# 1800: Vulcanimicrobium alpinus medium


## Main sol. 1800

0.44	g	KH2PO4	
0.1	g	(NH4)2SO4	
0.1	g	MgSO4 x 7 H2O	
0.3	g	Yeast extract (Bacto™ Yeast Extract recommended), <br>      can be increased to 0.3% = 3g/L	
0.5	g	Peptone	
0.05	g	CaCl2 x 2 H2O	
1	g/l	Glucose	
1	ml	**Trace element solution SL-8**	
1000	ml	Distilled water	

1. Adjust pH to 6.0.
2. aliquot 5 mL medium into 50 mL serum bottles
3. add 10% CO2 to gasphase (air)
4. Autoclave at 121 °C for 15 min.
5. add vitamins solutions from 1000 x stocks

0.25	ml/l	**Vitamin solution A (1000 x stock)**	
0.25	ml/l	**Vitamin solution B (1000 x stock)**	


## Vitamin solution A (1000 x stock)

10	mg	Biotin	
10	mg	Riboflavin	
100	mg	Thiamine HCl	
100	mg	Thiamine pyrophosphate	
100	mg	L-Ascorbic acid	
100	mg	Calcium pantothenate	
100	mg	Folic acid	
100	mg	Nicotinamide	
100	mg	Nicotinic acid	
100	mg	p-Aminobenzoic acid	
100	mg	Pyridoxine hydrochloride	
100	mg	Lipoic acid	
100	mg	beta-NAD	
100	ml	Potassium phosphate buffer (pH 7.2, 10 mM)	

1. Dissolve the vitamins in 10mM phosphate buffer, pH7.2.
2. Titrate with NaOH until vitamins are dissolved, filter sterilize afterwards, and freeze in appropriate aliquots until usage.


## Vitamin solution B (1000 x stock)

100	mg	Vitamin B12	
100	ml	Distilled water	

1. Dissolve the vitamin B12 in water.
2. Titrate with HCl until vitamin B12 is dissolved, filter sterilize afterwards, and freeze in appropriate aliquots until usage.


## Trace element solution SL-8

5.2	g	Na2-EDTA	
1.5	g	FeCl2 x 4 H2O	
70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
62	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
17	mg	CuCl2 x 2 H2O	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoSO4 x 2 H2O	
1000	ml	Distilled water	

1. pH 6.8