

Representative Results:

Figure 1. This figure shows force-displacement data for a single cell. The slope of this line is the bending stiffness of the cell.