# 1690: NITROSPIRA INOPINATA MEDIUM


## Main sol. 1690

1	g	KH2PO4	
1.5	g	KCl	
1	g	MgSO4 x 7 H2O	
11.68	g	NaCl	
1000	ml	Distilled water (MilliQ)	
4	g	CaCO3	

1. Dilute stock solution with MilliQ water (1+19 v/v), add 4 g CaCO3 per litre and autoclave. After autoclaving add sterile solutions:

1	ml	**Trace element solution SL-10**	
1	ml	**Selenite-tungstate solution**	
1	ml	**Borate-copper solution**	
10	ml	**Ammonium chloride solution**	

2. Complete medium can be stored at 4°C.
3. Check pH 7.6-8.0


## Borate-copper solution

54	mg	H3BO3	
18	mg	CuCl2 x 2 H2O	
1000	ml	Distilled water	

1. Incubate at 42°C without shaking. Activity and growth can be followed by measuring the depletion of ammonium or the production of nitrite and/or nitrate. Higher ammonium concentrations are inhibitive. For that reason our recommendation is follow nitrite content. As soon as nitrite has been produced and is depleting again (to form nitrate) another dose of ammonium can be added. Incubation time may be up to several weeks. We checked the nitrite content (absence/presence) every 5-7 days and added ammonium chloride about every 2-3 weeks. Don't expose cultures to intense light.


## Trace element solution SL-10

10	ml	HCl (25%)	
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	
990	ml	Distilled water	

1. First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml.


## Selenite-tungstate solution

0.5	g	NaOH	
3	mg	Na2SeO3 x 5 H2O	
4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	


## Ammonium chloride solution

0.8	g	Ammonium chloride	
100	ml	Distilled water