Fraquil Medium

Freshwater Trace Metal-Buffered Medium

CaCl21 mL
MgSO41 mL
NaHCO31 mL
K2HPO41 mL
NaNO31 mL
Trace metal mix1 mL
f/2 vitamin mix1 mL
Milli-Q Water993 mL
Use acid-cleaned polycarbonate bottles
Be sure to add the trace metal mix that will give the desired Fe concentration
After each nutrient is added, mix by gentle manual agitation
Filter sterilize through disposable 1 L filtration apparatus and dispense (in laminar flow hood) into acid-clean/microwave tyndalized polycarbonate tubes
(Tyndalization: bring milli-Q water just to boiling three separate times using microwave)

Individual Salt Stock Solutions:
CaCl2·2 H2O18.4 g
MgSO4·7 H2O18.5 g
NaHCO36.3 g
K2HPO40.87 g
NaNO34.25 g
Na2SiO3·9 H2O1.775g
Add 500 mL of milli-Q H2O to each acid-clean polyethylene bottle and then add the appropriate salt; dissolve salts by manual agitation
Silicate stock NOT required for growth of cyanobacteria !!

Individual Trace Metals Stocks Solutions:
CuSO4 · 5 H2O0.125 g500ml
(NH4)6Mo7O24 · 4 H2O0.133 g500 mL
CoCl2 · 6 H2O0.298 g500 mL
MnCl2 · 4 H2O0.455 g100 mL
ZnSO4 · 7 H2O0.115 g100 mL
FeCl3 · 7 H2O1.352 g500 mL
Add the indicated volume of milli-Q H2O to each acid-clean polyethylene bottle and then add the appropriate trace metal with gentle agitation following each addition

Trace Metals Mix
To create different media having known Fe concetration:

1 microM Fe (final) stock
milli-Q H2O443 mL
CuSO4·5 H2O500 microL
(NH4)6Mo7O24·4 H2O500 microL
CoCl2·6 H2O500 microL
MnCl2·4 H2O500 microL
ZnSO4·7 H2O500 microL
Na2EDTA (0.5 M)5 mL
FeCl3·7 H2O50 mL

100 nM Fe (final) stock
milli-Q H2O448 mL
CuSO4·5 H2O500 microL
(NH4)6Mo7O24·4 H2O500 microL
CoCl2·6 H2O500 microL
MnCl2·4 H2O500 microL
ZnSO4·7 H2O500 microL
Na2EDTA (0.5 M)5 mL
FeCl3·7 H2O5 mL

10 nM Fe (final) stock
milli-Q H2O492 mL
CuSO4·5 H2O500 microL
(NH4)6Mo7O24·4 H2O500 microL
CoCl2·6 H2O500 microL
MnCl2·4 H2O500 microL
ZnSO4·7 H2O500 microL
Na2EDTA (0.5 M)5 mL
FeCl3·7 H2O500 microL

Add the indicated volume of milli-Q H2O to each acid-clean polyethylene bottle and then add the indicated amount of each trace metal stock. Agitate manually following each addition. Ensure the solution is particularly well-mixed following addition of EDTA and prior to addition of Fe.

f/2 Vitamin Solution:
Vitamin B12 (1.0 g/L dH2O)1.0 ml
Biotin (0.1 g/L dH2O)10.0 ml
Thiamine HCl200.0 mg
Distilled water to1.0 L
Filter sterilize into plastic vials and store in refrigerator.

Note: Vitamin B12 and Biotin are obtained in a crystalline form. When preparing the Vitamin B12 Stock Solution allow for approximately 11% water of crystallization (For each 1.0 mg of Vitamin B12 add 0.89 ml dH2O). When preparing the Biotin Stock Solution allow for approximately 4% water of crystallization (For each 1.0 mg of Biotin add 9.6 ml dH2O).

Morel et al. 1975


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