As I wrote about before, my semester lab project for neurobiology has to do with regeneration. The idea is to damage the spinal cord and observe wonderful regeneration. This proposal was based on some articles I read about regeneration of zebrafish hearts, fins, tails, etc. Unfortunately I haven’t had much luck so far.
Last week, armed with an exacto knife, I performed my first round of spinal cord butchering on fifteen zebrafish that were only a few days old. The zebrafish are captured with a glass pipet and then immobilized using auger that’s just warm enough to be in liquid phase. They are then mounted on a slide under the stereoscope. I quickly discovered many faults in my methods, one of which being that I captured the fish in too many drops of water so when the auger was added in a test tube, the fish weren’t completely immobilized. I would then carefully approach a fish with the exacto knife on the slide under the scope and it would turn into a bucking bronco. Eventually I perfected the art of capturing the few day old zebrafish with the pipet and putting them in test tubes in only one drop of water, thus partially solving the immobilization issue.
The second problem I encountered is that there is nothing exact about an exacto knife under a stereoscope. Accomplishing this spinal cord severing is much like peeling an orange with a baseball bat in that it’s extremely difficult without making a mess. Even with the fish immobilized the tail doesn’t really stay put when pressure is applied with the seemingly crowbar sized exacto knife. The key to this dilemma, although I have yet to master it, is probably making the layer of auger on the slide as thin as humanly possible so that there isn’t as much room for movement.
All the zebrafish from my first attempt died. Two of them were alive for a day or so but barely. I did another round of zebrafish butchering with fifteen more fish yesterday (yes I enjoy spending my Sunday afternoon in the neurolab) and from what I could tell, all but two of them died immediately. I’ll have to see if the elite two are still swimming around today but if not, I’ll try yet another round of fifteen and see how it goes. If anyone has any ideas for instruments or methods that could improve my success, feel free to insert a comment.