Dispensing C. elegans to 96 well tracking plate using Integra VIAFILL
e.warren Warren
Abstract
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
Configure the Integra VIAFILL
Insert a small cassette into the machine.
Create a new program (alternatively open a perviously saved program "WORMS ELLIE")
Select
Mode: Repeat Dispense
Tubing: 8 channel small
Plate: 96
Volume: 10 μl
Map: Full
Speed: Fast
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
Save these settings.
Preparing C. elegans
Centrifuge larvae for 2 mins at 2500rpm (RCF:590, ascending 9; descending 7).
Remove supernatant with plastic Pasteur pipette.
Add 15 ml M9.
repeat steps 3 to 5.
Transfer the worm solution to a sterile glass bottle.
Gently mix the worm suspension and pipette 10 μl to a microscope slide or cover glass and count the number of worms present.
Dilute the worm suspension to achieve your desired worm density (e.g. 3 worms per 10 μl), by diluting in M9 buffer.
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.
Dispensing worms
Select the VIAFILL program.
Select the volume you wish to dispense (10 μl).
Place the end of the tubing from the cassette into the worm solution .
Press "Prime" to prime the tubing.
Place a 96 well tracking plate (containing NGM agar and desired bacterial lawns/compound conditions) in the stage.
Press "start" to dispense worms to each well of the plate, making sure to swirl the bottle of worms.
Repeat steps 15 and 16 until all plates have been dispensed.
Allow the plates to dry with the lids off inside a biosafety cabinet for 1 hour.
Cleaning the cassette
Press "recover" to recover the worm solution into the bottle.
Place the tubing ends into hot water.
Prime several times so that the water runs through .
Recover the water and remove the cassette from the machine.
Place the cassette into an empty tip box for autoclaving, and autoclave.
