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DSMZ

DSMZ 334a . METHANOTHRIX SOEHNGENII MEDIUM

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

Culture medium recipe

Vol.
×
Medium for Methanothrix thermoacetophila DSM 6194

* Modification: Supplement medium with 0.14 g/l coenzyme M (2-mercaptoethanesulfonate) added after autoclaving from a sterile anoxic stock solution. Adjust pH of complete medium to 7.0.

Growth conditions: 7-14 days anaerobic 55 °C

Main sol. 334a #

ml
Equipment needed: Centrifuge Gassing station Serum bottle Autoclave Filter
Compound Amount Unit Conc. [g/L] Conc. [mM]
Solution A 942 ml - -
Solution B 25 ml - -
Solution C 10 ml - -
Solution D 10 ml - -
Solution E 1 ml - -
Solution F 1 ml - -
Solution G 1 ml - -
Solution H 10 ml - -
1 Sparge solution A with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, dispense under same gas atmosphere into anoxic serum vials or balch tubes (e.g., 20 ml medium in 50 ml bottles) and autoclave. Prior to inoculation complete the medium by adding sterile solutions C - H prepared under 100% N2 gas and solution B prepared under 80% N2 and 20% CO2 gas atmosphere. Solutions E, F and G are sterilized by filtration. Adjust the pH of the complete medium to 7.2.
2 Notes: Medium is only stable for a few weeks and has to be prepared freshly. Use 20% (v/v) inoculum. Sodium sulfide should be as pure as possible (use only clean crystals for the preparation of stock solutions). It has been noted that impurities of sulfide stock solutions can inhibit growth.

Solution A #

942
Compound Amount Unit Conc. [g/L] Conc. [mM]
KH2PO4 0.22 g 0.234 1.716
Na2HPO4 x 2 H2O 0.86 g 0.913 6.431
Na-acetate 6.80 g 7.219 87.997
Na2-EDTA
(0.1% w/v) 		1.00 ml 0.001 0.003
Trace elements solution 10.00 ml - -
Sodium resazurin
(0.1% w/v) 		0.50 ml 5.3e-4 0.002
Cell-free culture supernatant of S. associata 100.00 ml - -
2-Mercaptoethanesulfonate* 0.14 g - -
Distilled water 830.00 ml - -

Solution B #

25
Compound Amount Unit Conc. [g/L] Conc. [mM]
Na2CO3 1.25 g 50 471.752
Distilled water 25.00 ml - -

Solution C #

10
Compound Amount Unit Conc. [g/L] Conc. [mM]
NaCl 0.30 g 30 513.347
CaCl2 x 2 H2O 0.11 g 11 74.822
MgCl2 x 6 H2O 0.10 g 10 49.188
Distilled water 10.00 ml - -

Solution D #

10
Compound Amount Unit Conc. [g/L] Conc. [mM]
NH4Cl 0.3 g 30 560.852
Distilled water 10.0 ml - -

Solution E #

1
Compound Amount Unit Conc. [g/L] Conc. [mM]
Wolin's vitamin solution (10x) 1 ml - -

Solution F #

1
Compound Amount Unit Conc. [g/L] Conc. [mM]
Butyl vinyl ether
(ALDRICH 110299) 		5 mg 5 49.92
Methanol 1 ml 792 24717.6

Solution G #

1
Compound Amount Unit Conc. [g/L] Conc. [mM]
L-α-Phosphatidylcholine
(SIGMA P3556) 		5 mg 5 -
Methanol 1 ml 792 24717.6

Solution H #

10
Compound Amount Unit Conc. [g/L] Conc. [mM]
Na2S x 9 H2O 0.5 g 50 208.175
Distilled water 10.0 ml - -

Trace elements solution #

1000
Solution occurs in 1 other media (first defined in Medium 334).
Compound Amount Unit Conc. [g/L] Conc. [mM]
Nitrilotriacetic acid
(NTA) 		12.80 g 12.8 66.967
FeCl3 x 6 H2O 1.35 g 1.35 4.995
MnCl2 x 4 H2O 0.10 g 0.1 0.505
CoCl2 x 6 H2O 0.03 g 0.03 0.231
CaCl2 x 2 H2O 0.10 g 0.1 0.68
ZnCl2 0.10 g 0.1 0.734
CuCl2 0.03 g 0.03 0.223
H3BO3 0.01 g 0.01 0.162
Na2MoO4 x 2 H2O 0.03 g 0.03 0.124
NiCl2 x 6 H2O 0.12 g 0.12 0.926
NaCl 1.00 g 1 17.112
Na2SeO3 x 5 H2O 0.03 g 0.03 0.114
Distilled water 1000.00 ml - -
1 First dissolve NTA in 200 ml of distilled water and adjust pH to 6.5 with KOH, then dissolve mineral salts. Finally adjust pH to 6.5 with KOH and make up to 1000.00 ml.

Cell-free culture supernatant of S. associata #

1000
1 Cultivate Sphaerochaeta associata DSM 26261 in DSM medium 119 supplemented with 1.00 g/l glucose at 28°C until stationary phase is reached (7-10 days). Centrifuge culture at 3500 xg for 30 min, then sterilize supernatant by filtration and add it to the anoxic solution A before autoclaving.
2 Note: It is also possible to add 50.00 ml/l of a grown S. associata culture to solution A before autoclaving, provided bacterial DNA from dead cells is not a problem in the downstream processing of the Methanothrix culture.

Wolin's vitamin solution (10x) #

1000
Solution occurs in 306 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: 10.10.22
Source: DSMZ
Taxonomic range:  Archaea
Medium type: Complex medium
Final pH: * 7.0
Equipment needed: Autoclave, Centrifuge, Filter, Gassing station, Serum bottle
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Gas composition

  • 80 % N2
  • 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: 3
Strains with modifications: 2
Methanothrix thermoacetophila DSM 6194

  7.0
  0.14 g 2-Mercaptoethanesulfonate

Molecular composition
* modified beta

Compound Conc. [g/L] Conc. [mM]
Na-acetate 6.8 82.893
Na2CO3 1.25 11.794
Na2HPO4 x 2 H2O 0.86 6.058
Methanol 0.792 24.718
Na2S x 9 H2O 0.5 2.082
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Cultivation metadata from 3 strains BacDive

phyla 1 5 strains
oxygen anaerobe 5 strains
temperature mesophilic 1 strain
temperature thermophilic 1 strain