2-chamber 3-electrode H-cell set up

2-chamber, 3-electrode set-up procedure
by HMJensen with help from Ben Gross in the El-Naggar Lab, significant revisions by MA TerAvest
Updated: 01/29/2016

Materials Needed
Please refer to pdf Electrochememical system setup 20160129.
Before getting into it, check the following:

  • Reference electrodes: Does the Ag wire appear white? Or black? If white, see the video protocol “refreshing reference electrode”
  • Graphite electrodes: cut to desired size, saturate with milli-Q water, store in Milli-Q
  • Pt wire electrodes: Are the electrode wires shiny? Dull? If dull, then use fine sand paper to remove any corrosion.
  • Cation exchange membrane: cut to size and store in 4% NaCl
  • Glass chamber set-up: Check that all of the components and tubing are clean.
  • Media: Check that all of the media solutions are available.


  1. Prepare your solutions for working and counter chamber (typically M9 medium base).
  2. Assemble the 2-chamber system: (see video procedure reactor_assembly)
  3. Insert the Pt cathode electrode on the left-hand chamber through the top septum.
  4. Insert the graphite anode into the right-hand chamber through the top septum.
  5. On the side ports of each half, screw down caps with septa.
  6. Place a stir bar in the working chamber.
  7. Very lightly grease the glass edges of the reactor halves. Over-greasing leads to grease on electrodes and is hard to clean.
  8. Place the membrane in the center of the o-ring gasket.
  9. Use the circle clamp to secure the two sides to one another. DO NOT OVER TIGHTEN.
  10. Fill the counter chamber with 140 mL of your solution (usually M9 base).
  11. Fill the working chamber with 135 mL of your solution (usually M9 base)
  12. Place the septa screw caps on the tops of each the anode and cathode side and leave a quarter to a half turn unscrewed for autoclaving.
  13. Autoclave the assembled cells. Let the media cool after autoclaving.
  14. Add supplements to the working chamber as needed (sterile technique!) Usually:
    • 1.4 mL Mineral supplement
    • 1.35 mL Vitamin supplement
    • 1.35 mL Amino Acid supplement
  15. Insert reference electrode into side port of working chamber (clean with ethanol first)
  16. Set up cells in their positions and start gas flow and potentiostat (see video protocol start_N2&potentiostat)
  17. Sparge and take background measurements for >5 hours.
  18. Inject bacteria

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