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Main sol. 710

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Internal Solution-ID: S1460

Equipment needed: Autoclave Hungate tubes Filter Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
Tryptone
(BD Bacto) 		1.00 g 0.998 -
Peptone
(meat) 		1.00 g 0.998 -
Yeast extract
(BD Bacto) 		1.00 g 0.998 -
K2HPO4 1.60 g 1.597 9.168
NaH2PO4 x 2 H2O 1.00 g 0.998 8.318
NH4Cl 0.50 g 0.499 9.329
MgSO4 x 6 H2O 0.16 g 0.16 1.327
Trace element solution SL-11 1.00 ml - -
Sodium resazurin
(0.1% w/v) 		0.50 ml 5.0e-4 0.002
CaCl2 x 2 H2O 0.06 g 0.06 0.407
NaHCO3 1.00 g 0.998 11.88
D-Glucose 5.00 g 4.99 27.698
Wolin's vitamin solution (10x) 1.00 ml - -
L-Cysteine HCl x H2O 0.30 g 0.299 1.705
Na2S x 9 H2O 0.30 g 0.299 1.247
Distilled water 1000.00 ml - -
1 Dissolve ingredients (except calcium chloride, bicarbonate, glucose, vitamins, cysteine and sulfide), adjust pH to 7.0 and sparge medium with 100% N2 gas 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 sterilization add calcium chloride, glucose, cysteine and sulfide from sterile anoxic stock solutions autoclaved under 100% N2 gas atmosphere. Add bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Vitamins are prepared under 100% N2 gas and sterilized by filtration. The pH of the complete medium should be at 7.0.
2 Note: Supplementation of medium with 10.00 g/l MOPS buffer (pH 6.9 - 7.0; 10% (w/v) anoxic stock solution) may enhance the buffer capacity of the medium.
710 THERMOANAEROBACTER (KoKo) MEDIUM