# 1210c: THERMOSULFURIPHILUS MEDIUM


## Main sol. 1210c

18	g	NaCl	
4	g	MgCl2 x 6 H2O	
0.33	g	KCl	
0.33	g	CaCl2 x 2 H2O	
0.5	g	(NH4)2SO4	
1	g	KNO3	
1	ml	**Trace element solution SL-10**	
1	ml	**Selenite-tungstate solution**	
0.5	ml	Sodium resazurin (0.1 % w/v)	
5	g	Sulfur (powder)	
1	g	Na2CO3	
0.33	g	KH2PO4	
3.5	g	Na2S2O3 x 5 H2O	
1	ml	**Wolin's vitamin solution (10x)**	
1000	ml	Distilled water	

1. Dissolve ingredients except sulfur, carbonate, hydrogen phosphate, thiosulfate, and vitamins, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials containing already the appropriate amount of sulfur and autoclave at <b>110°C</b> for 20 min. Add hydrogen phosphate, thiosulfate, and vitamins from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Stock solutions of thiosulfate and vitamins should be sterilized by filtration. The pH of the complete medium should be 6.5 - 6.8.


## Trace element solution SL-10

10	ml	HCl (25%)	
1.5	g	FeCl2 x 4 H2O	
70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
6	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 x 2 H2O	
990	ml	Distilled water	

1. First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml.


## Selenite-tungstate solution

0.5	g	NaOH	
3	mg	Na2SeO3 x 5 H2O	
4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	


## 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