# 1630a: NITROSOPUMILUS MEDIUM


## Main sol. 1630a

26	g	NaCl	
5	g	MgSO4 x 7 H2O	
5	g	MgCl2 x 6 H2O	
1.5	g	CaCl2 x 2 H2O	
0.1	g	KBr	
2	ml	KH2PO4 (0.1% w/v)	
10	ml	**HEPES buffer solution**	
0.05	g	NH4Cl	
1	ml	**Trace element solution SL-11**	
0.05	g	Na-pyruvate	
0.25	g	NaHCO3	
10	ml	**Wolin's vitamin solution**	
1000	ml	Distilled water	

1. Dissolve ingredients (except trace elements, HEPES buffer, ammonium chloride, pyruvate, bicarbonate and vitamins), dispense medium into Erlenmeyr flasks with screw caps (e.g., 20 ml medium per 100 ml flask) and autoclave. Add HEPES buffer and ammonium chloride from stock solutions sterilized by autoclaving. Trace elements, pyruvate, bicarbonate and vitamins are added from stock solutions sterilized by filtration. Adjust pH of the complete medium to 7.2 using sterile solutions of bicarbonate (5% w/v) or 1 N HCl.
2. Note: Use at least 5% (v/v) inoculum and incubate in the dark without shaking.


## HEPES buffer solution

24	g	NaOH (pellets)	
238.4	g	HEPES (free acid)	
1000	ml	Distilled water	

1. Fist dissolve NaOH in ca 600 ml water, then add HEPES, adjust pH to 7.5 and fill up to 1000 ml volume.


## Trace element solution SL-11

5.2	g	Na2-EDTA x 2 H2O	
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	
1000	ml	Distilled water	

1. Dissolve EDTA in 800 ml distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add other compounds. Finally adjust pH to 6.0 and bring volume to 1000 ml. 


## Wolin's vitamin solution

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 D-(+)-pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water