CHAPTER 4

Tetrahymena as a Laboratory Organism:

Usefd Strains, Cell Culture, and Cell
Line Maintenance
Eduardo Orias, Eileen P. Hamilton, and Judith D. Orias
Department of Molecular, Cellular and Developmental Biology
University of California at Santa Barbara
Santa Barbara, California 93106

I. Introduction
11. Useful Tetrahymena Strains
A. Tetrahymena Species and Strain Nomenclature
B.
Inbred Wild-Type Strains
C.
Generally Useful Mutant Strains
D.
Heterokaryons
E.
Star Strains
F. Panels of Strains Used for Mapping
G. Strains &om Species Closely Related to Tefrahymena themophila
111. Culture Media
A. General Considerations
B. Rich Axenic Nutrient Media
C. Chemically Defined Media
D. Bacterized Media
E. Starvation Media
F. Growth Media for Phagocytosis-Deficient Cells
IV. Culturing Tetrahymena Cells
A. General Considerations
B. Culture Vessels and Conditions
C. Dealing with Culture Contamination
V. Cell Line Storage
A. General Considerations
B. Maintenance of Stock Cultures
C. Storing Tetrahymena Cell Lines under Liquid Nitrogen
References
METHODS IN CELL BIOLOGY, VOL. 62
Copynghr 0 1999 by Academc P m r . AU righa of reproduction in any form reserved 189
0091-679W99 130.00
190 Orias ct al.

I. Introduction
A key to the usefulness of Tetruhymenu as a laboratory organism is its excep-
tionally fast growth rate under simple and inexpensive culture conditions. With
a doubling time under 2 h, Tetruhymenu is one of the fastest multiplying free-
living eukaryotic cells. The genetic domestication of Tetruhymenu thermophilu
added another dimension to the utility of this organism. In this chapter, we
describe useful inbred and mutant strains, growth media, and some basic methods
for laboratory storage and culture of Tetruhymenucells. Supplementaryinforma-
tion on these topics can be found in Orias and Bruns (1975).

II. Useful Tetruhymena Strains

A. Tetruhyrnenu Species and Strain Nomenclature
The present day Tetruhymenu thermophilu was originally a member of what
eventually was found to be a large species complex then known as Tetruhymenu
pyriformis (see Chapter 1). The discovery of sexually isolated groups within
this species complex led to the definition of varieties, later renamed syngens.
Subsequent advances in molecular discrimination between these groups allowed
the assignment of individual species names to the syngens (Nanney and McCoy,
1976). Tetruhymenu thermophilu started out as variety 1 and later syngen 1 of
Tetruhymenapyriformis, before acquiring its present name. The species Tetruhy-
menu pyriformis was named by Furgason (1940) after a careful investigation of
oral morphology. Prior to that time, the classificationwas in a state of confusion,
and Tetruhymenu species were referred to by a variety of other genus names
(e.g., Colpidium and Glaucoma).
The earliest and much of the subsequent physiological,biochemical, and molec-
ular work with Tetruhymenu used strain GL of Tetruhymenu pyriformis, which
retains the pyriformis species name in the new taxonomy. This strain lacks a
micronucleus and does not conjugate (i.e., has no sexual stage in its life cycle).
Many Tetruhymenu cell and molecular biologists have chosen to work with T.
thermophilu rather than pyriformis for reasons that include (1) the advantages
of laboratory mutants and genetic analysis and (2) an interest in the biology of
nuclear dimorphism. However, cell division is much easier to synchronize in
pyriformis than in thermophilu.
Wild-type isolates of Tetruhymenu thermophilu are given a two letter prefix,
derived from the location of isolation, followed by a serial number (e.g., WH52).
Strains generated as progeny of laboratory crosses are named with a two letter
prefix, specific for each laboratory, followed by a serial number (e.g., SB210).
While informal names are generally used when clones are first isolated (e.g.,
based on an experiment number and 96-well plate coordinate), strains important
enough to use for further work andor to name in a publication should be given
4. Tetrahymena as a Laboratory Organism 191

a proper name according to the preceding convention (and should be stored

under liquid nitrogen without delay). Appendix I and Allen et al. (1998) should
be consulted for rules and conventions for describing MIC and MAC genotypes
and phenotypes of Tetrahymena strains.

B. Inbred Wild-Type Strains

A program of inbreeding Tetrahymena thennophila was carried out mainly in
the 1950s and 1960s through the efforts of David L. Nanney and Sally L. Allen.
A list of the resulting inbred strains can be found in Allen and Gibson (1973),
Orias and Bruns (1975), and Allen et al. (1984). Cultures of the inbred strains
are available from the American Type Culture Collection (ATCC).
By agreement among Tetrahymena researchers, laboratory mutants are gener-
ated in the genetic background of inbred strain B. Differences between inbred
strain B and C3 have been used to identify DNA polymorphisms and to construct
a map of the genome (Orias, 1997, 1998). They have also been used to isolate
and characterize mutations affecting the amplification or replicative maintenance
of the rDNA (i.e., the MAC chromosome piece that carries the 18s and 28s
rRNA genes) (Larson et al., 1986).

C. Generally Useful Mutant Strains

Several laboratory mutant strains have been found to be generally useful by
Tetrahymena cell and molecular biologists. Most of the mutants described in this
chapter are currently available from the Orias laboratory. A list of other labora-
tory mutants can be found in Bruns and Cassidy-Hanley (1993). Please note that
if a mutant is extensively maintained under conditions where it grows slower
than wild type, reversion can occur in the MAC, and wild-type revertants can
eventually replace the mutant cells in the culture. (Reversions in the MIC are
generally of no concern for purposes other than breeding because they are neither
expressed nor selected for.) The 45-ploidy of the MAC is no impediment to
natural selection for cells carrying a reverted allele because assortment (see
Chapter 3) allows for an increase in the ratio of revertant-to-mutant alleles in
the MAC of some descendants,with a consequent growth advantage and natural
selection for those cells. Thus, it is important always to maintain stocks of such
mutants under the most permissive conditions and, if necessary, to go back to
frozen stocks. It is also important to verify that the mutants still have the adver-
tised phenotype before and after experiments.
1. Mutants with blocked exocytosis. In wild-type strains, the sticky material
discharged by mucocysts can hinder the purification of macromolecules or cell
organelles. Strain SB255 and its derivative SB715 carry a mutation that prevents
mucocyst discharge (Orias et al., 1983). These strains are used to isolate cilia,
purify dyneins, and make cortex preparations for in vitro assay of motor proteins
(e.g., Dentler, 1995; Johnson, 1986; Lombillo et al., 1993).
192 Orias er al.

2. Mutants blocked in phagocytosis. Phagocytosis (food vacuole formation)

at the oral apparatus and excretion of food vacuole contents at the cytroproct
constitute a route for the entry of nutrients and exit of waste products and
possibly enzymes. Mutants with a temperature-sensitive block in oral apparatus
development and, as a consequence, in phagocytosis (NP1, Orias and Pollock,
1975; SJlOO series, Suhr-Jessen and Orias, 1979; 118G,Tiedtke et aZ., 1988) have
been used to investigate entry routes of compounds and exit routes of hydrolytic
enzymes (e.g., Silberstein,1979).These mutants are also useful in another context.
Certain experimentally produced cellular malfunctions (e.g., ciliary loss or paraly-
sis or the loss of the micronucleus) can directly or indirectly inactivate phagocyto-
sis. Media designed for phagocytosis-deficient cells (see Section 1II.F) can be
used to distinguish whether the failure of the cells to grow is mechanistically
related to the malfunctions or is merely a secondary consequence of starvation
(e.g., Haremaki et aZ., 1996). When doing such tests, a mutant with temperature-
sensitive phagocytosis a good positive control. Be aware that NP1 sooner or
later reverts to the wild-type phenotype when maintained indefinitely at 37°C
(i.e., under the conditions that block phagocytosis) (Orias and Rasmussen, 1976).
Another mutant with the potential for general utility is blocked in the secretion
of lysosomal enzymes (Huenseler et aL, 1987).

D. Heterokaryons
Heterokaryons are strains in which the MIC and MAC differ genetically. One
very useful aspect of these strains is their ability to harbor lethal mutations in
homozygous form in the MIC. Such heterokaryonsare viable because their MAC,
the site of gene expression, is wildtype. Strains with germline knockouts of
essential genes can only be maintained as heterokaryons (see Chapters 27 and
28). Nullisomic strains are special heterokaryons in which both copies of entire
chromosome or chromosome arms are missing in the MIC (Bruns et aZ., 1982,
1983); they have been useful for mapping mutant genes and DNA sequences to
chromosome arms (see Chapter 10). Unisomic strains are special nullisomic
strains that contain a single chromosome in the MIC (P. J. Bruns and D. Cassidy-
Hanley, personal, communication); these strains are proving useful in mapping
DNA sequences to chromosome arms by fluorescence in situ hybridization
(FISH) (P. J. Bruns and D. Cassidy-Hanley, personal communication). Hetero-
karyons also find constant use in a variety of genetic work (Chapters 6 and 10).
Lists of nullisomic strains and other useful heterokaryons are given in Table I
of Chapters 10 and 6, respectively. These heterokaryons are generally available
from Tetrahyrnena genetics laboratories.

E. Star Strains
Star strains have a vestigial MIC, which is unable to contribute any genetic
information to sexual progeny. In a cross of a normal and a star strain, two
4. Tetruhymcna as a Laboratory Organism 193

successive rounds of mating (round I and round 11) occur (genomic exclusion;
Allen, 1967; see also Chapter 3). The round I exconjugants have a new MIC
that is homozygous for the entire genome present in one meiotic product from
the normal mate but retain their parental MAC.
During round I conjugation, star strains do not generate any meiotic products
or gametic nuclei. As a consequence, star strains have been useful in the study
of the cell biology of fertilization in Tetrahymena (e.g., Takagi et al., 1991). Star
strains are also potentially useful in conjugation rescue experiments when one
wants to exclude any possibility that the restoration of the wild-type phenotype
in the mutant cell is due to the transfer of wild-type genes from the mate (Satir
et al., 1986; see also Chapter 9). Conjugation with a star strain is also frequently
used to generate heterokaryons and for a variety of other purposes in genetic
procedures (see Chapters 6, 10, and 28). Table I gives a list of star strains, along
with their relative usefulness for various genetic applications.

F. Panels of Strains Used for Mapping

Panels of meiotic segregants and terminal assortants derived from heterozy-
gous progeny of inbred strains B X C3 have been used to map genetic loci
and DNA polymorphisms to the micronuclear and macronuclear genomes (see
Chapter 10). Those strains will be placed in the ATCC; meanwhile, you should
contact the Orias lab if you need to use these panels.

G. Strains from Species Closely Related to Tetrahymena themtophila

An extensive collection of species with a variable degree of genetic relationship
to Tetrahymena thermuphila is available mainly through the efforts of Ellen

Table I
Usefd Star Strains
Non-wild
Strain" Phenotype Utilityb

A*III 6mp-r Best for making homozygous heterokaryons;

acceptable for CR, not good for UPC or
SCGE
A*V NR Best for UPC
B*VI NR Best for UPC
B*VII NR Acceptable for UPC
C*III NR Best for SCGE acceptable for UPC

The first letter indicates inbred strain background (e.g., inbred strain
A for A*III).
See more detailed discussion in Cole and Bruns (1992).
Key: 6mp-r, resistant to 6-methylpurine; CR, conjugation rescue (see
Chapter 9); NR, none reported, wild type expected; SCGE, short circuit
genomic exclusion; UPC, uniparental cytogamy.
194 Orias ct al.

Simon and the late Alfred Elliott. These strains are particularly useful for detect-
ing functionally important regions in RNA and protein molecules through com-
parative DNA sequence analysis. For example, they have been used to determine
functionally important features (sequence and secondary structure) of the telo-
merase RNA molecule (Romero and Blackburn, 1991). The thoroughly investi-
gated rRNA-based phylogeny of these species (Nanney et al., 1989) allows com-
parison at different scales of evolutionary distance and varying rates of sequence
change. A list of strains used for phylogeny determination are listed in Preparata
et al. (1989); they are available from the ATCC.

III. Culture Media

A. General Considerations
Tetrahyrnenacells possess two efficient and sufficient routes of nutrient uptake
(Rasmussen and Orias, 1975): phagocytosis of particulate matter and active
transport of nutrients in solution. Tetrahyrnena cells (like many other ciliates)
were at first grown in bacterized infusions of vegetable material (such as hay or
baked lettuce leaf infusions). Later it became the first animal(-like) eukaryote
to be grown axenically (i.e., in the absence of other organisms, such as bacteria)
(Lwoff, 1923).Transplantable growth in a chemically defined medium was subse-
quently achieved by Kidder and Dewey (1951). Recent work has shown that
multiplication of Tetrahyrnena cells is under the control of an autocrine cytokine
system (reviewed in Rasmussen et aZ., 1996). This is a matter of practical signifi-
cance only when using chemically defined media.
De-ionized and/or distilled HzO of high purity as well as dedicated glassware
should be used to make media, as Tetruhyrnena cell are sensitive to impurities
in the water and soap residue on the glassware. Most types of glassware that
have been washed and rinsed in an automatic dishwasher are generally adequate
for storing media and growing cells. Potential exceptions are Erlenmeyer flasks
and other glassware with “bottle necks”; past experience in our lab has led us
to wash and rinse such containers exclusively by hand.

B. Rich Axenic Nutrient Media

Proteose peptone (PP), a peptic digest of beef extract manufactured by Difco,
has been the traditional basis for rich axenicmedia for growing Tetrahyrnenacells.
An excellent, general-purpose rich axenic medium is SSP medium (Gorovsky et
al., 1975). It consists of 2% PP, 0.1% yeast extract (Difco), 0.2% glucose, and
0.003%sequestrene (Fe-EDTA, Ciba-Geigy Chemical Co., Ardsley, NY).In this
medium, cells grow with a doubling time of 2.5 h at 30” C and in shaking cultures
can attain concentrations of at least 106 cells/ml. At 35”C,a minimum doubling
time (just under 2 h) is attained.
4. Tehahytnena as a Laboratory Organism 195

To prepare the medium, the components are dissolved in a small fraction of

the finalvolume. Insolubleparticles that can interfere with electronic cell counting
are eliminated either by centrifuging the medium at 7000 g for 30 min or by
filtering through Whatman No. 1filter paper. The concentrated medium may be
aliquoted and stored frozen at -20°C. It is then diluted to the desired concentra-
tion and sterilized by autoclaving (filter sterilizationis not advised;see Rasmussen
and Modeweg-Hansen, 1973). The sterile medium lasts for months at room
temperature if stored in the dark, as some of the required vitamins are light sen-
sitive.
When sterile conditions for the handling of cells must be relaxed (e.g., isolating
single cells by hand or replica plating on an open bench), penicillin G and
streptomycin sulfate (each at 250 pg/ml final concentration) are added in order
to discourage the growth of contaminating bacteria. A filter-sterilized stock
solution containing a mixture of both antibiotics at 1000times the final concentra-
tion is kept at -20°C (not at 4°C) and added aseptically to the culture medium
just before use. Amphotericin B (Fungizone, GIBCO) is added in some labs to
discourage the growth of yeasts and other fungal contaminants; published final
concentrations vary from 0.025 to 25 pg/ml.
In PP (even at the 2% concentration most commonly used), growth is limited
by iron. The required iron (ferrous or femc) can be supplied by supplementation
with salts (e.g., chloride), chelated salts (e.g., citrate or EDTA), or yeast extract.
Ferric or ferrous chloride have the disadvantage that they precipitate during
autoclaving of the medium. This precipitate does not affect doubling time, but
the particles can interfere with electronic cell counting. Precipitation can be
avoided by adding the ferric chloride from a filter-sterilized stock solution after
the medium has cooled or just before using it. If the doubling time in PP media
not supplemented with iron salts exceeds 2.5 h at 30”C, iron supplementation is
the first measure to try in order to increase the growth rate.
There are many variations of PP medium in use. For example, the glucose
and yeast extract can be omitted without affecting the doubling time of the cells.
Much of the ordinary genetic work in our lab has been done using 2% PP with
10 p M FeC1,; more recently the latter was replaced with 90 p M Fe-EDTA
because of the convenience of being able to add it before autoclaving. Fe EDTA
is prepared as a filter-sterilized 1000-fold concentrated solution and is stored
at 4°C.
“Modified Neffs” medium (Cassidy-Hanley et aZ., 1997) is used in DNA-
mediated transformation experiments. Its composition is as follows: 0.25% PP,
0.25% yeast extract, 0.5% glucose, 33 pM FeC13 supplemented with penicillin G
and streptomycin sulfate at 250 pg/ml each, and 1.25 p g / d of amphotericin B
(Fungizone, GIBCO). To avoid precipitate formation, the FeC13is first dissolved
by stirring in one-fourth of the final volume of H20; the glucose, yeast extract,
and PP are dissolved next. The remainder of the H 2 0 is then added, distributed
to bottles, and autoclaved.
196 Orias ct al.

The proteose peptone concentration in growth media can be reduced to 1%

without significant change in the doubling time, but its prolonged use can lead
to adverse effects. We have found that using 1%PP supplemented with 5 pM
FeC13 as an all-purpose medium for genetic work and stock maintenance greatly
increased the rate at which cells became infertile in crosses (unpublished obser-
vation).
The biotech potential of Tetrahymena (Munro, 1985; Wheatley et al., 1994)
has motivated searches for inexpensive growth media and conditions that opti-
mize yields under industrial-scale conditions (Kiy and Tiedtke, 1992a; Ethuin et
al., 1995). A doubling time of 1.4 h at 30°C has been reached in a growth medium
composed of 2% dried skimmed milk, 0.5% yeast extract, and 0.003% sequestrene,
and maximum cell concentrations of 2 X lo7 have been achieved using this
medium in a perfused bioreactor (Kiy and Tiedtke, 1992b).

C. Chemically Defined Media

Defined media are used in special circumstances (i.e., when it is particularly
important to control the chemical composition of the medium). Tetrahymena
and humans have very similar nutritional requirements, a feature that has made
this organism useful for testing the suitability of foodstuffs for human consump-
tion (e.g., Koehler et al., 1987).
The composition of a useful nutritionally complete, chemically defined medium
(Szablewski er al., 1991) and instructions for preparing it are shown in Table 11.
In this medium, cells typically grow with an identical doubling time as that
obtained in proteose peptone medium (2 h at 37°C) and reach concentrations of
up to 106celldml. A minimal defined medium useful for working with auxotrophic
mutants (Sanford and Orias, 1981) can be obtained by leaving out amino acid
mixtures B-E in Table 11. In this medium, cells grow a little slower (doubling
time of 2.5 h at 30°C).
The preceding media are considered nutritionally complete because Tetrahy-
menu cells can grow indefinitely in them when propagated by inoculating with
at least 2500 cells/ml. However, when the medium is inoculated at low cell
concentrations(below 250 cells/ml),the cells fail to multiply. An extensive investi-
gation of this phenomenon by Leif Rasmussen and his collaborators has led to
the conclusion that the multiplication of the Tetrahymenu cell is dependent on
an autocrine stimulation system. While the natural cytokine is not yet known,
any one of a number of compounds (e.g., hemin; see Table I1 and a longer list
in Rasmussen et al., 1996) circumvent the need for its action and allow a small
inoculum of cells, even a single cell, to multiply in defined medium (Christensen
and Rasmussen, 1992). (PP and bacterized growth media have adequate concen-
tration of growth factors so that even single cell isolates grow well in these media.)

D. Bacterized Media
Bacterized medium is used when one wants cells fist to grow and, without
further manipulation, to subsequently starve and become competent to mate. It
4. Tehahymma as a Laboratory Organism 197

Table I1
Composition of Standard Synthetic Medium for Tehahymena Cell and Concentration (mglml) of
Components in Stock Solutions
Amino Acid Solution A Amino Add Solntion E VitpminS Solution c
L-Arg-HC1 12 L-TYr 8 Thiamin-HC1 0.05
L-His-HC1 . H 2 0 8 (do not prepare) hydoxal-HC1 0.01
L-Ile 8 Nudeosidea Solution Nicotinic acid 0.09
L-Leu 8 Adenosine 0.2 D-Pantothenic
L-LYs-HCI 8 Cytidine 0.2 Acid, hemi Ca-salt 0.08
L-Met 6 Guanosine 0.2 Vitamins Solution D
L-Phe 6 Uridine 0.2 Folinic acid, Ca salt 0.01
L-Ser 6 Salts and Chelator Solution Trace Metala Solution
L-Thr 8 KzHPo4 * 3Hzo 25 FeClz . 6H20 1
~-Trp 6 Mzpo4 25 ms04 4Hzo 0.16
L-Val 4 MgSO4 .7Hzo 50 Co (NO& . 6Hz0 0.05
Amino Acid Solution B CaClz * 2HzO 1 ZnS04 . 7 H 2 0 0.45
I.-Glu 4 Tri-Potassium C U S O *~5HzO 0.03
Amino Acid Solution C Citrate 65 m )c iMe024 .4Hzo 0.01
L-Asn . H20 8 VitpminS Solution A Glucose Solution
L-Pro 8 Na Riboflavin Glucose 250
Amino Acid Solution D Phosphate * 2H20 0.05
L-Ala 6 Vitamins Solution B
L-ASP 8 DL-6,8-Thioctic
L-G~u 16 Acid 0.01
GlY 16

From Szablewski et al. (1991).

1. Relative concentrations and special preparation instructions for various stock solutions: 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: Ten-fold concentrated.
Salts and chelator solution: 1Wfold concentrated. Vitamins solutions: 100-fold concentrated; A store frozen; B: dissolve
in 1 ml absolute ethanol, dilute in 100 ml HzO; C adjust pH to 7. Trace metals solution: 100-fold concentrated, adjust
pH to -2 with 1 N HCl. Glucose solution: 50-fold concentrated.
2. Preparation of the final medium: Begin by dissolving Tyr at 60°C to give 0.2 m g / d in the final medium, cool, and
proceed to add the remaining solutions. The pH may be adjusted as required. The medium may be sterilizedby autoclaving
or by filtration; if autoclaving is used, glucose should be added aseptically after cooling.
3. Modifications for special purposes: (a) Amino acid solution A contains all the required amino acids; omission of
amino acid solutions B-E yields a minimal defined medium (Szablewski et al., 1991). (b) When inocula of less than 2500
celWtn1 of final medium will be used, the medium should be supplemented with hemin at a final concentration of
7.5 pM;a stock solution is prepared by dissolving in 0.01 N NaOH and autoclaving (Christensen and Rasmussen, 1992).
(c) For growingphugocytosk-deficientceh, the final concentrations of FeC13,CuS04;and folinic acid should be increased
to 1 mM, 25 pM and 1 &ml, respectively.

is used mainly for certain genetic work (e.g., mating-type tests or crossing cells
in 96-well plates; see Chapter 6). The most convenient medium for these purposes
is 2% bacterized peptone (Simon and Hwang, 1967). Maximal growth rate and
cell concentrations of 2 X 10'' cells/ml are typically obtained using this culture
medium.
198 Orias ct al.

A “100%” bacterized peptone (100% BP) is made by taking a flask of SSP

medium (or any other variation of PP medium) with no antibiotics added, inocu-
lating it with colony from a streak of Klebsiellu pneumoniue (formerly known
as Aerobucter uerogenes), and shaking overnight at 30°C or 37°C. This 100%BP
culture is kept in the refrigerator for no more than 1 week. To make 2% BP,
the 100%BP culture is diluted 50-fold with sterile water on the same day it is
needed. In order to keep the PP concentration at a level that allows starved cells
to mate, the 2% BP is inoculated by making at least a 50-fold dilution of a
Tetruhymenu culture grown in any of the standard 2% PP media. Bacterial cells
remaining in the 2% BP medium after the Tetruhymenu cells have starved can
be readily eliminated by adding penicillin and streptomycin to the subsequent
growth medium.
Early genetic work and stock maintenance for Tetruhymenu was done using
a bacterized infusion of rye leaves, marketed under the name of Cerophyl by
Cerophyl Laboratories Inc. (Nanney, 1953). Occasionally, special experiments
still call for the use of this medium [e.g., to reproduce early observations on
temperature effects on mating type frequency distribution (E. Orias, unpublished
observations) or to overproduce mucocysts in cells starved in unbacterized Cero-
phyl (Pesciotta and Satir, 1985)l. Cerophyl medium is prepared by adding 1.5 g
to 1liter of boiling distilled H20, filtering through Whatman #1filter paper after
cooling, and then autoclaving. The medium is finally inoculated from a Klebsiellu
streak and incubated overnight at 37°C.

E. Starvation Media
Many experimental procedures require starving Tetruhymenu cells. A large
variety of salts media and even sterile distilled water have been used for this
purpose. Cells can remain alive for more than a week in starvation medium.
There is an extensive literature on the physiology, biochemistry, and molecular
biology of starved Tetruhymenu cells. One relevant consequence of starvation is
the development of sexual reactivity, a process known as initiation. However,
high ionic strength of the starvation medium blocks initiation and thus mating
(Bruns and Brussard, 1974).
Three starvation media used for making Tetruhymenu crosses are:
Dryl’s medium (Dryl, 1959): It contains, per liter, 0.59 g of Na citrate-2H20
(2 mM), 0.14 g of NaH2P04 - H20 (1 mM), 0.14 g of Na2HP04 (1 mM),
and 0.13 g of CaClz (1.5 mM). To avoid precipitation of Ca phosphate, the
CaC12 solution is autoclaved separately from the mixture of sodium salts,
and the two solutions are mixed aseptically after cooling. One-hundred-fold
concentrated stock solutions can be prepared and stored in the refrigerator.
Tris buffer (Bruns and Brussard, 1974): 10 mM Tris HCl, pH 7.5.
NKC solution (Sugai and Hiwatashi, 1974): 0.2% NaC1, 0.008% KCl, and
0.12% CaC12.
4. Tefrahymena as a Laboratory Organism 199

F. Growth Media for Phagocytosis-Deficient Cells

The availability of a mutant with temperature-sensitive phagocytosis (Orias
and Pollock, 1975) led to the discovery that without phagocytosis Tetruhymenu
cells do not grow beyond two to three doublings in ordinary PP medium. The
mutant also allowed the development of axenic growth media in which cells
grow indefinitely without phagocytosis. EPP medium (Orias and Rasmussen,
1976) is one such medium that has the following composition per liter of
medium: PP, 20 g (2%); Na3 citrate 2H20, 0.6 g (2 mM); FeC13, 0.27 g (1 mM);
CuS04 . 5H20, 7.2 mg (30 p M ) ; and folinic acid, Ca salt (Lederle), 1 mg
(1.7 p M ) . A chemically defined medium for phagocytosis-deficient cells is for-
mulated by taking a standard chemically defined medium, such as the one in
Table 11, and increasing the concentrations of FeC13, CuSo4 and folinic acid to
1 mM, 25 pM, and 1 pg/ml, respectively. In bacterized medium, phagocytosis is
absolutely required for multiplication.

IV.Culturing Tetruhyrnenu Cells

A. General Considerations
Tetruhymenu can be cultured in standing containers or under forced aeration
(e.g., in a shaker or a tube rotator); a high surface-to-volume ratio is important
for maintaining adequate gas exchange in standing cultures. Cells that have
reached high concentration often must be transferred to conditions that are less
favorable for gas exchange (e.g., from a shaking culture to a centrifuge tube for
harvesting). Massive cell death (“culture crash”) will follow if one does not move
swiftly until the cells are returned to favorable conditions or are lysed under
experimental control or are transferred to ice bath temperature.
When Tetruhymenu cells need to be cultured for hundreds of fissions at 30°C,
as in certain genetic procedures, it is important to avoid subculturing the cells
at such high dilutions and rapid intervals that the cells remain essentially in
continuous exponential growth. Under these conditions, telomeres will lengthen
at a constant rate per fission, eventually resulting in a decreased growth rate and
replacement by mutants unable to undergo telomere lengthening (Larson et
uL, 1987).

B. Culture Vessels and Conditions

Tetruhymenu cells are routinely cultured in the laboratory in volumes ranging
from 30 p1 to several liters. They can be grown in drops on petri plates, wells
in plastic plates (e.g., 96-well plates), test tubes, flasks, carboys, and fermentors.
In stationary (i.e., nonshaking) cultures, maximal growth rates are obtained if
gas exchange conditions are adequate (e.g., a height of up to 5 mm in a cylindrical
container). When the need for large culture volumes make the depth limitation
200 Orias ct al.

impractical, the cultures must be rotated or shaken. In SSP medium cells can be
shaken at speeds up to 200 rpm without cell damage. Advanced techniques have
been used for efficiently growing Tetrahymena in industrial scale volumes (Kiy
and Tiedtke, 1992b). Cloning cells and replica plating involve specialized culture
methods that are described next.

1. Single Cell Isolation (Cloning)

Cells are cloned by physically isolating them into separate drops of growth
medium. Roughly 30-1.11 drops are placed directly on sterile standard-size Petri
dishes (100 X 15 mm) in a 6 X 8 drop grid pattern that matches the wells of
half of a 96-well plate (see illustration in Roberts and Orias, 1973). Drops can
be placed on the plate using a sterile Pasteur pipette, a pipetman (single or
multichannel), or a “dropmaker” [i.e., a block with 48 thick (1/4 in. diameter)
metal rods that picks up liquid and deposits it in the correct pattern]. The
dropmaker is sterilized as described later for the replicator.
Micropipettes are used to pick up cells under a dissecting microscope and then
release them, one to each drop. Micropipettes can readily be drawn by hand
over a Bunsen burner flame starting with glass tubing or a Pasteur pipette or by
using a micropipette puller. The micropipette is sterilized before each use by
drawing and expelling near-boiling water in a beaker kept on a hot plate by the
microscope. The suction for loading cells can be created manually with a bulb
or by mouth-using tubing to connect a mouthpiece to the pipette (Orias and
Bruns, 1975). The plates are carefully placed in a moist chamber (to prevent
drying up of the drops) and are incubated (without shaking) at 30°C. After 2 to
3 days, the number of cells has increased enough to allow replica plating to 96-
well plates. Colony formation on nutrient agar plates has been used rarely for
cloning, but special conditions are needed to discourage cells from swimming
from one colony to another (Gardonio et aZ., 1975).

2. Replica Plating (Replication)

This is done with “replicators,” consisting of 48 slender (1/16 in. diameter)
metal rods driven into a wooden block in a grid pattern that matches the well
pattern in a 96-well plate. The rods are sterilized before each use by dipping
them first in distilled H20 (to avoid the accumulation of burnt peptone residue),
then dipping them in 95% ethanol kept in a large petri plate, and finally quickly
passing the rods through a flame to burn off the ethanol, which can kill the cells.
After cooling, the rods are 6rst dipped in the drop cultures of the master plate
and then into the wells of the 96-well replica plate, previously filled with 25-
200 pl of medium depending on the needs of the experiment. It is most efficient
to work with two replicators, so that one cools while the other is in use. Two
48-rod replicators can be mechanically joined to speed up replication between
96-well plates. Cells with wild-type growth rate, maintained at 30”C, can be
4. Tefrahymma as a Laboratory Organism 201

replicated again after 1to 3 days. When the objective is to make serial replications
at maximum rate (e.g., to accumulate fissions and reach sexual maturity), continu-
ous daily replication can lead to loss of lines, perhaps because of telomere
elongation problems. In such case, it is safer and convenient to use a Monday,
Tuesday, Thursday, Friday weekly replication schedule.
A safety note: The ethanol used to sterilize the replicator will immediately
catch on fire if a drop of burning ethanol falls on it. The flame at first can be
nearly colorless and hard to detect except for the heat. To avoid this hazard,
keep the ethanol in a glass, rather than plastic, container (e.g., a 14-cm Petri
dish) and always direct the replicator with burning ethanol away from the ethanol
container. If the ethanol catches on fire, smother it quickly by covering the
container with its glass cover, which should be kept within reach.
For reasons of economy and conservation, we reuse 96-well plates. We wash
them in a dishwasher, rinse them by hand with pure water, and dry them standing
on edge at 30°C or 37°C (they melt in a conventional glassware drying oven or
in the autoclave). The plates are sterilized for 1 h under germicidal UV lamps
in a light-tight enclosure (to avoid eye and skin damage by UV). To sterilize,
place ten plates on an aluminum baking tray, irradiate for 30 min, quickly turn
them over with a minimum of manipulation, and irradiate for another 30 min.
Sterile plates are stored in a container previously sterilized by rubbing with
ethanol.

C. Dealing with Culture Contamination

Whenever possible, use the standard sterile technique (flaming pipettes, tips
and bottle necks, dipping replicators in alcohol, etc.). Some frequently used
procedures do not lend themselves to these extremes of care (e.g., doing single-
cell isolations, replica plating, or examining plates under the microscope). To
avoid contamination, some prefer to work in a sterile hood. In our lab, we
have traditionally done this work on an open bench. We add penicillin and
streptomycin to the medium, we keep plates uncovered as little as possible, and
we periodically wash the moisture chambers in which plates are incubated and
“dust” the microscope and the bench with an ethanol-soaked cloth or paper
towel.
In spite of all precautions, cultures occasionallyget contaminatedwith bacteria
or fungi. Generally, the contaminants grow faster than the Tetruhymenu and are
detected because they change the macroscopic appearance of the culture. It is
important to detect and remedy contamination quickly. When it is vital to save a
given contaminated culture, the procedure in the next section should be followed.
Often, however, the contaminated culture can simply be discarded because back-
ups are available (e.g., an earlier culture or a frozen stock).
Contamination of a well culture in a 96-well plate is particularly dangerous
because the contamination can spread to adjacent wells. In this case, the contami-
nated culture is aspirated off using a suitable pipette, the well is rinsed with
202 Orias ct al.

ethanol, and as much as possible of the ethanol is aspirated off. If ethanol is left
in the well, during subsequent incubation its vapor will kill the Tetruhymenu
cells in the adjacent wells.
When one needs to clean a culture contaminated with antibiotic-resistant
microbes, the following serial dilution procedure (Lwoff, 1923) works most of
the time.
Isolate several cells singly into individual drops of PP medium on a sterile
plastic Petri plate.
Transfer each cell to a new drop of medium serially every 30 min. (Live
spores can survive inside food vacuoles; if the cells are transferred at too
short intervals, contaminated food vacuoles may remain in the cell after
transfer to the last drop, recontaminatingthe culturewhen they are excreted.)
After five transfers, incubate the drops at 30°C overnight.
Transfer to growth medium in test tubes samples of drops that have Tetruhy-
menu cells and look free of contamination.
To decisively test for contamination, spot a sample of the culture on a PP
nutrient agar plate and incubate 1 or 2 days at 30°C. It may be necessary to
examine the plate under a dissecting microscope in order to distinguish
between colonies of contaminating microbes (generally opaque and circular)
and Tetruhymena cells (flattened but translucent).

V. Cell Line Storage

A. General Considerations
Tetruhymena cell lines are potentially immortal when transferred using rela-
tively large inocula (e.g., at least lo00 cells). Some Tetruhymenu clones are still
alive after more than 50 years of laboratory culture. On the other hand, because
the micronuclear genome is not expressed, there is no selection against chromo-
some losses and other DNA rearrangements and point mutations. Sooner or
later Tetruhymenu cell lines become sterile (i.e., unable to transmit germline
genetic information to sexual progeny) (reviewed by Nanney, 1974; see also
Allen et ul., 1984, Orias et al., 1999). Sterile cells can pair with fertile cells and
may well generate living exconjugants, but these will have retained the parental
macronucleus and phenotype and will have a MIC of uncertain genotype. Sterility
is reached progressively, and it is not possible to predict how quickly it will
happen for any given clone. To ensure the long-term maintenance of cell lines
with a desired germline genotype and with high fertility, cells are stored frozen
under liquid nitrogen.
Wild-type cells or cells with a MAC that is genetically homogeneous for a
trait of interest need not be maintained frozen if one is interested in only MAC
genes or their expressed products or the cell’s phenotype. However, cells with
4. Tetrahymma as a Laboratory Organism 203

a genetically mixed MAC generally cannot be maintained with any guarantee

that the MAC will remain mixed. This is because assortment during successive
cell multiplication constantly changes allele ratios and eventually leads to the
fixation of one allele (see Chapter 3). Even storing cells with a mixed MAC
frozen under LN, does not guarantee the recovery of a representative population
because only a relatively small fraction of cells survives freezing and thawing.
In maintaining cell lines with particular genotypes of interest, it is important
that the cells have no opportunity to inadvertently undergo conjugation. Members
of a clone whose progenitor was a cell pure for mating type are guaranteed
not to conjugate with one another under any known conditions. The normal
mechanism of mating type determination often generates progeny cells with a
mixture of mating type determinants in their macronucleus (reviewed in Orias,
1981). However, cell lines have more than 95% probability of being pure for
mating type if subcloned after having undergone at least 100 fissions from the
previous conjugation. Cells in PP-based media are guaranteed not to conjugate
in stationary phase even with a mixture of mating types in the culture. Cultures
containing more than one mating type will likely conjugate in bacterized or
chemically defined media when they run out of bacteria or of at least one
required nutrient.

B. Maintenance of Stock Cultures

Stock cultures are maintained for everyday work, (e.g., to ensure a constant
supply of reasonably healthy cells with which to inoculate cultures for an experi-
ment or to maintain progeny of laboratory crosses until they found to be impor-
tant enough to store frozen under LN,). This method can also be used for longer-
term maintenance of clones, if integrity of the germline is irrelevant and LN2
freezing is not convenient. Tetruhyrnenu cells survive in ponds whose surface
freezes in winter, and in the lab they remain viable after being kept for up to
several hours at 0°C. Nevertheless, a method for the long-term maintenance of
viable stock cultures at refrigerator temperature has not been developed.
Methods for maintaining stock cultures vary in detail from lab to lab. In
our lab, we maintain stocks in aluminum-capped Wasserman (4-in.) test tubes
containing 3 ml of 2% PP-based medium, supplemented with penicillin and
streptomycin, and inoculated with about 30 pl from the previous stock culture.
Stocks can be maintained at 18°C to room temperature and transferred once
every 2 to 4 weeks. We save at least the previous transfer as insurance against
contamination of the new culture or its failure to grow, which occurs infrequently.

C. Storing Tetruhyrnenu Cell Lines under Liquid Nitrogen

1. General Considerations
The long-term storage of live-frozen Tetruhymenucells is required to safeguard
cell lines and to prevent the genetic deterioration of the micronuclear genome,
204 Orias et al.

which occurs during cell multiplication. For this reason, it is important to freeze
newly generated clones as soon as they are determined to be useful for further
work. Tetrahymenacultures were first successfullyfrozen live in vials by Hwang et
al. (1964) and stored under LN2without loss of fertility (Simon and Hwang, 1967).
Freezing and storingculturesin Teflon tubules (Flacks, 1979)quadrupledthe num-
ber of cultures that can be maintained frozen in a given amount of space; below
we describe a slight modification of this method. A quicker freezing method with
simpler equipment requirements has been described (Cassidy-Hanleyet al., 1995;
Chapter 5),but thismethoddoesnot exploit the reductionof storage space afforded
by freezing in tubules. Both Flacks (1979) and Cassidy-Hanleyet al. (1995) should
be consulted for additional valuable information on freezing Tetrahymenacells.

2. Equipment and Materials Needed

a. For Freezing the Cells
Note: Some of these materials are shown in Fig. 1A.
8-in. screw cap tubes with 30 ml of PPY medium (2% PP and 0.1% yeast
extract, Difco; no other supplements are added).
Prelabeled sterile 50-ml conical centrifuge tubes (e.g., Fisher Scientific, Cata-
log # 05-538-55).
Sterile 20% aqueous dimethylsulfoxide (DMSO) solution. The solution is
prepared by making a fivefold dilution of the DMSO (SpectrAR, highest
grade; Fisher, certified A.C.S. grade) with sterile distilled H20.
Glass vials, 1 dram.
Cryovials-NUNCcryotube vials, sterile, 1.8 ml capacity, Nalge International,
Catalog # 363401. Available also from Fisher Scientific, Cat. # 12-565-17ON.
Cryovials are labeled with an autoclave-proof permanent marker; one can
also use NUNC cry0 pens (Fisher Scientific, Catalog # 12-565-237).
Critoseal (vinyl plastic putty for sealing hematocrit tubes), Oxford Labware,
St. Louis, MO 63103, Catalog # 8889-215003.
1-in. tubules of Teflon tubing, ID 3/32 in., OD 5/32in., Cole-Parmer, Chicago
IL 60648, Catalog # H-06406-63. The tubing comes in a 15-ft roll, and 1-in.
lengths are cut with a sharp safety blade. One end (only) of the tubule is
plugged by pushing it into the Critoseal putty. Four plugged tubules are
packed inside a 1-dram glass vial, plugged-end up. The vial is capped with
aluminum foil and autoclaved. Just before use, the glass vial is inverted over
the mouth of the prelabeled cryovial, so that the tubules slide into it, plugged-
end down. Twelve tubules (three vials) will be used for each culture to
be frozen.
Sterile Pasteur pipettes.
Well-insulated gloves, to safely handle objects at LN2 temperature.
Freezing apparatus capable of regulating freezing rate. We still use a 30-year-
old Linde Biological Freezing System, which consists of a BF-4 Controller
4. Tetrahymena as a Laboratory Organism 205

Fig. 1 Freezing cells in liquid nitrogen. (A) Materials used for freezing and storage: (1)aluminum
cane holding four cryovials; (2) empty cane; (3) cry0 sleeve to keep vials from falling off the cane
when submerged in liquid nitrogen; (4) two tubules, the top one with left end plugged with Critoseal;
( 5 ) glass vial with four Teflon tubules, plugged end up; (6) cryovial with four tubules, plugged end
down, ready to be filled with cells; (7) Critoseal tray; and (8) ruler to indicate scale. (B) Diagram
showing temperature probe assembly. The probe is the solid-filled segment. The long branch of the
probe has been threaded through a hole in the cryovial cap and has been inserted into a tubule
containingcells.The short branch will monitor chamber temperature. The hatched segment represents
the cable connectionto the strip chart recorder. (C) Three canes hanging inside the freezing chamber,
ready for a freezing run. The cane on the right holds the temperature probe assembly. The other
two canes hold cryovials containing tubules with cells to be frozen.

and the BF-4-1 Freezing Chamber. A temperature probe in the chamber is

connected to a Leeds and Northrop Speedomax H, Model S strip chart
recorder, whose output is visually monitored and is used to manually control
the cooling rate. A dedicated LN2tank supplies LN2to the freezing chamber
under regulation by the BF-4 Controller. One freezing run uses 7-10 liters
of LN2.We recommend filling the LN2 tank at least 24 h in advance, so that
adequate pressure (6-10 lb/in?) has been built up for the run.
Aluminum canes to hold the cryovials, Fisher Scientific,Catalog # 12-565-181.
Cry0 sleeves (to sheath the canes and prevent cryovials from falling off to
the bottom of the LN2 tank), Fisher Scientific, Catalog # 15-350-25.
LN2storage tank (e.g., Taylor-WhartonSuper 30A, Cat. # 35VHC), equipped
with an alarm (e.g., thermalert Model 610 for Super 30A, Gordinier Electron-
ics, Roseville, MI), for long-term storage of the frozen cultures.
A bookkeeping system for recording the location (tank, canister, and cane
numbers) of each clone successfully frozen.
206 Orias cf af.

b. For Thawing Cells

Preheated 35°C shaker bath.
Sterile Pastew pipettes.

3. Procedure
a. Inoculation of Cultures to be Frozen
Inoculate a stock culture (e.g., 4-in. test tube with 3 ml of your standard
2% PP-based medium with penicillin and streptomycin) and incubate at
room temperature.
Three days later, inoculate the 8-in. screw cap tube with 0.5 cc of the stock
culture. Incubate in a tube rotator (60 rpm) at room temperature (about
22°C) for 2 days. The culture should not be allowed to reach late stationary
phase. You may have to vary the size of the inoculum depending on the
conditions available to you and the growth rate of the clone.

b. Freezing Procedure
Pour culture into a 50 ml conical centrifuge tube and centrifuge 30 s at
2000 rpm.
Aspirate the supernatant with a sterile Pasteur pipette attached to a vacuum
line and trap; leave a total of 2.5 ml of cells and medium.
Resuspend the cells and add 2 ml of sterile 20% DMSO;gently vortex the
mixture and let the culture stand for 30 min before freezing starts. This time
period is critical.
Using a sterile Pasteur pipette, mix the cells and load the tubules of each
cryovial with cells (roughly 60 p1 per tubule), and screw-cap each cryovial.
Avoid leaving any bubbles in the tubules. Some practice may be required
for this operation, which should be completed within the 30 min allowed
after adding the DMSO.
Place each cryovial in one of the four lowest positions of a cane and then
place upright in the freezing chamber (see Fig. 1C).
To monitor temperature during freezing, prepare a cryovial loaded exactly
as the others. Remove the protective sheath from the temperature recorder
probe, and insert the probe half way into one of the tubules by threading
the probe through a hole in the center of the cryovial’s cap (see Fig. 1B).
Place the cryovial containing the probe on a cane at the third position from
the bottom. With masking tape, secure the cryovial to the cane, being careful
that the tape does not touch the temperature probe, and place the cane in
the freezing chamber (see Fig. 1C). Since this sample is used only to regulate
temperature, sterile technique is not necessary during its preparation.
Close and latch the freezing chamber cover.
4. Tctrahymrna as a Laboratory Organism 207

Using the controller, activate the flow of LN2 into the chamber. Set the flow
rate to lower the temperature of the sample by 6-10°C/min.
When the sample temperature reaches about -1O"C, the temperature
"jumps" up to about -8"C, as the sample freezes and the heat of fusion is
released. At this point, change the flow rate setting of the controller to one
previously determined to give a cooling rate of 2-3"C/min. The temperature
will drift down a few degrees without any LN2 influx, after which the control-
ler takes over to give the desired cooling rate.
After the temperature has reached -4O"C, open the chamber, and quickly
transfer the canes to a canister immersed in a LN2-containing storage tank.
Wipe the temperature recorder probe clean, and replace the sheath on
the probe.
After the cultures have been in the storage tank for at least 15 min, thaw
the four tubules of one cryovial and test for survival (see the next section).
The freezing is considered successful if all four tubule cultures survive.
It is also recommended that at least one thawed culture from each frozen
clone be tested for contamination by spotting on a nutrient agar plate (see
Section 1V.C).
Transfer the successfully frozen cryovials one by one to prelabeled canes,
which are sheathed with cry0 sleeves and returned to the LN2 tank.
If fewer than all four tubule cultures survive or if the survivors are contami-
nated, repeat the entire freezing procedure, starting with the original
stock culture.
c. Thawing Frozen Cultures
Please note: Speed (and practice) are very important in the following opera-
tions.
Remove one cryovial from the cane and put the cane back under LN2.
Remove one tubule from the cryovial using sterile forceps (sterilized by
dipping in ethanol, which is eliminated by flaming).
Drop the tubule into a 4-in. test tube, containing 3 ml of PPY with penicillin
and streptomycin, that has been prewarmed while standing in a 35°C shaker
water bath.
Turn the shaker on, and return the cryovial containing the remaining frozen
tubules to its cane and to the LN2 tank.
After 1.5-2 min of shaking at 35"C, pick up the test tube, load a sterile
Pasteur pipette with warm medium from the tube, insert it into the tubule,
and flush it. This step is important in order to dilute the DMSO concentration
in the vicinity of the cells. A good way to be sure that the tubule contents
have been flushed out is to create bubbles at the bottom of the tubule.
Please note: If you are thawing test vials, thaw the four tubules of the cryovial
one by one and discard the cryovial.
208 Orias ct al.

Incubate the test tube at 30°C and subculture it as soon as swimming cells
are observed (1-3 days later).

4.Precautions in Freezing and Thawing Cells

Dispensing LN, and handling objects at LN2 temperature should be ap-
proached with insulated gloves and great care; permanent skin damage and
local loss of tactile sense can result from carelessness.
Responsible care must be taken to periodically refill the LN, tanks; generally,
once every 2 weeks is generously safe for a well-insulated tank. Storage of
duplicate cultures in two separate tanks is a worthwhile precaution.
An ultrafreezer at -80°C does not provide adequate conditions for the long-
term storage of viable Tetrahyrnena cells (Cassidy-Hanley et al., 1995).
The cryoprotectant used for freezing the cells is dimethylsulfoxide (DMSO),
a chemical that is harmful to growing cells at the concentrations needed for
its cryoprotective activity. The concentration and time of treatment given
here are critical to ensure maximum survival (Hacks, 1979).
Sterile technique should be maintained during the entire freezing and thawing
procedures. A clone may ultimately be lost if frozen while contaminated. It
is thus important to look for contamination when test-thawing newly frozen
cultures, while the original stock culture is still available.
The fraction of cells that survive the freezing-thawing procedure is generally
small, on order of 1%.While this is not a general problem given the great
excess of Tetrahyrnena cells that can be frozen in one tubule (roughly 60,000),
it does mean that there is no guaranteethat any genetic heterogeneity present
in the original culture (e. g., due to phenotypic assortment) will be preserved
among the thawed survivors.

Acknowledgments
We thank Sally L. Allen and Arno Tiedtke for helpful comments on the manuscript. We thank
the National Institutes of Health (current grant RR-09231), the National Science Foundation, and
the American Cancer Society for the support of our Tetrahymena research over the years.

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