University of Malaya
From the SelectedWorks of Tan Tian Chye
2007
Evidence of plasmotomy in Blastocystis hominis
Tan Tian Chye, University of Malaya
Available at: http://works.bepress.com/tan_tianchye/5/
Parasitol Res (2007) 101:1521–1525
DOI 10.1007/s00436-007-0670-0
ORIGINAL PAPER
Evidence of plasmotomy in Blastocystis hominis
T. C. Tan & K. G. Suresh
Received: 4 July 2007 / Accepted: 9 July 2007 / Published online: 16 August 2007
# Springer-Verlag 2007
Abstract Blastocystis hominis has been regarded as an
enigmatic parasite as many aspects of its basic biology
remain uncertain. Many reproductive processes have been
suggested for the organism; however, to date, only the
binary fission has been proven. Plasmotomy is one of the
modes of reproduction previously suggested to be seen in in
vitro cultures. The present study provides trichrome and
acridine orange staining evidence for the existence of
nucleic acid suggestive of division of nucleus into
multinucleate forms with the respective cytoplasm dividing
giving rise to two or three progeny B. hominis. Transmission electron micrographs further confirmed that these
daughter cells had respective surrounding surface coat,
mitochondria, and vacuoles.
tally proven mainly because of the lack of suitable animal
models (Alexeieff 1911; Boreham and Stenzel 1993; Singh
et al. 1995; Tan 2004). Although several methods for
division of B. hominis cells such as binary fission,
plasmotomy, schizogony, and sporogony (Zierdt 1991;
Govind et al. 2002) have been described, binary fission is
the only plausible mode of reproduction (Stenzel and
Boreham 1996; Tan and Stenzel 2003). We previously
described amoeboid forms isolated from cultures of parasites obtained from symptomatic patients (Tan and Suresh
2006), suggesting its role in contributing toward pathogenicity. In this present study, we further provide evidence for
plasmotomy to be one of the reproductive processes
involved in B. hominis as suggested by others (Zierdt
1991; Zaman 1997; Zhang et al. 2007), whose evidence
were only confined to light microscopical observations.
Introduction
Blastocystis hominis is a highly polymorphic organism with
various morphological forms being reported in the literature
including vacuolar, granular, amoeboid, cyst, avacuolar,
and multivacuolar forms (Stenzel and Boreham 1996; Tan
et al. 2002). The true life cycle has not been conclusively
elucidated, and the modes of reproduction are still uncertain
(Stenzel and Boreham 1996).
To date, a total of five life cycles have been proposed for
Blastocystis; however, none of them have been experimen-
Materials and methods
T. C. Tan : K. G. Suresh (*)
Department of Parasitology, Faculty of Medicine,
University of Malaya,
Kuala Lumpur, Malaysia
e-mail: [email protected]
In vitro culture of B. hominis isolates
T. C. Tan
e-mail: [email protected]
Source of B. hominis isolates
In our previous study (Tan and Suresh 2006), the amoeboid
form of B. hominis was seen in ten symptomatic isolates.
Three out of the ten isolates, i.e., isolates S1, S8, and S10,
were randomly selected for detailed study to assess if
plasmotomy occurs in B. hominis.
The parasites were isolated from patients by in vitro
cultivation using Jones’s (1946) medium supplemented
with 10% horse serum (Suresh et al. 1994a; Zaman 1997).
Subsequently, after isolation, the parasites were maintained
in Jones’ medium by consecutive subculture every 3–4 days
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Parasitol Res (2007) 101:1521–1525
for at least 1 month before the morphological and
ultrastructural studies.
Trichrome staining
The trichrome technique of Wheatley (1951) was used.
Briefly, a direct smear was prepared from days-2 to -6
cultures of each isolate. The smears were air-dried at room
temperature and subsequently fixed in Schaudinn’s fixative
for at least 1 h at room temperature. The fixed smears were
dipped through three changes of 70% ethanol for 1 min each
(iodine was added to the first change of 70% ethanol) and
followed by staining in trichrome stain for 2–8 min. The
stained smears were differentiated in 90% ethanol containing
0.005% glacial acetic acid for 10–20 s followed by a brief
rinse in 95% ethanol. The smears were transferred into 100%
ethanol (1 min) and then xylene (1 min). The stained smears
were eventually mounted with DePeX mounting medium.
Acridine orange staining
Parasites from days-2 to -10 culture of each isolate were
stained with acridine orange solution according to the
method previously described by Suresh et al. (1994b).
Briefly, 5 ml of 0.1% acridine orange stock solution was
diluted with 45 ml of phosphate buffered saline pH 7.4
before use. A drop of culture sediment containing parasites
was mixed thoroughly on a clean glass slide with a drop of
diluted acridine orange. The preparation was viewed with a
fluorescence microscope (Leitz Wetzlar, Germany) using
incident light transmission at ×400 magnification.
TEM study
The contents from the day-5 culture were washed three
times using PBS, pH 7.4, and centrifuged at 500×g for
5 min. The pelleted cells were resuspended overnight in 4%
glutaraldehaye in 0.1 M sodium cacodylate buffer, pH 7.3
at 4°C, washed thoroughly with cacodylate buffer, and
postfixed for 30 min in 1% osmium tetroxide in cacodylate
buffer. The fixed cells were dehydrated in ascending series
of ethanols and embedded in epoxy resin. Semithin sections
were stained with toluidine blue. Ultrathin sections were cut
using an ultramicrotome, contrasted with uranyl acetate and
lead citrate and viewed using a TEM (LEO Libra 120).
Results
The amoeboid form of B. hominis was irregular in shape
with a prominent nucleus at the central zone and multiple
extended pseudopodia at the periphery (Fig. 1a). Phase
Fig. 1 Amoeboid form of B. hominis is seen undergoing plasmotomy.
a Phase contrast microscopy showing prospective progenies (arrow)
pinching off from the mother cells. Bar, 20 μm. b Transmission
electron micrograph showing a progeny of B. hominis pinching off
from the mother cell (arrow). Note: The progeny contains an electrondense mitochondrion (m), multiple small vacuoles (v) and is
completely surrounded by surface coat (sc). Bar, 2 μm
Parasitol Res (2007) 101:1521–1525
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Fig. 2 Trichome staining of B.
hominis. a The central zone of
the vacuolar form of B. hominis
is stained green, while the peripheral nuclei is stained reddish
brown. b The central zone of the
amoeboid form is stained with
intense reddish brown, while the
peripheral region is stained
green. Note: Three prospective
daughter cells appear to pinch
out from the mother cell. Reddish brown material is seen in
the periphery of one of the
prospective daughter cells (arrow). Bar, 20 μm
contrast microscopy revealed the pinching of cytoplasm
from the body of the irregular amoebic forms (Fig. 1a).
Ultrastructural studies using TEM showed that a long
irregular mass of cytoplasm was seen to be pinched off
from the mother cell with electron dense surface coat
surrounding the prospective progenies of B. hominis. The
Fig. 3 Acridine orange staining
of B. hominis. a The nuclei and
central body of the vacuolar
form of B. hominis is stained
bright and dull yellow-green,
respectively. b Three daughter
cells (arrow) appear to pinch out
from the amoeboid form by
plasmotomy. Note: The entire
cell body is stained flaming redorange. c Progeny cell with a
prominent bright yellow stained
nuclei (arrow) at the periphery
pinched from mother cell. Note
the thin band of cytoplasm connecting the progeny to the
mother cell. Bar, 10 μm
body of the mother cell showed intense electron dense
material. These progenies further showed multiple vacuoles
and mitochondrion (Fig. 1b).
Under trichrome staining, the cell body of the vacuolar
form of B. hominis is stained green, while the nuclei at the
peripheral region is stained reddish brown (Fig. 2a).
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However, the central body of the amoeboid form showed
intense reddish brown staining (Fig. 2b). Acridine orange
stained the nuclei and the central body of vacuolar forms
bright and dull yellow green, respectively (Fig. 3a). The
entire body of the amoeboid form is stained with intense
flaming red-orange (Fig. 3b,c).
Discussion
Reproductive processes in Blastocystis has always been in
controversy especially so when the proposed binary fission
as the only mode of reproduction accepted cannot account
for the high growth rate of parasites seen in in vitro cultures
of B. hominis. In the present study, plasmotomy was observed
only in the amoeboid form of B. hominis, which concurs with
findings from previous studies (Zierdt 1991; Zaman 1997).
However, the report of plasmotomy by Zhang et al. (2007) in
vacuolar forms needs further substantial evidence to provide
support for this reproductive mechanism.
In the present study, using trichrome staining, the
amoeboid forms undergoing plasmotomy showed intense
reddish brown staining in the central body, which is
suggestive of high nucleic acid level, which could lead to
active protein synthesis before the progeny being pinched
off from the mother cell. This was further confirmed with
acridine orange staining, which showed intense yellow
orange coloration in the central body indicative that the
organism is in a active state (Villar et al. 1998) and that
there is high level of proteins and RNA synthesis before the
cell division (Suresh et al. 1994b).
‘Plasmotomy’ is defined as the multinucleate body that
divides into two or more small, multinucleate individuals,
the cytoplasmic division taking place independently of
nuclear division (Kormos and Kormos 1958). This typical
mechanism of plasmotomy in Pelomyxa gruberi was
described by Frolov et al. (2006), who showed that
uninucleate individuals and mass appearance of multinucleate stages lead to micropopulations. In the present study,
we observed the accumulation of the nucleic acid at the
central zone of the amoeboid form of B. hominis as
evidenced by the reddish brown coloration and intense
flaming red-orange in both trichrome and acridine orange
staining, respectively, indicating high levels of nucleic acid
(Fig. 2b). This is suggestive that this process is leading to
the formation of multiple nuclei during plasmotomy.
The amoeboid form, which is generally shown to be
bigger in its size (5–65 μm; Tan and Suresh 2006) than the
vacuolar forms, undergoes plasmotomy to produce multiple
smaller progenies for the purposes of propagation of its
species to survive in adverse conditions as evidenced by
observing the occurrence of this reproductive process in
Parasitol Res (2007) 101:1521–1525
older cultures, which are known to be deprived of essential
nutrients.
In our previous study (Tan and Suresh 2006), we
provided evidence for the existence of amoeboid forms
seen only in cultures of B. hominis isolated from symptomatic patients. It is tempting to speculate that plasmo
tomy could be a reproductive mechanism seen only in
isolates that cause symptoms and may represent a particular
subtype of the organism; however, this would need further
studies to confirm.
Recent phylogenetic studies have shown that B. hominis
could be placed within the Stramenopiles (Silberman et al.
1996). Nevertheless, the Stramenopiles is a diverse group
of organism includes slime nets, water moulds, and brown
algae. It is rather difficult to find any morphological
similarity between B. hominis and other members of the
Stramenopiles. However, the ability of B. hominis to
reproduce by plasmotomy may possibly indicate its
phylogenetic affinity to the Stramenopiles. Protoopalina
intestinalis, an Opalinid (a member of the Stramenopile
group), which undergoes plasmotomy, was recently confirmed to be the closest sister group of B. hominis in the
Stramenopiles (Kostka et al. 2004).
Our present findings show that plasmotomy in B.
hominis confirm our previous suggestions (Govind et al.
2002) that Blastocystis does reproduce by method other
than binary fission.
Acknowledgment The study was funded by the Intensification of
Research in Priority Areas grant R&D 06-02-03-1012 from the
Malaysian government.
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