# 1630c: modified Nitrosospaera medium


## Main sol. 1630c

1	g	NaCl	
0.4	g	MgCl2 x 6 H2O	
0.1	g	CaCl2 x 2 H2O	
0.2	g	KH2PO4	
0.5	g	KCl	
0.05	g	Na2SO4	
1	ml	**Trace element solution (Nitrososphaera)**	
1	ml	**FeNaEDTA solution (7.5 mM)**	
2	ml	**NaHCO3 buffer (1M)**	
10	ml	**HEPES buffer solution**	
2	ml	**NH4Cl (1 M)**	
1	ml	**Sodiumpyruvate (1 M)**	
1000	ml	Double distilled water	

1. Incubate all used glass ware and magnetic stirring rods in 0.1 M HCl and rinse 3x with double distilled water to remove residual detergents (always check visual for soap bubbles).
2. Autoclave the Basic-FWM and cool to room temperature. This medium should be free of any precipitations!
3. Aseptically, add per L of BASIC-FWM the following solutions from sterile stocks (do not store complemented FWM):
- Trace element solution (Nitrososphaera)
- FeNaEDTA solution
- NaHCO3 solution
- HEPES buffer solution
- NH4Cl solution
- Sodiumpyruvate solution
4. Adjust pH to 7.5.


## Trace element solution (Nitrososphaera)

8	mL	HCl (12.5M)	
30	mg	H3BO3	
100	mg	MnCl2 x 4 H2O	
190	mg	CoCl2 x 6 H2O	
24	mg	NiCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
144	mg	ZnSO4 x 7 H2O	
36	mg	Na2MoO4 x 2 H2O	
1000	ml	Double distilled water	

1. This solution contains no iron! Sterilise by autoclaving or filtration (0.2 µm) and store in the dark at 4 °C.


## 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.


## FeNaEDTA solution (7.5 mM)

2753	mg	FeNa-EDTA	
1000	ml	Double distilled water	

1. Sterilise by filtration (0.2 µm) and store in the dark at 4 °C.


## NaHCO3 buffer (1M)

4.2	g	NaHCO3	
50	ml	Double distilled water	

1. Dissolve 4.2 g of NaHCO3 in 50 mL Double distilled water (use a small beaker with stirring rod to dissolve) and sterilize by filtration (0.2 µm) in the laminar flow hood. Store in a sterile 50 mL falcon tube at 4 °C. Avoid high headspace to liquid ratios to ensure stable concentration.


## NH4Cl (1 M)

2.67	g	NH4Cl	
50	ml	Double distilled water	

1. Dissolve 2.67 g ammonium chloride in 50 mL Double distilled water.
2. Filtrate (0.2 µm).


## Sodiumpyruvate (1 M)

5.5	g	Sodium pyruvate	
50	ml	Double distilled water	

1. Dissolve 5.5 g in 50 mL and filter it through 0.2 µm sterile filter in a sterile falcon tube. Store at 4 °C