| Selenite-tungstate solution |
1.0 |
ml |
| Composition |
capacity |
unit |
| NaOH |
0.4 |
g |
| Na2SeO3·5H_2O |
6 |
mg |
| Na2WO4·2H_2O |
8 |
mg |
| Distilled water |
1.0 |
L |
|
| NH_4Cl |
0.54 |
g |
| Yeast extract |
5.0 |
g |
| MgCl_2·6H_2O |
3.0 |
g |
| NaCl |
20.0 |
g |
| KH2PO4 |
0.14 |
g |
| CaCl_2·2H_2O |
0.15 |
g |
| Trace element solution |
1.0 |
ml |
| Composition |
capacity |
unit |
| ZnCl_2 |
70 |
mg |
| MnCl_2·4H_2O |
100 |
mg |
| H3BO3 |
6 |
mg |
| CoCl_2·6H_2O |
190 |
mg |
| CuCl_2·2H_2O |
2.0 |
mg |
| NiCl_2·6H_2O |
24 |
mg |
| Na2MoO4·2H_2O |
36 |
mg |
| Distilled water |
1.0 |
L |
|
| FeCl_2 solution |
1.0 |
ml |
| Composition |
capacity |
unit |
| 25% HCl (7.7 M) |
10.0 |
ml |
| FeCl_2·4H_2O |
1.5 |
g |
| Distilled water |
990 |
ml |
|
| NaHCO_3 |
2.5 |
g |
| Resazurin |
1.0 |
mg |
| Distilled water |
1.0 |
L |
| Mix components, except NaHCO3, and adjust pH 7.0. Bring to a boil for 5-10 seconds, then, cool down under a stream of N2-CO2 (80:20, v/v), add NaHCO3, and autoclave under the same gas mixture. After cooling. aseptically and anaerobically, add the following solutions (autoclaved or ·filter-sterilized, and stored under a N2 atmosphere): |
|
|
| Trace vitamins |
2.0 |
ml |
| Composition |
capacity |
unit |
| Biotin |
2.0 |
mg |
| Folic acid |
2.0 |
mg |
| Pyridoxine·HCl |
10.0 |
mg |
| Thiamine·HCl |
5.0 |
mg |
| Riboflavin |
5.0 |
mg |
| Nicotinic acid |
5.0 |
mg |
| Calcium pantothenate |
5.0 |
mg |
| Vitamin B12 |
0.1 |
mg |
| p-Aminobenzoic acid |
5.0 |
mg |
| Lipoic acid |
5.0 |
mg |
| Distilled water |
1.0 |
L |
|
| Culture supernatant |
400 |
ml |
| Composition |
capacity |
unit |
| rumen fluid or human fecal extract |
1.0 |
% |
|
| Distribute the medium into culture vessels (e.g., 10 ml in Hungate tubes) under the same gas mixture and seal with butyl rubber stoppers. Finally, add per liter the following solutions (autoclaved and stored under a N2 atmosphere): |
|
|
| 5% L-Cysteine·HCl·H2O solution |
5.0 |
ml |
| 5% Na_2S·9H_2O solution |
5.0 |
ml |
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