TITLE: Dispensing (non-L1) C. elegans to 96 well imaging plates using Integra VIAFILL
AUTHORS: e.warren Warren, Bonnie Evans

[DESCRIPTION]
Protocol for dispensing later larval stage C. elegans into 96 well plates using the Interga VIAFILL dispenser. A bleach synchronised C. elegans population should be "re-fed" onto agar plates in advance. 

[STEPS]
SECTION: Configure the Integra VIAFILL
1. Insert a small cassette into the machine.
SECTION: Configure the Integra VIAFILL
2. Create a new program (or use existing program) with the following settings:
SECTION: Configure the Integra VIAFILL
2.1. Mode: Repeat Dispense
Tubing: 8 channel small
Plate: 96 
Volume: 10 μl
Map: Full
Speed: Fast
SECTION: Configure the Integra VIAFILL
2.2. To adust the height, put a plate into the stage and press the "Height" button on the display. The plate clearance, dispense and pre-dispense heights can all be easily adjusted using the arrows. Click the back arrow to return to the program screen.
SECTION: Configure the Integra VIAFILL
2.3. Save these settings.
SECTION: Preparing C. elegans
4. Centrifuge for 2 minutes at 1500 rpm (RCF:210, ascending 9; descending 7) – program 1
SECTION: Preparing C. elegans
5. Remove supernatant with plastic Pasteur pipette.
SECTION: Preparing C. elegans
6. Add 15 ml M9.
SECTION: Preparing C. elegans
8. Transfer the worm solution to a sterile conical flask.
SECTION: Preparing C. elegans
9. Gently mix the worm suspension by swirling the flask, and pipette ten 10 μl droplets to a microscope slide or cover glass. Count the number of worms present in each droplet. 
SECTION: Dispense C. elegans with the VIAFILL
13. Select the VIAFILL program.
SECTION: Dispense C. elegans with the VIAFILL
14. Select the volume you wish to dispense (10 μl).
SECTION: Dispense C. elegans with the VIAFILL
15. Place the end of the tubing from the cassette into the worm solution.
SECTION: Dispense C. elegans with the VIAFILL
16. Press "Prime" to prime the tubing. Check all tips of the cassette are dispensing correctly. 

SECTION: Dispense C. elegans with the VIAFILL
20. Place a 96-well imaging plate in the stage and press "Start". 

SECTION: Cleaning the cassette
22. Press "Recover" to recover the worm solution into the bottle.
 

SECTION: Cleaning the cassette
23. Place the tubing ends into water.
SECTION: Cleaning the cassette
24. "Prime" the tubing several times so that the water runs through.
SECTION: Cleaning the cassette
25. Remove the end of the tubing from the water and "Prime" with air to remove any remaining liquid.
SECTION: Cleaning the cassette
26. Remove the cassette from the machine, place in an empty tip box, and autoclave.
SECTION: Preparing C. elegans
7. Repeat steps 3 to 6. 
SECTION: Dispense C. elegans with the VIAFILL
21. Once all imaging plates have been filled, dry with the lids off under laminar flow for 1 to 2 hours.
SECTION: Preparing C. elegans
3. Wash worms off plates with several ml of M9 solution and transfer to 15ml falcon tubes.
SECTION: Preparing C. elegans
10. Dilute the worm suspension to achieve your desired worm density (30 to 60 worms per 100 μl) by adding M9 buffer. 
SECTION: Dispense C. elegans with the VIAFILL
11. Add a sterile stir bar in the conical flask, and place on a magnetic stirrer.
SECTION: Dispense C. elegans with the VIAFILL
12. Turn the magnetic stirrer onto the lowest setting to agitate the worm suspension.
SECTION: Dispense C. elegans with the VIAFILL
17. Test dispense 96 droplets onto a plate lid. 
SECTION: Dispense C. elegans with the VIAFILL
18. Count the number of worms in 10 random droplets. Dilute worm solution with M9 to the correct desired density again if necessary. 
SECTION: Dispense C. elegans with the VIAFILL
19. Press "Recover" and re-prime the tubing before dispensing onto imaging plates