TITLE: Dispensing C. elegans to 96 well tracking plate using Integra VIAFILL
AUTHORS: e.warren

[DESCRIPTION]
Protocol for dispensing C. elegans into 96 well plates using the Interga VIAFILL dispenser. Bleach synchronized C. elegans should be prepared in advance. The X,Y,Z positions for dispensing can be adjusted according to the multiwell plates being used.

[STEPS]
SECTION: Configure the Integra VIAFILL
1. Insert a small cassette into the machine.
SECTION: Configure the Integra VIAFILL
2. Create a new program (alternatively open a perviously saved program "WORMS ELLIE")
SECTION: Configure the Integra VIAFILL
2.1. Select
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
3. 
Centrifuge larvae for 2 mins at 2500rpm (RCF:590, ascending 9; descending 7).
SECTION: Preparing C. elegans
4. Remove supernatant with plastic Pasteur pipette.
SECTION: Preparing C. elegans
5. Add 15 ml M9.
SECTION: Preparing C. elegans
8. Gently mix the worm suspension and pipette 10 μl to a microscope slide or cover glass and count the number of worms present.
SECTION: Preparing C. elegans
7. Transfer the worm solution to a sterile glass bottle.

 

SECTION: Preparing C. elegans
10. 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. Calculate the average number of worms per droplet. If correct use the suspension as is, if not repeat steps 8 and 9 until you obtain a suspension of the correct average worm density.
SECTION: Dispensing worms
11. Select the VIAFILL program.
SECTION: Dispensing worms
12. Select the volume you wish to dispense (10 μl).
SECTION: Dispensing worms
13. Place the end of the tubing from the cassette into the worm solution .
 

SECTION: Dispensing worms
14. Press "Prime" to prime the tubing.

SECTION: Dispensing worms
15. Place a 96 well tracking plate (containing NGM agar and desired bacterial lawns/compound conditions) in the stage.
SECTION: Dispensing worms
16. Press "start" to dispense worms to each well of the plate, making sure to swirl the bottle of worms.
SECTION: Dispensing worms
17. Repeat steps 15 and 16 until all plates have been dispensed.
SECTION: Cleaning the cassette
19. Press "recover" to recover the worm solution into the bottle.
 

SECTION: Cleaning the cassette
20. Place the tubing ends into hot water.
SECTION: Cleaning the cassette
21. Prime several times so that the water runs through .
SECTION: Cleaning the cassette
22. Recover the water and remove the cassette from the machine.
SECTION: Cleaning the cassette
23. Place the cassette into an empty tip box for autoclaving, and autoclave.
SECTION: Preparing C. elegans
6. repeat steps 3 to 5.
SECTION: Preparing C. elegans
9. Dilute the worm suspension to achieve your desired worm density (e.g. 3 worms per 10 μl), by diluting in M9 buffer. 

 

SECTION: Dispensing worms
18. Allow the plates to dry with the lids off inside a biosafety cabinet for 1 hour.