Jpn. J. Protozool. Vol. 38, No. 2.

(2005) 153

Original

How Paramecium cells die under a cover glass?

Yoshiomi TAKAGI1,*, So KITSUNEZAKI2, Tae OHKIDO1,$ and Rie KOMORI1,#

1
Department of Biological Sciences, Nara Women’s University, Nara 630-8506, Japan,
2
Department of Physics, Nara Women’s University, Nara 630-8506, Japan

SUMMARY were broken, and the cytoplasmic contents flew out.

Until the last moment of the cell rupture, and
The dying process of Paramecium tetraurelia sometimes even after that, ciliary beating was ob-
cells under a cover glass without supporting pillars served somewhere locally on the cell membrane.
was described. The initiation of the process was Among the cilia, those at the cytopharynx were the
defined by the cessation of swimming, and the first to stop beating so that food vacuole (FV) for-
termination by cell rupture. The process required 6 mation stopped somewhat earlier than the cellular
min on average, ranging from 1 to 11 min. The first disorganization. Cessation of the contractility of
symptom of death was the formation of a small contractile vacuoles (CVs) occurred a little bit later
bleb, a local swelling of the outer cell membrane. on average than the cessation of FV formation.
The number of blebs increased, each bleb grew, and Anterior and posterior CVs were not related to each
neighboring blebs fused to form a larger bleb. By other in the timing of loss of contractility and in the
supplying water, the blebs disappeared only when preceding pulsating cycles. Finally, we calculated
their size and numbers were relatively small, in- the physical pressure to burst the cell at about 950
dicative of a commitment point to death. Finally atm and estimated the actual pressure to be at
the outer membrane and then the inner membrane 470~2,000 atm.

*Corresponding author
Tel: 075-791-6846, E-mail: [email protected]

Present addresses:
*
1-1 Kitadacho, Kamitakano, Sakyo-ku, Kyoto 606-0036
$
2-277 Mozu-Honmachi, Sakai, Osaka 591-8036
#
Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences at Kagawa Campus,
Tokushima Bunri University, Shido, Kagawa 769-2193

Received: 20 June 2005; Accepted: 24 Aug 2005.

154 How Paramecium cells die under a cover glass?

INTRODUCTION that of (b) is death by heat or pressure. It remains

uncertain whether (a) and (b) are fundamentally
People used to say that biology is the science different phenomena or not. It is natural, among
of living organisms, not dead substances. Items living organisms, that a dead body will eventually
destined for death or extinction such as the tail of a disappear (even cells killed by desiccation cannot
tadpole during metamorphosis, the cuticle of an be a permanent preparation unless treated with
insect pupa during molting, or the macronucleus of chemical fixative). For example, when we studied
the ciliate during sexual process were not of inter- death by chemicals, we would observe many dead
est until the concept of apoptosis (Kerr et al., cells some hours after the treatment, but no corpses
1972) became widely accepted. That caused a on the next day. It should be asked, therefore, how
revolutionary change in biology by introducing cells die and how long the dead cells keep their
death as an essential part of the living system. shape.
However, there are still few investigations on the The length of time during which we can ob-
dying process of protozoan cells, although the dy- serve dead cells varies according to the situation.
ing process in apoptotic cells has been described But we have no answer to such questions as “Are
abundantly, and the mechanism of genetically con- there any relationships between the cause of death
trolled cellular death is now one of the most fasci- and the process of death, between the kind of
nating biological themes. chemicals and the process of death, and between
We found ourselves unable to answer the the concentration of chemicals and the persistence
question of “how Paramecium cells die when they time of dead cells?”.
are at an extremity of senescence”, although we Before answering such questions, we should
had long engaged in examining aging and lifespan know the death process in detail. We here paid
in Paramecium (Takagi and Yoshida, 1980; Ta- attention to the simple occasion of death by pres-
kagi and Kanazawa, 1982; Takagi et al., 1987a, sure under a cover glass. We observed more than
1987b; Takagi, 1988, 1999; Komori et al., 2004, 100 samples of dying Paramecium cells under a
2005). We noticed that we had little knowledge cover glass without supporting pillars, depicted the
about the dying process of Paramecium, not only sequence of the death process, and estimated the
at senescence but also at more commonly occur- pressure required for cell rupture.
ring accidental death from physical, chemical and
biological causes, although we had frequently met
death of Paramecium caused by competition for MATERIALS AND METHODS
survival (Maruyama et at., 1996, 2001; Maruyama
and Takagi, 1997), by UV (Yamamoto et al., 1997, The wild-type stock 51 of Paramecium tetra-
2005), and by toxins contained in a component of urelia was used.
culture medium (Tokusumi and Takagi, 2000; Cells were cultured in 0.5% phosphate-
Mizobuchi et al., 2003). buffered wheat grass powder (Pines Int., Inc.,
We have noticed so far that there are 2 forms Lawrence, KS) infusion supplemented with 0.5
of death: (a) death leaving the shape of the dead mg/l stigmasterol and inoculated with Klebsiella
cell, and (b) death not leaving the shape of the pneumoniae 2 days before use (Mizobuchi et al.,
dead cell. A typical death form of (a) is death by 2003). A small sample of cells at the stationary
desiccation, which is identical to the familiar proc- phase of growth were transferred to SMBIII
ess of making permanent cell-preparations, and (Miyake, 1981), a saline solution, in wells of de-
 Jpn. J. Protozool. Vol. 38, No. 2. (2005) 155

pression slide for washing. broken, and the cytoplasmic contents flew out.
A 10 µl drop of the saline solution including a
definite number of less than 10 cells was put on a Dying process in detail
clean slide glass, and a cover glass of (1.8 cm)2 in <Cell morphology>
area and 0.1 g in weight was put on the drop with- After the cells stopped swimming, a small
out pillars. A single cell was chased until it died bleb, an extrusion of the outer cell membrane was
and the dying process was observed, paying spe- formed as the first symptom of the death process.
cial attention to cell morphology, ciliary beating, The region where the first bleb was formed was
food vacuole (FV) formation and contractile vacu- not fixed; every region on the cell surface ap-
ole (CV) activity. We observed more than 100 peared to have an equal chance to form a bleb.
dying cells in all. Video-recordings of cells were The number of blebs increased and small neigh-
used for the observation of CVs. boring blebs fused to form a larger bleb. A typical
Handling and observation were performed at morphological change is shown in Fig. 1. If water
room temperature, which was usually in the range was supplied from a cover glass edge when blebs
of 22~28ºC. were smaller in size and fewer in number, the
blebs disappeared: the cell restored its normal
shape and began to swim normally. The point of
RESULTS no-return or the commitment point appeared to
exist somewhere during the bleb-growing process.
Sequence of death The cells at stages B and C in Fig. 1 would re-
Paramecium cells which were swimming ac- cover, but thereafter would not. Cilia were regu-
tively under a cover glass without pillars stopped larly located on the surface of the blebs, i.e., on the
moving before long. Although the time to the outer membrane. Trichocyst-discharging was
stoppage varied greatly among cells because of rarely observed during the dying process except at
such factors as inconsistent vigor of each cell, vari- the moment of cell rupture. When observed, it was
able rates of loss of water, contamination by debris outside the blebs.
that supported the cover glass and so on, the period Concomitant with the enlargement of blebs,
between the cessation of swimming and the cell enlargement of intracellular vacuoles occurred.
rupture was 6 min on average, ranging from 1 to We were unable to identify each vacuole enlarged
11 min. except CVs which we will mention later.
The first symptom of death was the formation <Ciliary beating >
of a small bleb, a local swelling of the outer cell Paramecium cells stopped swimming, al-
membrane. The number of blebs increased, each though cilia all over the cell surface continued to
bleb grew, and neighboring blebs fused to form a beat. Sometimes cells that stopped forward or
larger bleb. Cessation of ciliary beating at the backward swimming rotated at the resting position.
cytopharynx (buccal cavity), which means the ces- This made it easy to observe the subsequent proc-
sation of FV formation, tended to occur somewhat esses leading to death. Cilia on a part of the cell
earlier than the cessation of the contractility of surface continued to beat until the moment of cell
CVs. Ciliary beating was observed until the last rupture, and sometimes continued to beat after the
moment of the cell rupture at least somewhere cytoplasm began to flow out (Fig. 1F).
locally on the cell surface. Finally the outer cell <Food vacuole (FV) formation>
membrane and then the inner cell membrane were Although FVs under formation were not al-
156 How Paramecium cells die under a cover glass?

Fig. 1. Video-images of a typical dying cell under a cover glass without pillars. A: Normal cell that stopped swim-
ming. aCV: anterior contractile vacuole, pCV: posterior contractile vacuole, cp: cytopharynx. B: Four small blebs
(arrow heads) were formed. C: The blebs grew. D: Some blebs fused to form an enlarged bleb and new blebs were
formed. CVs also enlarged. E: The blebs grew to a single outer cell membrane. CVs were somewhat deformed.
F: The outer cell membrane at the posterior half ruptured. Cilia at the regions indicated by arrows were still beat-
ing.

ways discernible, cells would have the ability of counted the pulsation cycles of the anterior CV
FV formation as long as the ciliary beating along (aCV) and the posterior CV (pCV) until the cells
the cytopharynx (buccal cavity) continued. The collapsed. Three representative examples are
cilia on this region were the first to stop beating. shown in Fig. 2.
FVs that were present in the cytoplasm tended to In the cell of Fig. 2A, aCV pulsated more
enlarge prior to death. slowly and 3 times less frequently than pCV dur-
<Contractile vacuoles (CVs)> ing the observation period. The pulsating cycle of
The CV complex is an osmoregulatory organ- 9~10 s in aCV and that of 6~8 s in pCV remained
elle composed of many vesicles (Allen and Fok, for a time before death. The last pulsating cycle of
1988; Allen, 2000). We here call the easily visible aCV was 12 s, and that of pCV was 17 s. The total
complex of central vesicle and radial arms the CV. time of pulsation was 131 s for aCV and 125 s for
We recorded dying cells in a video recorder and pCV. The pCV, therefore, lost contractility earlier
 Jpn. J. Protozool. Vol. 38, No. 2. (2005) 157

than aCV, and aCV pulsated 1 time more after

pCV stopped pulsation.
In the cell of Fig. 2B, the pulsating cycle was
almost identical in both CVs: 5~8 s in aCV and
5~6 s in pCV. However, pCV suddenly increased
the cycle and lost contractility earlier than aCV:
the total time of pulsation was 202 s for aCV and
163 s for pCV. The last pulsating cycle of aCV
was 13 s, and that of pCV was 43 s. The aCV pul-
sated 9 times more than pCV during the observa-
tion period, and 4 times more after pCV stopped
pulsation.
In the cell of Fig. 2C, pCV was pulsating more
slowly and lost contractility earlier than aCV: the
total time of pulsation was 276 s for aCV and 127
s for pCV. The abrupt increase in the pulsating
cycle in pCV was a symptom of the loss of con-
tractility as in other cases, whereas contractility
was restored in aCV after an abrupt increase in the
pulsating cycle. The last pulsating cycle of aCV
was 26 s, and that of pCV was 31 s. The aCV pul-
sated 14 times more than pCV during the observa-
tion period, and 9 times more after pCV stopped
pulsation.
In all of the above 3 cases, pCV stopped pulsa-
tion earlier than aCV; 6 s earlier in A, 39 s earlier
in B and 149 s earlier in C. This was, however,
not a general rule as shown in Table 1, in which
we show the difference of pulsation in time and in
frequency between aCV and pCV during the ob-
servation period. Twelve cells, including those in
Fig. 2, were from video-recordings used to suc-
cessfully count pulsating cycles in both CVs.
Positive numbers mean that aCV pulsated for a
Fig. 2. Contractility of aCV (rectangles) and pCV longer time and more frequently than pCV, and
(circles) in 3 representative cells (A~C) when they negative numbers mean the reverse. The results
were to die. Abscissa: Number of pulsations until the
last moment of death. Start of the counting was arbi- show no correlation between aCV and pCV in
trary. Ordinate: Pulsating cycles in s. their pulsation when the cells are at death; aCV (or
pCV) pulsated for a longer time and more fre-
quently than pCV (or aCV) in some cells, while
aCV (or pCV) pulsated for a longer time but less
frequently than pCV (or aCV) in other cells. In 2
158 How Paramecium cells die under a cover glass?

Table 1. Discrepancy in pulsation between

aCV and pCV.

Cell Difference of pulsation in

time and in frequency between
aCV and pCV during the ob-
servation period
Time in s The number of
pulsation Fig. 3. Schematic depiction of a cell in water that
supports the cover glass showing possible forces in ar-
No. 1 154 10 rows. f: force supported by paramecia, mg: weight of
cover glass, P: hydrostatic pressure of water, P0: at-
No. 2 149 14
mospheric pressure, L: side length of cover glass, d:
No. 3 (aCV-1) 87 0 depth of water or the pressed width of the cell, α:
contact angle between water and a cover glass, γ :
No. 3 (aCV-2) -18 1 surface tension of water.
No. 4 39 9
No. 5 (pCV-1) 21 5
No. 5 (pCV-2) -37 4
Estimation of pressure at death
No. 6 18 4
As water under a cover glass decreases due to
No. 7 6 -3
desiccation, Paramecium cells bear the force aris-
No. 8 3 1 ing from the pressure difference between the water
No. 9 -20 1 and the atmosphere as well as the weight of the
No. 10 -36 -2 cover glass. We here assume that the cover glass
No. 11 -47 -2 is supported evenly by N cells of paramecia. Fig.
No. 12 -215 -1
3 is a schematic depiction of a cross section, where
the thickness of water is represented by d that is
Positive numbers indicate that aCV pulsated for a equal to the pressed width of the cell, and the
longer time and more frequently than pCV during the
observation period, and negative numbers indicate the cover glass is rectangular with mass m and length
reverse. In No. 1 cell, for example, aCV pulsated 154 of a side L.
s longer and 10 times more than pCV. No.3 and No.
5 cells had 2 aCVs and 2 pCVs, respectively. In No. As water begins to evaporate from the sides of
5 cell, for example, aCV pulsated 21 s more than the cover glass, the lateral interface of water be-
pCV-1 but 37 s less than pCV-2, and aCV pulsated 5
and 4 times more than pCV-1 and pCV-2, respec-
comes concave to the atmosphere. A cross-section
tively. A, B and C cells in Fig. 2 are identical to No. of the interface is approximated as a circular arc
7, 4 and 2 cells in Table 1, respectively. because the thickness d is sufficiently smaller than
the capillary length in general. Using the contact
angle between water and a glass plate, α, the aver-
of 12 cells, 1 of the CVs, aCV in 1 case and pCV age curvature of the interface κ is expressed as κ =
in the other case, duplicated during the death proc- 2 cosα / d. The difference between the hydrostatic
ess, and the resulting CVs resided close to each pressure of water P and the atmospheric pressure
other. Even in such a case, the 2 CVs pulsated P0 is derived from Laplace's theorem as
independently.
CVs enlarged and deformed prior to the cell P - P0 = γκ = 2 γ cosα / d , (1)
rupture, maintaining the ability of pulsation.
where γ is the surface tension of water (de
 Jpn. J. Protozool. Vol. 38, No. 2. (2005) 159

Gennes et al., 2004). g, g ≈ 980 cm/s2, γ ≈ 70 dyn/cm and L ≈ 1.8 cm.

The vertical force supported by N paramecia, Adopting α ≈ 20° as a typical value of the contact
f, is caused by the weight of the cover glass m, the angle, although it depends on the state of the glass
pressure difference P - P0 and the surface tension surfaces, we obtained an estimation of P* as
acting on the 4 sides of the cover glass, 4γL. Rep-
resenting the acceleration due to gravity by g, we P* ≈ 9.5 × 108 dyn/cm2 ≈ 9.5 × 102 atm. (5)
obtain the equation
In this case, the pressure caused by the weight
f = mg + L2 (P - P0) + 4γL sinα. (2) of the cover glass, mg/NS ≈ 0.3 atm, is very small
compared with this value.
Substituting Eq. (1), we find that the third term
is negligibly small compared with the second term
in the case of d << L. The equation is approxi- DISCUSSION
mated as
By carefully observing death process of Para-
2
f ≈ mg + 2γL cosα / d. (3) mecium cells under a cover glass without support-
ing pillars, we found that ciliary beating continued
We represent a typical contact area between until the last moment of cell rupture or even after
each Paramecium and the cover glass by S, and the cytoplasm began to flow out. This may not be
assume the intracellular pressure of paramecia as P surprising, since ciliary beating has been shown to
+ P*. P*, the difference between the inside and continue in non-living cell models such as the Tri-
outside pressures, works to burst paramecia. Re- ton model (Naitoh and Kaneko, 1972).
garding each Paramecium as a fluid cell sur- Cessation of CV pulsation tended to occur
rounded by a soft membrane, the inside pressure is later than the cessation of FV formation. Pulsation
uniform everywhere in the cell. The equilibrium of CVs continued until immediately before the cell
equation of forces acting on the contact areas is rupture. Enlarged and deformed CVs could still
expressed by f = NSP*, where the surface tension pulsate. In contrast to the ciliary beating, however,
of the membranes is negligible because the contact CV pulsation was never observed after the cyto-
areas are approximately flat. Therefore P* is esti- plasm began to flow out, although autonomous
mated by the equation pulsation of CV could occur in vitro (Tani et al.,
2001). We found that aCV and pCV pulsated in-
P* = f/NS ≈ ( mg + 2γL2 cosα / d) / NS. (4) dependently with different cycles and frequencies
in the cells at death, although the 2 CVs appeared
As an example, we calculated P* assuming to pulsate with regular intervals in intact cells. It
that N = 10, S ≈ 3.0 × 10-5 cm2 and d ≈ 15 µm = has been suggested that no fibrous network system
1.5 × 10-3 cm: A P. tetraurelia cell was considered surrounds the CV (Naitoh et al., 1997; Tani et al.,
as a rotatory ellipse with length of 100 µm and 2001). This means that the independency between
width of 30 µm; when it was pressed to half, the aCV and pCV pulsation is of an original nature,
projection area of the original ellipse would be and is not due to disconnection at death. Also
expanded; the contact area of the cell to the cover found was an occasional occurrence of an abrupt
glass (S) was assumed to be similar to the projec- duplication of CV during the dying process. Both
tion area of the original ellipse. We used m ≈ 10-1 aCV and pCV were duplicated, and the resulting
160 How Paramecium cells die under a cover glass?

CVs pulsated independently (Table 1). logical chemicals at higher concentrations and
We estimated that the Paramecium cell mem- death by starvation, which occur more slowly than
brane was broken only when the difference be- the present case.
tween the outside and inside pressures of the cell
was as high as ~950 atm. The value of P* calcu- ACKNOWLEDGMENTS
lated by Eq. (4) would change if the assumption of
N, S and d differed. For the evaluation of S, the We are grateful to Dr. Yoshihiro Taniguchi of
most puzzling value, we assumed that S would be Ritsumeikan University for sharing his experimen-
similar to the projection area (A) of the original tal data on biological tolerance to high pressure, and
ellipse with long axis of 100 µm and short axis of to Dr. Terue Harumoto of Nara Women’s Univer-
30 µm. We calculated A (2.4 ×10-5 cm2), corrected sity for her help in video-recording. We also thank
by computer-aided calculation of the surface area the anonymous referees for their fruitful comments.
of photographed cells (4.3 ×10-5 cm2), and adopted
the value of S = 3.0 ×10-5 cm2. However, the ac-
tual contact area of the cell to the cover glass REFERENCES
might be in the range of 2S~S/2 so that the actual
value of P* might be in the range of 470~2,000 Allen, R. D. (2000) The contractile vacuole and its
atm. membrane dynamics. Bioessays, 22, 1035-
N is also a factor that affects P*. Although we 1042.
assumed that the pressure was supported evenly by Allen, R. D. and Fok, A. K. (1988) Membrane
N cells, debris of similar size, which was not com- dynamics of the contractile vacuole complex
pletely eliminated, would also support the pres- of Paramecium. J. Protozool., 35, 63-71.
sure. We therefore consider that P* with the range de Gennes, P.-G., Brochard-Wyart, F. and Quere,
of 470~2,000 atm would be reasonable. D. (2004) Capillarity and wetting phenomena:
In classic studies of the effects of hydrostatic drops, bubbles, pearls, waves. Springer, New
pressure introduced by Wichterman (1953), Para- York.
mecium cells became immobile at 600 atm but Kerr, J. F., Wyllie, A. H. and Currie, A. R. (1972)
they could recover even after exposure to 800 atm. Apoptosis: a basic biological phenomenon
A recent experimental study by Dr. Y. Taniguchi with wide-ranging implications in tissue kinet-
of Ritsumeikan University showed that P. multim- ics. Br. J. Cancer, 26, 239-257.
icronucleatum cells could endure hydrostatic pres- Komori, R., Harumoto, T., Fujisawa, H. and Takagi,
sure of ~1,000 atm (Takagi, personal communica- Y. (2004) A Paramecium tetraurelia mutant
tion). Such information suggests that P. that has long autogamy immaturity period and
tetraurelia cells in this study were exposed to short clonal life span. Mech. Ageing Dev., 125,
600~800 atm when they stopped swimming at the 603-613.
beginning and to more than 1,000 atm when they Komori, R., Sato, H., Harumoto, T. and Takagi, Y.
were ruptured. It should be emphasized, however, (2005) A new mutation in the timing of
that P* is different from the hydrostatic pressure. autogamy in Paramecium tetraurelia. Mech.
The present observations on the death process Ageing Dev., 126, 752-759.
are a typical case of accidental death which takes Maruyama, C. Takahashi, R., Tokusumi, Y. and
place in a short time. It is of interest to know how Takagi, Y. (1996) Conditions of survival and
cells die in other cases such as death by physio- extinction of Paramecium multimicronuclea-
 Jpn. J. Protozool. Vol. 38, No. 2. (2005) 161

tum cultured with P. tetraurelia in a closed 191.

environment. Eur. J. Protistol., 32, 94-101. Takagi, Y. and Kanazawa, N. (1982) Age-
Maruyama, C. and Takagi, Y. (1997) Survival or associated change in macronuclear DNA con-
extinction of Paramecium multimicronuclea- tent in Paramecium caudatum. J. Cell Sci., 54,
tum cultured with P. tetraurelia is associated 137-147.
with the ability or inability to form food vacu- Takagi, Y., Nobuoka, T. and Doi, M. (1987a)
oles. Eur. J. Protistol., 33, 274-283. Clonal lifespan of Paramecium tetraurelia:
Maruyama, C., Fujisawa, H. and Takagi, Y. (2001) effect of selection on its extension and use of
Age-associated survival and extinction in fissions for its determination. J. Cell Sci., 88,
mixed cultures of Paramecium. Eur. J. Pro- 129-138.
tistol., 37, 303-312. Takagi, Y., Suzuki, T. and Shimada, C. (1987b)
Miyake, A. (1981) Cell interaction by gamones in Isolation of a Paramecium tetraurelia mutant
Blepharisma. In: Sexual interaction in eu- with short clonal life-span and with novel life-
karyotic microbes. O’Day, D. H. and P. A. cycle features. Zool. Sci., 4, 73-80.
Horgen (eds.). Academic Press, New York, pp. Tani, T., Allen, R. D. and Naitoh, Y. (2001) Cellu-
95-129. lar membranes that undergo cyclic change in
Mizobuchi, N., Yokoigawa, K., Harumoto, T., tension: Direct measurement of force genera-
Fujisawa, H. and Takagi, Y. (2003) Catalase is tion by an in vitro contractile vacuole of Para-
the bacteria-derived detoxifying substance mecium multimicronucleatum. J. Cell Sci. 114,
against paramecia-killing toxin in wheat grass 785-795.
powder infusion. J. Eukaryot. Microbiol., 50, Tokusumi, Y. and Takagi, Y. (2000) Ectosymbi-
299-303. otic role of food bacteria for Paramecium:
Naitoh, Y. and Kaneko, H. (1972) Reactivated Bacterial detoxification of paramecia-killing
Triton-extracted models of Paramecium: toxin contained in wheat grass powder. Zool.
Modification of ciliary movement by calcium Sci., 17, 341-348.
ions. Science, 176, 523-524. Yamamoto, N., Hayashihara, K. and Takagi, Y.
Naitoh, Y., Tominaga, T., Ishida, M., Fok, A., (1997) Changes in UV sensitivity with cell
Aihara, M. and Allen, R. (1997) How does the cycle, clonal age, and cultural age in Parame-
contractile vacuole of Paramecium multim- cium tetraurelia. Zool. Sci., 14, 747-752.
icronucleatum expel fluid? Modelling the ex- Yamamoto, N., Komori, R. and Takagi, Y. (2005)
pulsion mechanism. J. Exp. Biol., 200, 13-21. Abrupt increase in UV sensitivity at late log-
Takagi, Y. (1988) Aging. In: Paramecium. Görtz, phase of growth in Paramecium tetraurelia. J.
H-D. (ed.). Springer-Verlag. Berlin, pp. 131- Eukaryot. Microbiol., 52, 218-222.
140. Wichterman, R. (1953) The Biology of Parame-
Takagi, Y. (1999) Clonal life cycle of Paramecium cium. The Blakiston Company, Inc., New
in the context of evolutionally acquired mor- York.
tality. In: Cell Immortalization. Macieira-
Coelho, A. (ed.). Springer-Verlag, Berlin, pp.
81-101.
Takagi, Y. and Yoshida, M. (1980) Clonal death
associated with the number of fissions in
Paramecium caudatum. J. Cell Sci., 41, 177-