Main sol. 1681

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