Eggs and embryos are mostly water, but they also contain a fair amount of protein. Protein behaves differently under different conditions. We have all seen a raw egg, the white being mostly water and protein. Cook the egg and the white part becomes opaque white and solid. The same thing occurs to the proteins in sea urchin eggs when we fix them. Besides heat, there are numerous other ways to fix proteins. Other methods include aldehydes, alcohols and acids. Carnoy's fixative uses a combination of these last two methods.

The Formula:

3 parts 95% Ethanol to 1 part Glacial Acetic Acid

Clearing Solution:

45% Glacial Acetic Acid in distilled water.

Procedure

1. Let eggs settle in a test tube and pour off as much of the seawater as you can.
2. Add the Carnoy's fixative, let sit one hour.
3. Pour off the fixative and add fresh fixative, let sit at least one hour, best if overnight.
4. Pour off the fixative and add the Clearing Solution.
5. View under a phase microscope. (sorry, a normal microscope will not show much)

Material in Carnoy's will only last a few days, this is not a way of making a permanent mount.

Viewing

You should be able to see the following:

• Cortical granules in most species (not in L. pictus though)
• Sperm nuclei in the fertilized egg
• Nuclear centering and fusion
• Mitotic apparatus, chromosomes and chromosome condensation

Questions

This is really a method allowing you to see the effects of other changes. Using this technique you could see the effects of artificial activation or different seawater formulations.