| Popis: |
Publisher Summary This chapter presents a simple method for long-term storage of Tetrahymena thermophila, based on freezing starved cells. Starving cells before freezing greatly enhances the recovery of cells and eliminates the need for complicated freezing apparatuses or protocols. Tetrahymena thermophila and other ciliates require that growth be minimized or even eliminated during long-term storage: extended growth leads to strain degradation. Cultures of fresh exconjugants exhibit normal growth rates and can undergo normal conjugation, following a strain-specific period of immaturity. However, fertility begins to drop within a year or less of continuous growth in the laboratory; matings increasingly tend to undergo the abortive pathway called genomic exclusion. Cells still form pairs in these matings, but they fail to contribute any genome to the progeny. In addition, because they are not expressed and are therefore not selected against, micronuclear deletions accumulate with clonal age. The successful freezing of eukaryotic cells in liquid nitrogen is dependent on stringent control of a number of parameters, including: (1) the physiological state of the cells at the time of freezing; (2) the cryoprotectant and medium in which the cells are frozen; (3) the rate of cooling; (4) the temperature at which the cells are ultimately stored; and (5) the rate and temperature of thawing. |
