# 1712: AROMATOLEUM MEDIUM


## Main sol. 1712

0.5	g	KH2PO4	
0.3	g	NH4Cl	
0.5	g	MgSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.6	g	NaNO3	
950	mL	Distilled water	

1. The medium was autoclaved and removed from the autoclave at 85°C. Oxygen dissolution was prevented by exchanging the gas-phase with N2. The medium was complemented by addition of the following stock solutions: 

1	mL/L	**Trace element solution**	
1	mL/L	**Vitamin mixture**	
1	mL/L	**Vitamin B12 solution**	
40	mL/L	**Bicarbonate solution (1M)**	

2. After adding bicarbonate the gas was changed from N2 to N2/CO2 (90/10 %, v/v) and the pH was adjusted to 7.2 - 7.4 with sterile 2 M HCL. 

4	mL/L	**Ascorbate solution (1M)**	
4	mL/L	**Benzoate solution (1M)**	


## Trace element solution

5.2	g	Na2-EDTA	
2.1	g	FeSO4 x 7 H2O	
0.03	g	H3BO3	
0.1	g	MnCl2 x 4 H2O	
0.19	g	CoCl2 x 6 H2O	
0.024	g	NiCl2 x 6 H2O	
0.029	g	CuSO4 x 5 H2O	
0.144	g	ZnSO4 x 7 H2O	
0.036	g	Na2MoO4 x 2 H2O	

1. The trace element solution was prepared by dissolving the salts in 800 mL warm H2Obidest
2. NaOH was used to adjust the pH to 6.5. The solution was added up to 1000 mL with H2Obidest and autoclaved.


## Vitamin mixture

100	mL	Na2HPO4-NaH2PO4 buffer, 25 mM, pH 7.1	
4	mg	4-Aminobenzoic Acid	
1	mg	D-(+)-biotin	
10	mg	Nicotinic acid	
5	mg	Calcium D-(+)-pantothenate	
15	mg	Pyridoxal hydrochloride	
1.5	mg	DL-alpha-lipoic acid	
4	mg	Folic acid	
25	mg	2-Mercaptoethanesulfonic acid, sodium salt	

1. The solution was filter-sterilized and stored at 4°C in the dark.


## Vitamin B12 solution

2.5	mg	Cyanocobalamine	
50	mL	Double distilled water	

1. The solution was filter-sterilized and stored at 4°C in the dark.


## Bicarbonate solution (1M)

84	g	NaHCO3	
1000	mL	Double distilled water	

1. The solution was dispensed in the required portions (40 mL per 1 L medium). The head space of the bottles was flushed with CO2. Saturation of CO2 in the solution was achieved by repeated flushing and shaking of the bottles. Finally, the bottles were sealed with rubber stoppers and aluminum crimps and autoclaved.


## Ascorbate solution (1M)

17.6	g	Ascorbic acid	
100	mL	Double distilled water	

1. In an ice bath ascorbic acid was dissolved in 80 mL anoxic H2Obidest (autoclaved, cooled under N2 atmosphere) while flushing with N2. The pH was adjusted to 7.0 with NaOH and the solution added up to 100 ml with anoxic H2Obidest. After filter-sterilization the solution was stored under N2 atmosphere in a special flask (which can be flushed with gas) at 4°C in the dark.


## Benzoate solution (1M)

14.4	g	Sodiumbenzoate	
100	mL	Double distilled water	

1. The solution was filter-sterilized and stored at room temperature.