# 1058c: THIOHALORHABDUS DENITRIFICANS MEDIUM


## Main sol. 1058c

180	g	NaCl	
1.5	g	K2HPO4	
0.5	g	NH4Cl	
1	ml	**Trace elements solution (Pfennig & Lippert,1966)**	
0.05	g	CaCl2 x 2 H2O	
0.5	g	MgSO4 x 7 H2O	
5	g	Na2S2O3 x 5 H2O	
5	g	NaHCO3	
1	ml	**Seven vitamins solution**	
1000	ml	Distilled water	

1. Dissolve sodium chloride, potassium hydrogenphosphate and ammonium chloride, then sparge solution with 80% N2 and 20% CO2 gas mixture for at least 30 - 45 min to remove dissolved oxygen and to saturate the solution with CO2. Dispense solution in vials suitable for anaerobic cultures (e.g. Balch-type tubes) to 30% volume under 80% N2 and 20% CO2 gas atmosphere. Close vials with butyl rubber septa and autoclave. Add trace elements, calcium chloride, magnesium sulfate, thiosulfate, and vitamins from sterile stock solutions prepared under 100% N2 gas and bicarbonate from a sterile stock solution prepared under 80% N2 and 20% CO2 gas mixture. Thiosulfate and vitamins are sterilized by filtration. Adjust pH of the medium to 7.5 - 7.8 using a sterile stock solution of sodium carbonate (5% w/v).
2. After inoculation, add sterile air (with hypodermic needle through the rubber closure) to a concentration of ca. 4% (v/v) O2 (e.g., add 7 ml of air to a Balch-type tube with a total volume of 27 ml).
3. Note: Use at least 10% (v/v) as inoculum.


## Trace elements solution (Pfennig & Lippert,1966)

5	g	EDTA	
2.2	g	FeSO4 x 7 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.03	g	MnCl2 x 4 H2O	
0.03	g	H3BO3	
0.2	g	CoCl2 x 6 H2O	
0.03	g	CuCl2 x 2 H2O	
0.03	g	NiCl2 x 6 H2O	
0.03	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. pH 3.0-4.0


## 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