Comparative Physiology @ Guelph
Myxicola Axoplasm Isolation for mechanical testing
- Prepare anaesthetic (0.2% MS-222/0.4% NaHCO3 in 100 mL ASW)
Remove slime cocoon from worm, blot dry, weigh
Can get usable pieces of axon from worms down to ~0.8 g. Below that just doesn’t work
Place worm in anaesthetic for 30 minutes at 5 degrees C. Do this in fridge, not on ice
While worm is being anaesthetized:
- Turn on chller and make sure it is hooked up to microscope stage.
- Prepare bucket of ice
- Set out dissection tools – 3 fine forceps, fine scissors, eyelash tools, Loctite gel
- Set out distilled water, ethanol squirt bottles, Kimwipes
- Keep bottle of Ca-free dissection buffer in ice nearby
- Beaker for waste water
- Right before worm is ready, nest smaller dissection block in ice on tray
- Stretch out worm dorsal side up and extended on paraffin – pin both ends (Dorsal side is opposite triangular eyespot and opposite to dark ventral groove, which swerves to dorsal side only for the 9 segments closest to the anterior
- Cut through muscle layer with scissors, being careful to cut all the way through, but not so far that the GI tract is ruptured.
- Rinse liberally with dissection buffer
- Pin animal open using about 30 dissection pins, severing connections as necessary using pins.
- Rinse again
- Sever lateral ligaments using scissors, then cut GI tract at anterior end, and quickly pull it up and away from the animal. Rinse very quickly after this to get rid of proteases.
- Tease away dark blood vessel on top of axon with fine forceps, being extremely careful not to nick the axon membrane
- Rinse thoroughly
- Blot dry with Kimwipes and filter paper, remove most ice from tray
- Very carefully cut lateral projections of axon
- Insert scissors under axon membrane and very carefully cut open top (Only do this for 6 segments or so)
- Place a small drop of Loctite gel on axoplasm end and allow to harden
- Gently lift hardened end with forceps, while holding scissors in left hand
- Place a drop of mineral oil on axon to keep hydrated
- Pull up a usable stretch of axon, cut with scissors
- Very quickly transfer to stretching apparatus to prevent drying
- Measure resting length of axon