# 991: CALDISPHAERA MEDIUM


## Main sol. 991

1.3	g	(NH4)2SO4	
0.28	g	KH2PO4	
0.25	g	MgSO4 x 7 H2O	
0.07	g	CaCl2 x 2 H2O	
0.02	g	FeCl3 x 6 H2O	
10	ml	**Allen's trace element solution**	
2.94	g	Trisodium citrate x 2 H2O	
0.5	ml	Sodium resazurin (0.1% w/v)	
10	g	Sulfur (powdered)	
0.5	g	Yeast extract (OXOID)	
1	ml	**Wolin's vitamin solution (10x)**	
0.5	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients (except sulfur, yeast extract, vitamins and sulfide), adjust pH to 3.5 with 10 N H2SO4 and sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials (e.g., 20 ml medium in 100 ml bottles) containing already the appropriate amount of sulfur. Sterilize medium by heating cultivation vessels in a water bath to 90 - 100°C for 1 - 2 hours on each of 3 successive days. Add yeast extract, vitamins (sterilized by filtration) and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Adjust pH of complete medium to 4.0 - 4.5, if necessary.
2. After inoculation pressurize vials to 1 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture.


## Allen's trace element solution

180	mg	MnCl2 x 4 H2O	
450	mg	Na2B4O7 x 10 H2O	
22	mg	ZnSO4 x 7 H2O	
5	mg	CuCl2 x 2 H2O	
3	mg	Na2MoO4 x 2 H2O	
3	mg	VOSO4 x 2 H2O	
1	mg	CoSO4 x 7 H2O	
1000	ml	Distilled water	

1. Adjust pH of final solution to 2 with 1 N HCl.


## 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