# J448: KA22 MEDIUM


## Main sol. J448

30	g	Sea Salt (Sigma)	
2.54	g	Mg(NO3)2 x 6 H2O	
0.3	g	KH2PO4	
1	g	NaHCO3	
1.95	g	MES	
12	g	Sulfur (powder)	
10	ml	**Trace minerals**	
0.5	ml	**Trace vitamins**	
0.5	g	Na2S x 9 H2O	
1	mg	Resazurin	

1. Mix ingredients except sulfur, NaHCO3 and Na2S x 9H2O, adjust pH to 6.5 and autoclave. Filter-sterilize 8% NaHCO3 solution and add to the medium. Steam sulfur for 3 hr on each of 3 successive days. Separately autoclave 5% Na2S x 9H2O solution under a N2 atmosphere. Aseptically distribute the medium into culture vessels containing appropriate amount of sulfur (e.g., 20 ml culture medium in 120 ml serum bottle containing 0.24 g sulfur) under a H2-CO2 (4:1, v/v) gas mixture and seal with butyl rubber stoppers. Prior to inoculation, add the sterile Na2S x 9H2O solution and readjust pH to 6.5, if necessary. Pressurize the inoculated bottles to 200 kPa H2-CO2 (4:1, v/v).


## Trace minerals

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x n H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.1	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.01	g	AlK(SO4)2	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. Dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH solution. Then proceed to add minerals. Adjust final pH to 7.0.


## Trace vitamins

2	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Thiamine HCl	
5	mg	Riboflavin	
5	mg	Nicotinic acid	
5	mg	Calcium pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	Lipoic acid	
1000	ml	Distilled water