|
KH2PO4 |
0.227 |
g |
|
K2HPO4 |
0.348 |
g |
|
MgSO4·7H2O |
0.5 |
g |
|
NH4Cl |
0.5 |
g |
|
CaCl2·2H2O |
0.25 |
g |
|
NaCl |
2.25 |
g |
|
Bacto Yeast Extract (Difco) |
2 |
g |
|
Hipolypepton (Nihon seiyaku) |
2 |
g |
|
Trace elements solution* |
10 |
ml |
|
Vitamin solution** |
10 |
ml |
|
Methanol |
10 |
ml |
|
NaHCO3 |
0.8 |
g |
|
Resazurin |
1 |
mg |
|
Cystein-HCl |
0.5 |
g |
|
Na2S·9H2O |
0.5 |
g |
|
Distilled water |
1 |
L |
|
|
|
Mix ingredients except vitamin solution, NaHCO3, cysteine-HCl and Na2S·9H2O, and adjust pH to 7 with NaOH. Dispense the medium into suitable culture vessels under a stream of N2/CO2 (80/20) and seal with butyl rubber stoppers. Separately autoclave cysteine-HCl and Na2S·9H2O as 5% solutions under a N2 atmosphere. Sterilize vitamin and 8% NaHCO3 solutions by filtration. Prior to inoculation, aseptically and anaerobically add vitamine, NaHCO3, cysteine-HCl and Na2S·9H2O solutions. Adjust pH to 7.0. |
|
|
|
|
|
|
|
|
|
*Trace elements solution |
|
|
|
Nitrilotriacetic acid (NTA) |
12.8 |
g |
|
|
|
FeCl3·6H2O |
1.35 |
g |
|
|
|
MnCl2·4H2O |
0.1 |
g |
|
|
|
CoCl2·6H2O |
0.024 |
g |
|
|
|
CaCl2·2H2O |
0.1 |
g |
|
|
|
ZnCl2 |
0.1 |
g |
|
|
|
CuCl2·2H2O |
0.025 |
g |
|
|
|
H3BO3 |
0.01 |
g |
|
|
|
Na2MoO4·2H2O |
0.024 |
g |
|
|
|
NaCl |
1 |
g |
|
|
|
NiCl2·6H2O |
0.12 |
g |
|
|
|
Na2SeO4 |
0.004 |
g |
|
|
|
Na2WO4 |
0.004 |
g |
|
|
|
Distilled water |
1 |
L |
|
|
|
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with NaOH, and then add minerals. Final pH 7.0. |
|
|
|
|
|
|
|
|
|
**Vitamin solution |
|
|
|
Biotin |
2 |
mg |
|
|
|
Folic acid |
2 |
mg |
|
|
|
Pyridoxine-HCl |
10 |
mg |
|
|
|
Thiamine-HCl |
5 |
mg |
|
|
|
Riboflavin |
5 |
mg |
|
|
|
Nicotinic acid |
5 |
mg |
|
|
|
Ca-pantothenate |
5 |
mg |
|
|
|
p-Aminobenzoic acid |
1 |
mg |
|
|
|
Vitamin B12 |
0.01 |
mg |
|
|
|
Distilled water |
1 |
L |
|