Main sol. 1030

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-Mercaptoethanesulfonate (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