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Main sol. 1681

Internal Solution-ID: S3470

Equipment needed: Filter Hungate tubes Autoclave Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
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 1.0e-3 -
Yeast extract 1.00 g 1 -
Polypeptone 10.00 g 10 -
NaHCO3 3.52 g 3.52 41.901
Colloidal chitin 5.00 g 5 -
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.
1681 Mineral salt medium with colloidal chitin