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Main sol. 924

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Internal Solution-ID: S1899

Equipment needed: Autoclave Filter Gassing station Serum bottle
Compound Amount Unit Conc. [g/L] Conc. [mM]
NaCl 10.00 g 9.891 169.254
MgCl2 x 6 H2O 1.00 g 0.989 4.865
KCl 0.50 g 0.495 6.634
CaCl2 x 2 H2O 0.40 g 0.396 2.691
K2HPO4 0.40 g 0.396 2.272
NH4Cl 1.00 g 0.989 18.492
Modified Wolin's mineral solution 10.00 ml - -
Yeast extract 2.00 g 1.978 -
Trypticase peptone
(BD BBL) 		2.00 g 1.978 -
Sodium resazurin
(0.1% w/v) 		0.50 ml 4.9e-4 0.002
NaHCO3 4.00 g 3.956 47.097
Na-acetate 1.60 g 1.583 19.292
Na-formate 6.80 g 6.726 98.902
Wolin's vitamin solution (10x) 1.00 ml - -
L-Cysteine HCl x H2O 0.25 g 0.247 1.408
Na2S x 9 H2O 0.25 g 0.247 1.03
Distilled water 1000.00 ml - -
1 Dissolve ingredients (except bicarbonate, acetate, formate, vitamins and reducing agents), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add solid bicarbonate, adjust pH to 6.8 - 7.0, dispense medium under 80% N2 and 20% CO2 gas atmosphere into anoxic serum bottles and autoclave. Add acetate, formate, vitamins (sterilized by filtration), cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. The pH of the complete medium should be 7.2.
924 METHANOCALCULUS MB MEDIUM