How-To-Do-It
Investigating Phagocytosis in Tetrahymena
An Experimental System Suitable for Introductory &
Advanced Instruction
Donna M. Bozzone
Tetrahymena, a freshwater ciliated
protozoan, is in many ways an ideal
candidate for the study of cells by
students. Readily obtained and cultured, Tetrahymena can be used to
study ciliary motion, organelle structure and function, cell morphology,
cell proliferation, and cell behaviors
(Leick & Hellung-Larsen 1992; Wheatley et al. 1994). Because of the ease
with which Tetrahymena can be grown
and manipulated, it is especially suitable for use in inquiry-based labs and
student projects.
Phagocytosis in Tetrahymena is an
especially dramatic cell behavior that
students can observe directly with a
compound microscope. When hungry
Tetrahymena encounter food, they use
their cilia to sweep it into each cell’s
oral groove. This process can be visualized by feeding stained yeast cells
India ink (Keenan 1984) or Chlorella
to Tetrahymena. Phagocytosis can be
quantitated by counting the number
of vacuoles that form in a defined
time period.
In this paper, I describe how our
students use the India ink method,
described below, to investigate phagocytosis and vacuole formation in Tetrahymena first to learn the basis of microscope use, and then to pursue various
experimental projects.
Procedures
Observation of Phagocytosis
Student Instructions
1. Tetrahymena grown in 2% proteose peptone for 48 to 72 hours
will be provided.
Donna M. Bozzone, Ph.D., is Associate
Professor and Chair of Biology at St.
Michael’s College, Colchester, VT
05439; e-mail: dbozzone@smcvt.edu.
2. Pipette 3 ml of Tetrahymena and
3 ml of 1% India ink into a test
tube. Mix gently.
3. Using a compound microscope,
observe the behaviors of the cells.
In particular, pay attention to
how the cells swim and eat.
Record your observations.
4. Sample cells at 0, 2, 5, 10, 20 and
30 minutes. To sample, pipette 20
microliters of cell ink suspension into a microfuge tube containing 10 microliters of 3% glutaraldehyde. Exposing cells to
glutaraldehyde, a fixative, will
kill the cells. Consequently, you
can work at your own pace once
the cells have been sampled. Also
note that the ‘‘0 minute’’ time
point refers to the first sample
you are able to collect right after
the combination of the cells and
ink. In real time, it will be less
than one minute.
5. Place a drop of sample cells on
a slide and, using a compound
microscope at 100 to 200X total
magnification, count the number
of vacuoles containing ink per
cell for at least 20 cells for each
time point.
6. Prepare a table and graph to display your results.
7. Design an experiment in which
you use this assay to investigate
some aspect of phagocytosis.
Information for Instructors. This simple procedure is an excellent one for
introducing students to the basics of
microscope use. Tetrahymena are best
observed at either 100 or 200X total
magnification. Since the vacuoles that
form are full of ink, they are easy
to identify (Figure 1). In 1% ink, the
movement of the cilia can be observed
as can the formation of the vacuoles.
Moreover, students can also see the
movement of the vacuoles inside the
136 THE AMERICAN BIOLOGY TEACHER, VOLUME 62, NO. 2, FEBRUARY 2000
cell as new ones form, and if they
are sufficiently patient, sometimes they
can even see vacuoles ‘‘void’’ their
contents into the extracellular space.
If you have a video camera that can
be attached to your microscope, a permanent record of student work is
possible.
Aseptic technique should be used
to grow Tetrahymena. To culture, inoculate 1 ml into 25 ml of 2% proteose
peptone in a 125 ml Erlenmeyer flask
(the ratio of medium volume to total
flask volume should be 1:5 or lower).
Incubation at room temperature, (20°
to 22° C) is best. Tetrahymena can be
purchased from many suppliers; I use
Carolina Biological. Be sure to order
the ‘‘bacteria-free’’ cultures.
To make 2% proteose peptone, add
2 g proteose peptone (Difco) per 100
ml distilled or deionized water. Autoclave 20 minutes on liquid cycle or
slow exhaust. This medium should be
stored in the refrigerator and used
within two months.
The type of ink you use for this
procedure matters a great deal because
some inks have detergent in them;
these won’t work. I use Hunt-Speedball. All dilutions are made in distilled
or deionized water.
Examples of Student Projects
Using the basic protocol described
above, students have designed and
implemented many projects that
explored some aspect of phagocytosis.
The three projects presented below
show examples of questions students
asked and answered experimentally.
Does the Concentration of Ink Affect
the Rate of Vacuole Formation? In
this experiment, Tetrahymena were fed
1, 5 or 10% ink. Cells were sampled
at 2, 10, 20 and 30 minutes. As shown
in Table 1, the rate and degree of
phagocytosis were inversely propor-
(b)
(a)
Figure 1. a) Tetrahymena fixed in 3% glutaraldehyde. b) Tetrahymena fed 1% India ink for 10 minutes and fixed in 3%
glutaraldehyde.
Table 1. Phagocytosis of different concentrations of India ink by Tetrahymena.
Time (min)
2
10
20
30
1% Ink
2.6
3.2
5.1
5.2
Mean Number of Vacuoles/Cell
5% Ink
2.1
1.7
2.1
2
tional to the concentration of ink. This
observation was a surprise to the students who did this experiment and
they did additional research to try to
explain why the higher concentrations
of ink resulted in fewer vacuoles.
While they did not determine the specific mechanism that accounted for this
phenomenon, they did articulate additional testable questions to further
explore this result. For example, they
wondered whether the high concentration of particulates in the ink might
have damaged the cilia around the
oral groove so that material could not
be swept in efficiently. To test this
they proposed exposing cells to 5 and
10% ink for 10 minutes, and then
washing the cells to see if they would
now eat 1% ink at the control rate.
Does the Nutritional State of Tetrahymena Affect the Rate at Which Food
Vacuoles Form? In this project, stu-
10% Ink
0.7
1.3
1.3
1.1
dents wanted to see whether Tetrahymena would eat faster if they had been
deprived of food for a while. To test
this, a well-fed and a starved population of Tetrahymena were each fed 1%
India ink and sampled at 0, 5, 10 and
20 minutes. The number of vacuoles
per cell was determined for each cell
sample. As shown in Table 2, the population of cells that were starved exhibited a reduced rate of vacuole formation as compared to the well-fed control. At first, this result seemed
counterintuitive to the students but
after additional research they reasoned
that perhaps starved cells will only
eat actual food while well-fed cells
will ingest anything that will fit into
the oral groove, even ink particles.
Again, students were able to design a
logical experiment to test this hypothesis: Would starved and well-fed Tetrahymena eat Chlorella, or yeast, at a
similar rate?
In this experiment, the well-fed cells
were Tetrahymena that had been grown
for 48 to 72 hours in 2% proteose
peptone. To starve Tetrahymena, centrifuge cells from a 48 to 72-hour culture
(1000 rpm in a tabletop centrifuge for
4 minutes), and resuspend the pellet
in dilute salt solution. Centrifuge and
wash the cells two more times. Resuspend the pellet in a volume of dilute
salt solution equal to the volume of
medium you initially removed. The
resuspended cells should be incubated
Table 2. Phagocytosis in starved and well-fed Tetrahymena.
Time (min)
0
5
10
20
Mean Number of Vacuoles/Cell
Well-fed cells
Starved cells
0.88
0
5.3
1.8
6.4
3.1
8.5
4.4
PHAGOCYTOSIS IN TETRAHYMENA 137
at room temperature. To make the
dilute salt solution, add the following
to each liter of distilled or deionized
water.
• 6 mg KCl
• 4 mg CaHPO4 (or CaCl2)
• 2 mg MgSO4-7H2O
Autoclave 20 minutes at slow exhaust.
Is the Cytoskeleton Involved in the
Formation of Vacuoles? Observations
of the formation of vacuoles and the
movement of those vacuoles within
the cell led two groups of students
to wonder what role the cytoskeleton
plays in this process. To determine
whether microfilaments or microtubules were essential for phagocytosis,
Tetrahymena were exposed to cytochalasin B or colchicine, respectively.
More specifically, 2 ml of 48 to 72hour cultures were incubated in a test
tube with either 20 microliters of cytochalasin B or colchicine for 10 minutes.
After this incubation, 2 ml of 1% ink
were added to each tube. Cells were
sampled every 10 minutes for 30
minutes and vacuole formation was
quantitated. Table 3 shows the results
of this experiment. Cytochalasin B dramatically inhibited vacuole formation.
Ink was ingested by the cells, but the
vacuole that formed never pinched off
and moved into the cell. Colchicine
also decreased the rate of vacuole formation. Students hypothesized that its
effect was due to the loss of microtubule tracks within the cells. Without
these tracks, the vacuoles could not
move efficiently into the cell body.
Since cytochalasin B is insoluble in
water, it is dissolved in a small amount
of ethanol (see below). Consequently,
we ran a control experiment to verify
that cytochalasin B, and not ethanol,
was causing the effects observed
(Table 4). Additional questions raised
by these students include: Are the
effects of the drugs reversible? Is the
effect of the drugs dose-dependent?
Because this experiment uses cytochalasin B and colchicine, potentially
hazardous materials, students should
be carefully supervised during this
experiment and appropriate precautions should be taken. To prepare a
stock solution of cytochalasin B, add
Table 4. Control experiment demonstrating phagocytosis in the presence of
ethanol, the solvent used in the cytochalasin B experiment.
Time (min)
0
10
20
30
1% Ink
1.2
6.7
8.0
8.5
Mean Number of Vacuoles/Cell
Ethanol 1% Ink
0.7
6.3
8.5
8.8
5 mg per ml of 70% ethanol. The final
concentration used in the experiment
above was 50 micrograms per ml. To
prepare a stock solution of colchicine,
add 400 mg per ml of distilled or
deionized water. The final concentration used was 4 mg per ml. Both stock
solutions should be refrigerated.
Additional Possibilities
Using the simple procedure of measuring phagocytosis and vacuole
formation by feeding India ink to
Tetrahymena, students can design and
implement experiments of varying
complexity. In addition to the explorations described in this paper, examples
of other testable questions generated
by students include:
• Does the rate of phagocytosis vary
with temperature?
• Does the velocity with which Tetrahymena are agitated affect the rate
of phagocytosis?
• Will Tetrahymena select specific
items for ingestion when presented
with mixtures of food?
Invariably, an experiment done to
answer a particular question leads to
more questions and ideas for experiments. It is helpful to note that many
of the papers that describe basic studies
of phagocytosis and vacuole formation
are available to provide context for the
work that students do (Ricketts 1972;
Nilsson et al. 1973; Hoffman et al. 1974).
Also, phagocytosis in Tetrahymena has,
in recent years, become a model for
learning about pharmacology and cell
membrane function (Stefanidou et al.
1990; Csaba & Darvas 1992; Renaud et
al. 1995). Consequently, the projects
students do have a connection to cur-
rent research problems. The ease with
which the procedure can be done along
with the vast array of potential testable
questions that can be pursued make
studying phagocytosis in Tetrahymena
an experimental system rich with
possibilities.
Acknowledgments
This work was supported in part by
N S F - I L I g r a n t # 9 2 0 5 1 7 4 a n d S t.
Michael’s College. I would also like
to thank two anonymous reviewers
f o r t h e i r he lp fu l c om me nt s a nd
suggestions.
References
Csaba, G. & Darvas, Z. (1992). Insulin
antagonizes the phagocytosis stimulating action of histamine in Tetrahymena. Bioscience Reports, 12(1), 23–27.
Hoffman, E.K., Rasmussen, L. & Zeuthen, E. (1974). Cytochalasin B:
Aspects of phagocytosis in nutrient
uptake in Tetrahymena. Journal of Cell
Science, 15, 403–406.
Keenan, K. (1984). Uptake of particles
by the ciliate Tetrahymena. In C.L.
Harris (Ed.), Tested Studies for Laboratory Teaching. Proceedings of the
Fourth Workshop/Conference of the
Association for Biology Laboratory Education (ABLE).
Leick, V. & Hellung-Larsen, P. (1992).
Chemosensory behavior of Tetrahymena. BioEssays, 14(1), 61–66.
Nilsson, J.R., Ricketts, T.R. & Zeuthen,
E. (1973). Effects of cytochalasin B
on cell division and vacuole formation in Tetrahymena pyriformis. Experimental Cell Research, 79, 456–459.
Renaud, F.L., Colon, I., Lebron, J.,
Ortiz, N., Rodriguez, F. & Cadilla,
Table 3. Effects of cytochalasin B and colchicine on phagocytosis in Tetrahymena.
Time (min)
0
10
20
30
1% Ink
0
6.9
7.6
9.3
Mean Number of Vacuoles/Cell
Colchicine 1% Ink
0
3.9
5.1
5.2
138 THE AMERICAN BIOLOGY TEACHER, VOLUME 62, NO. 2, FEBRUARY 2000
Cytochalasin B 1% Ink
0.1
1
1
1
C. (1995). A novel opioid mechanism
seems to modulate phagocytosis in
Tetrahymena. Journal of Eukaryotic
Microbiology, 42(3), 205–207.
Ricketts, T.R. (1972). The induction of
endocytosis in starved Tetrahymena
pyriformis. Journal of Protozoology,
19, 373–375.
Stefanidou, M., Georgiou, M., Marvelias, C. & Koutselinis, A. (1990). The
effects of morphine, cocaine,
amphetamine and hashish on the
phagocytosis of the protozoan Tetrahymena pyriformis strain W. Toxicology In Vitro, 4(6), 779–781.
Wheatley, D.N., Rasmussen, L. & Tiedtke, A. (1994). Tetrahymena: A model
for growth, cell cycle and nutritional
studies with biotechnological potential. BioEssays, 16(5), 367–371.
PHAGOCYTOSIS IN TETRAHYMENA 139