825a .
METHANOBACTERIUM II MEDIUM (N2/CO2)
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Selected strain: Methanosarcina mazei DSM 10132
This strain uses the standard medium without modifications.
Growth conditions: anaerobic
37 °C
Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
6.8 - 7.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
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NH4Cl | 1.0 | g | ||
|
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K2HPO4 | 0.3 | g | ||
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KH2PO4 | 0.3 | g | ||
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MgCl2 x 6 H2O | 0.2 | g | ||
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CaCl2 x 2 H2O | 0.1 | g | ||
|
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KCl | 0.1 | g | ||
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NaCl | 0.6 | g | ||
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Modified Wolin's mineral solution | 10.0 | ml | ||
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Na-acetate | 0.5 | g | ||
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Yeast extract | 1.0 | g | ||
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Sodium resazurin
(0.1% w/v) |
0.5 | ml | ||
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NaHCO3 | 4.0 | g | ||
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Methanol
(15% v/v) |
10.0 | ml | ||
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Wolin's vitamin solution (10x) | 1.0 | ml | ||
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L-Cysteine HCl x H2O | 0.5 | g | ||
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Na2S x 9 H2O | 0.5 | g | ||
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Distilled water | 1000.0 | ml | ||
| 1 Dissolve ingredients except bicarbonate, methanol, vitamins, cysteine and sulfide. Sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve bicarbonate, adjust pH to 7.0, then dispense medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add methanol (15% v/v solution), vitamins (sterilized by filtration), cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Prior to use check pH of complete medium and adjust to 6.8 - 7.0, if necessary. | |||||
Modified Wolin's mineral solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid | 1.50 | g | ||
|
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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 | ||
|
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FeSO4 x 7 H2O | 0.10 | g | ||
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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 | ||
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H3BO3 | 0.01 | g | ||
|
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Na2MoO4 x 2 H2O | 0.01 | g | ||
|
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NiCl2 x 6 H2O | 0.03 | g | ||
|
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Na2SeO3 x 5 H2O | 0.30 | mg | ||
|
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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. | |||||
Wolin's vitamin solution (10x) #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 20 | mg |
|
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Folic acid | 20 | mg |
|
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Pyridoxine hydrochloride | 100 | mg |
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Thiamine HCl | 50 | mg |
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Riboflavin | 50 | mg |
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Nicotinic acid | 50 | mg |
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Calcium D-(+)-pantothenate | 50 | mg |
|
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Vitamin B12 | 1 | mg |
|
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p-Aminobenzoic acid | 50 | mg |
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(DL)-alpha-Lipoic acid | 50 | mg |
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Distilled water | 1000 | ml |