Nematode dye filling

Nematode dye filling
(Ed Hedgecock [modified by M. Herman/K. Williams/S.M. Hettenbach])

Thumb plate

1. Transfer worms into a known amount of M9 in a labeled depression.
2. Add DiO(2 mg/ml in DMF) at 10 l/ml of M9.
3. Let sit for 2 hours.
4. Transfer worms with a Pasteur pipet in as little liquid as possible to a feeding plate.
5. Allow the plates to sit at room temperature (RT) or at 20°C for 30 minutes to 2 hours to pass the dye out of the digestive tract. Worms dyed late in the afternoon can be stored at 15°C overnight (o/n) and viewed the next morning.

Microfuge tube

1. Label 1.7 ml microfuge tube with strain name.
2. Squirt ~1 ml M9 onto plate with a pasteur pipet. Rinse plate with M9 using a pasteur pipet and transfer solution to labeled microfuge tube.
3. Spin for 10-20 seconds in a mini centrifuge and carefully remove supernatant (s/n). Worm pellet will be very loose.
4. Wash once with M9. Optional -- don't do if pellet is very small.
5. Add 1 ml M9.
6. Add DiO (2 mg/ml in DMF) at 5 l/ml of M9.
7. Rock or rotate the tubes for 2 hours at RT or 20°C.
8. Spin for 10-20 seconds in the mini centrifuge and carefully remove s/n. Worm pellet will be very loose.
9. Wash once with M9. NOT optional.
10. Allow the plates to sit at RT or at 20°C for 30 minutes to 2 hours to pass the dye out of the digestive tract. Worms dyed late in the afternoon can be stored at 15°C o/n and viewed the next morning.

15 ml tube

1. Label 15 ml centrifuge (c/f) tube with strain name.
2. Squirt ~1-2 ml M9 onto plate with a pasteur pipet. Rinse plate with M9 using a pasteur pipet and transfer solution to c/f tube.
3. Spin pulse on high in a clinical c/f and carefully remove s/n. Worm pellet will be very loose.
4. Wash once with M9.
5. Add 2 ml M9.
6. Add DiO (2 mg/ml in DMF) at 5 l/ml of M9.
7. Rock the tubes for 2 hours at RT on a lab quake rocker.
8. Spin pulse on high in a clinical c/f and carefully remove s/n. Worm pellet will be very loose.
9. Wash once with M9.
10. Transfer worm pellet to labeled plate(s). Use enough plates that worms have sufficient food.
11. Allow the plates to sit at RT or at 20°C for 10 minutes to 2 hours to pass the dye out of the digestive tract. Worms dyed late in the afternoon can be stored at 15°C o/n and viewed the next morning.

Viewing

Big Scope

1. Make pads using 5% agar with 10mM NaN₃ (10 l of 1M NaN₃ /ml of 5% agar -- our test tubes contain 4 ml of 5% agar).
2. Apply 4-6 l of M9 to one end of the pad.
3. Transfer 25-50 worms to the M9, adding more M9 if it starts to evaporate.
4. Put cover slip on and view with upright and fluorescence.

Dissecting Scope

1. View worms on feeding plate using dissecting microscope.
2. Use white light on lowest setting.
3. Adjust intensity of white light using mirror. Do not turn lamp source on and off.