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334a. METHANOTHRIX SOEHNGENII MEDIUM

Culture medium recipe

Vol.
×
Equipment needed: Filter Autoclave
# Compound Amount Unit Conc. [g/L] Conc. [mM]
1 Solution A 960 ml - -
2 Solution B 25 ml - -
3 Solution C 10 ml - -
4 Solution D 10 ml - -
5 Solution E 10 ml - -
6 Solution F 1 ml - -
7 Solution G 1 ml - -
8 Solution H 10 ml - -
9 , might need strain adjustment 10 ml/l - -
Instructions:
  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.
# Compound Amount Unit Conc. [g/L] Conc. [mM]
1 0.22 g 0.229 1.681
2 0.86 g 0.894 5.023
3 6.80 g 7.069 86.167
4
(0.1% w/v)
1.00 ml 0.001 0.003
5 Trace elements solution 10.00 ml - -
6
(0.1% w/v)
0.50 ml 5.2e-4 0.002
7 Cell-free culture supernatant of S. associata DSM 26261T 100.00 ml - -
8 850.00 ml 881.992 48957.9
# Compound Amount Unit Conc. [g/L] Conc. [mM]
1 1.25 g 50 471.752
2 25.00 ml 998.207 55408.8
# Compound Amount Unit Conc. [g/L] Conc. [mM]
1 0.30 g 30 513.325
2 0.11 g 11 74.822
3 0.10 g 10 49.188
4 10.00 ml 998.207 55408.8
# Compound Amount Unit Conc. [g/L] Conc. [mM]
1 0.3 g 30 560.84
2 10.0 ml 998.207 55408.8
# Compound Amount Unit Conc. [g/L] Conc. [mM]
1 Vitamin solution 10 ml - -
# Compound Amount Unit Conc. [g/L] Conc. [mM]
1
(ALDRICH 110299)
5 mg 5 49.92
2 1 ml 790 24655.2
# Compound Amount Unit Conc. [g/L] Conc. [mM]
1
(SIGMA P3556)
5 mg 5 -
2 1 ml 790 24655.2
# Compound Amount Unit Conc. [g/L] Conc. [mM]
1 0.5 g 50 208.175
2 10.0 ml 998.207 55408.8
Solution occurs in 1 other media (first defined in medium 334 )
# Compound Amount Unit Conc. [g/L] Conc. [mM]
1
(NTA)
12.80 g 12.8 66.967
2 1.35 g 1.35 4.995
3 0.10 g 0.1 0.505
4 0.03 g 0.03 0.126
5 0.10 g 0.1 0.68
6 0.10 g 0.1 0.734
7 0.03 g 0.03 0.223
8 0.01 g 0.01 0.162
9 0.03 g 0.03 0.124
10 0.12 g 0.12 0.505
11 1.00 g 1 17.111
12 0.03 g 0.03 0.114
13 1000.00 ml 998.207 55408.8
Instructions:
  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.
Equipment needed: Filter Centrifuge
# Compound Amount Unit Conc. [g/L] Conc. [mM]
1 Main sol. 119 1000 ml - -
2 1 g/l 1 5.551
Instructions:
  1. Cultivate T 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.
Solution occurs in 366 other media (first defined in medium 141 )
# Compound Amount Unit Conc. [g/L] Conc. [mM]
1 2.0 mg 0.002 0.008
2 2.0 mg 0.002 0.005
3 10.0 mg 0.01 0.049
4 5.0 mg 0.005 0.015
5 5.0 mg 0.005 0.013
6 5.0 mg 0.005 0.041
7 5.0 mg 0.005 0.01
8 0.1 mg 1.0e-4 6.3e-5
9 5.0 mg 0.005 0.036
10 5.0 mg 0.005 0.024
11 1000.0 ml 998.207 55408.8
Solution occurs in 1 other media (first defined in medium 119 )
Equipment needed: Hungate tubes Autoclave
# Compound Amount Unit Conc. [g/L] Conc. [mM]
1 0.50 g 0.498 3.66
2 0.40 g 0.398 1.616
3 0.40 g 0.398 6.817
4 0.40 g 0.398 7.448
5 0.05 g 0.05 0.339
6
(0.1% w/v in 0.1 N H2SO4)
2.00 ml 0.002 0.007
7 Trace element solution SL-10 1.00 ml - -
8
(OXOID)
1.00 g 0.996 -
9 , might need strain adjustment 1.00 g 0.996 12.142
10 2.00 g 1.992 29.292
11 Sludge fluid 50.00 ml - -
12 Fatty acid mixture 20.00 ml - -
13
(0.1% w/v)
0.50 ml 5.0e-4 0.002
14 4.00 g 3.984 47.426
15 0.50 g 0.498 2.835
16 0.50 g 0.498 2.073
17 930.00 ml 924.634 51324.9
19 , might need strain adjustment 2.00 g/l 2 14.08
20 , might need strain adjustment 2.00 g/l 2 -
22 Vitamin solution , might need strain adjustment 10.00 ml/l - -
Instructions:
  1. Dissolve ingredients except bicarbonate, cysteine and sulfide. Sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 minto make it anoxic. Add and dissolve bicarbonate, adjust pH to 6.8 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: The ferrous sulfate solution is not stable and should be freshly prepared. 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.
Solution occurs in 8 other media (first defined in medium 119 )
Equipment needed: Autoclave Anaerobic workbench Centrifuge
Instructions:
  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 room temperature in the dark.
Solution occurs in 10 other media (first defined in medium 119 )
# Compound Amount Unit Conc. [g/L] Conc. [mM]
1 23 ml - -
2 27 ml - -
3 27 ml 22.989 225.092
4 27 ml - -
5 1000 ml 904.173 50189.2
Instructions:
  1. Adjust pH to 7.0 - 7.3 with 2 N NaOH.
Solution occurs in 32 other media (first defined in medium 1310 )
Equipment needed: Centrifuge Filter Autoclave
Instructions:
  1. Rumen fluid from cow or sheep (obtained from fistulated animals or abattoir refuse) is filtered through muslin, autoclaved at 121°C for 15 minand 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 239 other media (first defined in medium 320 )
# Compound Amount Unit Conc. [g/L] Conc. [mM]
2
(25%; 7.7 M)
10.0 ml 3.17 86.943
3 1.5 g 1.5 7.545
4 70.0 mg 0.07 0.514
5 100.0 mg 0.1 0.505
6 6.0 mg 0.006 0.097
7 190.0 mg 0.19 0.799
8 2.0 mg 0.002 0.012
9 24.0 mg 0.024 0.101
10 36.0 mg 0.036 0.149
11 990.0 ml 988.225 54854.8
Instructions:
  1. First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.0 ml.

Metadata

Media status: Official DSMZ medium
Source: DSMZ
Taxonomic range:  Archaea
Medium type: Complex medium
Final pH: 7.2
Equipment needed: Anaerobic workbench, Autoclave, Centrifuge, Filter, Hungate tubes

Associated strains

All strains for this medium: 4
Strains with modifications: 4

Molecular composition

Compound Conc. [g/L] Conc. [mM]
Distilled water 989.02 54898.9
Sodium acetate 6.607 80.546
Methanol 1.538 48.014
Na2CO3 1.217 11.484
Na2HPO4 x 2 H2O 0.836 4.695
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Cultivation metadata from 4 strains BacDive

phyla 1 4 strains
oxygen anaerobe 4 strains
temperature thermophilic 3 strains
temperature mesophilic 1 strain

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