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
2

Department of Biological Sciences, Nara Women’s University, Nara 630-8506, Japan,
Department of Physics, Nara Women’s University, Nara 630-8506, JapanSUMMARY
The dying process of Paramecium tetraurelia
cells under a cover glass without supporting pillars
was described. The initiation of the process was
defined by the cessation of swimming, and thetermination by cell rupture. The process required 6
min on average, ranging from 1 to 11 min. The firstsymptom of death was the formation of a small
bleb, a local swelling of the outer cell membrane.
The number of blebs increased, each bleb grew, andneighboring blebs fused to form a larger bleb. By
supplying water, the blebs disappeared only when
their size and numbers were relatively small, indicative of a commitment point to death. Finallythe outer membrane and then the inner membrane

were broken, and the cytoplasmic contents flew out.
Until the last moment of the cell rupture, andsometimes even after that, ciliary beating was observed somewhere locally on the cell membrane.
Among the cilia, those at the cytopharynx were thefirst to stop beating so that food vacuole (FV) formation stopped somewhat earlier than the cellulardisorganization. Cessation of the contractility ofcontractile vacuoles (CVs) occurred a little bit lateron average than the cessation of FV formation.
Anterior and posterior CVs were not related to eachother in the timing of loss of contractility and in thepreceding pulsating cycles. Finally, we calculated
the physical pressure to burst the cell at about 950
atm and estimated the actual pressure to be at
470~2,000 atm.

*Corresponding author
Tel: 075-791-6846, E-mail: takagi@cc.nara-wu.ac.jp
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
People used to say that biology is the scienceof living organisms, not dead substances. Items
destined for death or extinction such as the tail of atadpole during metamorphosis, the cuticle of an
insect pupa during molting, or the macronucleus of
the ciliate during sexual process were not of interest until the concept of apoptosis (Kerr et al.,1972) became widely accepted. That caused a
revolutionary change in biology by introducing
death as an essential part of the living system.
However, there are still few investigations on the
dying process of protozoan cells, although the dying process in apoptotic cells has been describedabundantly, and the mechanism of genetically controlled cellular death is now one of the most fascinating biological themes.
We found ourselves unable to answer thequestion of “how Paramecium cells die when they
are at an extremity of senescence”, although we
had long engaged in examining aging and lifespanin Paramecium (Takagi and Yoshida, 1980; Takagi and Kanazawa, 1982; Takagi et al., 1987a,
1987b; Takagi, 1988, 1999; Komori et al., 2004,
2005). We noticed that we had little knowledge
about the dying process of Paramecium, not onlyat senescence but also at more commonly occurring accidental death from physical, chemical andbiological causes, although we had frequently met
death of Paramecium caused by competition forsurvival (Maruyama et at., 1996, 2001; Maruyama
and Takagi, 1997), by UV (Yamamoto et al., 1997,2005), and by toxins contained in a component of
culture medium (Tokusumi and Takagi, 2000;
Mizobuchi et al., 2003).
We have noticed so far that there are 2 forms
of death: (a) death leaving the shape of the deadcell, and (b) death not leaving the shape of thedead cell. A typical death form of (a) is death bydesiccation, which is identical to the familiar process of making permanent cell-preparations, andthat of (b) is death by heat or pressure. It remains
uncertain whether (a) and (b) are fundamentallydifferent phenomena or not. It is natural, amongliving organisms, that a dead body will eventuallydisappear (even cells killed by desiccation cannot
be a permanent preparation unless treated withchemical fixative). For example, when we studied
death by chemicals, we would observe many deadcells some hours after the treatment, but no corpses
on the next day. It should be asked, therefore, howcells die and how long the dead cells keep theirshape.
The length of time during which we can observe dead cells varies according to the situation.
But we have no answer to such questions as “Arethere any relationships between the cause of deathand the process of death, between the kind ofchemicals and the process of death, and between
the concentration of chemicals and the persistence
time of dead cells?”.
Before answering such questions, we should
know the death process in detail. We here paid
attention to the simple occasion of death by pressure under a cover glass. We observed more than
100 samples of dying Paramecium cells under acover glass without supporting pillars, depicted thesequence of the death process, and estimated thepressure required for cell rupture.

MATERIALS AND METHODS
The wild-type stock 51 of Paramecium tetraurelia was used.
Cells were cultured in 0.5% phosphatebuffered wheat grass powder (Pines Int., Inc.,
Lawrence, KS) infusion supplemented with 0.5
mg/l stigmasterol and inoculated with Klebsiellapneumoniae 2 days before use (Mizobuchi et al.,2003). A small sample of cells at the stationary
phase of growth were transferred to SMBIII(Miyake, 1981), a saline solution, in wells of de-Jpn. J. Protozool. Vol. 38, No. 2. (2005)

pression slide for washing.
A 10 µl drop of the saline solution including adefinite number of less than 10 cells was put on aclean slide glass, and a cover glass of (1.8 cm)2 inarea and 0.1 g in weight was put on the drop without pillars. A single cell was chased until it diedand the dying process was observed, paying special attention to cell morphology, ciliary beating,food vacuole (FV) formation and contractile vacuole (CV) activity. We observed more than 100
dying cells in all. Video-recordings of cells wereused for the observation of CVs.
Handling and observation were performed atroom temperature, which was usually in the rangeof 22~28ºC.

RESULTS
Sequence of death
Paramecium cells which were swimming actively under a cover glass without pillars stopped
moving before long. Although the time to thestoppage varied greatly among cells because ofsuch factors as inconsistent vigor of each cell, variable rates of loss of water, contamination by debris
that supported the cover glass and so on, the period
between the cessation of swimming and the cell
rupture was 6 min on average, ranging from 1 to
11 min.
The first symptom of death was the formationof a small bleb, a local swelling of the outer cell
membrane. The number of blebs increased, eachbleb grew, and neighboring blebs fused to form alarger bleb. Cessation of ciliary beating at thecytopharynx (buccal cavity), which means the cessation of FV formation, tended to occur somewhat
earlier than the cessation of the contractility ofCVs. Ciliary beating was observed until the last
moment of the cell rupture at least somewhere
locally on the cell surface. Finally the outer cell
membrane and then the inner cell membrane were155

broken, and the cytoplasmic contents flew out.
Dying process in detail<Cell morphology>
After the cells stopped swimming, a small
bleb, an extrusion of the outer cell membrane wasformed as the first symptom of the death process.
The region where the first bleb was formed was
not fixed; every region on the cell surface appeared to have an equal chance to form a bleb.
The number of blebs increased and small neighboring blebs fused to form a larger bleb. A typicalmorphological change is shown in Fig. 1. If water
was supplied from a cover glass edge when blebs
were smaller in size and fewer in number, theblebs disappeared: the cell restored its normal
shape and began to swim normally. The point of
no-return or the commitment point appeared toexist somewhere during the bleb-growing process.
The cells at stages B and C in Fig. 1 would recover, but thereafter would not. Cilia were regularly located on the surface of the blebs, i.e., on theouter membrane. Trichocyst-discharging was
rarely observed during the dying process except at
the moment of cell rupture. When observed, it wasoutside the blebs.
Concomitant with the enlargement of blebs,
enlargement of intracellular vacuoles occurred.
We were unable to identify each vacuole enlarged
except CVs which we will mention later.
<Ciliary beating >Paramecium cells stopped swimming, although cilia all over the cell surface continued tobeat. Sometimes cells that stopped forward or
backward swimming rotated at the resting position.
This made it easy to observe the subsequent processes leading to death. Cilia on a part of the cellsurface continued to beat until the moment of cell
rupture, and sometimes continued to beat after thecytoplasm began to flow out (Fig. 1F).
<Food vacuole (FV) formation>
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 swimming. 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 wereformed. 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 beating.

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

Fig. 2. Contractility of aCV (rectangles) and pCV(circles) in 3 representative cells (A~C) when they
were to die. Abscissa: Number of pulsations until thelast moment of death. Start of the counting was arbitrary. Ordinate: Pulsating cycles in s.

157

than aCV, and aCV pulsated 1 time more afterpCV stopped pulsation.
In the cell of Fig. 2B, the pulsating cycle wasalmost identical in both CVs: 5~8 s in aCV and5~6 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 pulsated 9 times more than pCV during the observation 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: thetotal 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 contractility as in other cases, whereas contractility
was restored in aCV after an abrupt increase in thepulsating cycle. The last pulsating cycle of aCV
was 26 s, and that of pCV was 31 s. The aCV pulsated 14 times more than pCV during the observation period, and 9 times more after pCV stopped
pulsation.
In all of the above 3 cases, pCV stopped pulsation 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 observation period. Twelve cells, including those in
Fig. 2, were from video-recordings used to successfully count pulsating cycles in both CVs.
Positive numbers mean that aCV pulsated for alonger time and more frequently than pCV, andnegative numbers mean the reverse. The results
show no correlation between aCV and pCV intheir pulsation when the cells are at death; aCV (orpCV) pulsated for a longer time and more frequently than pCV (or aCV) in some cells, whileaCV (or pCV) pulsated for a longer time but lessfrequently 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 intime and in frequency between
aCV and pCV during the observation period
Time in s

The number ofpulsation

No. 1

154

10

No. 2

149

14

No. 3 (aCV-1)

87

0

No. 3 (aCV-2)

-18

1

No. 4

39

9

No. 5 (pCV-1)

215

No. 5 (pCV-2)

-37

4

No. 6

18

4

No. 7

6

-3

No. 8

3

1

No. 9

-20

1

No. 10-36-2

No. 11-47-2

No. 12-215-1

Positive numbers indicate that aCV pulsated for alonger time and more frequently than pCV during theobservation period, and negative numbers indicate thereverse. In No. 1 cell, for example, aCV pulsated 154
s longer and 10 times more than pCV. No.3 and No.
5 cells had 2 aCVs and 2 pCVs, respectively. In No.
5 cell, for example, aCV pulsated 21 s more thanpCV-1 but 37 s less than pCV-2, and aCV pulsated 5
and 4 times more than pCV-1 and pCV-2, respectively. A, B and C cells in Fig. 2 are identical to No.
7, 4 and 2 cells in Table 1, respectively.

of 12 cells, 1 of the CVs, aCV in 1 case and pCVin the other case, duplicated during the death process, and the resulting CVs resided close to eachother. Even in such a case, the 2 CVs pulsatedindependently.
CVs enlarged and deformed prior to the cell
rupture, maintaining the ability of pulsation.

Fig. 3. Schematic depiction of a cell in water that
supports the cover glass showing possible forces in arrows. f: force supported by paramecia, mg: weight ofcover glass, P: hydrostatic pressure of water, P0: atmospheric pressure, L: side length of cover glass, d:depth of water or the pressed width of the cell, α:
contact angle between water and a cover glass, γ :surface tension of water.

Estimation of pressure at death
As water under a cover glass decreases due todesiccation, Paramecium cells bear the force arising from the pressure difference between the waterand the atmosphere as well as the weight of thecover glass. We here assume that the cover glass
is supported evenly by N cells of paramecia. Fig.
3 is a schematic depiction of a cross section, where
the thickness of water is represented by d that isequal to the pressed width of the cell, and thecover glass is rectangular with mass m and lengthof a side L.
As water begins to evaporate from the sides of
the cover glass, the lateral interface of water becomes concave to the atmosphere. A cross-sectionof the interface is approximated as a circular arc
because the thickness d is sufficiently smaller thanthe capillary length in general. Using the contact
angle between water and a glass plate, α, the average curvature of the interface κ is expressed as κ =2 cosα / d. The difference between the hydrostaticpressure of water P and the atmospheric pressure
P0 is derived from Laplace's theorem as
P - P0 = γκ = 2 γ cosα / d ,(1)where γ is the surface tension of water (deJpn. J. Protozool. Vol. 38, No. 2. (2005)

Gennes et al., 2004).
The vertical force supported by N paramecia,
f, is caused by the weight of the cover glass m, thepressure difference P - P0 and the surface tension
acting on the 4 sides of the cover glass, 4γL. Representing the acceleration due to gravity by g, we
obtain the equationf = mg + L2 (P - P0) + 4γL sinα.

(2)

Substituting Eq. (1), we find that the third term
is negligibly small compared with the second termin the case of d << L. The equation is approximated as2

f ≈ mg + 2γL cosα / d.

(3)

We represent a typical contact area between
each Paramecium and the cover glass by S, andassume the intracellular pressure of paramecia as P
+ P*. P*, the difference between the inside andoutside pressures, works to burst paramecia. Regarding each Paramecium as a fluid cell surrounded by a soft membrane, the inside pressure isuniform everywhere in the cell. The equilibrium
equation of forces acting on the contact areas isexpressed by f = NSP*, where the surface tensionof the membranes is negligible because the contact
areas are approximately flat. Therefore P* is estimated by the equation
P* = f/NS ≈ ( mg + 2γL2 cosα / d) / NS. (4)As an example, we calculated P* assumingthat N = 10, S ≈ 3.0 × 10-5 cm2 and d ≈ 15 µm =1.5 × 10-3 cm: A P. tetraurelia cell was considered
as a rotatory ellipse with length of 100 µm andwidth of 30 µm; when it was pressed to half, theprojection area of the original ellipse would be
expanded; the contact area of the cell to the coverglass (S) was assumed to be similar to the projection area of the original ellipse. We used m ≈ 10-1

159

g, g ≈ 980 cm/s2, γ ≈ 70 dyn/cm and L ≈ 1.8 cm.
Adopting α ≈ 20° as a typical value of the contact
angle, although it depends on the state of the glass
surfaces, we obtained an estimation of P* as
P* ≈ 9.5 × 108 dyn/cm2 ≈ 9.5 × 102 atm.

(5)

In this case, the pressure caused by the weightof the cover glass, mg/NS ≈ 0.3 atm, is very small
compared with this value.

DISCUSSION
By carefully observing death process of Paramecium cells under a cover glass without supporting pillars, we found that ciliary beating continued
until the last moment of cell rupture or even after
the cytoplasm began to flow out. This may not be
surprising, since ciliary beating has been shown tocontinue in non-living cell models such as the Triton model (Naitoh and Kaneko, 1972).
Cessation of CV pulsation tended to occur
later than the cessation of FV formation. Pulsationof CVs continued until immediately before the cell
rupture. Enlarged and deformed CVs could still
pulsate. In contrast to the ciliary beating, however,CV pulsation was never observed after the cytoplasm began to flow out, although autonomous
pulsation of CV could occur in vitro (Tani et al.,2001). We found that aCV and pCV pulsated independently with different cycles and frequencies
in the cells at death, although the 2 CVs appeared
to pulsate with regular intervals in intact cells. It
has been suggested that no fibrous network systemsurrounds the CV (Naitoh et al., 1997; Tani et al.,2001). This means that the independency between
aCV and pCV pulsation is of an original nature,and is not due to disconnection at death. Also
found was an occasional occurrence of an abrupt
duplication of CV during the dying process. BothaCV and pCV were duplicated, and the resulting

160

How Paramecium cells die under a cover glass?

CVs pulsated independently (Table 1).
We estimated that the Paramecium cell membrane was broken only when the difference between the outside and inside pressures of the cell
was as high as ~950 atm. The value of P* calculated by Eq. (4) would change if the assumption of
N, S and d differed. For the evaluation of S, themost puzzling value, we assumed that S would be
similar to the projection area (A) of the original
ellipse with long axis of 100 µm and short axis of
30 µm. We calculated A (2.4 ×10-5 cm2), correctedby computer-aided calculation of the surface areaof photographed cells (4.3 ×10-5 cm2), and adopted
the value of S = 3.0 ×10-5 cm2. However, the actual contact area of the cell to the cover glass
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
atm.
N is also a factor that affects P*. Although weassumed that the pressure was supported evenly by
N cells, debris of similar size, which was not completely eliminated, would also support the pressure. We therefore consider that P* with the rangeof 470~2,000 atm would be reasonable.
In classic studies of the effects of hydrostaticpressure introduced by Wichterman (1953), Paramecium cells became immobile at 600 atm but
they could recover even after exposure to 800 atm.
A recent experimental study by Dr. Y. Taniguchi
of Ritsumeikan University showed that P. multimicronucleatum cells could endure hydrostatic pressure of ~1,000 atm (Takagi, personal communication).
Such information suggests that P.
tetraurelia cells in this study were exposed to600~800 atm when they stopped swimming at the
beginning and to more than 1,000 atm when they
were ruptured. It should be emphasized, however,that P* is different from the hydrostatic pressure.
The present observations on the death process
are a typical case of accidental death which takes
place in a short time. It is of interest to know how
cells die in other cases such as death by physio-

logical chemicals at higher concentrations anddeath by starvation, which occur more slowly thanthe present case.
ACKNOWLEDGMENTS
We are grateful to Dr. Yoshihiro Taniguchi of
Ritsumeikan University for sharing his experimental data on biological tolerance to high pressure, andto Dr. Terue Harumoto of Nara Women’s University for her help in video-recording. We also thankthe anonymous referees for their fruitful comments.

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