# 142: THIOMICROSPIRA PELOPHILA MEDIUM


## Main sol. 142

25	g	NaCl	
1	g	(NH4)2SO4	
1.5	g	MgSO4 x 7 H2O	
0.42	g	CaCl2 x 2 H2O	
0.2	ml	**Trace element solution (Vishniac & Santer, 1957)**	
4	ml	Bromothymol blue (0.1% w/v)	
0.5	g	K2HPO4	
5	g	Na2S2O3 x 5 H2O	
1	ml	**Seven vitamins solution**	
1000	ml	Distilled water	

1. Dissolve ingredients (except hydrogenphosphate, thiosulfate and vitamins), adjust pH to 7.2 and autoclave. K2HPO4 and Na2S2O3 are autoclaved separately each in 10% of the final volume. Filter sterilize the vitamins solution. Adjust pH of the complete medium to 7.2 with sterile 0.4% (w/v) Na2CO3 solution.  Acidification of the medium during growth causes the pH indicator bromothymol blue to turn from blue to yellow.
2. Note: Growth of most Thiomicrospira strains is more reliable if the medium is prepared under a 80% N2 and 20% CO2 gas atmosphere to make it anoxic and then filled under air atmosphere into Hungate-type tubes (5 ml per vial). The pH is adjusted with a sterile stock solution of Na2CO3 (5% w/v) after autoclaving.


## Trace element solution (Vishniac & Santer, 1957)

50	g	Na2-EDTA	
22	g	ZnSO4 x 7 H2O	
5.54	g	CaCl2 x 2 H2O	
5.06	g	MnCl2 x 4 H2O	
5	g	FeSO4 x 7 H2O	
1.1	g	(NH4)6Mo7O24 x 4 H2O	
1.57	g	CuSO4 x 5 H2O	
1.61	g	CoCl2 x 6 H2O	
1000	ml	Distilled water	

1. Dissolve EDTA in distilled water, adjust pH to 7 using 2 N KOH, then add remaining compounds. Adjust pH of final solution to 6.0 with KOH.


## Seven vitamins solution

100	mg	Vitamin B12	
80	mg	p-Aminobenzoic acid	
20	mg	D-(+)-biotin	
200	mg	Nicotinic acid	
100	mg	Calcium pantothenate	
300	mg	Pyridoxine hydrochloride	
200	mg	Thiamine-HCl x 2 H2O	
1000	ml	Distilled water