# J513: CALDOCOCCUS MEDIUM


## Main sol. J513

0.1	g	NaCl	
0.1	g	CaCl2 x 2 H2O	
0.05	g	FeSO4 x 7 H2O	
0.1	g	Yeast extract (BD-Difco)	
0.1	g	Casamino acids (BD-Difco)	
0.8	g	Peptone	
10	ml	**Trace vitamins**	
1	ml	**Trace mineral solution**	
2	g	Sulfur	
1	mg	Resazurin	
0.5	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Mix ingredients except vitamins, sulfur and Na2S x 9H2O, adjust pH to 2.7 with H2SO4 and autoclave. Separately autoclave 5% (w/v) Na2S x 9H2O solution under a N2 atmosphere. Steam sulfur for 3 hr on each of 3 successive days. Filter-sterilize Trace vitamins solution. Distribute the medium into culture vessels (e.g., 20 ml per 100 ml serum bottles) containing Trace vitamins and sulfur, replace the gas phase with a H2-CO2 (80:20) gas atmosphere and seal with butyl rubber stoppers. Prior to inoculation, add sterile Na2S x 9H2O solution and adjust pH to 3.0 with sterile 1 N H2SO4. Pressurize the inoculated bottle to 200 kPa H2-CO2 (80:20).


## Trace mineral solution

5	g	MnSO4 x n H2O	
1	g	CoSO4 x 7 H2O	
1	g	ZnSO4 x 7 H2O	
0.1	g	CuSO4 x 5 H2O	
0.1	g	H3BO3	
0.1	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	


## 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