DSMZ 322 . METHANOSPHAERA (MCB-3) MEDIUM

Official DSMZ medium: This medium was manually curated by experts from the DSMZ.

Culture medium recipe

Vol.
×
Equipment needed: Autoclave Hungate tubes Filter Gassing station Centrifuge
Compound Amount Unit Conc. [g/L] Conc. [mM]
Clarified rumen fluid 100.00 ml - -
Trypticase peptone
(BD BBL)
2.00 g 1.955 -
Yeast extract 2.00 g 1.955 -
Na-acetate 0.50 g 0.489 5.958
Na-formate 0.50 g 0.489 7.187
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
(0.1% w/v in 0.1 N H2SO4)
3.00 ml 0.003 0.011
K2HPO4 0.60 g 0.587 3.367
KH2PO4 2.80 g 2.737 20.113
(NH4)2SO4 0.30 g 0.293 2.219
NH4Cl 1.00 g 0.978 18.275
NaCl 0.60 g 0.587 10.036
MgSO4 x 7 H2O 0.15 g 0.147 0.595
CaCl2 x 2 H2O 0.08 g 0.078 0.532
Sodium resazurin
(0.1% w/v)
0.50 ml 4.9e-4 0.002
NaHCO3 4.00 g 3.91 46.545
Methanol 5.00 ml 3.871 120.809
Wolin's vitamin solution (10x) 2.00 ml - -
L-Cysteine HCl x H2O 0.50 g 0.489 2.783
Na2S x 9 H2O 0.50 g 0.489 2.035
Distilled water 900.00 ml - -
1 Dissolve ingredients except bicarbonate, methanol, 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, 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, vitamins (sterilized by filtration), cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. 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.
Solution occurs in 27 other media (first defined in Medium 1310).
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.
Solution occurs in 166 other media (first defined in Medium 141).
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.
Solution occurs in 299 other media (first defined in Medium 120).
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 - -

Metadata

Last modified: 27.10.22
Source: DSMZ
Taxonomic range:  Archaea
Medium type: Complex medium
Final pH: 6.7 - 6.9
Equipment needed: Autoclave, Centrifuge, Filter, Gassing station, Hungate tubes
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Gas composition

  • 80 % H2
  • 20 % CO2

Detailed instructions for the cultivation of anaerobes including important warnings can be found in the cultivation instructions .

Associated strains

All strains for this medium: 1

Molecular composition

Compound Conc. [g/L] Conc. [mM]
NaHCO3 3.91 46.545
Methanol 3.871 120.809
KH2PO4 2.737 20.113
Trypticase peptone 1.955 -
Yeast extract 1.955 -
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Cultivation metadata from 1 strains BacDive

phyla 1 1 strain
oxygen anaerobe 1 strain
temperature mesophilic 1 strain