UO Lockery Lab
---------------------------------------------------------------------------------
How to glue nematodes for electrophysiology
---------------------------------------------------------------------------------

Chamber: We drill a 0.75 in hole in the center of a 1 mm thick rectangular glass plate (Bio-Rad #165-2907) cut to a size that is convenient for the microscope. We add feet to the plate by gluing a small square coverslip under the plate at each corner using cyanoacrylate (Vetbond, World Precision Instruments). The glue layer should be infinitely thin to avoid thermal expansion/contraction of the bond, leading to vertical drift during recording.

Glue: Nexaband S/C, by Tri-point Medical, 919-876-7800. The glue is available from World Precision Instruments. Alternatively, call Tri-Point--they will tell you your nearest distributor. You might need to get a veterinarian to buy it, depending on the supplier. Don't use Vetbond because it is more toxic to worms than Nexaband S/C.

Animals: We record mostly from L1 and L2 animals, hatched off onto seedless, but otherwise standard, plates. Wash the worms into a 1.5 mL epindorf, spin (1000 rpm 1 min), and place 0.3 to 0.6 uL onto the center of a 1.5% moist agarose pad (10 uL of agarose) on a #0 cover slip. Disperse the worms and the fluid by blowing air from a mouth tube fitted with a pipette with an opening of about .3 mm. Blow until the worms are moving normally, not flailing in fluid. The main thing is to avoid a meniscus of water around the worm. On humid days this is a problem because of condensation during the cooling procedure, see below.

Glueing: Make a pipette with an opening of about 15 um and mount it in a manipulator and connect it to a mouth tube. Thin walled glass allows more control over when and where the glue comes out. Suck glue into the tip. Experiment with the height of the column of glue to get the size glue drop you want. Drop size can be fine tuned during gluing by very gently sucking or blowing on the mouth tube as the droplet forms.

Put the worm-pad-coverslip on top of a culture medium flask (50 ml, Nunc 136196) filled with water and kept in the fridge (4 deg C). The worms will stop moving. You slide the flask around to get to a worm you like, then touch the glue pipette tip to the agarose near (approx. 1 worm diam from) the worm. You should get a drop of glue that will spread to the worm and then polymerize. Adjust the volume of glue in the drop so that it contacts the worm along one fifth of its length. If the pad is too wet the tip of the pipette may fill w/ water, making it seem clogged. This is generally not the case, however, and the pipette can be cleared by sucking or blowing on the pipette (mount it in an electrode holder w/ a side-port for a vac. tube).

Tack the worm down in several places. The less glue the better, since it seems to disrupt water balance. Wash the chamber ~6 times with distilled water to remove unglued worms and bacteria. Wash three more time with physiological saline. Sometimes air bubbles stick to the glue. They can be removed by a small get of air from a mouth tube. We find it helps much to have an xy stage for the cold bottle.

Updated, including WPI as supplier 10/25/01.
Updated, including modification for Nexaband S/C 2/15/00.


Home Research Publications Procedures Neuron-Specific Promoters Gallery People Links

Last modified 06/12/02 by amiller@uoneuro.uoregon.edu
Home Research Publications of the Lockery Lab Proceudres from the Lockery Lab Neuron-Specific Promoters Gallery People in the Lockery Lab Useful Links