# 480b: TTD-MEDIUM


## Main sol. 480b

0.33	g	NH4Cl	
0.33	g	CaCl2 x 2 H2O	
0.33	g	MgCl2 x 6 H2O	
0.33	g	KCl	
0.33	g	KH2PO4	
1	ml	**Trace element solution SL-10**	
0.5	ml	Sodium resazurin (0.1% w/v)	
10	g	Sulfur (powder)	
1000	ml	Distilled water	

1. Add to the medium 0.5% casitone and 2.5% NaCl (marine source). Adjust pH to 5.7 with hydrochloric acid before sterilization. Sterilize the medium by heating for 1 h at 90 - 100°C on three successive days. Before use add 30.0 ml/l of 5% (w/v) sterile, anaerobic NaHCO3-solution, vitamins and sulfide. Final pH should be 6.5.

0.5	%	Casitone	
2.5	%	NaCl	
30	ml	NaHCO3 (5% w/v)	
10	ml	**Wolin's vitamin solution**	
0.5	g	Na2S x 9 H2O	


## 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

2	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Thiamine HCl	
5	mg	Riboflavin	
5	mg	Nicotinic acid	
5	mg	Calcium D-(+)-pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water