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DSMZ

DSMZ 119 . METHANOBACTERIUM MEDIUM

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

Culture medium recipe

Vol.
×
Medium for Methanobrevibacter olleyae DSM 16632

* Modification: Replace sludge fluid with the same volume of clarified rumen fluid (see medium 1310) and supplement medium with 2.00 g/l Trypticase peptone and 10.00 ml/l of Wolin's vitamin solution (see medium 141).

Growth conditions: anaerobic 37 °C

Main sol. 119 #

ml
Equipment needed: Hungate tubes Centrifuge Autoclave Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
KH2PO4 0.50 g 0.499 3.663
MgSO4 x 7 H2O 0.40 g 0.399 1.618
NaCl 0.40 g 0.399 6.824
NH4Cl 0.40 g 0.399 7.456
CaCl2 x 2 H2O 0.05 g 0.05 0.339
Trace element solution SL-10 1.00 ml - -
Yeast extract
(OXOID) 		1.00 g 0.997 -
Na-acetate 1.00 g 0.997 12.154
Na-formate 2.00 g 1.994 29.321
FeSO4 x 7 H2O solution (0.1% w/v) 2.00 ml - -
Sludge fluid* 50.00 ml - -
Fatty acid mixture 20.00 ml - -
Sodium resazurin
(0.1% w/v) 		0.50 ml 5.0e-4 0.002
NaHCO3 4.00 g 3.988 47.473
L-Cysteine HCl x H2O 0.50 g 0.499 2.838
Na2S x 9 H2O 0.50 g 0.499 2.076
Trypticase peptone* 2.00 g - -
Clarified rumen fluid* 50.00 ml - -
Wolin's vitamin solution* 10.00 ml - -
Distilled water 930.00 ml - -
1 Dissolve ingredients except bicarbonate, cysteine and sulfide. Sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. 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. Add cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Prior to use check pH of complete medium and adjust to 6.8 - 7.0, if necessary.
2 Note: After growth has started and the culture is becoming turbid add sterile 80% H2 and 20% CO2 gas mixture to 0.5 - 1 bar overpressure.

Sludge fluid #

1000
Solution occurs in 7 other media (first defined in Medium 119).
Compound Amount Unit Conc. [g/L] Conc. [mM]
Yeast extract 4 g 4 -
Sludge 1000 ml - -
1 Add 0.4% yeast extract to sludge from an anaerobic digester, and after gassing with nitrogen gas for a few minutes incubate it at 37°C for 24 hours. Then centrifuge the sludge at 13000 g and autoclave the resulting, clear supernatant in screw-capped vessels under nitrogen gas. The sludge fluid can be stored at 8-12°C in the dark.

Fatty acid mixture #

1000
Solution occurs in 6 other media (first defined in Medium 119).
Compound Amount Unit Conc. [g/L] Conc. [mM]
Isobutyric acid 23 ml - -
DL-2-Methylbutyric acid 27 ml - -
Valeric acid 27 ml - -
Isovaleric acid 27 ml - -
Distilled water 896 ml - -
1 Adjust pH to 7.5 with concentrated NaOH.

Trace element solution SL-10 #

1000
Solution occurs in 265 other media (first defined in Medium 320).
Compound Amount Unit Conc. [g/L] Conc. [mM]
HCl
(25%) 		10.0 ml 2.5 68.568
FeCl2 x 4 H2O 1.5 g 1.5 7.545
ZnCl2 70.0 mg 0.07 0.514
MnCl2 x 4 H2O 100.0 mg 0.1 0.505
H3BO3 6.0 mg 0.006 0.097
CoCl2 x 6 H2O 190.0 mg 0.19 1.463
CuCl2 x 2 H2O 2.0 mg 0.002 0.012
NiCl2 x 6 H2O 24.0 mg 0.024 0.185
Na2MoO4 x 2 H2O 36.0 mg 0.036 0.149
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.

FeSO4 x 7 H2O solution (0.1% w/v) #

1000
Solution occurs in 13 other media (first defined in Medium 119).
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.

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.

Wolin's vitamin solution * #

1000
Solution occurs in 53 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 0.021
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: 15.09.22
Source: DSMZ
Taxonomic range:  Archaea
Taxonomic range:  Bacteria
Medium type: Complex medium
Final pH: 6.8 - 7.0
Equipment needed: Autoclave, Centrifuge, 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: 81
Strains with modifications: 19
Methanobrevibacter olleyae DSM 16632

+  50.0 ml Clarified rumen fluid
+  10.0 ml Wolin's vitamin solution
-  Sludge fluid
  2.00 g Trypticase peptone

Molecular composition
* modified beta

Compound Conc. [g/L] Conc. [mM]
NaHCO3 3.988 47.473
Na-formate 1.994 29.321
Trypticase peptone 1.994 -
Na-acetate 0.997 12.154
KH2PO4 0.499 3.663
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Cultivation metadata from 81 strains BacDive

pH 6 - 8 3 strains
pH 4 - 6 1 strain
pH 8 - 10 1 strain
phyla 3 83 strains
oxygen anaerobe 78 strains
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