# 465e: MINERAL MEDIUM WITH SULFOBENZOIC ACID


## Main sol. 465e

1000	ml	**Main sol. 465**	
2.5	ml	**Vitamin solution**	
50	ml/l	2-Sulfobenzoic acid (20 mg/ml)	

1. Prepare medium 465 supplemented with vitamin solution (see medium 462). Prepare a stock solution of 2-sulfobenzoic acid (20 mg/ml) and sterilize by filtration. Add 50 ml of this stock solution to 1 l of the medium.


## Main sol. 465

3.5	g	Na2HPO4 x 2 H2O	
1	g	KH2PO4	
0.5	g	(NH4)2SO4	
0.1	g	MgCl2 x 6 H2O	
0.05	g	Ca(NO3)2 x 4 H2O	
1	ml	**Trace element solution SL-4**	
1000	ml	Distilled water	

1. pH 7.25
2. Rehydrate and cultivate lyophilized cells in the complex medium recommended for the specific strain (e.g. medium 1, 220, 535 or 830). After this reactivation, cultivate on mineral medium with the appropriate carbon source.


## Vitamin solution

1	mg	p-Aminobenzoate	
0.2	mg	Biotin	
2	mg	Nicotinic acid	
1	mg	Thiamine-HCl x 2 H2O	
0.5	mg	Calcium pantothenate	
5	mg	Pyridoxamine	
2	mg	Vitamin B12	
100	ml	Distilled water	


## Trace element solution SL-4

0.5	g	Na2-EDTA	
0.2	g	FeSO4 x 7 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.03	g	MnCl2 x 4 H2O	
0.3	g	H3BO3	
0.2	g	CoCl2 x 6 H2O	
0.01	g	CuCl2 x 2 H2O	
0.02	g	NiCl2 x 6 H2O	
0.03	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve EDTA in distilled water and adjust pH to 7.0 using 2 N NaOH; then add other compounds.


## Main sol. 1

5	g	Peptone	
3	g	Meat extract	
15	g	Agar, for solid medium	
1000	ml	Distilled water	

1. Adjust pH to 7.0.
2. For Bacillus strains the addition of 10.0 mg MnSO4 x H2O is recommended for sporulation.


## Main sol. 220

15	g	Casein peptone	
5	g	Soy peptone	
5	g	NaCl	
15	g	Agar	
1000	ml	Distilled water	

1. Adjust pH to 7.3. Medium is identical with Tryptone Soya Agar (Oxoid CM 131).


## Main sol. 535

30	g	Trypticase soy broth (BBL 11768, Oxoid CM129 or Merck 5459)	
20	g	Agar	
1000	ml	Distilled water	

1. pH 7.3
2. Autoclave at 121°C for 15 min.


## Main sol. 830

0.5	g	Yeast extract	
0.5	g	Proteose peptone (Difco no. 3)	
0.5	g	Casamino acids	
0.5	g	Glucose	
0.5	g	Starch (soluble)	
0.3	g	Na-pyruvate	
0.3	g	K2HPO4	
0.05	g	MgSO4 x 7 H2O	
15	g	Agar	
1000	ml	Distilled water	

1. Final pH 7.2; adjust with crystalline K2HPO4 or KH2PO4 before adding agar. Add agar, heat medium to boiling to dissolve agar, and autoclave for 15 min at 121°C.