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Main sol. 1030

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Internal Solution-ID: S2089

Equipment needed: Autoclave Hungate tubes Filter Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
NH4Cl 1.0 g 0.978 18.293
MgCl2 x 6 H2O 1.0 g 0.978 4.813
CaCl2 x 2 H2O 0.4 g 0.391 2.662
KH2PO4 0.4 g 0.391 2.876
Wolfe's mineral elixir 1.0 ml - -
Yeast extract 2.0 g 1.957 -
Trypticase peptone
(BD BBL) 		2.0 g 1.957 -
Sodium resazurin
(0.1% w/v) 		0.5 ml 4.9e-4 0.002
Na2CO3 1.0 g 0.978 9.232
Na-crotonate solution (1 M) 20.0 ml - -
2-Mercaptoethanesulfonic acid
(coenzyme M) 		0.5 g 0.489 3.44
Wolin's vitamin solution (10x) 1.0 ml - -
Na2S x 9 H2O 0.3 g 0.294 1.222
Distilled water 1000.0 ml - -
1 Dissolve ingredients (except carbonate, crotonate, coenzyme M, vitamins and sulfide), then 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 Hungate-type tubes or serum vials (e.g., 5 ml medium in Balch-type tubes) and autoclave. Add crotonate, coenzyme M, vitamins 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 mixture. Stock solutions of crotonate, coenzyme M and vitamins should be sterilized by filtration. Adjust pH of the complete medium to 7.0 - 7.2.
2 Note: Use 10% (v/v) as inoculum.
1030 SMITHELLA MEDIUM