|
KH2PO4 |
0.2 |
g |
|
NH4Cl |
0.25 |
g |
|
NaCl |
26 |
g |
|
MgCl2·6H2O |
11.2 |
g |
|
KCl |
0.7 |
g |
|
CaCl2·2H2O |
1.4 |
g |
|
Na2SO4 |
4 |
g |
|
KBr |
0.1 |
g |
|
NaHCO3 |
2.5 |
g |
|
Sodium lactate |
2.3 |
g |
|
Resazurin |
0.5 |
g |
|
Trace element solution SL-10* |
1 |
ml |
|
Selenite-tungstate solution** |
1 |
ml |
|
Vitamin solution*** |
1 |
ml |
|
Sulfide solution**** |
10 |
ml |
|
Distilled water |
1 |
L |
|
|
|
Dissolve ingredients except dihydrogenphosphate, ammonium chloride, bicarbonate, lactate, vitamins and sulfide, and autoclave it in a cultivation device for anaerobes. After sterilization and cooling under a nitrogen atmosphere, add dihydrogenphosphate, ammonium chloride, bicarbonate, lactate, vitamins and sulfide from sterile stock solutions. Adjust the final pH of the medium to 7.2-7.4. Dispense the medium under N2/CO2 (80/20) gas mixture in culture vessels. |
|
|
|
|
|
|
|
|
|
*Trace element solution SL-10 |
|
|
|
HCl (25%; 7.7 M) |
10 |
ml |
|
|
|
FeCl2·4H2O |
1.5 |
g |
|
|
|
ZnCl2 |
7 |
mg |
|
|
|
MnCl2·4H2O |
100 |
mg |
|
|
|
H3BO3 |
6 |
mg |
|
|
|
CoCl2·6H2O |
190 |
mg |
|
|
|
CuCl2·2H2O |
2 |
mg |
|
|
|
NiCl2·6H2O |
24 |
mg |
|
|
|
Na2MoO4·2H2O |
36 |
mg |
|
|
|
Distilled water |
990 |
ml |
|
|
|
|
|
|
|
|
|
**Selenite-tungstate solution |
|
|
|
NaOH |
0.5 |
g |
|
|
|
Na2SeO3·5H2O |
3 |
mg |
|
|
|
Na2WO4·2H2O |
4 |
mg |
|
|
|
distilled water |
1 |
L |
|
|
|
Sterilized by autoclaving. |
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|
|
|
|
|
|
|
|
***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 |
|
|
|
Vitamin B12 |
0.1 |
mg |
|
|
|
p-Aminobenzoic acid |
5 |
mg |
|
|
|
Lipoic acid |
5 |
mg |
|
|
|
Distilled water |
1 |
L |
|
|
|
|
|
|
|
|
|
****Neutralized sulfide solution |
|
|
|
Na2S·9H2O |
1.5 |
g |
|
|
|
Distilled water |
100 |
ml |
|
|
|
The sulfide solution is prepared in a vessel sealed with a butyl rubber stopper. Autoclave at 121℃ for 15 min under a N2 atmosphere. After cooling to room temperature, the pH is adjusted to about 7.3 by adding of sterile 2 N H2SO4 drop-wise with a syringe without opening the bottle. Appearance of yellow colour indicates the pH is about 8. The vessel should be shaken continuously to avoid precipitation of elemental sulfur. The final solution should be transparent yellow. |