Main sol. 135a

Equipment needed: Autoclave Hungate tubes Filter Gassing station

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
NaCl	20.00	g	19.589	335.192
NH₄Cl	1.00	g	0.979	18.311
KH₂PO₄	0.33	g	0.323	2.375
K₂HPO₄	0.45	g	0.441	2.53
MgSO₄ x 7 H₂O	0.10	g	0.098	0.397
Modified Wolin's mineral solution	20.00	ml	-	-
Yeast extract	2.00	g	1.959	-
Resazurin (0.1% w/v)	0.50	ml	4.9e-4	0.002
NaHCO₃	5.00	g	4.897	58.295
Ethylene glycol	1.25	g	1.224	19.725
Wolin's vitamin solution (10x)	1.00	ml	-	-
L-Cysteine HCl x H₂O	0.50	g	0.49	2.788
Na₂S x 9 H₂O	0.50	g	0.49	2.039
Distilled water	1000.00	ml	-	-
1 Dissolve ingredients except bicarbonate, ethylene glycol, vitamins, cysteine and sulfide, bring to the boil and cool to room temperature under 80% N₂ and 20% CO₂ gas mixture. Add bicarbonate (solid) and equilibrate the medium with the gas until a pH of around 7.4 is reached. Then distribute under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Before use add ethylene glycol, vitamins (sterilized by filtration), cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N₂ gas. Adjust pH of complete medium to 7.0 - 7.2, if necessary.

135a	ACETOBACTERIUM MEDIUM (MARINE)