Main sol. 1811
Internal Solution-ID: S6317
Equipment needed: Filter
Autoclave
Anaerobic workbench
| Compound | Amount | Unit | Conc. [g/L] | Conc. [mM] | |
|---|---|---|---|---|---|
| Modified Brock's salt base solution | 1000.00 | ml | - | - | |
| Trisodium citrate x 2 H2O | 2.94 | g | 2.911 | 9.898 | |
| Resazurin | 1.00 | mg | 9.9e-4 | 0.004 | |
| 1 Combine Modified Brock's salt base solution, trisodium citrate and resazurin. | |||||
| 2 Adjust pH to 3.5 with 10N H2SO4. | |||||
| 3 Autoclave under N2 atmosphere. | |||||
| 4 Autoclave at 121 °C for 15 min. | |||||
| 5 Separately autoclave yeast extract and Na2S x 9H2O as 10% and 5% solutions, respectively, under a N2 atmosphere. Filter-sterilize Trace vitamins. Steam sulfur for 3 hr on each of 3 successive days. | |||||
| Trace vitamins | 10.00 | ml | - | - | |
|
Yeast extract
(BD-Difco, 10% solution in water) |
0.50 | g | 0.495 | - | |
|
Sulfur
(powder) |
10.00 | g | 9.901 | 308.731 | |
| 6 Aseptically mix components except Na2S x 9H2O in suitable culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) while gassing with H2-CO2 (80:20) gas mixture and seal with butyl rubber stoppers. | |||||
|
Na2S x 9 H2O
(5% solution in water) |
0.50 | g | 0.495 | 2.061 | |
| 7 Prior to inoculation, aseptically and anaerobically add the Na2S x 9H2O solution. | |||||
| 8 Adjust pH to 4.0-4.5. | |||||
| 9 When the indicator (resazurin) turns to colorless, inoculate the culture and pressurize the vessels to 100 kPa H2-CO2 (80:20). | |||||