141 .
METHANOGENIUM MEDIUM (H2/CO2)
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
6.8 - 7.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
KCl | 0.34 | g | ||
|
|
MgCl2 x 6 H2O | 4.00 | g | ||
|
|
MgSO4 x 7 H2O | 3.45 | g | ||
|
|
NH4Cl | 0.25 | g | ||
|
|
CaCl2 x 2 H2O | 0.14 | g | ||
|
|
K2HPO4 | 0.14 | g | ||
|
|
NaCl | 18.00 | g | ||
|
|
Modified Wolin's mineral solution | 10.00 | ml | ||
|
|
Fe(NH4)2(SO4)2 x 6 H2O
(0.1% w/v) |
2.00 | ml | ||
|
|
Na-acetate | 1.00 | g | ||
|
|
Yeast extract
(OXOID) |
2.00 | g | ||
|
|
Trypticase peptone
(BD BBL) |
2.00 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
Wolin's vitamin solution | 10.00 | ml | ||
|
|
NaHCO3 | 5.00 | g | ||
|
|
L-Cysteine HCl x H2O | 0.50 | g | ||
|
|
Na2S x 9 H2O | 0.50 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Note: If the medium is being used without overpressure then adjust pH with a small amount of sterile anoxic 1 N HCl, if necessary. | |||||
| 2 Dissolve ingredients (except bicarbonate, vitamins, cysteine and sulfide), sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve bicarbonate and adjust pH to 6.5, then dispense medium under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. After sterilization add cysteine and sulfide from sterile anoxic stock solutions autoclaved under 100% N2 gas. Vitamins are prepared under 100% N2 gas atmosphere and sterilized by filtration. Adjust pH of complete medium to 6.8 - 7.0, if necessary. | |||||
| 3 For incubation use sterile 80% H2 and 20% CO2 gas mixture at two atmospheres of pressure. | |||||
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. | |||||
Wolin's vitamin solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 2.0 | mg |
|
|
Folic acid | 2.0 | mg |
|
|
Pyridoxine hydrochloride | 10.0 | mg |
|
|
Thiamine HCl | 5.0 | mg |
|
|
Riboflavin | 5.0 | mg |
|
|
Nicotinic acid | 5.0 | mg |
|
|
Calcium D-(+)-pantothenate | 5.0 | mg |
|
|
Vitamin B12 | 0.1 | mg |
|
|
p-Aminobenzoic acid | 5.0 | mg |
|
|
(DL)-alpha-Lipoic acid | 5.0 | mg |
|
|
Distilled water | 1000.0 | ml |