230 .
METHANOSARCINA MEDIUM
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Equipment needed:
Anaerobic workbench
Autoclave
Final pH:
n.d.
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
KH2PO4 | 0.227 | g | ||
|
|
K2HPO4 | 0.348 | g | ||
|
|
NH4Cl | 0.500 | g | ||
|
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MgSO4 x 7 H2O | 0.500 | g | ||
|
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CaCl2 x 2 H2O | 0.250 | g | ||
|
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NaCl | 2.250 | g | ||
|
|
FeSO4 x 7 H2O | 2.000 | mg | ||
|
|
Yeast extract
(BD-Difco) |
2.000 | g | ||
|
|
Casitone
(BD-Difco) |
2.000 | g | ||
|
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Trace vitamins | 10.000 | ml | ||
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FeCl2 solution | 1.000 | ml | ||
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Trace element solution | 1.000 | ml | ||
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NaHCO3 | 0.850 | g | ||
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Methanol | 10.000 | ml | ||
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L-Cysteine HCl x H2O | 0.300 | g | ||
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Na2S x 9 H2O | 0.300 | g | ||
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Resazurin | 1.000 | mg | ||
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Distilled water | 980.000 | ml | ||
| 1 Mix ingredients, except NaHCO3, methanol, cysteine x HCl and Na2S x 9H2O, and bring to a boil for 5-10 sec. Cool down under a stream of N2-CO2 (80:20), and add NaHCO3 and methanol to the medium. Dispense the medium into suitable culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under a stream of N2-CO2 (80:20) and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight. Separately autoclave cysteine x HCl and Na2S x 9H2O as 5% solutions, respectively, under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add the cysteine x HCl and Na2S x 9H2O solutions. | |||||
Trace vitamins #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 2.0 | mg |
|
|
Folic acid | 2.0 | mg |
|
|
Pyridoxine hydrochloride | 10.0 | mg |
|
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Thiamine HCl | 5.0 | mg |
|
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Riboflavin | 5.0 | mg |
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Nicotinic acid | 5.0 | mg |
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Calcium pantothenate | 5.0 | mg |
|
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Vitamin B12 | 0.1 | mg |
|
|
p-Aminobenzoic acid | 5.0 | mg |
|
|
Lipoic acid | 5.0 | mg |
|
|
Distilled water | 1000.0 | ml |
FeCl2 solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
HCl
(7.7 M, 25%) |
10.0 | ml |
|
|
FeCl2 x 4 H2O | 1.5 | g |
|
|
Distilled water | 990.0 | ml |
Trace element solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
ZnCl2 | 70 | mg |
|
|
MnCl2 x 4 H2O | 100 | mg |
|
|
H3BO3 | 6 | mg |
|
|
CoCl2 x 6 H2O | 190 | mg |
|
|
CuCl2 x 2 H2O | 2 | mg |
|
|
NiCl2 x 6 H2O | 24 | mg |
|
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Na2MoO4 x 2 H2O | 36 | mg |
|
|
Distilled water | 1000 | ml |