814 .
METHANOBACTERIUM SUBTERRANEUM MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final gas composition:
100 % N2
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Final pH:
8.0 - 8.5
Select a strain (optional):
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 | ||
|
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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 | ||
|
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Na2S x 9 H2O | 0.25 | g | ||
|
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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 | ||
|
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MnSO4 x H2O | 0.50 | g | ||
|
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NaCl | 1.00 | g | ||
|
|
FeSO4 x 7 H2O | 0.10 | g | ||
|
|
CoSO4 x 7 H2O | 0.18 | g | ||
|
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CaCl2 x 2 H2O | 0.10 | g | ||
|
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ZnSO4 x 7 H2O | 0.18 | g | ||
|
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CuSO4 x 5 H2O | 0.01 | g | ||
|
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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 | ||
|
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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. | |||||
Seven vitamins solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Vitamin B12 | 100 | mg |
|
|
p-Aminobenzoic acid | 80 | mg |
|
|
D-(+)-biotin | 20 | mg |
|
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Nicotinic acid | 200 | mg |
|
|
Calcium pantothenate | 100 | mg |
|
|
Pyridoxine hydrochloride | 300 | mg |
|
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Thiamine-HCl x 2 H2O | 200 | mg |
|
|
Distilled water | 1000 | ml |