# 817: THERMOSPHAERA MEDIUM


## Main sol. 817

2.2	g	MgCl2 x 6 H2O	
0.9	g	NaCl	
17	mg	KCl	
12	mg	NH4Cl	
7	ml	CaCl2 x 2 H2O (0.1% w/v)	
7	ml	K2HPO4 x 3 H2O (0.1% w/v)	
0.5	ml	FeCl3 (0.01% w/v in 0.2 N HCl)	
1	g	Yeast extract (BD Bacto)	
1	g	Peptone (BD Bacto)	
0.5	ml	Sodium resazurin (0.1% w/v)	
0.5	g	Na2CO3	
1	ml	**Wolin's vitamin solution (10x)**	
0.5	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients except carbonate, vitamins and sulfide. Sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add vitamins and sulfide 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 atmosphere. Vitamins should be sterilized by filtration. The pH of the complete medium should be adjusted to 6.5.
2. After inoculation pressurize tubes or bottles (heavy walled) to 2 bar overpressure with sterile 80% N2 and 20% CO2 gas mixture.


## 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