# 480d: THERMOPROTEUS UZONIENSIS MEDIUM


## Main sol. 480d

1000	ml	**Main sol. 480**	
3	g/l	Casitone	

1. Use medium 480, but reduce the amount of Na acetate to 1.0 g/l and add 3.0 g/l casitone. Prepare sulphide and vitamin stock solutions anaerobically. Autoclave the sulphide solution but filter sterilise the vitamins. Prepare the medium anaerobically under N2:CO2 80:20. Adjust the pH to 5.7 with HCl. Sterilise the medium by heating for 3 hours on three successive days at 90-100°C. Add the vitamins and sulphide to the sterilised medium before use. The final pH should be 5.7-6.0. Dr. Bonch-Osmoloskaya supplied the information that the strain grows better on a peptone originally available in Russia. This product is no longer available, but it appears this product gave better growth than with highly purified products from Oxoid or Difco.


## 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	


## 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.


## Main sol. 480

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**	
0.1	g	Yeast extract	
0.5	ml	Sodium resazurin (0.1% w/v)	
10	g	Sulfur (powder)	
5	g	Na-acetate	
2.5	g	NaHCO3	
10	ml	**Wolin's vitamin solution**	
0.5	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients (except sulfur, acetate, bicarbonate, vitamins and sulfide) and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Adjust pH to 5.9, dispense under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials containing already the appropriate amount of sulfur and sterilize by heating for 2-3 hours in a boiling water bath on each of 3 successive days. Add acetate, vitamins (sterilized by filtration)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. Prior to use adjust pH of complete medium to 6.8 - 7.0, if necessary.