# 135: ACETOBACTERIUM MEDIUM


## Main sol. 135

1	g	NH4Cl	
0.33	g	KH2PO4	
0.45	g	K2HPO4	
0.1	g	MgSO4 x 7 H2O	
20	ml	**Modified Wolin's mineral solution**	
2	g	Yeast extract	
0.5	ml	Sodium resazurin (0.1% w/v)	
10	g	NaHCO3	
10	g	D-Fructose	
1	ml	**Wolin's vitamin solution (10x)**	
0.5	g	L-Cysteine HCl x H2O	
0.5	g	Na2S x 9 H2O	
1000	ml	Distilled water	

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.


## Modified Wolin's mineral solution

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.18	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.18	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.02	g	AlK(SO4)2 x 12 H2O	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
0.03	g	NiCl2 x 6 H2O	
0.3	mg	Na2SeO3 x 5 H2O	
0.4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.


## Wolin's vitamin solution (10x)

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	Calcium D-(+)-pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
50	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water