120b .
METHANOMICROCOCCUS MEDIUM
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* Medium for Methanimicrococcus hongohii DSM 114388
Supplement medium after autoclaving with 0.5 g/l DTT (DL-dithiothreitol) added from an anoxic stock solution sterilized by filtration. Omit Na2S x 9 H2O and L-cysteine HCl x H2O.
Growth conditions: 1-2 days
anaerobic
37 °C
Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
7.0 - 7.2
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
K2HPO4 | 0.35 | g | ||
|
|
KH2PO4 | 0.23 | g | ||
|
|
NH4Cl | 0.50 | g | ||
|
|
MgSO4 x 7 H2O | 0.50 | g | ||
|
|
CaCl2 x 2 H2O | 0.25 | g | ||
|
|
NaCl | 2.25 | g | ||
|
|
FeSO4 x 7 H2O solution (0.1% w/v) | 2.00 | ml | ||
|
|
Trace element solution SL-10 | 1.00 | ml | ||
|
|
Yeast extract
(OXOID) |
2.00 | g | ||
|
|
Casitone
(BD BBL) |
2.00 | g | ||
|
|
Na-acetate | 2.50 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
NaHCO3 | 2.50 | g | ||
|
|
2-Mercaptoethanesulfonate
(coenzyme M) |
0.14 | g | ||
|
|
Methanol
(50% v/v) |
20.00 | ml | ||
|
|
Wolin's vitamin solution (10x) | 1.00 | ml | ||
| L-Cysteine HCl x H2O* | 0.36 | g | |||
| Na2S x 9 H2O* | 0.36 | g | |||
|
|
DL-Dithiothreitol | 0.50 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients (except bicarbonate, coenzyme M, vitamins, methanol, cysteine and sulfide) and sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Then add and dissolve bicarbonate, adjust pH to 6.5 and 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. Methanol (50% v/v stock solution) and the reducing agents are each autoclaved separately under 100% N2 gas atmosphere as concentrated solutions in tightly closed tubes. Vitamins and coenzyme M are prepared under 100% N2 gas atmosphere and sterilized by filtration. Appropriate volumes of the stock solutions are injected into the sterile medium with hypodermic syringes. Adjust pH of the complete medium to 7.0 - 7.2, if necessary. | |||||
| 2 After inoculation, pressurize culture vessels with sterile 80% H2 and 20% CO2 gas mixture to 1 bar overpressure. | |||||
Trace element solution SL-10 #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
HCl
(25%) |
10.0 | ml | ||
|
|
FeCl2 x 4 H2O | 1.5 | g | ||
|
|
ZnCl2 | 70.0 | mg | ||
|
|
MnCl2 x 4 H2O | 100.0 | mg | ||
|
|
H3BO3 | 6.0 | mg | ||
|
|
CoCl2 x 6 H2O | 190.0 | mg | ||
|
|
CuCl2 x 2 H2O | 2.0 | mg | ||
|
|
NiCl2 x 6 H2O | 24.0 | mg | ||
|
|
Na2MoO4 x 2 H2O | 36.0 | mg | ||
|
|
Distilled water | 990.0 | ml | ||
| 1 First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml. | |||||
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 |
FeSO4 x 7 H2O solution (0.1% w/v) #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
FeSO4 x 7 H2O | 1 | g | ||
|
|
H2SO4
(0.1 N) |
1000 | ml | ||
| 1 The ferrous sulfate solution is not stable and should be freshly prepared. | |||||