Official DSMZ medium: This medium was manually curated by experts from the DSMZ.
Compound | Amount | Unit | Conc. [g/L] | Conc. [mM] | |
---|---|---|---|---|---|
Clarified rumen fluid | 100.00 | ml | - | - | |
Trypticase peptone
(BD BBL) |
2.00 | g | 1.946 | - | |
Yeast extract | 2.00 | g | 1.946 | - | |
Na-acetate | 0.50 | g | 0.486 | 5.929 | |
Na-formate | 0.50 | g | 0.486 | 7.152 | |
Modified Wolin's mineral solution | 10.00 | ml | - | - | |
Na2SeO4
(0.1% w/v) |
1.90 | ml | 0.002 | 0.01 | |
NiCl2 x 6 H2O
(0.1% w/v) |
0.70 | ml | 6.8e-4 | 0.005 | |
FeSO4 x 7 H2O solution (0.1% w/v) | 3.00 | ml | - | - | |
K2HPO4 | 0.60 | g | 0.584 | 3.351 | |
KH2PO4 | 2.80 | g | 2.724 | 20.015 | |
(NH4)2SO4 | 0.30 | g | 0.292 | 2.208 | |
NH4Cl | 1.00 | g | 0.973 | 18.186 | |
NaCl | 0.60 | g | 0.584 | 9.987 | |
MgSO4 x 7 H2O | 0.15 | g | 0.146 | 0.592 | |
CaCl2 x 2 H2O | 0.08 | g | 0.078 | 0.529 | |
Sodium resazurin
(0.1% w/v) |
0.50 | ml | 4.9e-4 | 0.002 | |
NaHCO3 | 4.00 | g | 3.891 | 46.318 | |
Methanol
(50% v/v) |
10.00 | ml | 7.704 | 240.443 | |
Wolin's vitamin solution (10x) | 2.00 | ml | - | - | |
DL-Dithiothreitol | 0.50 | g | 0.486 | 3.152 | |
Distilled water | 900.00 | ml | - | - | |
1 Dissolve ingredients except bicarbonate, methanol, and dithiothreitol (DTT), then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve bicarbonate, adjust pH of medium to 6.8 - 7.0, 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. Add methanol (50% v/v solution), vitamins, and DTT from sterile anoxic stock solutions prepared under 100% N2 gas. Vitamins and DTT should be sterilized by filtration. Prior to use check pH of complete medium and adjust to 6.7 - 6.9, if necessary. | |||||
2 After inoculation add sterile 80% H2 and 20% CO2 gas mixture to 1 bar overpressure. |
1 Rumen fluid from cow or sheep (obtained from fistulated animals or abattoir refuse) is filtered through muslin, autoclaved at 121°C for 15 min and then centrifuged at 27,000 g for 20 min. The supernatant is made anoxic by sparging with 100% N2 gas for 15 min, dispensed under same gas atmosphere into anoxic serum vials to 30% of volume and then stored frozen at -20°C. |
Compound | Amount | Unit | Conc. [g/L] | Conc. [mM] | |
---|---|---|---|---|---|
Nitrilotriacetic acid | 1.50 | g | 1.5 | 7.848 | |
MgSO4 x 7 H2O | 3.00 | g | 3 | 12.172 | |
MnSO4 x H2O | 0.50 | g | 0.5 | 2.958 | |
NaCl | 1.00 | g | 1 | 17.112 | |
FeSO4 x 7 H2O | 0.10 | g | 0.1 | 0.36 | |
CoSO4 x 7 H2O | 0.18 | g | 0.18 | 1.161 | |
CaCl2 x 2 H2O | 0.10 | g | 0.1 | 0.68 | |
ZnSO4 x 7 H2O | 0.18 | g | 0.18 | 0.626 | |
CuSO4 x 5 H2O | 0.01 | g | 0.01 | 0.04 | |
AlK(SO4)2 x 12 H2O | 0.02 | g | 0.02 | 0.042 | |
H3BO3 | 0.01 | g | 0.01 | 0.162 | |
Na2MoO4 x 2 H2O | 0.01 | g | 0.01 | 0.041 | |
NiCl2 x 6 H2O | 0.03 | g | 0.03 | 0.231 | |
Na2SeO3 x 5 H2O | 0.30 | mg | 3.0e-4 | 0.001 | |
Na2WO4 x 2 H2O | 0.40 | mg | 4.0e-4 | 0.001 | |
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. |
Compound | Amount | Unit | Conc. [g/L] | Conc. [mM] |
---|---|---|---|---|
Biotin | 20 | mg | 0.02 | 0.082 |
Folic acid | 20 | mg | 0.02 | 0.045 |
Pyridoxine hydrochloride | 100 | mg | 0.1 | 0.486 |
Thiamine HCl | 50 | mg | 0.05 | 0.148 |
Riboflavin | 50 | mg | 0.05 | 0.133 |
Nicotinic acid | 50 | mg | 0.05 | 0.406 |
Calcium D-(+)-pantothenate | 50 | mg | 0.05 | 0.21 |
Vitamin B12 | 1 | mg | 1.0e-3 | 7.4e-4 |
p-Aminobenzoic acid | 50 | mg | 0.05 | 0.365 |
(DL)-alpha-Lipoic acid | 50 | mg | 0.05 | 0.242 |
Distilled water | 1000 | ml | - | - |
Compound | Amount | Unit | Conc. [g/L] | Conc. [mM] | |
---|---|---|---|---|---|
FeSO4 x 7 H2O | 1 | g | 1 | 3.597 | |
H2SO4
(0.1 N) |
1000 | ml | - | - | |
1 The ferrous sulfate solution is not stable and should be freshly prepared. |
Last modified: | 13.09.23 |
Source: | DSMZ |
Synonyms: | METHANOSPHAERA (MCB-3) MEDIUM |
Taxonomic range: | Archaea |
Medium type: | Complex medium |
Final pH: | 6.7 - 6.9 |
Equipment needed: | Autoclave, Centrifuge, Filter, Gassing station, Hungate tubes |
Edit in Medium builder |
Detailed instructions for the cultivation of anaerobes including important warnings can be found in the cultivation instructions .
All strains for this medium: | 1 |
Compound | Conc. [g/L] | Conc. [mM] |
---|---|---|
Methanol | 7.704 | 240.443 |
NaHCO3 | 3.891 | 46.318 |
KH2PO4 | 2.724 | 20.015 |
Trypticase peptone | 1.946 | - |
Yeast extract | 1.946 | - |
Show more |