培養(yǎng)基配方
NH4Cl 1.00 g
K2HPO4 0.30 g
KH2PO4 0.30 g
MgCl2 x 6 H2O 0.20 g
CaCl2 x 2 H2O 0.10 g
KCl 0.10 g
NaCl 0.60 g
Yeast extract 0.50 g
Na-acetate x 3 H2O 0.50 g
Trace element solution SL-10 1.50 ml
Sodium resazurin (0.1% w/v) 0.50 ml
Na2CO3 1.50 g
3,4,5-Trimethoxycinnamic acid (TMC) 1.19 g
Wolin's vitamin solution (10x) 1.00 ml
L-Cysteine HCl x H2O 0.30 g
Na2S x 9 H2O 0.30 g
Distilled water 1000.00 ml
Dissolve ingredients (except carbonate, vitamins, TMC and reducing agents) and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After autoclaving add vitamins, TMC, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. The vitamin solution should be sterilized by filtration. The stock solution of TMC (6% w/v) should be neutralized with NaOH prior to sterilization by filtration. Adjust pH of complete medium to 7.0, if necessary.
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.
Wolin's vitamin solution (10x) (from medium 120)
Biotin 20.00 mg
Folic acid 20.00 mg
Pyridoxine hydrochloride 100.00 mg
Thiamine HCl 50.00 mg
Riboflavin 50.00 mg
Nicotinic acid 50.00 mg
Calcium D-(+)-pantothenate 50.00 mg
Vitamin B12 1.00 mg
p-Aminobenzoic acid 50.00 mg
(DL)-alpha-Lipoic acid 50.00 mg
Distilled water 1000.00 ml
