905 .
METHANOCALCULUS HALOTOLERANS MEDIUM
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Equipment needed:
Autoclave
Gassing station
Hungate tubes
Final pH:
7.2 - 7.6
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NH4Cl | 1.00 | g | ||
|
|
K2HPO4 | 0.30 | g | ||
|
|
KH2PO4 | 0.30 | g | ||
|
|
KCl | 0.17 | g | ||
|
|
NaCl | 50.00 | g | ||
|
|
Na-acetate | 0.50 | g | ||
|
|
Yeast extract
(OXOID) |
0.50 | g | ||
|
|
Trypticase
(BD BBL) |
0.50 | g | ||
|
|
Modified Wolin's mineral solution | 10.00 | ml | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
NaHCO3 | 2.00 | g | ||
|
|
L-Cysteine HCl x H2O | 0.50 | g | ||
|
|
CaCl2 x 2 H2O | 0.60 | g | ||
|
|
MgCl2 x 6 H2O | 3.20 | g | ||
|
|
Na2S x 9 H2O | 0.30 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients (except bicarbonate, cysteine, calcium chloride, magnesium chloride and sulfide), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve solid bicarbonate and cysteine, then adjust pH to 7.5 with 10 N KOH. Dispense medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials (to 30% of volume) and autoclave. Add calcium chloride, magnesium chloride and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. The pH of the complete medium should be between 7.2 and 7.6. | |||||
| 2 After inoculation pressurize vessels with sterile 80% H2 and 20% CO2 gas mixture to 1 bar overpressure. | |||||
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. | |||||