This week is the second to last week of the semester before finals and everything is coming down to the wire, including my neurobio lab project. PZ was so kind as to come in and help me out this past Sunday morning; the morning after the blizzard had quieted leaving everything covered in various quantities of snow. In going over my methods we found that I wasn't adding a drop or two of water on top of the auger layer with the immobilized zebrafish. The reason this is important is that so after the spinal cord severing is accomplished, the auger layer is separated allowing water to surround the fish immediately and preventing air exposure. The fish can then be pipetted up and put into a dish of water for observations. PZ also suggested using water with an increased concentration of calcium (14g/100mL) to facilitate better fish recovery. The fish should not be left however in the calcium water for an extended period of time because it can adversely affect development.
Repeating my methods and taking into practice the slight changes that PZ recommended, I found that after one day, four of eleven fish were still alive! After slicing up more than sixty fish with a 100% mortality rate after one day and wondering what on earth I could have been doing wrong, I was ecstatic. It's unfortunate that this success has come so late in the game and the writeup for this project won't show much for results other than how not to butcher zebrafish. I have learned quite a bit though about the interesting techniques I've been using and also about the differences in zebrafish at various stages of development. So with that, back to the lab I go to continue working with zebrafish.
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Ah, memories of my senior microbiology project. I was growing bacteria with naphthalene as the carbon source, and it took me most of the project to realize that the salicylic acid they were producing was acidifying the media and stopping further growth. (THe aspirin was giving them headaches!) I had time to do one quick set of experiments with various buffers, and poof! the course was over.
I do a lot of work with mouse development. When I recover embryos, they go straight into PBS (Phosphate-Buffered Saline, you can easily find a recipe by googling '10x PBS Recipe'). The pH and salinity is the same as what the embryos see, i.e., physiologic pH, and this helps the tissue survive if I am going after a specific subset of the tissue from the embryos. Calcium is not a common component of PBS, IIRC, but many recipes include a source of Ca2+ for maintenance of tissue survivial.
What my rambling post adds up to is this: will your embryos survive better if they are kept in PBS for the manipulation?
I think it is really great that PZ would come in and help you like that on a weekend. Good luck finishing the project.
Take a look at the fisheries literature about adding salt to the water when transporting freshwater fish. Fish Ringer's solution more or less.
Death and destruction to all programmers of spell checkers.
There actually is a word auger in English. It means "big drill". What you are looking for is agar (or actually agar-agar. AH-gahr.
See? I got so upset that I forgot to close the parenthesis (after agar-agar).
Ah, so you weren't putting the fish on a drill, then. :P
Awesome. If you want more of this (things going wrong), consider graduate school.
Also, that's incredibly nice of PZ to come in and help on a Sunday morning. Guess he gave up church for a week. Take advantage of his help! Jeezus, that's always been one of my mistakes - not accepting help from people who are bursting to offer it to you, as long as you're going to put the work in in return.
Also, 14g Ca/100ml? Wow. Zebrafish sure do like a lot of calcium.
Thank you, David Marjanović. This is the second BE post using that odd spelling for agar (agar agar, I was taught). Makes me wince every time, picturing those teeny drilled fish. Or imagining that it's actually an ice fishing expedition.
Otherwise, Blue Expo, an interesting post.
Death and destruction to all programmers of spell checkers.
There actually is a word auger in English. It means "big drill". What you are looking for is agar (or actually agar-agar. AH-gahr.
See? I got so upset that I forgot to close the parenthesis (after agar-agar).