# 465h: MINERALMEDIUM WITH 6-METHYLNICOTINATE


## Main sol. 465h

1000	ml	**Main sol. 465**	
1	ml	**Selenite/tungstate stock solution**	
5	g/l	6-methylnicotinate, adjust if required	
1	ml	**Vitamin solution**	

1. Supplement medium 465 with selenite, tungstate, 2.5 - 5 g/l 6-methylnicotinate from a stock solution (autoclave separately) and 1 ml/l vitamin solution (see medium 461).


## 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.


## Selenite/tungstate stock solution

500	mg	NaOH	
3	mg	Na2SeO3 x 5 H2O	
4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	

1. Autoclave separately. Add 1 ml per l medium.


## Vitamin solution

50	mg	Vitamin B12	
50	mg	Pantothenic acid	
50	mg	Riboflavin	
10	mg	Pyridoxamine hydrochloride	
20	mg	Biotin	
20	mg	Folic acid	
25	mg	Nicotinic acid	
25	mg	Nicotine amide	
50	mg	alpha-lipoic acid	
50	mg	p-Aminobenzoic acid	
50	mg	Thiamine-HCl x 2 H2O	
1000	ml	Distilled water	

1. Stir for some hours, filter sterilize the solution.


## 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.