# 977: VULCANITHERMUS MEDIUM


## Main sol. 977

0.33	g	NH4Cl	
0.33	g	MgCl2 x 6 H2O	
0.33	g	CaCl2 x 2 H2O	
0.33	g	KCl	
0.33	g	KNO3	
25	g	NaCl	
3.6	g	PIPES (12mM)	
0.5	g	Yeast extract	
1	g	Tryptone	
1	g	Sucrose	
1	ml	**Modified Wolin's mineral solution II**	
1	ml	**Wolin's vitamin solution**	
1000	ml	Distilled water	

1. Prepare the medium anaerobically, under nitrogen, omitting the CaCl2, MgCl2, KNO3, tryptone, yeast extract, vitamins and sucrose. The pH should be 6.8. Dispense the medium into vessels suitable for anaerobic growth (Hungate tubes or serum bottles) under an atmosphere of nitrogen and autoclave. To the sterile, cooled medium add, from sterile stock solutions the CaCl2, MgCl2, KNO3, tryptone, yeast extract, vitamins and sucrose. The CaCl2, MgCl2, KNO3, tryptone, yeast extract, and sucrose stock solutions should be autoclaved, while the vitamin solution is sterile filtered. This strain grows anaerobically in the presence of nitrate, but aerobic growth has been reported provided the oxygen concentration does not exceed 16% (v/v). Under anaerobic conditions a redox agent, such as sulphide is not required.


## Wolin's vitamin solution

2	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Thiamine HCl	
5	mg	Riboflavin	
5	mg	Nicotinic acid	
5	mg	Calcium D-(+)-pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water	


## Main sol. 534

30	g	NaCl	
7	g	MgSO4 x 7 H2O	
5.5	g	MgCl2 x 6 H2O	
0.65	g	KCl	
0.1	g	NaBr	
0.15	g	NH4Cl	
0.15	g	K2HPO4	
0.5	g	CaCl2 x 2 H2O	
10	ml	**Trace element solution**	
1000	ml	Distilled water	

1. Sparge medium with 100% N2 gas mixture for 20 min to make it anoxic.
2. Adjust pH to 6.5-6.8 with 1N H2SO4.
3. Fill 10 ml of medium in serum bottles (100 ml) under a protective H2/CO2 atmosphere and tightly stopper.
4. Autoclave at 121 °C for 15 min.
5. after autoclaving add the following filter-sterilized solutions.

30	ml/l	**Solution A** (gassed with CO2), 0.3 ml/10ml	
30	ml/l	**Solution B** (gassed with N2), 0.3 ml/10ml	

6. apply xx bar H2/CO2 (80%/20%) + yy % O2 
(Note: strain specific conditions need to be applied)
7. For more details on the medium see the original description (Huber et al. System. Appl. Microbiol. 15:340-351 (1992)).


## Trace element solution

1000	ml	**Modified Wolin's mineral solution II**	

1. To 1000.0 ml trace element solution in medium 141 add:  

2	g	(NH4)2Ni(SO4)2	
10	mg	Na2WO4	
10	mg	Na2SeO4	


## Solution A

2	g	NaHCO3	
30	ml	Distilled water	

1. prepared under CO2 in an ice-bath, filter-sterilise


## Solution B

2	g	Na2S2O3 x 5 H2O	
30	ml	Distilled water	

1. Prepared under N2,autoclave 121°C, 15 min


## Modified Wolin's mineral solution II

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.18	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.18	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.02	g	AlK(SO4)2 x 12 H2O	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
0.025	g	NiCl2 x 6 H2O	
0.3	mg	Na2SeO3 x 5 H2O	
1000	ml	Distilled water	

1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.