培養基配方
Final pH: 6.8 - 7.0
Final volume: 1002 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
Brain heart infusion (BD Bacto) 6.00 g
Proteose peptone (BD Difco) 6.00 g
Yeast extract (OXOID) 2.00 g
Na-acetate 1.00 g
Na-formate 2.00 g
Sodium resazurin (0.1% w/v) 0.50 ml
NaHCO3 4.00 g
Seven vitamins solution 1.00 ml
L-Cysteine HCl x H2O 0.50 g
Na2S x 9 H2O 0.50 g
Distilled water 1000.00 ml
1. Dissolve ingredients except bicarbonate, vitamins, 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 and adjust pH to 6.5. Then 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 vitamins, cysteine, and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. The vitamin solution should be sterilized by filtration. Before use, check the complete medium's pH and adjust it to 6.8 - 7.0, if necessary.
2. 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 862, DSM 1093, DSM 1125, DSM 11977, DSM 11978, DSM 11995, DSM 16643, DSM 116060, DSM 118696: Supplement medium after autoclaving with 0.50 g/l coenzyme M (2-mercaptoethanesulfonic acid) and 0.30 g/l DTT (DL-Dithiothreitol) added from filter-sterilized anoxic stock solutions prepared under N2. Omit sulfide and cysteine from the medium.
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 2640, DSM 2702, DSM 3823, DSM 4140, DSM 4179, DSM 4273, DSM 4274, DSM 10196: Increase amount of Na-acetate to 4.00 g/l.
For DSM 5522: Supplement medium with 5.0 g/l D-glucose added after autoclaving from a sterile anoxic stock solution.
For DSM 15163: Adjust pH of complete medium to 6.0.
For DSM 16632: Supplement medium after autoclaving with 50 ml/l clarified rumen fluid and 0.3 g/l DTT (DL-dithiothreitol). Omit Na2S x 9 H2O.
For DSM 115763, DSM 115764, DSM 115765: Supplement medium after autoclaving with 0.30 g/l DTT (DL-dithiothreitol), 10.00 ml/l methanol (50 % v/v), and 0.50 g/l coenzyme M (2-mercaptoethanesulfonic acid) added from filter-sterilized anoxic stock solutions prepared under N2. Omit Na2S x 9 H2O and L-cysteine HCl x H2O.
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.
Seven vitamins solution (from medium 503)
Vitamin B12 100.00 mg
p-Aminobenzoic acid 80.00 mg
D-(+)-biotin 20.00 mg
Nicotinic acid 200.00 mg
Calcium pantothenate 100.00 mg
Pyridoxine hydrochloride 300.00 mg
Thiamine-HCl x 2 H2O 200.00 mg
Distilled water 1000.00 ml
