# J1100: CTM MEDIUM


## Main sol. J1100

20	ml	**Solution 1 (50 x stock)**	
3	ml	**Solution 2**	
2	ml	**Solution 3**	
2.5	ml	**Solution 4**	
2.4	g	HEPES	

1. CTM medium basis:
2. Mix all ingredients in 1.0 L medium bottle. Add distilled water close to 1.0 L. Adjust pH to 7.0 with 2 M KOH and fill up to 1.0 L. Autoclave at 121C for 40 min. After autoclaving, seal medium bottle immediately in order to avoid oxygenation. Cool down to around 70C. Add 30 ml filter-sterilized Mixed solution (see below) to the autoclaved CTM medium basis for completing the CTM medium.


## Solution 1 (50 x stock)

3.75	g	MgSO4 x 7 H2O	
1.8	g	CaCl2 x 2 H2O	
0.3	g	Citric acid	
0.6	ml	Na2-EDTA (pH 8.0, 0.25 M)	
50	ml	**Trace metal solution**	
1000	ml	Distilled water	


## Trace metal solution

2.86	g	H3BO3	
1.81	g	MnCl2 x 4 H2O	
0.222	g	ZnSO4 x 7 H2O	
0.39	g	Na2MoO4 x 2 H2O	
0.079	g	CuSO4 x 5 H2O	
0.049	g	Co(NO3)2 x 6 H2O	
1000	ml	Distilled water	


## Solution 2

15.3	g	K2HPO4	
1000	ml	Distilled water	


## Solution 3

12	g	Ferric ammonium citrate	
1000	ml	Distilled water	


## Solution 4

14.6	g	2-Oxoglutarate	
1000	ml	Distilled water	


## Mixed solution

0.625	g	NaHCO3	
0.125	g	Sodium thioglycolate	
0.5	ml	**Solution 5**	
10	ml	**Solution 6**	
0.25	ml	**Vitamin solution A (1000 x stock)**	
0.25	ml	**Vitamin solution B (1000 x stock)**	

1. Add distilled water to bring final volume to 30 ml.


## Solution 5

10	g	Bacto peptone (BD-Difco)	
100	ml	Distilled water	


## Solution 6

100	mg	L-Alanine	
100	mg	L-Arginine	
100	mg	L-Asparagine	
100	mg	L-Aspartic acid	
100	mg	L-Cysteine	
100	mg	L-Glutamic acid	
100	mg	L-Glutamine	
100	mg	Glycine	
100	mg	L-Histidine	
100	mg	L-Isoleucine	
100	mg	L-Leucine	
100	mg	L-Lysine	
100	mg	L-Methionine	
100	mg	L-Phenylalanine	
100	mg	L-Proline	
100	mg	L-Serine	
100	mg	L-Threonine	
100	mg	L-Tryptophan	
100	mg	L-Tyrosine	
100	mg	L-Valine	
400	ml	Distilled water	


## Vitamin solution A (1000 x stock)

10	mg	Biotin	
10	mg	Riboflavin	
100	mg	Thiamine HCl	
100	mg	Thiamine pyrophosphate	
100	mg	L-Ascorbic acid	
100	mg	Calcium pantothenate	
100	mg	Folic acid	
100	mg	Nicotinamide	
100	mg	Nicotinic acid	
100	mg	p-Aminobenzoic acid	
100	mg	Pyridoxine hydrochloride	
100	mg	Lipoic acid	
100	mg	beta-NAD	
100	ml	Potassium phosphate buffer (pH 7.2, 10 mM)	


## Vitamin solution B (1000 x stock)

100	mg	Vitamin B12	
100	ml	Distilled water