培養基配方
Final pH: 6.8 - 7.0
Final volume: 1003 ml
KH2PO4 0.50 g
MgSO4 x 7 H2O 0.40 g
NaCl 0.40 g
NH4Cl 0.40 g
CaCl2 x 2 H2O 0.05 g
Trace element solution SL-10 1.00 ml
Yeast extract (OXOID) 1.00 g
Na-acetate 1.00 g
Na-formate 2.00 g
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
NaHCO3 4.00 g
L-Cysteine HCl x H2O 0.50 g
Na2S x 9 H2O 0.50 g
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.
For DSM 1093, DSM 1125, DSM 11977, DSM 11978, DSM 11995, DSM 16643: Supplement
medium after autoclaving with 1 ml/l Wolin's vitamin solution (10x, see medium 120), 0.30
g/l DL-dithiothreitol, and 0.15 g/l coenzyme M (2-mercaptoethanesulfonic acid) from anoxic
stock solutions sterilized by filtration. Omit Na2S x 9 H2O and L-Cysteine HCl x H2O.
For DSM 1535: Adjust pH of complete medium to 7.6.
For DSM 2030: Adjust pH to 6.5 and add sterile 80% H2 and 20% CO2 gas to 2 bar
overpressure after inoculation.
For DSM 3267, DSM 11074, DSM 11075: Adjust pH of complete medium to 8.0 - 8.3.
For DSM 5522: Supplement medium with 5.0 g/l D-glucose added after autoclaving from a
sterile anoxic stock solution.
For DSM 6216: Increase amount of Na-acetate to 3.00 g/l.
For DSM 7057: Supplement medium with 2.00 g/l Na2SO4.
For DSM 7095, DSM 28100: Supplement medium with 0.3 g DL-Dithiothreitol. Omit Na2S x
9 H2O.
For DSM 9575: Add sterile 80% H2 and 20% CO2 gas mixture to 2 bar overpressure after
inoculation.
For DSM 15163: Supplement medium with 0.3 g DL-Dithiothreitol. Omit Na2S x 9 H2O.
Adjust pH of final medium to 6.0.
For DSM 16632: 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).
For DSM 25824: Supplement medium after autoclaving with 0.10 g/l
2-mercaptoethanesulfonic acid (coenzyme M) added from an anoxic stock solution
sterilized by filtration and add sterile 80% H2 and 20% CO2 gas mixture to 1 bar
overpressure after inoculation.
For DSM 25939: Adjust pH of the complete medium to 7.2 and add sterile 80% H2 and 20%
CO2 gas mixture to 1 bar overpressure after inoculation.
For DSM 25945: Adjust pH of the complete medium to 7.4 and add sterile 80% H2 and 20%
CO2 gas mixture to 1 bar overpressure after inoculation.
For DSM 102889: Adjust pH of complete medium to 8.5 - 9.0.
For DSM 108286: Adjust pH of final medium to 6.0.
Sludge fluid (from medium 119)
Yeast extract 4.00 g
Sludge 1000.00 ml
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 (from medium 119)
Isobutyric acid 23.00 ml
DL-2-Methylbutyric acid 27.00 ml
Valeric acid 27.00 ml
Isovaleric acid 27.00 ml
Distilled water 896.00 ml
Adjust pH to 7.5 with concentrated NaOH.
Trace element solution SL-10 (from medium 320)
HCl (25%) 10.00 ml
FeCl2 x 4 H2O 1.50 g
ZnCl2 70.00 mg
MnCl2 x 4 H2O 100.00 mg
H3BO3 6.00 mg
CoCl2 x 6 H2O 190.00 mg
CuCl2 x 2 H2O 2.00 mg
NiCl2 x 6 H2O 24.00 mg
Na2MoO4 x 2 H2O 36.00 mg
Distilled water 990.00 ml
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) (from medium 119)
FeSO4 x 7 H2O 1.00 g
H2SO4 (0.1 N) 1000.00 ml
The ferrous sulfate solution is not stable and should be freshly prepared.
