# 916: DESULFOFABA HANSENII MEDIUM


## Main sol. 916

912	ml	**Solution A**	
50	ml	**Solution B**	
25	ml	**Solution C**	
10	ml	**Solution D**	
1	ml	**Solution E**	
10	ml	**Solution F**	

1. Sparge solution A 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 and autoclave. Solutions B and D are autoclaved under 100% N2 gas atmosphere. Solution C is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solutions E and F are prepared under 100% N2 gas and sterilized by filtration. To complete the medium appropriate amounts of solutions B to F are added to the sterile solution A in the sequence as indicated. The pH of the complete medium should be 7.0 - 7.2.
2. Note: Addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 and filter-sterilized) may stimulate growth at the beginning.


## Solution A

20	g	NaCl	
0.67	g	KCl	
0.1	g	NH4Cl	
0.01	g	KH2PO4	
0.02	g	MgSO4 x 7 H2O	
1.42	g	Na2SO4	
1	ml	**Trace element solution SL-10**	
1	ml	**Selenite-tungstate solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
910	ml	Distilled water	


## Solution B

10.6	g	MgCl2 x 6 H2O	
1.52	g	CaCl2 x 2 H2O	
50	ml	Distilled water	


## Solution C

1.25	g	Na2CO3	
25	ml	Distilled water	


## Solution D

0.96	g	Na-propionate	
10	ml	Distilled water	


## Solution E

1	ml	**Wolin's vitamin solution (10x)**	


## Solution F

0.52	g	FeCl2 x 4 H2O	
10	ml	Distilled water	

1. Adjust to pH 2 with HCl.


## 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