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			ESAW/HESNW Culture Medium The medium given below can be made with either artificial seawater or natural seawater. Artificial Seawater Make two separate solutions: Solution I and Solution II. Add salts in order to distilled deionized water (DDW). When the salts in each are completely dissolved mix solutions together. Dilute to total volume with DDW. Table 1. ESAW Salts Solution I: Anhydrous salts Molecular Weight (g/mol) Amount to weigh: (g/l solution) Concentration (mM) NaCl 58.44 20.756 362.7 Na2SO4 142.04 3.477 25.0 KCl 74.56 0.587 8.03 NaHCO3 84 0.17 2.067 KBr 119.01 0.0845 0.725 H3BO3 61.83 0.022 0.372 NaF 41.99 0.0027 0.0657 Solution II - Hydrated salts MgCl2 . 6H2O 203.33 9.395 47.18 CaCl2 . 2H2O 147.03 1.316 9.134 SrCl2 . 6H2O 266.64 0.0214 0.082 Table 2. Nutrient Enrichment Stocks Enrichment Stocks Stock Conc.(g/L) Final Conc.(µM) 1 NaNO3 46.67 549.1 2 * Na2glycerophosphate 6.67 21.8 3 Na2SiO3 . 9H2O 15.00 105.6 4 ** Na2EDTA . 2H2O 3.64 9.81 *** Fe(NH4)2(SO4)2 . 6H2O 2.34 5.97 FeCl3 . 6H2O 0.16 0.592 5 MnSO4 . 4H2O 0.54 2.42 ZnSO4 . 7H2O 0.073 0.254 CoSO4 . 7H2O 0.016 0.0569 Na2MoO4 . 2H4O 0.126 0.520 ** Na2EDTA . 2H2O 1.89 5.05 6 H3BO3 3.80 61.46 7 Na2SeO3 0.00173 0.001 Enrichment stocks can be refrigerated. Adjust Solution #5 to pH 6 using solid NaCO3 (about 2 g). Solution # 4 can be heated to dissolve the iron. Notes: * Can be replaced with an equimolar stock of Na2HPO4 ** Add before the trace metals. *** Can be replaced with an equimolar stock of FeCl3 Table 3. Vitamin Stocks Vitamin Stock Stock Conc.(g/L) Final Conc.(mM) Thiamine 0.1 2.97 x 10-1 Vitamin B12 0.002 1.47 x 10-3 Biotin 0.001 4.09 x 10-3 Vitamin stocks should be stored frozen. TO MAKE 1 LITRE OF HESNW 1) Filter natural seawater through 0.45 µm membrane filter with a glass fiber prefilter. 2) Use a flask that has been acid-washed in 10% HCl and rinsed in distilled water before first use. 3) Pour 1 L filtered seawater or artificial seawater into flask. Add the following while mixing with a stir-bar: a) 6 ml Combined Stock (Enrichment stocks #1,2,4-7) (OR 1 ml of each individual stock solution) b) 2 ml Silica Stock (Enrichment stock #3) c) 2 ml Vitamin Stock 4) To reduce precipitation during autoclaving add: d) 1.44 ml of 1N HCl e) 0.12 g Sodium bicarbonate 5) Autoclave 20 minutes for one litre, ~ 30 minutes for 2 litres. Autoclaving for longer periods will result in more precipitation. Flasks should not be filled up more than 2/3 of their capacity, to avoid boiling over during sterilization. 6) Let stand for 2 days to allow pH to equilibrate (should be ~ pH 8.2). 7) Filter aseptically through a glass fiber filter to remove precipitate. Some taxa appear to be sensitive to it (optional). Reference Harrison, P.J., R.E. Waters and F.J.R. Taylor. 1980. A broad spectrum artificial medium for coastal and open ocean phytoplankton. J. Phycol. 16:28-35. Return to top
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