# 1114: THIOFABA MEDIUM


## Main sol. 1114

972	ml	**Solution A**	
10	ml	**Solution B**	
10	ml	**Solution C**	
10	ml	**Solution D**	
1	ml	**Solution E**	

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 serum vials (to 20% of volume) and autoclave. Solution B is autoclaved separately under 100% N2 gas and solution C under 80% N2 and 20% CO2 gas atmosphere. Solutions D and E are prepared under 100% N2 gas atmosphere and sterilized by filtration. To complete the medium appropriate amounts of solutions B to E are added to the sterile solution A in the sequence as indicated. The pH of the complete medium should be 6.5.
2. After inoculation cultivation vials are pressurized with an amount of sterile air that is equivalent to 30 vol% of the cultivation vial.


## Solution A

0.75	g	MgCl2 x 6 H2O	
0.15	g	CaCl2 x 2 H2O	
0.54	g	NH4Cl	
2	ml	**Modified Wolin's mineral solution**	
970	ml	Distilled water	


## Solution B

1.19	g	KH2PO4	
0.21	g	K2HPO4	
10	ml	Distilled water	


## Solution C

0.5	g	Na2CO3	
10	ml	Distilled water	


## Solution D

1.25	g	Na2S2O3 x 5 H2O	
10	ml	Distilled water	


## Solution E

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


## Modified Wolin's mineral solution

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.18	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.18	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.02	g	AlK(SO4)2 x 12 H2O	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
0.03	g	NiCl2 x 6 H2O	
0.3	mg	Na2SeO3 x 5 H2O	
0.4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.


## 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