The medium given below can be made with either artificial seawater or natural 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.
|Amount to weigh:
|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|
|Enrichment Stocks||Stock Conc.(g/L)||Final Conc.(µM)|
|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|
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.
* Can be replaced with an equimolar stock of Na2HPO4
** Add before the trace metals.
*** Can be replaced with an equimolar stock of FeCl3
|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).
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.