Compound | Amount | Unit | |||
---|---|---|---|---|---|
|
Solution A | 966.0 | ml | ||
|
Solution B | 20.0 | ml | ||
|
Solution C | 11.0 | ml | ||
|
Solution D | 10.0 | ml | ||
|
Solution E | 1.0 | ml | ||
|
Solution F | 1.0 | ml | ||
|
Solution G | 1.0 | ml | ||
|
Solution H | 1.7 | ml | ||
1 Sparge solution A with 100% N2 gas for 30 - 45 min to make it anoxic. Thereafter, dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Anoxic stock solutions C, D, E, F, and H are prepared under 100% N2 gas atmosphere and solution G under 80% N2 and 20% CO2 gas atmosphere. Filter sterilize solutions D, F and G. To complete the medium appropriate amounts of solutions B to H are added to the sterile solution A in the sequence as indicated. Adjust pH of the complete medium to 7.2 - 7.5, if necessary. |
Compound | Amount | Unit | |
---|---|---|---|
|
Na2SO4 | 3.00 | g |
|
KH2PO4 | 0.20 | g |
|
NH4Cl | 0.30 | g |
|
CaCl2 x 2 H2O | 0.15 | g |
|
MgCl2 x 6 H2O | 0.40 | g |
|
NaCl | 1.00 | g |
|
Trace element solution SL-10 | 1.00 | ml |
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml |
|
Distilled water | 965.00 | ml |
Compound | Amount | Unit | |||
---|---|---|---|---|---|
|
HCl
(25%) |
10.0 | ml | ||
|
FeCl2 x 4 H2O | 1.5 | g | ||
|
ZnCl2 | 70.0 | mg | ||
|
MnCl2 x 4 H2O | 100.0 | mg | ||
|
H3BO3 | 6.0 | mg | ||
|
CoCl2 x 6 H2O | 190.0 | mg | ||
|
CuCl2 x 2 H2O | 2.0 | mg | ||
|
NiCl2 x 6 H2O | 24.0 | mg | ||
|
Na2MoO4 x 2 H2O | 36.0 | mg | ||
|
Distilled water | 990.0 | ml | ||
1 First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml. |
1 Rumen fluid from cow or sheep (obtained from fistulated animals or abattoir refuse) is filtered through muslin, autoclaved at 121°C for 15 min and then centrifuged at 27,000 g for 20 min. The supernatant is made anoxic by sparging with 100% N2 gas for 15 min, dispensed under same gas atmosphere into anoxic serum vials to 30% of volume and then stored frozen at -20°C. |
Compound | Amount | Unit | |
---|---|---|---|
|
Biotin | 20 | mg |
|
Folic acid | 20 | mg |
|
Pyridoxine hydrochloride | 100 | mg |
|
Thiamine HCl | 50 | mg |
|
Riboflavin | 50 | mg |
|
Nicotinic acid | 50 | mg |
|
Calcium D-(+)-pantothenate | 50 | mg |
|
Vitamin B12 | 1 | mg |
|
p-Aminobenzoic acid | 50 | mg |
|
(DL)-alpha-Lipoic acid | 50 | mg |
|
Distilled water | 1000 | ml |
Compound | Amount | Unit | |||
---|---|---|---|---|---|
|
NaHCO3 | 50 | g | ||
|
Na2S2O4 | 50 | g | ||
|
Distilled water | 1000 | ml | ||
1 Dissolve NaHCO3 in water and make the solution anoxic by sparging with 80% N2 and 20% CO2 gas mixture. Then dissolve the Na-dithionite and filter sterilize the solution into anoxic Hungate tubes. Store the prepared solution in the dark and refrigerated. Prepare only small amounts of stock solution, as Na-dithionite decomposes rapidly. |