# 1529: GEOGLOBUS AHANGARI MEDIUM


## Main sol. 1529

11.2	g	Ferric citrate (Aldrich F6129)	
19	g	NaCl	
9	g	MgCl2 x 6 H2O	
0.15	g	MgSO4 x 7 H2O	
0.3	g	CaCl2 x 2 H2O	
0.1	g	KCl	
0.6	g	KH2PO4	
0.1	g	(NH4)2SO4	
0.05	g	NaBr	
0.02	g	SrCl2	
1	ml	**Trace element solution SL-10**	
2.5	g	NaHCO3	
0.1	g	Yeast extract	
1	g	Na-pyruvate	
0.2	g	FeCl2 x 4 H2O	
1	ml	**Wolin's vitamin solution (10x)**	
0.1	g	L-Cysteine HCl x H2O	
1000	ml	Distilled water	

1. First, dissolve ferric citrate in water by heating, adjust to pH 5.8, then cool to room temperature. Add other compounds (except bicarbonate, yeast extract, pyruvate, vitamins, cysteine, and ferrous chloride) and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Then add cysteine and ferrous chloride and adjust pH to 6.8 - 7.0 with bicarbonate. Dispense medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Before inoculation add yeast extract, pyruvate, and vitamins, from sterile anoxic stock solutions prepared under 100% N2 gas. Stock solutions of pyruvate and vitamins should be sterilized by filtration. Adjust the pH of the complete medium to 6.8 - 7.0.
2. Note: Use 5 - 10% (v/v) inoculum. The color of the medium supernatant changes from orange-brown to yellow-greenish during growth.


## Trace element solution SL-10

10	ml	HCl (25%)	
1.5	g	FeCl2 x 4 H2O	
70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
6	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 x 2 H2O	
990	ml	Distilled water	

1. First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml.


## 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