# 1630e: Synthetic Crenarchaeota Medium (SCM)


## Basic salt water medium

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	**NaHCO3 buffer (1M)**	
10	ml	**HEPES buffer solution**	
5	ml	**KH2PO4 solution (2.93 nM)**	
1	ml	**Trace element solution (Nitrososphaera)**	
1	ml	**FeNaEDTA solution (7.5 mM)**	
2	ml	**NH4Cl (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 salt water medium and cool to room temperature. This medium should be free of any precipitations!
3. Aseptically, add per L of basic salt water medium the following solutions from sterile stocks (do not store complemented SCM): Trace element solution (Nitrososphaera), FeNaEDTA solution, NaHCO3 solution, HEPES buffer solution, NH4Cl solution, KH2PO4 solution
4. Adjust pH to 7.6.


## 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 (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.


## 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).


## KH2PO4 solution (2.93 nM)

0.4	g	KH2PO4	
1000	ml	Double distilled water	


## alpha-ketoglutamate solution (20mM)

20	mM	alpha-ketoglutamate	
1000	ml	Double distilled water