1681 .
Mineral salt medium with colloidal chitin
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
7.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Solution A | 50.00 | ml | ||
|
|
Solution B | 50.00 | ml | ||
|
|
Modified Wolin's mineral solution | 10.00 | ml | ||
|
|
Wolin's vitamin solution | 10.00 | ml | ||
|
|
Sodium resazurin
(0.1% w/v) |
1.00 | ml | ||
|
|
Yeast extract | 1.00 | g | ||
|
|
Polypeptone | 10.00 | g | ||
|
|
NaHCO3 | 3.52 | g | ||
|
|
Colloidal chitin | 5.00 | g | ||
|
|
Distilled water | 890.00 | ml | ||
| 1 Reducing agents: Flush each solution under N2 for 30 minutes, stopper and autoclave. L-Cysteine-HCL x H2O solution (10%): add 12.5 µl per 10 ml medium Na2S x 9 H2O solution (3%): add 40 µl per 10 ml medium. | |||||
| 2 Colloidal chitin preparation: Cut 5 gram commercial crab (shrimp) shell flakes into small pieces, and dissolve in 100 ml of 12M HCL while stirring in a fume hood at room temperature. Add chitin-HCL mixture slowly into pre-cooled water to obtain the colloidal chitin, centrifuge and wash in pre-cooled water till the colloidal chitin reaches a pH of 6 to 6.5. Mix 0.5% with MS medium as the sole carbon source. As an alternative commercial available chitin powder can be used. | |||||
| 3 Prepare MS medium with chitin: Dissolve the ingredients of MS medium (except NaHCO3 and vitamins), boil medium under CO2 until the resazurin indicator changes from pink to colourless. Cool down under CO2, add NaHCO3 and adjust pH to 7.0. Switch to N2 to prevent further lowering of the pH. Fill up into Hungate tubes under N2 and autoclave. Before use add vitamins (sterilized by filtration) and reducing agents. | |||||
Solution A #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
KH2PO4 | 8 | g |
|
|
NH4Cl | 5 | g |
|
|
MgCl2 x 6 H2O | 4 | g |
|
|
CaCl2 x 2 H2O | 1 | g |
|
|
Distilled water | 1000 | ml |
Wolin's vitamin solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 2.0 | mg |
|
|
Folic acid | 2.0 | mg |
|
|
Pyridoxine hydrochloride | 10.0 | mg |
|
|
Thiamine HCl | 5.0 | mg |
|
|
Riboflavin | 5.0 | mg |
|
|
Nicotinic acid | 5.0 | mg |
|
|
Calcium D-(+)-pantothenate | 5.0 | mg |
|
|
Vitamin B12 | 0.1 | mg |
|
|
p-Aminobenzoic acid | 5.0 | mg |
|
|
(DL)-alpha-Lipoic acid | 5.0 | mg |
|
|
Distilled water | 1000.0 | ml |
Modified Wolin's mineral solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid | 1.50 | g | ||
|
|
MgSO4 x 7 H2O | 3.00 | g | ||
|
|
MnSO4 x H2O | 0.50 | g | ||
|
|
NaCl | 1.00 | g | ||
|
|
FeSO4 x 7 H2O | 0.10 | g | ||
|
|
CoSO4 x 7 H2O | 0.18 | g | ||
|
|
CaCl2 x 2 H2O | 0.10 | g | ||
|
|
ZnSO4 x 7 H2O | 0.18 | g | ||
|
|
CuSO4 x 5 H2O | 0.01 | g | ||
|
|
AlK(SO4)2 x 12 H2O | 0.02 | g | ||
|
|
H3BO3 | 0.01 | g | ||
|
|
Na2MoO4 x 2 H2O | 0.01 | g | ||
|
|
NiCl2 x 6 H2O | 0.03 | g | ||
|
|
Na2SeO3 x 5 H2O | 0.30 | mg | ||
|
|
Na2WO4 x 2 H2O | 0.40 | mg | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH. | |||||