# 1604: ATHALASSOTOGA MEDIUM


## Main sol. 1604

2.4	g	Ferric citrate monohydrate	
0.2	g	(NH4)2SO4	
3	g	KH2PO4	
0.5	g	MgSO4 x 7 H2O	
0.25	g	CaCl2 x 2 H2O	
0.5	g	Yeast extract (OXOID)	
10	ml	**Allen's trace element solution**	
0.26	g	FeCl2 x 4 H2O	
1	ml	**Wolin's vitamin solution (10x)**	
0.04	g	L-Cysteine HCl x H2O	
1000	ml	Distilled water	

1. First dissolve ferric citrate in water by heating and adjust to pH 6.0, then add other medium ingredients except ferrous chloride, vitamins and cysteine. Sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, then dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add ferrous chloride (dissolved in 0.1 N HCl), vitamins (sterilized by filtration) and cysteine from sterile anoxic stock solutions prepared under 100% N2 gas. Prior to inoculation check pH and adjust to 6.0, if necessary.


## Allen's trace element solution

180	mg	MnCl2 x 4 H2O	
450	mg	Na2B4O7 x 10 H2O	
22	mg	ZnSO4 x 7 H2O	
5	mg	CuCl2 x 2 H2O	
3	mg	Na2MoO4 x 2 H2O	
3	mg	VOSO4 x 2 H2O	
1	mg	CoSO4 x 7 H2O	
1000	ml	Distilled water	

1. Adjust pH of final solution to 2 with 1 N HCl.


## 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