# 540: ACETITOMACULUM MEDIUM


## Main sol. 540

0.2	g	KH2PO4	
0.1	g	Na2SO4	
0.3	g	NH4Cl	
0.5	g	KCl	
7	g	NaCl	
1.2	g	MgCl2 x 6 H2O	
0.15	g	CaCl2 x 2 H2O	
1.5	g	Yeast extract	
10	ml	**Modified Wolin's mineral solution**	
1	ml	NiCl2 x 6 H2O (0.1% w/v)	
0.1	ml	Na2WO4 x 2 H2O (0.1% w/v)	
0.5	ml	Ascorbic acid (0.01% w/v)	
0.5	ml	Myo-inositol (0.01% w/v)	
0.5	ml	Niacinamide (0.01% w/v)	
0.5	ml	Choline chloride (0.01% w/v)	
0.5	ml	Sodium resazurin (0.1% w/v)	
4	g	NaHCO3	
5	g	D-Glucose	
1	ml	**Wolin's vitamin solution (10x)**	
0.3	g	L-Cysteine HCl x H2O	
0.3	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients (except bicarbonate, glucose, vitamins and reducing agents), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add solid bicarbonate and equilibrate pH to 6.8 with 80% N2 and 20% CO2 gas mixture, then dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add glucose, vitamins, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. Vitamins should be sterilized by filtration. Adjust pH of complete medium to 6.8 - 7.0, if necessary.


## 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