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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.


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