# Sterilizing Lines

## Protocol

1. In three large beakers, prepare 500 mL of 10% bleach, 200 mL of 10% bleach and 300 mL 70% ethanol.
2. Using the switches on the eVOLVER device, turn on the 5 V power supply on the eVOLVER, wait for >3 seconds, then turn on the 12 V power supply.
3. If running several vials from the same media bottle, connect multiple media input lines with tubing splitters. Custom tubing splitters can be [constructed ](../../guides/making-media-bottles-and-splitters)with Luer components.
4. Submerge the media input lines (clear, media in) in the first bleach beaker, and the media efflux lines (blue) in the second bleach beaker. Place the downstream end of the media input lines (clear, to vial) in the second beaker with the efflux lines. Since the lines may be different lengths, a hairclip is useful to keep the ends of each lines close to eachother in order to ensure all remain submerged.
5. Add 1 L bleach into large empty waste carboy, which will sterilize waste generated during experiment. Consult with a safety coordinator to ensure proper disposal of waste.
6. Using the switches on the eVOLVER device, turn on the 5 V power supply on the eVOLVER, wait for 5 seconds, then turn on the 12 V power supply.
7. If running several vials from the same media bottle, connect multiple media input lines with tubing splitters (drawer under eVOLVER computer) before sterilizing. Custom sizes can be made if necessary.
8. Submerge the media input lines in the first bleach beaker, and the media efflux lines in the second bleach beaker.
9. Place the downstream end of the media input lines in the second beaker with the efflux lines. Place waste lines into waste carboy.
10. Open the eVOLVER electron app, select the appropriate eVOLVER from the dropdown menu.
11. Navigate to setup, then fluidics. Highlight the appropriate vials, select the influx and the efflux pumps, and run pumps for 20 seconds to fill lines with 10% bleach.
12. While running, ensure by visual inspection that all lines have been filled and that pumps are operating normally. Allow bleach to sit in the lines for at least 30 minutes to sterilize. This is a good pause point, can wait up to overnight. This is a good time to make code edits if necessary.
13. Run pumps again using the touchscreen app until lines are no longer submerged, pushing air through the lines to get as much of the bleach out as possible.
14. Place media input lines in the ethanol beaker (or pour ethanol into the first beaker) and repeat as in above, filling the lines with ethanol and flushing with air. Note: ethanol sterilizes quickly, so no need to wait 30 mins here.
15. It is also recommended to dip the downstream ends of the influx lines in ethanol to rinse off residual bleach, then drape on side of beaker rather than resubmerging in the remaining bleach. Residual bleach can affect culture viability, though washing with ethanol and media should be sufficient.
16. Attach media input lines to the media bottles with the Luer connectors and run the pumps until the media fully runs through the lines (10-20 seconds, depending on tubing), flushing out any residual ethanol.
