Main sol. 1210b

Equipment needed: Autoclave Hungate tubes Centrifuge Filter Gassing station

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
NaCl	18.00	g	17.946	307.087
MgCl₂ x 6 H₂O	4.00	g	3.988	19.616
KCl	0.33	g	0.329	4.413
CaCl₂ x 2 H₂O	0.33	g	0.329	2.238
(NH₄)₂SO₄	0.50	g	0.499	3.773
Trace element solution SL-10	1.00	ml	-	-
Selenite-tungstate solution	1.00	ml	-	-
Sodium resazurin (0.1 % w/v)	0.50	ml	5.0e-4	0.002
Na₂CO₃	1.00	g	0.997	9.407
KH₂PO₄	0.33	g	0.329	2.418
FeCl₂ x 4 H₂O	0.52	g	0.518	2.608
Yeast extract	0.20	g	0.199	-
Wolin's vitamin solution (10x)	1.00	ml	-	-
L-Cysteine HCl x H₂O	0.10	g	0.1	0.568
Amorphous Fe(OH)3	100.00	ml	-	-
Distilled water	900.00	ml	-	-
1 Dissolve ingredients except carbonate, hydrogenphosphate, ferrous chloride, yeast extract, vitamins, cysteine and ferric iron hydroxide sludge. Suspend pellet of ferric iron hydroxide in medium and sparge with 80% H₂ and 20% CO₂ gas mixture for 30 - 45 min to make it anoxic. Thereafter, dispense suspension under 80% H₂ and 20% CO₂ gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave for 30 min. Add hydrogenphosphate, ferrous chloride (dissolved in 0.1 N HCl), yeast extract, cysteine and vitamins 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. Vitamins and ferrous chloride should be sterilized by filtration. The pH of the complete medium should be at 5.5.

PYRODICTIUM DELANEYI MEDIUM