DSMZ 814 . METHANOBACTERIUM SUBTERRANEUM MEDIUM

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Equipment needed: Autoclave Filter Gassing station Hungate tubes

Final gas composition: 100 % N2 read more

Final pH: 8.0 - 8.5

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ml
Compound Amount Unit
NH4Cl 0.40 g
MgCl2 x 6 H2O 0.03 g
NaCl 0.45 g
K2HPO4 0.50 g
FeSO4 x 7 H2O
(0.1% w/v in 0.1 N H2SO4)
3.00 ml
Modified Wolin's mineral solution 10.00 ml
Tryptone
(BD Bacto)
1.00 g
Na-formate 3.20 g
Na-acetate 1.00 g
Sodium resazurin
(0.1% w/v)
0.50 ml
Na2CO3 1.00 g
Seven vitamins solution 1.00 ml
L-Cysteine HCl x H2O 0.25 g
Na2S x 9 H2O 0.25 g
Distilled water 1000.00 ml
1 Dissolve ingredients except carbonate, vitamins, cysteine and sulfide, then sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add vitamins (sterilized by filtration), cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Prior to use adjust pH of complete medium to 8.0 - 8.5.

Modified Wolin's mineral solution #

1000
Compound Amount Unit
Nitrilotriacetic acid 1.50 g
MgSO4 x 7 H2O 3.00 g
MnSO4 x H2O 0.50 g
NaCl 1.00 g
FeSO4 x 7 H2O 0.10 g
CoSO4 x 7 H2O 0.18 g
CaCl2 x 2 H2O 0.10 g
ZnSO4 x 7 H2O 0.18 g
CuSO4 x 5 H2O 0.01 g
AlK(SO4)2 x 12 H2O 0.02 g
H3BO3 0.01 g
Na2MoO4 x 2 H2O 0.01 g
NiCl2 x 6 H2O 0.03 g
Na2SeO3 x 5 H2O 0.30 mg
Na2WO4 x 2 H2O 0.40 mg
Distilled water 1000.00 ml
1 First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.