520 .
RUMINOCLOSTRIDIUM CELLULOLYTICUM (CM3) MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
7.2
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
(NH4)2SO4 | 1.30 | g | ||
|
|
KH2PO4 | 1.50 | g | ||
|
|
K2HPO4 x 3 H2O | 2.90 | g | ||
|
|
FeSO4 x 7 H2O
(0.1% w/v in 0.1 N H2SO4) |
1.25 | ml | ||
|
|
Trace element solution SL-10 | 1.00 | ml | ||
|
|
Yeast extract | 2.00 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
MgCl2 x 6 H2O | 0.20 | g | ||
|
|
CaCl2 x 2 H2O | 75.00 | mg | ||
|
|
Cellobiose | 6.00 | g | ||
|
|
Cellulose
, MN 301
optional |
10.00 | g | ||
|
|
Na2CO3 | 1.50 | g | ||
|
|
L-Cysteine HCl x H2O | 0.50 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients except magnesium chloride, calcium chloride, cellobiose, cysteine and carbonate, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After autoclaving add magnesium chloride, calcium chloride and cellobiose from anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2. Cellobiose has to be sterilized by filtration. Prior to inoculation add cysteine from a sterile anoxic stock solution prepared under 100% N2 gas and adjust pH to 7.2. | |||||
| 2 Note: Some strains can be adapted to cellulose as substrate using 10.00 g/l cellulose powder MN 301 (MACHEREY-NAGEL). | |||||
Trace element solution SL-10 #
1000
| 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. | |||||