Appendix 7 - Cytological Preparations

Pairs of acetone/alcohol-cleaned, circular coverslips (22 mm) are readied. A small aliquot of eggs in seawater is taken up by a braking (controlled) mouth pipette and a single small drop placed in the center of a coverslip. Another identical sized drop of Kahle's fixative (Appendix 8) is placed on a second coverslip, then flipped without the drop spreading and carefully placed on the coverslip with eggs to form a sandwich. If desired, stages can be flattened with a 14 g weight added simultaneously with fixation. The double coverslip is carefully slipped into a coverslip staining jar containing more fixative. Coverslips with adherent eggs can be separated with a needle probe and subjected to cytochemical tests, autoradiography, etc.

Preparation for scanning microscopy

Oocytes or embryonic stages can be fixed in 2% glutaraldehyde prepared in micromembrane filtered (0.45 µm) seawater, rinsed four times in filtered seawater and stored in 0.05 M cacodylate buffer at 4°C. Post-fixation in 1% osmium tetroxide in cacodylate buffer for 1 h is followed by three washes of buffer. Cells are allowed to settle onto coverslips coated with polylysine and excess buffer is drawn off. Preparations are dehydrated in acetone, critical point dried in CO₂ and coated with gold palladium by vacuum evaporation (Tweedell, 1976).

Preparation of adults for in vivo oocyte studies by sectioning

Animals can be force fed alternate materials that have been screened through wire mesh to the equivalent size of large sand grains. The most successful materials were ground white rice or a protein cereal such as Kellogg's Concentrate. These materials are placed in 5-inch open end glass tubes, l/2 inch diameter, that were covered at one end with several layers of cheese cloth. Tubes are half filled with the feeding material and placed cloth end down in a test tube rack. Intact adult animals are inserted head down in the tube and a flow of slowly running seawater inserted from the top. Animals are allowed to feed for 24 to 48 hours, then placed in running seawater for 24 hours to purge the excess contents. After removal from their tests, the adult animals are narcotized, fixed in Kahle's fixative and double embedded in methyl-benzoate-celloidin/paraffin (see below) before sectioning and staining in Jenner-Giemsa (Appendix 8).

Double embedding

1. Dehydrate whole "purged" animals very slowly in progressive alcohol series.
2. From 100% ethyl alcohol, pass whole animal into methyl benzoate/celloidin solution (see Appendix 8) for 24 hours. Repeat in fresh solution for 12 hours or until animal is totally transparent.
3. Pass into 2 or 3 changes of fresh 100% benzene
4. Infiltrate in 50:50 benzene:paraffin in hot water bath
5. Change to fresh paraffin and place in vacuum oven for 20 min.
6. Embed and section as usual.