Bold 3N Medium

NaNO3 solution (25 g/L) 30 ml
P-IV metal solution 6 ml
CaCl2 2H2O solution (2.5 g/L) 10 ml
MgSO4 7H2O solution (7.5 g/L) 10 ml
K2HPO4 solution (7.5 g/L) 10 ml
KH2PO4 solution (17.5 g/L) 10 ml
Vitamin B12 solution 1 ml
Soilwater: GR+ medium 40 ml
Deionized water to 1 L

Vitamins are added after autoclaving.

P-IV metal solution:
Na2EDTA 2H2O 0.75 g
FeCl3 6H2O 0.097 g
MnCl2 4H2O 0.041 g
ZnCl2 0.005 g
CoCl2 6H2O 0.002 g
Na2MoO4 2H2O 0.004 g
Deionized water to 1 L

To approximately 950 ml of dH2O, add ingredients in order listed stirring continuously. The EDTA should be fully dissoled before adding other components. Bring to 1 L with dH2O and store at 4 C.

Vitamin B12 solution:

HEPES buffer 2.4 g
Vitamin B12 0.027 g
Deionized water to 200 ml

Prepare 200 ml of HEPES buffer at pH 7.8 and fully dissolve vitamin. Filter-sterilize and store in dark at -20 C.

Soilwater: GR+ Medium:

Greenhouse soil 1 tsp
CaCO3 1 mg
Deionized water to 200 ml

Combine ingredients. Cover container and steam for 2 consecutive days, 3 h each day with gradually rising temperature to 95 C in 15 min, then to 98 C for 3 h. Cool at room temperature and store at 4 C.

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