441 .
DIAZOTROPHIC MEDIUM (RBA)
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Selected strain: Azotobacter chroococcum DSM 379
This strain uses the standard medium without modifications.
Growth conditions: 2-3 days
aerobic
30 °C
Equipment needed:
Autoclave
Filter
Final pH:
n.d.
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Solution A | 953 | ml | ||
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Solution B | 50 | ml | ||
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Standard vitamin solution | 5 | ml | ||
| 1 Sterilize solution A separately at 121°C for 15 min., cool to 50°C and then mix aseptically with filter-sterilized solution B and 5.0 ml of filter-sterilized standard vitamin solution (see medium 428). | |||||
| 2 RBA is an ammonium-free medium which has successfully been used for the isolation, growth and purity check of a broad spectrum of nitrogen fixing bacteria (Ref. 3363). For microaerophilic nitrogen-fixing bacteria use semisolid medium with 0.3% end concentration of agar and incubate the liquid cultures under 10% (v/v) air and 90% (v/v) N2. | |||||
Solution A #
953
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
KH2PO4 | 0.10 | g | ||
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K2HPO4 | 0.90 | g | ||
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NaCl | 0.10 | g | ||
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CaCl2 x 2 H2O | 0.10 | g | ||
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MgSO4 x 7 H2O | 0.10 | g | ||
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Na2MoO4 x 2 H2O | 5.00 | mg | ||
|
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NaVO3 x H2O | 5.00 | mg | ||
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MnSO4 x H2O | 5.00 | mg | ||
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FeSO4 x 7 H2O | 0.01 | g | ||
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Yeast extract | 0.05 | g | ||
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Trace element solution SL-6 | 3.00 | ml | ||
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Distilled water | 950.00 | ml | ||
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Agar , if required | 15.00 | g | ||
| 1 Adjust pH to 7.3. | |||||
Solution B #
50
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Disodium succinate | 1 | g | ||
|
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DL-Malate | 2 | g | ||
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Na-pyruvate | 1 | g | ||
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D-Mannitol | 2 | g | ||
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D-Glucose | 2 | g | ||
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Distilled water | 50 | ml | ||
| 1 Adjust pH to 7.3. | |||||
Trace element solution SL-6 #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
ZnSO4 x 7 H2O | 0.10 | g |
|
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MnCl2 x 4 H2O | 0.03 | g |
|
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H3BO3 | 0.30 | g |
|
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CoCl2 x 6 H2O | 0.20 | g |
|
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CuCl2 x 2 H2O | 0.01 | g |
|
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NiCl2 x 6 H2O | 0.02 | g |
|
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Na2MoO4 x 2 H2O | 0.03 | g |
|
|
Distilled water | 1000.00 | ml |
Standard vitamin solution #
100
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Riboflavin | 10.0 | mg |
|
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Thiamine-HCl x 2 H2O | 50.0 | mg |
|
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Nicotinic acid | 50.0 | mg |
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Pyridoxine hydrochloride | 50.0 | mg |
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Calcium pantothenate | 50.0 | mg |
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Biotin | 0.1 | mg |
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Folic acid | 0.2 | mg |
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Vitamin B12 | 1.0 | mg |
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Distilled water | 100.0 | ml |