Medium data

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301   THERMODESULFOBACTERIUM HYDROGENOPHILUM MEDIUM

NaCl  30.0     g
MgCl2·6H2O  3.0     g
Na2SO4  4.0     g
KCl  0.5     g
NH4Cl  0.25    g
KH2PO4  0.2     g
PIPES  3.46    g
CaCl2·2H2O  0.15    g
Yeast extract (Difco)  0.5     g
Sodium acetate  0.83    g
Trace element solution (see below)  1.0     ml
Na2WO4·2H2O  0.2     mg
Na2SeO4  0.05    mg
Trace vitamins (see Medium No. 197)  10.0     ml
Vitamin B12  0.05    mg
Growth stimulating factors (see below)  1.0     ml
Resazurin  1.0     mg
Na2S·9H2O  0.5     g
Distilled water  1.0     L
Mix ingredients, except Na2S·9H2O, and adjust pH to 6.5 with HCl. Distribute the medium into culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under a stream of H2-CO2 (4:1, v/v) gas mixture and seal with butyl rubber stoppers, and autoclave. Neutralize Na2S·9H2O as a 5% solution, and autoclave under a N2 atmosphere. Add the sterile Na2S solution to the medium, and pressurize the bottles to 200 kPa H2-CO2 (4:1, v/v). After incubation at 70C for 1 hr, readjust pH to 6.5 with a sterile and anaerobic NaHCO3 stock solution.
Trace element solution:
Nitrilotriacetic acid  12.8     g
Fe2SO4·7H2O  0.42    g
H3BO3  0.01    g
MnCl2·4H2O  0.1     g
CoCl2·6H2O  0.17    g
CuCl2·2H2O  0.02    g
Zn2SO4·7H2O  0.21    g
Na2MoO4·2H2O  0.01    g
Dissolve nitrilotriacetic acid in 800 ml distilled water and adjust pH to 6.5 with NaOH. Then proceed to add minerals and bring volume to 1000 ml.
Growth stimulating factors:
Isobutyric acid  0.5     g
Valeric acid  0.5     g
2-Methyl-butyric acid  0.5     g
3-Methyl-butyric acid  0.5     g
Caproic acid  0.2     g
Succinic acid  0.6     g
Distilled water  100.0     ml
Adjust pH to 9.0 with NaOH.