Main sol. 135

Equipment needed: Hungate tubes Autoclave Filter Gassing station

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
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	-
Sodium resazurin (0.1% w/v)	0.50	ml	4.9e-4	0.002
NaHCO₃	10.00	g	9.794	116.589
D-Fructose	10.00	g	9.794	54.365
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, fructose, 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 in anoxic Hungate-type tubes or serum vials and autoclave. Before use adjust the pH to 7.5 - 8.0 by adding a sterile anoxic stock solution of sodium carbonate (5% w/v) prepared under 80% N₂ and 20% CO₂ gas mixture and add fructose, vitamins (sterilized by filtration), cysteine, and sulfide from anoxic sterile stock solutions prepared under 100% N₂.
2 Note: For autotrophic growth D-fructose is omitted and a gas atmosphere of 80% H₂ and 20% CO₂ is used.

135	ACETOBACTERIUM MEDIUM