Procedure
Animals are anesthetized with 0.04% tricaine methane sulfonate in filtered seawater ( = 3-aminobenzoic acid ethyl ester or "MS 222"; Sigma) or with 50% ethanol added dropwise to a small dish of seawater. The average coelomic fluid volume of organisms weighing 0.75 gr was 0.375 ml (excluding cells). From 0.05 to 0.1 ml can be injected with a microliter syringe (#27 ga needle) without appreciable fluid loss. In vivo incorporation into ovarian cells or into oocyte stages (3H uridine) can be produced by the injection of 5 to 10 µC of tritiated nucleosides (1.7C/mmole) with an optimum exposure of 9 to 12 hours. Injection is directly through the firm cephalic plaque into the coelom. This plaque can be located by the prominent flattened golden setae that are embedded at the base. Animals should be returned to running filtered seawater (Appendix 8) immediately for recovery (Tweedell, 1976).
In vivo exposure to isotopes
Oocytes or embryos are labeled in vitro by immersing them directly into membrane filtered seawater containing the isotopes. Pulses can be from 2 to 8 hours. At the moment of fixation in Kahle's fixative (Appendix 8) the cells are compressed by placing 45 grams pressure on the whole cells deposited on coverslips (Appendix 7). Thereafter the embryos are dehydrated up to 100% ethanol and air dried. The coverslips are cemented to slides with the cells facing upward. After dipping the slides in Kodak NTB2 or Ilford G-5 emulsions, they are exposed for 2 weeks. Whole cells/embryos are best stained in gallocyanin chrome-alum, pH 1.7 for 8 to 12 h and counterstained in eosin. Sections may be stained in Jenner-Giemsa solution (Appendix 8) .