Materials

1. Fertilization medium (FM): All chemicals should be tissue-culture grade. Prepare the following concentrated stocks, and filter each through a 0.4-|jm Millipore filter into a sterile plastic tube. Store frozen at -20°C.

a. 10X A stock: Dissolve 7.013 g NaCl (Sigma S5886), 1.0 g glucose (Sigma G6138), 0.201 g KCl (Sigma P5405), 21.3 mg Na2HPO4 (Sigma S5136), 0.102 g MgCl26H2O (Sigma M2393) in double-distilled or Milli-Q water. Bring to 100 mL final volume.

b. 10X B stock: Dissolve 2.106 g NaHO3 (Life Technologies 895-1810P), 1.0 g phenol red (Sigma P5530) in water. Bring to 100 mL final volume.

c. 100X C stock: Dissolve 55 mg sodium pyruvate (Sigma P5280) in 10 mL water.

d. 100X D stock: Dissolve 0.264 g CaC122H20 (Sigma C7902) in 10 mL water. To prepare fertilization medium, mix 10 mL of 10X A, 10 mL of 10X B, 1 mL of 100X C, and 1 mL of 100X D. Make up to 100 mL by adding 78 mL double-distilled water. Add 3.0 g BSA (Sigma A4161), and mix gently until dissolved. Pass through a 0.45-|im Millipore filter using a large disposable syringe. Store in sterile plastic containers at -20°C.

2. M16 medium.

3. 70% Ethanol in a squeeze bottle.

4. Light paraffin oil (Fluka).

5. 35-mm Sterile tissue-culture dishes.

6. Micropipet and sterile tips (e.g., Gilson, Villiers-le-Bel, France, Pipetman, P200).

7. Egg transfer and mouth-operated pipet system.

8. Dissecting scissors: 1 regular, 1 fine.

9. Watchmaker's forceps: 2X #5.

10. Stereomicroscope with understage illumination (e.g., Nikon; SMZ-1OTD).

11. Fiberoptic light source.

12. 37°C incubator gassed with 5% CO2.

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