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. Author manuscript; available in PMC: 2013 Mar 26.
Published in final edited form as: Methods Cell Biol. 2012;109:237–276. doi: 10.1016/B978-0-12-385967-9.00008-6

Table II.

Chemically Defined Synthetic Media: Composition, Concentration of Stock Solutions, and Preparation.

CDMA a CMDC b CDMA a CMDC b
Amino Acid Solution A mg/ml Salts and Chelator Solution mg/ml
L-Arg-HCl 12 2.4 K2HPO4 • 3H2O 25 5
L-His-HCl • H2O 8 1.6 KH2PO4 25 5
L-Ile 8 8 MgSO4 • 7H2O 50 10
L-Leu 8 8 CaCl2 • 2H2O 1 0.2
L-Lys-HCl 8 16 Tri-Potassium Citrate 65 13
L-Met 6 6 Vitamins (Solution A)
L-Phe 6 6 Na Riboflavin Phosphate • 2H2O 0.05 0.05
L-Ser 6 12 Vitamins (Solution B)
L-Thr 8 16 DL-6, 8-Thioctic Acid 0.01 0.01
L-Trp 6 12 Vitamins (Solution C)
L-Val 4 4 Thiamin-HCl 0.05 0.05
Amino Acid Solution B Prydoxal-HCl 0.01 0.01
L-Gln 4 0.8 Nicotinic acid 0.09 0.09
Amino Acid Solution C D-Pantothenic Acid, hemi Ca-salt 0.08 0.08
L-Asn • H2O 8 16 Vitamins (Solution D)
L-Pro 8 16 Folinic acid, Ca salt 0.01 0.01
Amino Acid Solution D Trace metals solution
L-Ala 6 12 FeCl2 • 6H2O 1 0.2
L-Asp 8 1.6 MnSO4 • 4H2O 0.16 0.032
L-Glu 16 0.8 Co (NO3)2 • 6H2O 0.05 0.01
Gly 16 32 ZnSO4 • 7H2O 0.45 0.09
Amino Acid Solution E CuSO4 • 5H2O 0.03 0.006
L-Tyr (Do not prepare ahead) 8 8 (NH4)6 Mo7O24 • 4H2O 0.01 0.002
Nucleoside solutions Glucose solution
Adenosine 0.2 0.2 Glucose 250 250
Cytidine 0.2 0.2
Guanosine 0.2 0.2
Uridine 0.2 0.2
a

CDMA is modified chemically defined media that supports rapid growth similar to that observed in PP media (Szablewski et al., 1991).

b

CDMC is a modification of CDMA that permits growth of low concentration inoculum (including single cells) without additional supplements (Hagemeister, Grave, Kristiansen, Assaad, and Hellung-Larsen, 1999).

1. Media preparation:

Unless otherwise noted, all ingredients are made up as stock solutions in high purity distilled water, sterilized by filtration, and stored at 4°C.
  • Amino acid solutions A – D are prepared as 40-fold concentrated stock solutions. Preparation notes: Solutions A and C: adjust pH to 7 and sterilize by filtration; Solution B: store frozen; Solution D: dissolve aspartic and glutamic acids in water with stirring, keep pH from dropping below 7 with 1N KOH, add alanine and glycine, adjust pH to 7, filter sterilize and store at 4°C.
  • Nucleoside solutions are prepared as 10-fold concentrated stock solutions.
  • Salts and Chelator solutions are prepared as 100-fold concentrated stock solutions.
  • Vitamins are prepared as 100-fold concentrated stock solutions and stored frozen. To make Vitamin Solution B, dl-6, 8-Thioctic Acid is dissolved in 1 ml absolute ethanol, then diluted in 100 ml H2O and filter sterilized.
  • Trace metals solution are prepared as a 100-fold concentrated solution, and adjusted to approximately pH 2 with 1 N HCl.
  • Glucose solution is prepared as a 50-fold concentrated solution.

To make the complete media, dissolve Tyr at 60°C, adjust to give a concentration of 0.2 mg/ml in the final medium, cool and then proceed to add the remaining solutions. The pH may be adjusted as required. The medium may be sterilized by autoclaving or by filtration; if autoclaving is used, glucose should be added aseptically after cooling.

2. Special purpose modifications:
  • Minimal defined media. Amino acid solution A contains all required amino acids. A minimal defined medium can be created by omitting amino acid solutions B – E.
  • Low cell inoculum in CDMA. When inocula of less than 2500 cells/ml of final medium will be used, CDMA should be supplemented with hemin at a final concentration of 7.5 μM. To prepare a stock solution, dissolve hemin in 0.01 N NaOH and autoclave ((Christensen and Rasmussen, 1992).
  • Phagocytosis deficient cells. For growing phagocytosis-deficient cells, the final concentrations of FeCl­, CuSO4 and folinic acid should be increased to 1mM, 25 μM and 1 mg/ml respectively (Orias and Rasmussen, 1979).

Christensen, S. T., and Rasmussen, L. (1992). Evidence for growth factors which control cell multiplication in tetrahymena thermophila. Acta Protozoologica, 31(4), 215–219.

Hagemeister, J. J., Grave, M., Kristiansen, T. B., Assaad, F., and Hellung-Larsen, P. (1999). Interface-mediated death of unconditioned tetrahymena cells: Effect of the medium composition. J Eukaryot Microbiol, 46(1), 6–11.

Orias, E., and Rasmussen, L. (1979). Dual capacity for nutrient uptake in tetrahymena. V. utilization of amino acids and proteins. J Cell Sci, 36, 343–353.

Szablewski, L., Andreasen, P.H., Tiedtke, A., Florin-Christensen, J., Florin-Christensen, M., and Rasmussen, L. (1991). Tetrahymena thermophila: Growth in synthetic nutrient medium in the presence and absence of glucose. Journal of Eukaryotic Microbiology, 38(1), 62–65.