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DSMZ

DSMZ 559 . TMBS4 MEDIUM

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Equipment needed: Autoclave Filter Gassing station Hungate tubes
Final gas composition: 100 % N2 read more
Final pH: 7.2 - 7.4
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ml
Compound Amount Unit
Solution A 932 ml
Solution B 30 ml
Solution C 10 ml
Solution D 2 ml
Solution E 10 ml
Solution F 10 ml
Solution G 10 ml
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 #

932
Compound Amount Unit
NaCl 1.00 g
MgCl2 x 6 H2O 0.40 g
CaCl2 x 2 H2O 0.15 g
KCl 0.50 g
NH4Cl 0.25 g
KH2PO4 0.20 g
Trace element solution SL-10 1.00 ml
Selenite-tungstate solution 1.00 ml
Resazurin 0.50 mg
Distilled water 930.00 ml

Solution B #

30
Compound Amount Unit
NaHCO3 , 5% w/v solution 30 ml

Solution C #

10
Compound Amount Unit
Vitamin solution 10 ml

Solution D #

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

Solution E #

10
Compound Amount Unit
Yeast extract , 10% w/v solution 10 ml

Solution F #

10
Compound Amount Unit
Syringate solution 6.0 %(w/v) 10 ml

Solution G #

10
Compound Amount Unit
Dithiothreitol , 100 mM solution 10 ml

Vitamin solution #

1000
Compound Amount Unit
Biotin 2 mg
Folic acid 2 mg
Pyridoxine hydrochloride 10 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 5 mg
Distilled water 1000 ml
1 Adjust final pH of solution to 7.2 using 50 mM sodium phosphate buffer.

Syringate solution 6.0 %(w/v) #

100
Compound Amount Unit
Syringic acid 6 %(w/v)
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 #

1000
Compound Amount Unit
HCl
(25%) 		10.0 ml
FeCl2 x 4 H2O 1.5 g
ZnCl2 70.0 mg
MnCl2 x 4 H2O 100.0 mg
H3BO3 6.0 mg
CoCl2 x 6 H2O 190.0 mg
CuCl2 x 2 H2O 2.0 mg
NiCl2 x 6 H2O 24.0 mg
Na2MoO4 x 2 H2O 36.0 mg
Distilled water 990.0 ml
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 #

1000
Compound Amount Unit
NaOH 0.5 g
Na2SeO3 x 5 H2O 3.0 mg
Na2WO4 x 2 H2O 4.0 mg
Distilled water 1000.0 ml