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. Configure X, Y, and Z settings for the multiwell plate by clicking on tool symbol -> stage alignment.
SECTION: Configure the Integra VIAFILL
2.1. Put the plate into the stage and then press 'Move' so that the plate moves so that it is under the dispensing cassette.
SECTION: Configure the Integra VIAFILL
2.2. Use the up and down arrows to move the dispensing tubes so that they hover just over the plate and make note of the height (this will be entered into the dispensing program). 
SECTION: Configure the Integra VIAFILL
2.3. Use the X, Y arrows to move the plate so that the dispensing tubes are in the correct position.
SECTION: Configure the Integra VIAFILL
2.4. 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
6. Gently mix the worm suspension and pipette 10 ul to a microscope slide or cover glass and count the number of worms present.
SECTION: Preparing C. elegans
7. Dilute the worm suspension to achieve your desired worm density (e.g. 3 worms per 10 μl), by diluting in M9 buffer. 
For example:
If you have 30 worms in one droplet, dilute your suspension 1 in 10 to obtain 3 worms per 10 ul
 

SECTION: Preparing C. elegans
8. 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 7 and 8 until you obtain a suspension of the correct average worm density.
SECTION: Dispensing worms
9. Select the VIAFILL program.
SECTION: Dispensing worms
10. Select the volume you wish to dispense (10 μl).
SECTION: Dispensing worms
11. Place the end of the tubing from the cassette into the worm solution .
 

SECTION: Dispensing worms
12. Press "Prine" to prime the tubing.

SECTION: Dispensing worms
13. Place a 96 well plate (containing agar and desired bacterial lawns/compound conditions) in the stage.
SECTION: Dispensing worms
14. Press run to dispense worms to each well of the plate, making sure to swirl the flask of worms.
SECTION: Dispensing worms
15. Repeat steps 13 and 14 until all plates have been dispensed.
SECTION: Cleaning the cassette
16. Recover the worm solution into the flask.
SECTION: Cleaning the cassette
17. Place the tubing ends into hot water.
SECTION: Cleaning the cassette
18. Prime several times so that the water runs through .
SECTION: Cleaning the cassette
19. Recover the water and remove the cassette from the machine.
SECTION: Cleaning the cassette
20. Place the cassette into an empty tip box for autoclaving, and autoclave.