# 792c: NAUTILIA NITRATIREDUCENS MEDIUM


## Main sol. 792c

958	ml	**Solution A**	
10	ml	**Solution B**	
20	ml	**Solution C**	
1	ml	**Solution D**	
15	ml	**Solution E**	

1. Sparge solution A with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic serum vials (e.g., 20 ml medium in 100 ml bottles) and autoclave. Solutions B and C are autoclaved separately under 80% N2 and 20% CO2 gas mixture. Solution D is prepared under 100% N2 gas atmosphere and sterilized by filtration. Solution E is autoclaved under 100% N2 gas atmosphere.
To complete the medium appropriate amounts of solutions B to E are added to solution A. Adjust pH of the complete medium to 6.5, if necessary.
2. After inoculation pressurize vials to 1 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture.
3. Note: Solution A has to be prepared freshly. It cannot be stored for a long period of time.


## Solution A

20	g	NaCl	
3.5	g	MgSO4 x 7 H2O	
2.75	g	MgCl2 x 6 H2O	
0.75	g	CaCl2 x 2 H2O	
0.33	g	KCl	
0.05	g	NaBr	
15	mg	H3BO3	
7	ml	SrCl2 x 6 H2O (0.1% w/v)	
0.5	ml	KI (0.01% w/v)	
1	ml	**Wolfe's mineral elixir**	
950	ml	Distilled water	


## Solution B

1	g	KNO3	
0.5	g	KH2PO4	
10	ml	Distilled water	


## Solution C

1	g	Na2CO3	
20	ml	Distilled water	


## Solution D

1	ml	**Wolin's vitamin solution (10x)**	


## Solution E

0.45	g	Na2S x 9 H2O	
15	ml	Distilled water	


## Wolfe's mineral elixir

30	g	MgSO4 x 7 H2O	
5	g	MnSO4 x H2O	
10	g	NaCl	
1	g	FeSO4 x 7 H2O	
1.8	g	CoCl2 x 6 H2O	
1	g	CaCl2 x 2 H2O	
1.8	g	ZnSO4 x 7 H2O	
0.1	g	CuSO4 x 5 H2O	
0.18	g	AlK(SO4)2 x 12 H2O	
0.1	g	H3BO3	
0.1	g	Na2MoO4 x 2 H2O	
2.8	g	(NH4)2Ni(SO4)2 x 6 H2O	
0.1	g	Na2WO4 x 2 H2O	
0.1	g	Na2SeO4	
1000	ml	Distilled water	

1. First adjust pH to 1.0 with diluted H2SO4, then add and dissolve the salts.


## Wolin's vitamin solution (10x)

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	Calcium D-(+)-pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
50	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water