T. thermophila B2086 (II), CU428 (VII), and CU427 (VI) were obtained from the National Tetrahymena Stock Center (http://tetrahymena.vet.cornell.edu/ , Cornell University, Ithaca, NY). Cells were cultured at 30 °C in a super proteose peptone medium (1% proteose peptone, 0.1% yeast extract, 0.2% glucose, and 0.003% EDTA-Fe). For starvation, log-phase cells were washed with 10 mmol/L Tris–HCl (pH 7.4) and resuspended in 10 mmol/L Tris–HCl (pH 7.4) at 30 °C for 16–24 h. The distinct mating type cells were mixed and initiated sexual development.
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