404 .
METHANOSPHAERA CUNICULI MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
7.0 - 7.2
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Mineral solution 1 | 40.0 | ml | ||
|
|
Minerals solution | 40.0 | ml | ||
|
|
Fe(NH4)2(SO4)2 x 7 H2O
(0.1% w/v) |
2.0 | ml | ||
|
|
NiCl2 x 6 H2O
(0.1% w/v) |
0.2 | ml | ||
|
|
Modified Wolin's mineral solution | 10.0 | ml | ||
|
|
Na-acetate x 3 H2O | 3.0 | g | ||
|
|
Yeast extract
(OXOID) |
1.0 | g | ||
|
|
Trypticase peptone
(BD BBL) |
1.0 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.5 | ml | ||
|
|
NaHCO3 | 3.0 | g | ||
|
|
Methanol
(50% v/v) |
10.0 | ml | ||
|
|
Wolin's vitamin solution (10x) | 1.0 | ml | ||
|
|
L-Cysteine HCl x H2O | 0.3 | g | ||
|
|
Na2S x 9 H2O | 0.3 | g | ||
|
|
Distilled water | 900.0 | ml | ||
| 1 Dissolve ingredients (except bicarbonate, methanol, vitamins, cysteine and sulfide), then 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 7.0, then distribute 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 methanol (50% v/v), vitamins, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. Vitamins are sterilized by filtration. Adjust pH of complete medium to 7.0 - 7.2. | |||||
| 2 After inoculation, pressurize culture bottles with sterile 80% H2 and 20% CO2 gas mixture to 2 bar overpressure. | |||||
Minerals solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
KH2PO4 | 6.00 | g |
|
|
(NH4)2SO4 | 6.00 | g |
|
|
NaCl | 12.00 | g |
|
|
MgSO4 x 7 H2O | 2.60 | g |
|
|
CaCl2 x 2 H2O | 0.16 | g |
|
|
Distilled water | 1000.00 | ml |
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 (10x) #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 20 | mg |
|
|
Folic acid | 20 | mg |
|
|
Pyridoxine hydrochloride | 100 | mg |
|
|
Thiamine HCl | 50 | mg |
|
|
Riboflavin | 50 | mg |
|
|
Nicotinic acid | 50 | mg |
|
|
Calcium D-(+)-pantothenate | 50 | mg |
|
|
Vitamin B12 | 1 | mg |
|
|
p-Aminobenzoic acid | 50 | mg |
|
|
(DL)-alpha-Lipoic acid | 50 | mg |
|
|
Distilled water | 1000 | ml |