Jpn. J. Protozool. Vol. 37, No. 2. (2004)

139

Original
A bacteria-free monoxenic culture of Paramecium bursaria: its growth
characteristics and the re-establishment of symbiosis with Chlorella inbacteria-free conditions
Gen OMURA1,2, Masaki ISHIDA3, Mikihiko ARIKAWA1,4, S. M. Mostafa Kamal KHAN1,
Yasutaka SUETOMO1, Soichiro KAKUTA1, Chisato YOSHIMURA5 and Toshinobu SUZAKI1,*
1

Department of Biology, Faculty of Science, Kobe University, Kobe 657-8501, Japan, 2Biological
Institute, Universität Stuttgart, D-70569 Stuttgart, Germany, 3School of Science Education, Nara
University of Education, Nara 630-8528, Japan, 4Graduate School of Human Culture, Nara
Women's University, Nara 630-8506, Japan, and 5Center for Environmental Management, Kobe
University, Kobe 657-8501, Japan.

SUMMARY
A bacteria-free monoxenic culture of Paramecium bursaria has been established. Chlorogonium elongatum was used as the sole food source
and large numbers of easily maintained P. bursaria
were obtained. Additional enhancement of thegrowth of P. bursaria was achieved by supplementing the culture medium with soybean lecithin.
The ability of monoxenically cultured P. bursaria
to re-establish its symbiotic relationship with
Chlorella in bacteria-free conditions was examined.

*Corresponding author
Tel: + 81 78 803 5722
Fax: + 81 78 803 5722
E-mail: suzaki@kobe-u.ac.jp
Received: 9 Nov. 2004; Accepted: 2 Nov. 2004.

Bacteria-free, monoxenic, Chlorella-free (white) P.
bursaria, and axenic isolated symbiotic Chlorella
cultures were established from bacteria-free monoxenic cultures of Chlorella-bearing (green) P.
bursaria. When the white paramecia and isolated
Chlorella were mixed in bacteria-free conditions,they re-established a symbiotic relationship, andnormal green paramecia were reconstructed. This
newly developed bacteria-free monoxenic culturingof P. bursaria will be useful in the study of protozoan symbiosis, as this method provides us withlarge numbers of cells that possess the ability to reestablish a symbiotic relationship with Chlorella.

INTRODUCTION
The ciliate Paramecium bursaria harbors
many symbiotic cells of the unicellular alga
Chlorella (Chlorophyta) in its cytoplasm. P. bur-

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Monoxenic culture of Paramecium bursaria

saria can be freed of its symbiotic Chlorella bycultivation in prolonged darkness with an excess
food supply (Siegel, 1960), or by treating cells withphotosynthesis inhibitors such as DCMU and methylviologen (Reisser, 1976; Hosoya et al., 1995) orwith the antibiotic cycloheximide (Weis, 1984).
Symbiotic Chlorella can also be isolated from hostP. bursaria (Loefer, 1936; Siegel, 1960; Weis,
1978; Takeda, 1995; Nishihara et al., 1998). Thus,
under laboratory conditions, both Chlorella-free(white) P. bursaria and isolated symbiotic Chlorella can be cultured independently. They can reestablish the symbiotic relationship when mixed,
resulting in reconstruction of green paramecia(Siegel, 1960; Karakashian, 1963; Bomford, 1965;
Karakashian, 1975; Weis, 1978; Weis and Ayala,
1979; Görtz, 1982; Reisser et al., 1982; Meier andWeissner, 1988; Nishihara et al., 1996; Takeda etal., 1998). Additionally, the white paramecium canbe artificially infected with other microorganisms
such as Scenedesmus (a unicellular alga), yeasts,
and bacteria (Bomford, 1965; Görtz, 1982). The
mechanism of the infection by microorganisms andsubsequent establishment of a symbiotic relationship is still not understood. In particular, investigations of the molecular basis of symbiosis have notbeen possible because of difficulties in preparing
the large numbers of P. bursaria cells essential forbiochemical and molecular biological experiments.
Axenic cultures of P. bursaria have been
described in the literature, although they only growslowly (Loefer, 1936), or need time-consuming andrather elaborate preparations such as using glutaraldehyde-killed bacteria in the culture medium(Weis, 1975). P. bursaria has therefore been
mainly cultured in non-sterile hay or lettuce infusions that are inoculated with food bacteria in advance (Sonneborn, 1950). Although cultures including living bacteria as a food source can provide
a good cell harvest, such cultures can be easily
contaminated. Especially in cultures of white P.
bursaria, cells are more vulnerable to accidental

infection by undesirable microorganisms (Görtz,1982). Therefore, a relatively easily maintained,bacteria-free culture has long been needed for thecultivation of P. bursaria.
In this study, we succeeded in establishing abacteria-free monoxenic culture with the flagellated
unicellular alga, Chlorogonium elongatum(Chlorophyta), as a sole food source. We havecharacterized some features of this novel culture
method, and further examined the ability of P.
bursaria to re-establish its symbiotic relationshipwith Chlorella in bacteria-free conditions.

MATERIALS AND METHODS
Conventional culture of P. bursaria
Paramecium bursaria (neither syngen nor
mating type identified) collected from a small pondon the campus of Kobe University (Kobe, Japan)
was pre-cultured in 0.01% Knop medium (0.24 mM
Ca(NO3)2, 0.14 mM KNO3, 0.06 mM MgSO4, 0.10
mM KH2PO4 ) with 10 mM HEPES-KOH (pH 7.0)
at 23°C under a 12:12 h light : dark cycle, and C.
elongatum was added as a food source. C. elongatum was routinely cultured in an axenic medium(Chlorogonium medium (CM): 1.36 mM CaCl2,
0.24 M sodium acetate, 0.02% polypeptone, 0.04%
tryptone and 0.04% yeast extract) at 20°C undercontinuous light (Sakaguchi and Suzaki, 1999).
Establishment of a bacteria-free monoxenic
culture of P. bursaria
Paramecium bursaria that had been precultured as described above was washed by transferring cells with a fine-tipped Pasteur pipettethrough about 15 ml of sterilized 0.01% Knop with
10 mM HEPES-KOH (pH 7.0) (KH) in successive
Petri dishes. About 20 cells were chosen from thepre-culture, washed, and finally transferred to adish that contained sterilized KH with antibiotics
that was then sealed with Parafilm. The optimal

Jpn. J. Protozool. Vol. 37, No. 2. (2004)

concentration was determined for the antibiotics
penicillin, kanamycin and streptomycin. P. bursaria was able to increase in number, consuming C.
elongatum, in KH that contained penicillin at concentrations less than 800 µg/ml. Kanamycin or
streptomycin, even at concentrations less than 50µg/ml, inhibited the proliferation of P. bursaria
significantly. Therefore, 800 µg/ml penicillin wasused in the experiments. The dish with penicillin
and washed P. bursaria was incubated for several
days to allow P. bursaria to increase in number
sufficiently for the next washing procedure. Cells
were washed again in sterilized KH and incubatedwith penicillin as described. After each incubation
with penicillin, the dish was observed with an inverted light microscope, and the procedure ofwashing and incubation was repeated until no contaminating bacteria were observed in the dish. A
part of P. bursaria was thereby sterilized, and was
finally transferred to a penicillin-free medium consisting of 0.01% Knop and 20% CM. An adequate
amount of axenically grown C. elongatum wasadded as a food source for the P. bursaria. The
culture was incubated at 23°C under a 12:12 hlight : dark cycle (light intensity, 2,000-3,000 lux),and subcultured weekly. The rest of P. bursaria
was again subjected to the washing procedure, anda part of the culture was successively transferred toanother penicillin-free culture medium. Cultures
were routinely checked for sterility by spreading adrop of the culture medium onto an agar plate (2%agar in culture medium) and checking for contamination after the plate was incubated. When any
contamination was found, the culture was discarded.
To enhance the final harvest of P. bursaria, themedium composition was modified by increasing
the concentration of CM or adding soybean lecithin(see details in Table 1). The axenic culture medium
for P. multimicronucleatum used by Fok and Allen(1979) was also tested as an alternative medium,
without added C. elongatum. To do this, P. bursaria from an aged culture, in which all food C.

141elongatum was consumed, was inoculated directly
to the axenic medium, and then incubated at 25°C
under 12:12 h light : dark cycle.
Establishment of a bacteria-free culture ofwhite P. bursaria and an axenic culture of isolated symbiotic Chlorella
A photosynthesis inhibitor, methylviologen(1, 1-dimethyl-4, 4-bipyridinium dichloride, Sigma) was aseptically added through a syringe fittedwith a disposable sterilized filter (pore size: 0.45µm) to culture medium (0.01% Knop + 20% CM) in
which green P. bursaria had been incubated for 7
days. The concentration of methylviologen that isenough to remove all the symbiotic Chlorella waspreliminarily determined to be 10 µg/ml. At thisconcentration, no symbiotic Chlorella cells wereobserved in the host P. bursaria after the paramecium was exposed to the reagent for 10 days. This
Chlorella-free (white) P. bursaria was subcultured
in the same culture medium as that used for green P.
bursaria. To isolate symbiotic Chlorella, green P.
bursaria in an aged culture where all food C. elongatum had been consumed was sonicated aseptically in a small sterilized test tube, and transferred
directly to an axenic culture medium for green algae(CA medium; Nishihara et al., 1998). Symbiotic
Chlorella thereby isolated was subcultured weekly
in CA medium and grown at 25°C under continuous
light.
Assessment of the ability of white P. bursaria
to re-establish the symbiotic relationship with
Chlorella in bacteria-free culture
White P. bursaria was taken from a monoxenic culture in which all food C. elongatum had
been digested. This corresponds to the 7th or 8th
day of culture as shown in Fig. 2. At this stage, thewhite P. bursaria was starved but still actively
swimming. Isolated symbiotic Chlorella wereprepared from an axenic culture in its early stationary phase. White P. bursaria was centrifugally

142

Monoxenic culture of Paramecium bursaria(200 g, 5 min) washed twice in sterilized KH, as
was symbiotic Chlorella (3,000 g, 5 min ×2). They
were then mixed well in a tube and incubated at 20°
C under continuous light. The final density of
Chlorella cells in the mixture was 106 cells/ml, andthe ratio of Chlorella to Paramecium was 10,000:1.
After 24 hr, the mixture was washed with sterilized
KH by passing the cells through a nylon mesh (11µm pore size) to remove free Chlorella cells. P.
bursaria cells retained on the mesh were recovered
and transferred to 0.01% Knop + 20% CM withadded C. elongatum. The culture was then incubated at 23°C under 12:12 h light : dark cycle. Allprocedures (including centrifugation, washing andtransfer of cells) were carried out aseptically.
Cultures were routinely checked for sterility
by spreading a drop of the culture medium onto anagar plate (2% agar and culture medium), andchecking for contamination after the plate wasincubated under an appropriate condition.

Fig. 1. Paramecium bursaria grown for 1 week in abacteria-free monoxenic medium consisting of 0.01%
Knop with 20% CM. Scale bar = 50 µm.

RESULTSGreen P. bursaria was readily sterilized bywashing and penicillin treatment. Sterilized P.
bursaria was inoculated, with food C. elongatum,
into culture medium consisting of 0.01% Knop +20% CM, and subsequently subcultured in the same
medium. In this culture medium, P. bursaria wasable to increase in number by consuming C. elongatum, and reached peak density after about one
week. P. bursaria grown in this bacteria-freemonoxenic culture retained several hundred healthy-looking Chlorella in the cytoplasm of each cell(Fig. 1). No contaminating bacteria or fungi wereobserved either in the culture solution or on solidified medium enriched with organic components tofacilitate their growth.
In the bacteria-free monoxenic culture, peakcell density was observed first for C. elongatum,
followed by free-living Chlorella cells, and then P.

Fig. 2. A growth curve of P. bursaria in a bacteria-freemonoxenic medium consisting of 0.01% Knop with 20%
CM. The profile and the time shift of the growth curveof each population are similar at each subculture.

bursaria (Fig. 2). The populations of C. elongatum
and free-living Chlorella were only transient; they
disappeared as the growth of P. bursaria proceeded. The population of P. bursaria remained at
a plateau level for at least a few weeks even without
any additional food supply, and the population of
free-living Chlorella gradually increased again.
The retention of the plateau level of P. bursaria wasdependent on the illumination conditions (Fig. 3),

Jpn. J. Protozool. Vol. 37, No. 2. (2004)

143

Table 1. Cell density (cells/ml) of P. bursaria in the media of different compositions.
0.01% Knop with
Mediumcomposition

20% CM (a)30% CM (b)40% CM (c)20% CM + 0.125
mg/ml soybean
lecithin (d)Cell density

1,500

2,000

2,500

5,000

Accumulation of Chlorella

+

++

+++

+

Final cell densities were determined at 7 days post-inoculation for a–c and 11 days for d. The relative
amount of accumulated free-living Chlorella in the medium is indicated by numbers of “+” marks (+,~104 cells/ml; ++, ~105 cells/ml; +++, ~106 cells/ml).

Fig. 3. A population profile of green P. bursaria in thedark. The medium consists of 0.01% Knop with 20%
CM. From day 8 after inoculation, the culture flask was
incubated in the dark.

Fig. 4. Light micrographs of green P. bursaria takenfrom the culture shown in Fig. 3. A: day 7. B: day 14.
C: day 20. Scale bar = 40 µm.

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Monoxenic culture of Paramecium bursaria

Fig. 5. Growth curves of P. bursaria in the complete
axenic medium for P. multimicronucleatum at full andhalf strength. In the full-strength medium (closed circles), the cell density reached a maximum of 2,500
cells/ml at 12 days after the inoculation, while smaller
numbers of cells were harvested in half-strength medium (open circles).

Fig. 6. White P. bursaria grown for 1 week in a bacteria-free monoxenic culture medium consisting of 0.01%
Knop with 20% CM. Food C. elongatum had been
completely digested and the cells were slightly starved.
Scale bar = 50 µm.

indicating that photosynthesis of symbiotic Chlorella contributes to survival of the host cell in thisbacteria-free culture medium. Microscope observations on P. bursaria kept in the dark showed thatthe host paramecium gradually lost its symbiotic
Chlorella (Fig. 4, A-C), suggesting that the symbiotic Chlorella was digested as an innate foodsource.
The peak cell density of green P. bursaria
was increased by raising the concentration of CM in
the culture medium (Table 1). The higher the concentration of CM, the more the growth of C. elongatum, providing more food for P. bursaria andresulting in higher peak cell densities at 7–8 days
after inoculation. At concentrations of CM above
30%, increased death of P. bursaria was observed
during its growth phase (data not shown), alongwith an accumulation of free-living Chlorella cells
in the medium after the growth of P. bursaria
reached its plateau level. In this situation, the culture medium appeared green due to the accumulated Chlorella cells, and the survival period of P.

bursaria was much shorter (data not shown). The
optimum concentration of CM is therefore 20%.
Addition of soybean lecithin to the culture
medium facilitated the growth of P. bursaria (Table
1). Although a few additional culture days wererequired to reach the peak cell density, P. bursaria
remained at the plateau level for a few weeks as it
did in the lecithin-free medium. In the presence oflecithin, however, accumulation of free-living
Chlorella cells was not observed during the plateau
phase. The opaque appearance of the culture medium due to the added lecithin gradually faded
through the incubation period, and the medium
cleared completely 1–2 days after all food C. elongatum were taken up by P. bursaria.
Further improvement of cell harvest was
expected in axenic culture conditions already developed for other species of the genus Paramecium
including P. multimicronucleatum. P. bursaria
increased in number in the axenic medium developed for P. multimicronucleatum, and the cell density of P. bursaria in this medium finally reachedJpn. J. Protozool. Vol. 37, No. 2. (2004)

Fig. 7. A growth curve of white P. bursaria in a bacteria-free monoxenic medium in 0.01% Knop with 20% CM.
In contrast to the Chlorella-bearing normal P. bursaria
shown in Fig. 2, the cell density of P. bursaria gradually
decreased after it reached its peak at 7–10 days after inoculation. The profile and the time shift of the growth
curve of each population are similar at each subculture.

Fig. 8. Isolated symbiotic Chlorella grown in CA medium. Scale bar = 50 µm.

about 2,500 cells/ml 12 days after inoculation (Fig.
5).
Chlorella-free (white) P. bursaria was prepared aseptically by exposing green P. bursaria
cultured in sterile monoxenic medium to the photosynthesis inhibitor methylviologen. During theexposure period, the paramecium gradually lost its

145

Fig. 9. Growth of white P. bursaria that was incubatedwith Chlorella for 24 h. The medium consists of 0.01%
Knop solution with 20% CM.

symbiotic Chlorella cells and, 10 days after thetreatment, no symbiotic Chlorella at all was observed in the host cells. P. bursaria thereby freed
from symbiotic Chlorella was cultured with food C.
elongatum in the bacteria-free monoxenic medium
used for green P. bursaria. In this culture, no
Chlorella was observed either in the host cell or in
the surrounding medium through successive subcultures, indicating that the paramecia were completely deprived of their symbiotic Chlorella (Fig.
6). The population density of white P. bursaria
increased and reached its peak cell density 6–7 days
after inoculation, and gradually decreased following a stationary period of about 2 days (Fig. 7). The
resulting peak cell density was almost identical tothat of green P. bursaria (Fig. 2). An axenic cultureof isolated symbiotic Chlorella was also successfully established directly from sonicated green P.
bursaria (Fig. 8). The population of isolated symbiotic Chlorella reached about 7 × 106 cells/ml
within 2 weeks under continuous light.
The ability of white P. bursaria to reestablish symbiosis with Chlorella was assayed inbacteria-free conditions. White P. bursaria thatwas fed on Chlorella cells for 24 hr also increasedin number in the bacteria-free monoxenic medium

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Monoxenic culture of Paramecium bursaria

mixtures of digested (brown) and apparently
healthy (green) Chlorella cells during observation.
On day 8 after the inoculation, all food C. elongatum were consumed, and the cytoplasm of theparamecia had cleared, leaving many healthylooking green Chlorella cells stabilized in the peripheral region of the host paramecia (Fig. 10B).
On day 14, after the population of Chlorella-fed
white P. bursaria reached a plateau level, many
green Chlorella cells were still observed in theparamecia (Fig. 10C). This Chlorella-fed white P.
bursaria was successively subcultured in the same
medium. The growth curve of established Chlorella-fed white P. bursaria was identical to that ofgreen P. bursaria, and no white paramecia emergedin the culture medium even after prolonged subculturing (data not shown).

DISCUSSION

Fig. 10. Light micrographs of P. bursaria taken from theculture shown in Fig. 9. A: day 0. B: day 8. C: day 14.
Scale bar = 40 µm.

(Fig. 9). The population of Chlorella-fed white P.
bursaria increased and reached peak cell density ina similar manner to both green and white P. bursaria, and afterwards did not decrease, but retained
a plateau level. Light microscope observations on
Chlorella-fed white P. bursaria were made during
the culture period. On day 0, just after the 24-hour
incubation with Chlorella finished, the cytoplasmof the paramecia was filled with abundant ingested
Chlorella cells. Many of the Chlorella cells werebeing digested, and the paramecia appearedbrownish (Fig. 10A). The paramecia indeed ejected

In this study, bacteria-free monoxenic cultures of P. bursaria was established, using C. elongatum as a food source. Peak cell density wasreached first by C. elongatum, followed by Chlorella growing free in the medium, and then P. bursaria reached its peak as the other two populations
declined. The profile of the growth curve was
identical for green and white P. bursaria. This
indicates that, at least in the presence of sufficient
external food, the growth of P. bursaria is not influenced by factors derived from its symbiotic
Chlorella. Similar growth profiles for P. bursaria
have been reported earlier (Karakashian, 1963;
Pado, 1965; Karakashian, 1975; Nishihara et al,1996), although they were not grown in bacteriafree conditions. After all food C. elongatum wasconsumed, white P. bursaria decreased in number,but green P. bursaria retained its cell density at aplateau level for a few weeks. In the dark, however,
Chlorella-bearing P. bursaria decreased in number,with a gradual loss of symbiotic intracellularJpn. J. Protozool. Vol. 37, No. 2. (2004)

Chlorella. Symbiotic Chlorella releases the majorpart of newly-fixed photosynthetic products as
maltose (Muscatine et al., 1967; Ziesenisz et al.,
1981; Dorling et al., 1997), and the host paramecium appears to use it (Muscatine et al., 1967;
Brown and Nielsen, 1974). After all external food
was consumed in the bacteria-free culture, the survival of P. bursaria was dependent on the presenceof intracellular symbiotic Chlorella, which probably provides photosynthetic products in the light,
and may be digested by the host paramecium in thedark.
For the first time, the population of Chlorella
living free in the surrounding medium was investigated throughout the culture cycle. Free-living
Chlorella floating in the medium has been reported
in an aged culture of P. bursaria in non-sterile
conditions (Siegel, 1960). It is speculated that thefree-floating Chlorella may have been liberated
from dead host cells or released from intact hostcells, although the origin of the algae was not identified. We observed green P. bursaria exocytotically releasing living Chlorella cells from the cytoproct after feeding on food C. elongatum (notshown). This phenomenon is under further study
and will be reported elsewhere. In our bacteria-free
culture, the cell density of free-living Chlorella firstincreased and then decreased markedly during theearly growth phase of P. bursaria, strongly suggesting that P. bursaria took up the free Chlorella
cells. Weis (1976) showed that Chlorella-bearing
P. bursaria can grow by feeding on extracellular
Chlorella from the surrounding medium, and about
2,000 Chlorella cells per paramecium per day are
required to allow growth with a generation time of
40 hr. In our bacteria-free culture, the ratio of freeliving Chlorella cells to green P. bursaria cells
was, at most, 10 at the transient peak cell density of
free-living Chlorella (for example, at day 4 in Fig.
2). Therefore, in our cultures, free-living Chlorella
would not contribute substantially to the growth of
P. bursaria, even if they were all digested.

147

The composition of our culture medium was
modified to improve the final cell harvest of P.
bursaria. At higher concentrations of CM, the peakcell density of P. bursaria increased, although
excess free-living Chlorella accumulated. Thisaccumulation could be due to increased exocytotic
release of intracellular Chlorella cells from living
paramecia, but is more likely due to release fromdead P. bursaria cells, which were found frequently
on the bottom of the culture vessel. Concentrated
CM may have direct detrimental effects on P. bursaria, or may contribute to excess growth of existing free Chlorella, which might then excrete factors
fatal to P. bursaria. We did not investigate thisfurther because we found that addition of soybean
lecithin was far superior for increasing paramecium
growth. When soybean lecithin was added to themedium, P. bursaria underwent additional fissions,and maintained its cell density at a plateau level
without excess accumulation of free-living Chlorella. Lecithin, phosphatidylethanolamine, or phosphatidylcholine has been previously used as a lipid
source in axenic media for Paramecium (Fok andAllen, 1979; Schönefeld et al., 1986). Weis (1975)
grew P. bursaria using an exogenous lipid source inan axenic medium. When our culture medium was
supplemented with soybean lecithin, P. bursaria
was probably able to use this as a source of lipid toachieve additional growth.
We also confirmed that P. bursaria is able togrow without food C. elongatum in a completely
axenic medium that was originally developed for P.
multimicronucleatum (Fok and Allen, 1979).
However, the final cell density, 2,500 cells/ml, wasonly the same as that obtained in our medium
without soybean lecithin. Considering the elaborate preparation procedures and complicated constituents for this complete axenic medium, we believe that our monoxenic culture method, with foodC. elongatum, is superior to the axenic method for
culturing large quantities of P. bursaria. Different
strains and species of Paramecium have different

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Monoxenic culture of Paramecium bursaria

nutritional requirements, and the amounts of, as
well as the ratios between, phospholipids and stigmasterol have been regarded as critical for ensuring
optimum growth of Paramecium (Soldo and van
Wagtendonk, 1969; Fok and Allen, 1979; Hennessey et al., 1983). Weis (1975) showed that, in theaxenic medium, P. bursaria requires a certain ratio
between TEM-4T (replaceable with phospholipids)
and stigmasterol for optimal growth. Therefore,
further modifications to both the axenic and ourmonoxenic medium would possibly promote better
growth of P. bursaria.
Using a bacteria-free monoxenic culture ofwhite P. bursaria and an axenic culture of isolated
symbiotic Chlorella, the ability of white P. bursaria to re-establish a symbiotic relationship with
its original symbiotic Chlorella was assayed inbacteria-free conditions. We employed two criteria
to judge the success of re-establishment: the morphology and the growth curve of Chlorella-fed
white P. bursaria. In normal P. bursaria, symbiotic Chlorella cells are stabilized in the peripheral
region of the host cell (Karakashian et al. 1968;
Weis 1976), and Chlorella thereby appears to escape the host’s digestive process (Karakashian andRudzinska, 1981; Karakashian and Karakashian,1973). The peripheral region of P. bursaria cells is,however, also able to harbor some species of bacteria and fungi that do not provide the host cell withany benefits (Görtz, 1982). Therefore, the retention of ingested Chlorella in the peripheral regionof the host cell seems not to be the result of reestablished symbiosis, but is a prerequisite for it.
Confirmation of the symbiotic relationship should
be possible by observing characteristics that occur
only in Chlorella-bearing P. bursaria. In our bacteria-free monoxenic culture medium, Chlorellafed white P. bursaria accumulated many Chlorella
cells in the peripheral region, and maintained its
cell density at a plateau level as green P. bursaria
does. This implies that photosynthetic products
from established intracellular symbiotic Chlorella

are being used by the paramecium, indicating that a
symbiotic relationship was successfully reestablished between white P. bursaria and Chlorella. This result also demonstrates that the symbiotic relationship between P. bursaria and its
Chlorella does not involve any other organisms,
such as co-existing bacteria.
We conclude that our bacteria-free monoxenic culture method for P. bursaria can provide
enough cells that are able to re-establish and maintain a symbiotic relationship with Chlorella toenable further studies on the molecular mechanismof the symbiotic relationship between P. bursaria
and Chlorella.

ACKNOWLEDGMENTS
We thank Cathy Busby for her critical reading of the manuscript. This work was partly supported by the River Environment Fund (REF) incharge of the Foundation of River and Watershed
Environment Management (FOREM) to TS andGerman Academic Exchange Service (DAAD)
fellowship for young scientists awarded to GO.

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