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DSMZ

JCM 574 . DB CHARACTERIZATION MEDIUM NO. 2

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Equipment needed: Autoclave Filter
Final pH: 7.5
Select a strain (optional):

ml
Compound Amount Unit
MDS salt water 833.00 ml
FeCl2 solution 1.00 ml
Trace element solution 1.00 ml
Peptone
(Oxoid) 		0.25 g
Yeast extract
(BD-Difco) 		0.05 g
1 Add components and bring volume to 980 ml with distilled water. For preparation of solid medium, add 20 g/L agar (if necessary, use washed agar as described below). Autoclave, and after cooling, add the following solutions (filter-sterilized or *autoclaved) to the medium:
NH4Cl
(1 M) 		5.00 ml
Potassium phosphate buffer
(*) 		2.00 ml
Vitamin solution 3.00 ml
Sodium pyruvate
(1 M) 		10.00 ml
2 Readjust pH to 7.0 - 7.5 with a sterilized 10% Na2CO3 solution, if necessary.

Washed agar #

1 Stir agar in distilled water and stand still for 30 min. Then, remove supernatant by decantation. Repeat the washing at least twice.

MDS salt water #

1000
Compound Amount Unit
NaCl 240 g
MgCl2 x 6 H2O 30 g
MgSO4 x 7 H2O 35 g
KCl 7 g
CaCl2
(1 M) 		5 ml
1 Add components to distilled water and bring volume to 1.0 L. Adjust pH to 7.5 with 1 M Tris base.

Potassium phosphate buffer #

100
Compound Amount Unit
K2HPO4
(1 M) 		83.4 ml
KH2PO4
(1 M) 		16.6 ml
1 Combine the two solutions and check that pH is close to 7.5. Add an equal volume of distilled water.

Vitamin solution #

1000
Compound Amount Unit
Biotin 3 mg
Folic acid 4 mg
Pyridoxine hydrochloride 50 mg
Thiamine HCl 33 mg
Riboflavin 10 mg
Nicotinic acid 33 mg
DL-Calcium pantothenate 17 mg
Vitamin B12 17 mg
p-Aminobenzoic acid 13 mg
Lipoic acid 10 mg
Distilled water 1000 ml

FeCl2 solution #

1000
Compound Amount Unit
HCl
(7.7 M, 25%) 		10.0 ml
FeCl2 x 4 H2O 1.5 g
Distilled water 990.0 ml

Trace element solution #

1000
Compound Amount Unit
ZnCl2 70 mg
MnCl2 x 4 H2O 100 mg
H3BO3 6 mg
CoCl2 x 6 H2O 190 mg
CuCl2 x 2 H2O 2 mg
NiCl2 x 6 H2O 24 mg
Na2MoO4 x 2 H2O 36 mg
Distilled water 1000 ml