Mix ingredients except Na2S·9H2O, adjust pH to 6.5 with NaOH, and autoclave under a N2 atmosphere. Separately autoclave and neutralize Na2S·9H2O as 5% solution under a N2 atmosphere. Anaerobically add Na2S·9H2O solution.
*Trace minerals
Nitrilotriacetic acid
1.5
g
MgSO4·7H2O
3
g
MnSO4·2H2O
0.5
g
NaCl
1
g
FeSO4·7H2O
0.1
g
CoSO4·7H2O
0.18
g
CaCl2·2H2O
0.1
g
ZnSO4·7H2O
0.18
g
CuSO4·5H2O
0.01
g
H3BO3
0.01
g
Na2MoO4·2H2O
0.01
g
NiCl2·6H2O
0.025
g
Na2SeO3·5H2O
0.8
mg
Na2WO4
0.8
mg
KAl(SO4)2·12H2O
4
mg
Distilled water
1
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Final pH 7.0 (with KOH).
**The sulfide is prepared in a vessel sealed with a butyl rubber stopper. Autoclave under a N2 atmosphere. After cooling, the pH is adjusted to about 7.3 by adding of sterile 4-0.2 N H2SO4 drop-wise with a syringe without opening the bottle. Appearance of yellow color indicate the pH is about 8. The vessel should be shaken continuously to avoid precipitation of elemental sulfur. The final solution should be transparent yellow.
