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Media Recipes

250 μg/ml penicillin G and streptomycin sulfate and 1.25 μg /ml amphotericin B (Fungizone-GIBCO) can be added to working cultures of any media if desired. This is not necessary if sterile conditions are maintained. We have also found 2 μl/ml Normocin™ (InvivoGen) useful in clearing up stubborn contamination.

In all media, growth can be limited by iron, which can be supplied by iron salts or chelated iron salts.

Rich Axenic Nutrient Media

Proteose peptone (PP) medium[show]

2% proteose peptone
10 μM FeCl3 or 90 μM sequestrene (Fe-EDTA)
Absence of glucose and yeast extract is reported not to affect doubling time.

See: Asai DL and Forney JD. 2000. Tetrahymena thermophila. methods in cell biology. San Diego: Academic Pres. Chapter 4.

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Modified Neff medium[show]

0.25% proteose peptone
0.25% yeast extract
0.5% glucose
33.3 μM FeCl3

To avoid precipitate formation, the FeCl3 is first dissolved in one quarter of the final H2O volume, and the glucose, yeast and PP are added and dissolved next. The remainder of the H2O is then added, bottled and autoclaved.

See: Cassidy-Hanley D, Bowen J, Lee JH, Cole E, VerPlank LA, Gaertig J, Gorovsky MA, Bruns PJ. 1997. Germline and somatic transformation of mating tetrahymena thermophila by particle bombardment. Genetics 146(1):135-47.

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SSP medium[show]

2% proteose peptone
0.1% yeast extract
0.2% glucose
0.003% sequestrene (Fe-EDTA), can be replaced with 33 μM FeCl3

See: Gorovsky MA, Yao MC, Keevert JB, Pleger GL. 1975. Isolation of micro- and macronuclei of tetrahymena pyriformis. Methods Cell Biol 9(0):311-27.

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PPY medium[show]

1% proteose peptone
0.15% yeast extract
0.01 mM FeCl3

See: Smith DL and Doerder FP. 1992. Multiple effects of mutation on expression of alternative cell surface protein genes in tetrahymena thermophila. Genetics 130(1):97-104.

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PPYG medium[show]

0.4% proteose peptone
0.2% yeast extract
1.0% glucose

See: Mori K., Yomo T., Kashiwagi A. 2011. Single-cell isolation and cloning of tetrahymena thermophila cells with a fluorescence-activated cell sorter. J.Eukaryotic Microbiol.Journal of Eukaryotic Microbiology 58(1):37-42.

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PPYS medium[show]

1% proteose peptone
0.15% yeast extract
0.01 mM FeCl3
0.2 M NaCl

See: Smith DL and Doerder FP. 1992. Multiple effects of mutation on expression of alternative cell surface protein genes in tetrahymena thermophila. Genetics 130(1):97-104.

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Chemically Defined Media

Standard synthetic medium[show]

Amino Acid Solution A mg/ml
L-Arg-HCl 12
L-His-HCl • H2O 8
L-Ile 8
L-Leu 8
L-Lys-HCl 8
L-Met 6
L-Phe 6
L-Ser 6
L-Thr 8
L-Trp 6
L-Val 4
Amino Acid Solution B  
L-Glu 4
Amino Acid Solution C  
L-Asn • H2O 8
L-Pro 8
Amino Acid Solution D mg/ml
L-Ala 6
L-Asp 8
L-Glu 16
Gly 16
Amino Acid Solution E  
L-Tyr 8
Nucleoside solutions  
Adenosine 0.2
Cytidine 0.2
Guanosine 0.2
Uridine 0.2
Salts and Chelator Solution  
K2HPO4 • 3H2O 25
KH2PO4 25
MgSO4 • 7H2O 50
CaCl2 • 2H2O 1
Tri-Potassium Citrate 65
Vitamins (Solution A) mg/ml
Na Riboflavin Phosphate • 2H2O 0.05
Vitamins (Solution B)  
DL-6, 8-Thioctic Acid 0.01
Vitamins (Solution C)  
Thiamin-HCl 0.05
Prydoxal-HCl 0.01
Nicotinic acid 0.09
D-Pantothenic Acid, hemi Ca-salt 0.08
Vitamins (Solution D)  
Folinic acid, Ca salt 0.01
Trace metals solution  
FeCl2 • 6H2O 1
MnSO4 • 4H2O 0.16
Co (NO3)2 • 6H2O 0.05
ZnSO4 • 7H2O 0.45
CuSO4 • 5H2O 0.03
(NH4)6 Mo7O24 • 4H2O 0.01
Glucose solution  
Glucose 250

  1. Unless otherwise specified, all stock solutions are made with distilled water, sterilized by filtration and stored at 4°C.
    • Amino acids solutions:
      • A-D: 40-fold concentrated.
      • A, C: adjust pH to 7.
      • B: store frozen.
      • D: dissolve Asp and Glu in water with stirring, use 1 N KOH to prevent the pH from dropping below 7, add Ala and Gly, and adjust pH to 7.
    • Nucleosides solution: 10-fold concentrated.
    • Salts and chelator solution: 100-fold concentrated.
    • Vitamins solutions: 100-fold concentrated.
      • A: store frozen.
      • B: dissolve in 1 ml absolute ethanol, dilute in 100 ml H2O.
      • C: adjust pH to 7.
    • Trace metals solution: 100-fold concentrated, adjust pH to approximately 2 with 1 N HCl.
    • Glucose solution: 50-fold concentrated.
  2. Begin by dissolving Tyr at 60°C to give 0.2 mg/ml in the final medium, cool and 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.
  3. Special purpose modifications:
    • Amino acid solution A contains all the required amino acids; omission of amino acid solutions B-E yields a minimal defined medium.
    • When inocula of less than 2500 cells/ml of final medium will be used, the medium should be supplemented with hemin at a final concentration of 7.5 μM; a stock solution is prepared by dissolving in 0.01 N NaOH and autoclaving.
    • For growing phagocytosis-deficient cells, the final concentrations of FeCl3, CuSO4 and folinic acid should be increased to 1mM, 25 μM and 1 μg/ml respectively.

See:

Szablewski L, Andreasen PH, Tiedtke A, Florin-Christensen J, Florin-Christensen M, Rasmussen L. 1991. Tetrahymena thermophila: Growth in synthetic nutrient medium in the presence and absence of glucose. J Eukaryot Microbiol 38(1):62-5.

Christensen ST and Rasmussen L. 1992. Evidence for growth factors which control cell multiplication in tetrahymena thermophila. Acta Protozoologica 31(4):215-219.

Sanford YM and Orias E. 1981. Phenylketonuric tetrahymena: Phenylalanine hydroxylase mutants and other tyrosine auxotrophs. Proc Natl Acad Sci U S A 78(12):7614-8.

Rasmussen L, Christensen ST, Schousboe P, Wheatley DN. 1996. Cell survival and multiplication. the overriding need for signals: From unicellular to multicellular systems. FEMS Microbiol Lett 137(2-3):123-8.

Asai DL and Forney JD. 2000. Tetrahymena thermophila. methods in cell biology. San Diego: Academic Pres. Chapter 4.

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Bacterized Media

100% BP[show]

Bacterized media may be used to first grow cells and then subsequently starve them, but they are somewhat difficult to prepare and are rarely used. For more information, see:

Simon EM and Hwang SW. 1967. Tetrahymena: Effect of freezing and subsequent thawing on breeding performance. Science 155(763):694-6.

Asai DL and Forney JD. 2000. Tetrahymena thermophila. methods in cell biology. San Diego: Academic Pres. Chapter 4.

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Starvation Media

Dryl's medium[show]

0.59 g of Na citrate-2H2O (2 mM)
0.14 g of NaH2PO4 • H2O (1 mM)
0.14 g of Na2HPO4 (1 mM)
0.13 g of CaCl2 (1.5 mM)
To avoid precipitation of the CA phosphate, the CaCl2 solution is autoclaved separately from the mixture of sodium salts, and the two solutions are mixed aseptically after cooling.

See: Dryl S. 1959. Antigenic transformation in paramecium aurelia after homologous antiserum treatment during autogamy and conjugation. J Protozool 6(25).

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Tris buffer[show]

10 mM Tris HCl, pH 7.5

See: Bruns PJ and Brussard TB. 1974. Pair formation in tetrahymena pyriformis, an inducible developmental system. J Exp Zool 188(3):337-44.

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NKC solution[show]

0.2% NaCl
0.008% KCl
0.012% CaCl2

See: Sugai T and Hiwatashi K. 1974. Cytologic and autoradiographic studies of the micronucleus at meiotic prophase in tetrahymena pyriformis. J Protozool 21(4):542-8.

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Growth Media for Phagocytosis-Deficient Cells

EPP medium[show]

Composition per liter of medium:

20g (2%) PP
0.6g (2mM) Na3 citrate 2H2O
0.27g (1 mM) FeCl3
7.2 mg (30 μM) CuSO4 • 5H2O
1 mg (1.7 μM) Ca salt (Lederle)

See: Orias E and Rasmussen L. 1976. Dual capacity for nutrient uptake in tetrahymena. IV. growth without food vacuoles and its implications. Exp Cell Res 102(1):127-37.

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Chemically defined medium[show]

Use the standard chemically defined medium as shown above. Then increase the concentrations of FeCl3, CuSO4 and folinic acid to 1 mM, 25 μM and 1 μg/ml respectively.

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Protocols

T. thermophila freezing protocol (Word document)
Tetrahymena in the Laboratory: Strain Resources, Methods for Culture, Maintenance, and Storage
Handling plasmids on filter paper (Word document)