# 1508: CALDERIHABITANS MEDIUM


## Main sol. 1508

21	g	Sea Salt (SIGMA)	
0.1	g	K2HPO4	
0.1	g	NH4Cl	
0.05	g	NaNO3	
0.05	g	Yeast extract	
0.01	g	Ferric citrate monohydrate	
1	ml	**Trace element solution SL-4**	
0.5	ml	Sodium resazurin (0.1% w/v)	
1.5	g	Na2CO3	
1	g	Na-pyruvate	
1	g	Na2S2O3 x 5 H2O	
1	ml	**Wolin's vitamin solution (10x)**	
0.25	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients except carbonate, pyruvate, thiosulfate, vitamins and sulfide, then sparge medium with 80% N2 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 and autoclave. Add pyruvate, thiosulfate, 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 mixture. Stock solutions of pyruvate, thiosulfate and vitamins should be sterilized by filtration. Prior to use adjust pH of complete medium to 7.3 - 7.5, if necessary.


## Trace element solution SL-4

0.5	g	Na2-EDTA	
0.2	g	FeSO4 x 7 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.03	g	MnCl2 x 4 H2O	
0.3	g	H3BO3	
0.2	g	CoCl2 x 6 H2O	
0.01	g	CuCl2 x 2 H2O	
0.02	g	NiCl2 x 6 H2O	
0.03	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve EDTA in distilled water and adjust pH to 7.0 using 2 N NaOH; then add other compounds.


## 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