# 902: CARCBOXYDIBRACHIUM MEDIUM


## Main sol. 902

20	g	NaCl	
0.7	g	KCl	
3.9	g	MgSO4 x 7 H2O	
0.4	g	CaCl2 x 2 H2O	
0.3	g	NH4Cl	
0.15	g	Na2HPO4	
0.03	g	Na2SiO3	
0.05	g	Yeast extract	
1	ml	**Trace element solution SL-10**	
0.5	ml	Sodium resazurin (0.1% w/v)	
0.5	g	NaHCO3	
2.5	g	Na-pyruvate	
1	ml	**Wolin's vitamin solution (10x)**	
0.45	g	L-Cysteine HCl x H2O	
0.45	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients except bicarbonate, pyruvate, vitamins and reducing agents, then adjust pH of solution to 7.0 with NaOH. Sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic, then dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add pyruvate, vitamins, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Vitamins should be sterilized by filtration. Adjust pH of complete medium to 7.0, if necessary.


## 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.


## 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