# 1525: KALLOTENUE MEDIUM


## Main sol. 1525

3.57	g	HEPES buffer (SIGMA)	
27	mg	NH4Cl	
60	mg	CaSO4 x 2 H2O	
0.1	g	MgSO4 x 7 H2O	
8	mg	NaCl	
0.1	g	KNO3	
0.69	g	NaNO3	
0.11	g	Na2HPO4	
10	ml	**Modified Wolin's mineral solution**	
2	g	D-Glucose	
1	g	Trypticase peptone (BD BBL)	
1	ml	**Wolin's vitamin solution (10x)**	
1000	ml	Distilled water	

1. Dissolve ingredients (except glucose, peptone and vitamins) and adjust pH to 8.1. Dispense medium under air atmosphere into culture vessels suitable for anaerobic cultivation (e.g., 10 ml medium per Balch-type tube), seal and autoclave. Add glucose, peptone and vitamins from sterile stock solutions sterilized by filtration. Adjust pH of complete medium to 7.5, if necessary.


## 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