# 559: TMBS4 MEDIUM


## Main sol. 559

932	ml	**Solution A**	
30	ml	**Solution B**	
10	ml	**Solution C**	
2	ml	**Solution D**	
10	ml	**Solution E**	
10	ml	**Solution F**	
10	ml	**Solution G**	

1. Boil solution A for 3 min., then cool to room temperature under 100% N2 gas atmosphere. Dispense the medium under same gas atmosphere in culture vessels and autoclave. Autoclave separately solutions E and F under 100% N2 and solution B under 80% N2 and 20% CO2 gas atmosphere. Solutions C, D, and G are prepared under N2 gas atmosphere and sterilized by filtration.
2. Solutions B to G are added to the sterile, cooled solution A in the sequence as indicated. The final pH of the medium should be 7.2-7.4. After inoculation add 25 mg sodium dithionite per liter medium from a 2.5% (w/v) solution, freshly prepared under N2 and filter-sterilized.


## Solution A

1	g	NaCl	
0.4	g	MgCl2 x 6 H2O	
0.15	g	CaCl2 x 2 H2O	
0.5	g	KCl	
0.25	g	NH4Cl	
0.2	g	KH2PO4	
1	ml	**Trace element solution SL-10**	
1	ml	**Selenite-tungstate solution**	
0.5	mg	Resazurin	
930	ml	Distilled water	


## Solution B

30	ml	NaHCO3, 5% w/v solution	


## Solution C

10	ml	**Vitamin solution**	


## Solution D

2	ml	Na-thiosulfate, 5% w/v solution	


## Solution E

10	ml	Yeast extract, 10% w/v solution	


## Solution F

10	ml	**Syringate solution 6.0 %(w/v)**	


## Solution G

10	ml	Dithiothreitol, 100 mM solution	


## Vitamin solution

2	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Thiamine HCl	
5	mg	Riboflavin	
5	mg	Nicotinic acid	
5	mg	Calcium D-(+)-pantothenate	
5	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water	

1. Adjust final pH of solution to 7.2 using 50 mM sodium phosphate buffer.


## Syringate solution 6.0 %(w/v)

6	%(w/v)	Syringic  acid	

1. Dissolve syringic acid in distilled water and neutralize solution with 2 N NaOH until a pH of 7.5 is reached. The final concentration of syringate in the solution should be 6% w/v.


## Trace element solution SL-10

10	ml	HCl (25%)	
1.5	g	FeCl2 x 4 H2O	
70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
6	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 x 2 H2O	
990	ml	Distilled water	

1. First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml.


## Selenite-tungstate solution

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