Cleaning Up After Experiment

Bleach (sodium hypochlorite) may create a small amount of chloroform when mixed with ethanol. In addition, many EHS policies prohibit mixing of ethanol and bleach waste streams. Considering this, you may want to move your efflux lines to a separate waste container when sterilizing tubing with ethanol, and move them back to your normal waste container when starting your experiment. Also consider using alternative sterilization agents such as Wescodyne and BacDown detergent.

Stopping Experiment and Bleaching

  1. In the GUI electron app or command line, click to stop your experiment.

  2. In large beakers, prepare 500mL of 10% bleach and 200 mL of 10% bleach.

  3. Disconnect media bottles and place lines into 10% bleach solution

  4. To use pumps to bleach the cultures, keep tubing connected, but move vials to a white eVOLVER rack. Prepare an additional 1L of 10% bleach and add to the media input beaker.

  5. Run 10% bleach through vials until final concentration in the vials reaches ~10% bleach (~60 seconds for 25mL volume). Disconnect influx and efflux lines from vials, set vials aside for 30 mins.

  6. To manually clean vials, disconnect influx and efflux lines and skip steps 39 and 40

  7. Submerge the disconnected influx and efflux lines in the 200mL bleach beaker, then run all pumps for 20 seconds to fill all lines with 10% bleach. Let sit for no less than 30 minutes, no more than 48 hours.

  8. Remove media input lines from bleach, and run air though the lines to remove bleach. If not planning on using the device again right away, run diH2O through all lines for 20 seconds to remove residual bleach from the lines. Dried bleach can form salt crystals and clog the lines. If this happens, break up the crystals manually using pliers to squeeze the line.

  9. Turn off the 12V power, then the 5V power.

  10. Proceed to vial & bottle cleaning protocol.

Clean Vials After Bleaching Cultures

  1. Prepare a beaker ~50% full of 10% bleach

  2. Remove vial lids and place in 10% bleach solution

  3. Using the magnetic stir bar grabber, remove the stir bars from each vial, place in the 10% bleach solution

  4. Let vial caps and stir bars sit for 30 minutes-overnight in the 10% bleach solution

  5. Meanwhile, dump bleached culture down the drain, rinse with diH2O

  6. Use a test tube brush to scrub inside of vial, particularly important if you grew bacterial cultures

  7. Place bleached/scrubbed vials right-side-up in the vial racks to indicate they have not yet been dishwashed

  8. Once vial lids/stir bars have been soaked for sufficient time, carefully pour out the 10% bleach solution, and replace with diH2O

  9. Let vial lids and stir bars soak for another 30 mins-overnight.

  10. Once vial lids/stir bars have been soaked for sufficient time, carefully pour out the diH2O.

  11. Run diH2O through influx and efflux straws for 2-3 seconds at the sink to remove any residual bleach

  12. Place stir bars in the stir bar tray.

  13. Once 60+ vials have accumulated, vials should be dishwashed.

  14. Place vials open side up in 3 white eVOLVER racks.

  15. Take one rack, cover the vials using the wire mesh from the bottom rack of the dishwasher, then flip upside down. Repeat for the other two racks.

  16. Add any other glassware or eVOLVER bottles to the top rack as desired, then run on cycle 3.

  17. Store vials upside down in vial boxes to indicate that they have been dishwashed.

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