# J378: ALKALIPHILUS CROTONATOXIDANS MEDIUM


## Main sol. J378

2	g	Yeast extract (BD-Difco)	
2	g	Tryptone (BD-Difco)	
0.86	g	Crotonic acid	
0.5	g	KH2PO4	
0.33	g	MgCl2 x 6 H2O	
0.4	g	NaCl	
0.4	g	NH4Cl	
0.05	g	CaCl2 x 2 H2O	
10	ml	**Trace metal solution**	
10	ml	**Trace vitamins**	
3.5	g	NaHCO3	
0.5	g	L-Cysteine HCl x H2O	
0.5	g	Na2S x 9 H2O	
1	mg	Resazurin	

1. Dissolve ingredients, except crotonic acid, NaHCO3, L-cysteine x HCl x H2O and Na2S x 9H2O, in 900 ml of distilled water and adjust pH to 7.0-7.5. Bring to a boil for 5-10 sec, then, cool down under a stream of N2-CO2 (80:20, v/v) and add NaHCO3 to the medium. Dispense 0.9 volume of the medium into suitable culture vessels under a stream of N2-CO2 (80:20, v/v) and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight. Add 0.1 volume of a filter-sterilized crotonic acid solution (0.86 g/100 ml, pH 7.0) to the medium. Autoclave cysteine x HCl and Na2S x 9H2O as 5% solutions, respectively, under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add the solutions of crotonic acid, L-cysteine x HCl x H2O and Na2S x 9H2O to the medium.


## Trace metal solution

1	g	MnSO4 x n H2O	
0.8	g	Fe(NH4)2(SO4)2 x 6 H2O	
0.2	g	CoCl2 x 6 H2O	
0.2	g	ZnSO4 x 7 H2O	
0.02	g	CuCl2 x 2 H2O	
0.02	g	NiCl2 x 6 H2O	
0.02	g	Na2MoO4 x 2 H2O	
0.02	g	Na2SeO4	
0.02	g	Na2WO4	
2	g	Nitrilotriacetic acid	
1000	ml	Distilled water	

1. Adjust pH to 6.0 with KOH.


## 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