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Main sol. 287

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Internal Solution-ID: S554

Equipment needed: Hungate tubes Filter Autoclave Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
K2HPO4 0.30 g 0.297 1.704
KH2PO4 0.30 g 0.297 2.181
(NH4)2SO4 0.30 g 0.297 2.246
NaCl 0.61 g 0.603 10.325
CaCl2 x H2O 0.14 g 0.138 1.248
MgSO4 x 7 H2O 0.13 g 0.129 0.522
Modified Wolin's mineral solution 10.00 ml - -
NH4Cl 2.70 g 2.671 49.928
Na-acetate 2.50 g 2.473 30.144
Sodium resazurin
(0.1% w/v) 		0.50 ml 4.9e-4 0.002
NaHCO3 5.00 g 4.946 58.871
Wolin's vitamin solution (10x) 1.00 ml - -
L-Cysteine HCl x H2O 0.30 g 0.297 1.689
Na2S x 9 H2O 0.30 g 0.297 1.235
Distilled water 1000.00 ml - -
1 Dissolve ingredients (except bicarbonate, vitamins, cysteine and sulfide) and 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 6.8 and then dispense medium under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum bottles and autoclave. After sterilization add cysteine and sulfide from sterile anoxic stock solutions autoclaved under 100% N2 gas. Vitamins are prepared under 100% N2 gas atmosphere and sterilized by filtration. Adjust pH of complete medium to 6.8 - 7.0. For incubation use sterile 80% H2 and 20% CO2 gas mixture at one atmospheres of pressure.
287 METHANOCULLEUS CHIKUGOENSIS MEDIUM