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DSMZ

DSMZ 322 . METHANOSPHAERA (MCB-3) MEDIUM

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

Culture medium recipe

Vol.
×

Main sol. 322 #

ml
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 2 -
Yeast extract 2.00 g 2 -
Na-acetate 0.50 g 0.5 6.095
Na-formate 0.50 g 0.5 7.352
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 7.0e-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 0.6 3.445
KH2PO4 2.80 g 2.8 20.575
(NH4)2SO4 0.30 g 0.3 2.27
NH4Cl 1.00 g 1 18.695
NaCl 0.60 g 0.6 10.267
MgSO4 x 7 H2O 0.15 g 0.15 0.609
CaCl2 x 2 H2O 0.08 g 0.08 0.544
Sodium resazurin
(0.1% w/v) 		0.50 ml 5.0e-4 -
NaHCO3 4.00 g 4 47.615
Methanol 5.00 ml - -
Wolin's vitamin solution 20.00 ml - -
L-Cysteine HCl x H2O 0.50 g 0.5 2.847
Na2S x 9 H2O 0.50 g 0.5 2.082
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 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. After inoculation add sterile 80% H2 and 20% CO2 gas mixture to 1 bar overpressure.

Clarified rumen fluid #

1000
Solution occurs in 28 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.

Modified Wolin's mineral solution #

1000
Solution occurs in 170 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.

Wolin's vitamin solution #

1000
Solution occurs in 357 other media (first defined in Medium 141).
Compound Amount Unit Conc. [g/L] Conc. [mM]
Biotin 2.0 mg 0.002 0.008
Folic acid 2.0 mg 0.002 0.005
Pyridoxine hydrochloride 10.0 mg 0.01 0.049
Thiamine HCl 5.0 mg 0.005 0.015
Riboflavin 5.0 mg 0.005 0.013
Nicotinic acid 5.0 mg 0.005 0.041
Calcium D-(+)-pantothenate 5.0 mg 0.005 -
Vitamin B12 0.1 mg 1.0e-4 7.4e-5
p-Aminobenzoic acid 5.0 mg 0.005 0.036
(DL)-alpha-Lipoic acid 5.0 mg 0.005 0.024
Distilled water 1000.0 ml - -

Metadata

Last modified: 25.02.22
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 4 47.615
KH2PO4 2.8 20.575
Trypticase peptone 2 -
Yeast extract 2 -
NH4Cl 1 18.695
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Cultivation metadata from 1 strains BacDive

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