Main sol. 399a

Equipment needed: Autoclave Gassing station Serum bottle Filter

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
KCl	0.34	g	0.339	4.547
MgCl₂ x 6 H₂O	4.00	g	3.988	19.616
MgSO₄ x 7 H₂O	0.14	g	0.14	0.566
NH₄Cl	0.25	g	0.249	4.66
CaCl₂ x 2 H₂O	0.14	g	0.14	0.949
K₂HPO₄	0.14	g	0.14	0.801
NaCl	18.00	g	17.946	307.087
Trace element solution SL-10	1.00	ml	-	-
Selenite-tungstate solution	1.00	ml	-	-
Yeast extract (OXOID)	0.50	g	0.499	-
Sodium resazurin (0.1% w/v)	0.50	ml	5.0e-4	0.002
Na₂CO₃	0.50	g	0.499	4.703
Na-pyruvate	1.00	g	0.997	9.06
Na₂SO₃	0.60	g	0.598	4.746
Wolin's vitamin solution (10x)	1.00	ml	-	-
DL-Dithiothreitol	0.50	g	0.499	3.231
Distilled water	1000.00	ml	-	-
1 Dissolve ingredients except for carbonate, pyruvate, sulfite (Na₂SO₃), vitamins, and dithiothreitol, then sparge medium with 80% N₂ and 20% CO₂ gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into serum vials (e.g., 20 ml per 100 ml bottle), seal, and autoclave. Before use add carbonate from a sterile anoxic stock solution prepared under 80% N₂ and 20% CO₂ gas atmosphere. Pyruvate, sulfite, vitamins, and dithiothreitol are added from sterile anoxic stock solutions prepared under 100% N₂ gas and sterilized by filtration. Adjust the pH of the complete medium to 6.5, if necessary.

399a	ARCHAEOGLOBUS NEPTUNIUS MEDIUM