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AF-6 Medium (modified*)

(Andersen et al. 2005, Kasai et al. 2009, NIES)

*CaCO3 is omitted and P-V tracemetals and MES buffer are used compared to
the original recipe by Kato (1982).

1. Stock solutions

Fe-citrate 0.2 g/100 mL
NaNO3 14 g/100 mL
Citric acid 0.2 g/100 mL
NH4NO3 2.2 g/100 mL
MgSO4•7H2O 3 g/100 mL
KH2PO4 1 g/100 mL
K2HPO4 0.5 g/100 mL
CaCl2•2H2O 1 g/100 mL
FeCl3•6H2O** 0.1 g/100 mL
Na2EDTA•2H2O** 0.8 g/100 mL

**These chemicals are sometimes mixed with the P-V metals (Andersen et al. 2005),
but kept separate here to avoid precipitation.

2. Trace metal solutions

MnCl2•4H2O 100 mg
ZnSO4•7H2O (4 g/100 mL) 0.5 mL
CoCl2•6H2O (0.8 g/100 mL) 0.5 mL
Na2MoO4•2H2O (2 g/100 mL) 0.5 mL
Na3VO4•nH2O (0.2 g/100 mL) 0.5 mL
H2SeO3 (0.4 g/100 mL) 0.5 mL
dH2O to 500 mL

3. AF-6 vitamin primary stock solutions

Biotin 0.01 g/100 mL
Cyanocobalamin (B12) 0.1 g/100 mL
Pyridoxine (B6) 0.01 g/100 mL

Note: Vitamin B12 and Biotin are obtained in a crystalline form. When preparing the Vitamin B12 Stock Solution allow for approximately 11% water of crystallization (For each 1.0 mg of Vitamin B12 add 0.89 ml dH2O). When preparing the Biotin Stock Solution allow for approximately 4% water of crystallization (For each 1.0 mg of Biotin add 9.6 ml dH2O).
Keep the vitamin solutions frozen. Bottles of polyethylene are recommended for storage of vitamins.

4. AF-6 vitamin primary stock solutions
1) Dissolve 10 mg Thiamine HCl (Vitamin B1) in ca. 80 mL dH2O in ca. 950 mL dH2O.
2) Add 20 mL of the biotin primary stock solution.
3) Add 1 mL of the cyanocobalamin primary stock solution.
4) Add 1 mL of the pyridoxine primary stock solution.
5) Fill with dH2O to 1000 mL.
The vitamin working stock solution is divided into to 10-mL lots in polyethylene vials and kept frozen until use.

5. Final preparation of the AF-6 medium
To 950 mL dH2O add 400 mg MES buffer, 1 mL of each of the stock solution (begin with Fe-citrate and citric acid), 1 ml of the trace metal solution and 1 mL AF-6 vitamin solution. Make final volume up to 1 L with dH2O. Adjust pH to 6.6 and autoclave.

Andersen, R.A., Berges, J.A., Harrison, P.J. & Watanabe, M.M. 1997. Recipes for freshwater and seawater media.
In: R. A. Andersen ed.: Algal culturing techniques. Elsevier Academic Press, Burlington, p. 429-538.
Kasai, F., Kawachi, M., Erata, M., Mori, F., Yumoto, K. Sato, M., & Ishimoto, M. 2009. NIES-Collection List of Straind, 8th Edition, Jpn. J. Phycol. (Sôrui) 57(1) Suppl.: 1-350.
Kato, S. 1982. Laboratory culture and morphology of Colacium vesiculosum Ehrb. (Euglenophyceae). [in Japanese]. Jap. J. Phycol. 30: 63-67.