Screenchip electropharyngeogram (EPG) recordings.

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Materials list is empty in the source — the heading is present with a single bullet containing nothing. Someone should fill it in.

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Advance preparation

Bleach synchronize worm strains and hatch on seeded 10 cm NGM plates.

Protocol

1. When worms are young adults, ~4 days after bleaching, they are the appropriate stage for the SC40 chips.

2. Wash and prepare worms:

   • Wash worms from 10 cm NGM plates into 1.5 mL Eppendorf tubes using 1 mL of M9.
   • Allow worms to settle for 2 minutes.
   • Remove the supernatant without disturbing the worm pellet.
   • Add 1 mL of M9 and invert to wash.
   • Repeat twice more, for a total of three M9 washes.

3. After the final wash, add the desired drugs plus 10 mM serotonin and wait 20 minutes.

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   Serotonin: 10.6 mg into 5 mL M9 for a final concentration of 10 mM. Incubate worms in solution for 20 minutes to activate pharyngeal pumping. Use serotonin solution within 4 hours. Chips are reusable for up to 8 hours, or 50–70 worms.

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4. While waiting for drugs to take effect, set up the EPG device.

   • Connect the computer, amplifier, and ScreenChip dock. Ensure cords are not touching each other, to prevent background signal.
   • Use a syringe to pull fresh M9 into the chip and run the noise test.

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   Unplugging the microscope is important to bring noise levels into an acceptable range. The outlet tubing can also increase background noise — if this is a problem, it can be unplugged from the chip before recording.

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5. Loading worms into the chip:

   • Place the short tube into the Eppendorf tube containing treated worms.
   • Gently pull back on the syringe until worms are visible in the waiting chamber. Ensure there is always media in the Eppendorf tube and refill as needed.

6. Recording:

   • Save data to a folder labeled with the date, e.g. 20190224, with each strain assigned its own sub-folder.
   • Using the syringe, gently pull back on the plunger and position a worm into the recording channel.
   • Once in position, eliminate background noise as necessary — unplug the microscope, remove outlet tubing, check that cords are not touching or moving — wait 30 seconds, then press record.
   • Record for a total of 2 minutes, ensuring the worm does not drift from the recording channel. If drift occurs, stop the recording, reposition the worm, and repeat.
   • Use experiment notes to record worm orientation, drug concentration, strain, worm number, and initials.
   • Remove the worm from the recording channel and position the next one.

7. Clean-up:

   • Discard any chips where worms remain, or which have been used to record more than 50 worms.
   • Clean the tubing by filling the syringe and passing 5 mL of M9 through into a waste container.
   • Pass air through the tubing to help dry it and remove any remaining debris.

8. Analysis:

   • Open the NemAnalysis program and select the folder containing data to analyze.
   • Click Customize Analysis and change settings by clicking individual File Settings, using the brute force method. Analysis parameter numbers may need adjusting for each experiment, strain, or treatment.
   • Allow the program to analyze files, going through each as it completes to check for accuracy — high signal-to-noise ratio, worm orientation correlating to EPG peaks.
   • Once finished analyzing a strain, export the information to Excel. Change "experiment date" and "analyzed date" to YYYYMMDD format, and save the document as a CSV file.

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Note on step 8. The source lettered these sub-steps a, c, d, e — skipping b. Either a sub-step is missing between opening NemAnalysis and customizing the analysis, or it is just a lettering slip.

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