Part III: Operating Principles
Chapter 8 - Tetrahymena in the Laboratory: Strain Resources, Methods for Culture, Maintenance, and Storage

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Abstract

The ciliated protozoan Tetrahymena thermophila has been an important model system for biological research for many years. During that time, a variety of useful strains, including highly inbred stocks, a collection of diverse mutant strains, and wild cultivars from a variety of geographical locations have been identified. In addition, thanks to the efforts of many different laboratories, optimal conditions for growth, maintenance, and storage of Tetrahymena have been worked out. To facilitate the efficient use of Tetrahymena, especially by those new to the system, this chapter presents a brief description of many available Tetrahymena strains and lists possible resources for obtaining viable cultures of T. thermophila and other Tetrahymena species. Descriptions of commonly used media, methods for cell culture and maintenance, and protocols for short- and long-term storage are also presented.

Introduction

The increasing use of Tetrahymena for both research and educational purposes has been facilitated by the ease with which it can be grown and maintained in a wide range of conditions, from single cells in hanging drops to multiliter cultures grown in large bioreactors. Sexual reproduction is dependably controlled by transfer to nonnutritive media, and simple selection schemes are available for the identification of sexual progeny. Under optimal conditions, Tetrahymena has a rapid growth rate, with a doubling time of less than 2 h. However, slowly growing vegetative cultures can be maintained on the bench for several months with very limited loss of function or fertility, and strains can be stored for years in liquid nitrogen. A number of mutant and inbred strains of Tetrahymena thermophila, the species most commonly used for physiological, biochemical, and molecular research, are readily available. A number of other Tetrahymena species, many of which can be maintained under conditions similar to those used to culture T. thermophila, are also easily obtainable. This chapter provides basic information on the most frequently utilized T. thermophila strains, current sources for obtaining T. thermophila strains and other Tetrahymena species, and methods for growing, maintaining, mating, and storing Tetrahymena cultures.

Section snippets

Tetrahymena thermophila Strains

T. thermophila provides a rich resource of useful strains, including highly inbred stocks derived from wild isolates over half a century ago (Allen and Gibson, 1973; and Chapter 2 in this volume), a collection of diverse mutant strains, and wild cultivars from a variety of geographical locations. T. thermophila strains are generally named according to location of origin. Wild-type isolates are given a two-letter prefix based on location of origin (e.g., WH for isolates originally collected at

Other Tetrahymena Species

Although, as discussed above, T. thermophila is the primary species of choice for Tetrahymena research, significant work has been carried out using other Tetrahymena species. Tetrahymena ssp. are useful indicators for ecotoxicity tests (Gerhardt et al., 2010), and Tetrahymena pyriformis GL, an amicronucleate strain used for much of the early Tetrahymena research, is still frequently used for toxicological studies (Artemenko et al., 2011, Sauvant et al., 1995, Sauvant et al., 1997, Sauvant et

Cell Culture Media

Originally cultured in bacterized hay or vegetable matter infusions, Tetrahymena was the first animal-like eukaryotic cell to be grown axenically (Lwoff, 1923). Tetrahymena has two separate nutrient uptake systems; phagocytosis, which in Tetrahymena involves intake of particulate matter via a highly specialized oral apparatus and subsequent nutrient digestion in food vacuoles, and a surface uptake system that transports nutrients in solution into the cell (Orias and Rasmussen, 1976, Rasmussen

Basic Information

Tetrahymena can be easily cultured using a wide variety of media, containers, and conditions, as long as basic requirements for nutrition, aeration, temperature, and cell concentration are met. Culture vessels must be kept meticulously clean, and the use of dedicated flasks for cell growth is strongly recommended. The same stringent criteria described above (Section IV.A) for preparing glassware used in making media should be applied to all culture vessels. High surface-to-volume ratios should

Serial Transfer

Tetrahymena can be maintained for years by serial transfer, provided that a reasonable cell inoculum (a minimum of ∼1000 cells) is used for each transfer. However, prolonged vegetative growth can lead to both micronuclear and macronuclear genetic changes over time. In micronucleate strains like T. thermophila, the transcriptionally inactive germinal micronucleus is not subject to direct selection and can accumulate chromosomal changes, including whole chromosome loss, deletions, and lethal

Acknowledgments

D.C.H. is supported by NIH NCRR SEPA grant 5 R25 RR025126-03 and by NIH Grant No. P40 RR019688-01A1. The Tetrahymena Stock Center is funded by NIH Grant No. P40 RR019688-01A1. Past support by NIH, NSF, and USDA is gratefully acknowledged for providing funding for the development of many of these techniques. DCH is a founding member of Tetragenetics, Inc., Ithaca, NY, a company developing and utilizing Tetrahymena as a platform for the manufacture of biotechnological products. Neither this

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