KCTC Media No. 270 : Minimal Salts (MS) Medium with colloidal chitin

조성	용량	단위
Solution A	50	ml/L
조성 용량 단위 KH2PO4 8.0 g/L MgCl_2·6H2O 4.0 g/L NH4Cl 5.0 g/L CaCl2·2H2O 1.0 g/L
Solution B	50	ml/L
조성 용량 단위 K2HPO4 8.0 g/L
Trace metal solution	10.0	ml/L
조성 용량 단위 Trace metal solution (100X) Nitrilotriacetic acid 12.8 g/L FeSO4·7H2O 0.42 g/L MnCl2·4H2O 0.1 g/L CoCl2·6H2O 0.17 g/L CuCl2·2H2O 0.02 g/L ZnSO4·7H2O 0.21 g/L Na2MoO4·2H2O 0.01 g/L
Wolfe's vitamin solution	10.0	ml/L
조성 용량 단위 Wolfe's vitamin solution (100X) Biotin 2.0 mg/L folic acid 2.0 mg/L pyridoxine HCl 10.0 mg/L thiamine HCl 5.0 mg/L riboflavin 5.0 mg/L nicotinic acid 5.0 mg/L calcium D-(+)pantothenate 5.0 mg/L cyanocobalamine 0.1 mg/L p-aminobenzoic acid 5.0 mg/L thioctic acid 5.0 mg/L
Yeast extracrt	1.0	g/L
Resazurin(0.1%)	1.0	ml/L
NaHCO3	3.52	g/L
Polypeptone	10.0	g/L
Colloidal chitin	5.0	g/L

** ph : 7-7.2

** memo : Adjust it with NaOH Final concentration of ~0.5% colloidal chitin was mixed with MS medium
as sole carbon substrate to grow the strain.

** 추가조치 사항 : *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.

*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.

*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.