Appendix 4: Seawater Formulae

Natural Seawater

Because Chaetopterus oocytes do not undergo GVBD in artificial seawaters, it is essential to have a quantity of natural seawater present. It should be Millipore-filtered (0.22 µm) (MFSW). Oocytes may either be spawned directly into MFSW, or they may be suspended 10-15 min in MFSW after being spawned in an artificial seawater. Fertilization and all of subsequent development occur as well in ASW as in MFSW.

Artificial Seawaters

The formulae presented are MBL formulae (Cavanaugh, 1975) with Tris buffer added. Other artificial seawaters can also be used.

Tris-buffered MBL Artificial Seawater (TMBL-ASW); for 1 liter:

INGREDIENT	AMOUNT/VOL	FINAL CONC.
NaCl	84.6 ml 5M	24.72 g/liter
KCl	9.0 ml 1M	0.67 g/l
CaCl2.2H2O	9.27 ml 1M	1.36 g/l
MgCl2.6H2O	4.68 ml 4.9M	4.66 g/l
MgSO4.7H2O	12.75 ml 2M	6.29 g/l
NaHCO3	2.15 ml 1M	0.18 g/l
Tris.HCl, pH 8.2	5 ml 1M	0.005M
H2O	to 1 liter	-

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Tris-buffered MBL Calcium-free ASW (CaF-TMBL-ASW); for 1 liter:

INGREDIENT	AMOUNT/VOL	FINAL CONC.
NaCl	87.4 ml 5M	25.53 g/l
KCl	9.0 ml 1M	0.67 g/l
MgCl2.6H2O	4.68 ml 4.9M	4.66 g/l
MgSO4.7H2O	12.75 ml 2M	6.29 g/l
NaHCO3	2.15 ml 1M	0.18 g/l
Tris.HCl, pH 8.2	5 ml 1M	0.005M
H2O	to 1 liter	-

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Tris-buffered MBL Calcium- and Magnesium-free ASW (CaMgF-TMBL-ASW); for 1 liter:

INGREDIENT	AMOUNT/VOL	FINAL CONC.
NaCl	92.2 ml 5M	27.0 g/l
KCl	9.0 ml 1M	0.67 g/l
Na2SO4	7.04 ml 1M	1.0 g/l
NaHCO3	2.15 ml 1M	0.18 g/l
Tris.HCl, pH 8.2	5 ml 1M	0.005M
H2O	to 1 liter	-

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