Parasitol Res (1999) 85: 791 ± 793
ORIGINAL PAPER
M. Pirehma á K. Suresh á S. Sivanandam
A. Khairul Anuar á K. Ramakrishnan
G. Suresh Kumar
Field's stain ± a rapid staining method for Acanthamoeba spp.
Received: 24 March 1999 / Accepted: 16 April 1999
Abstract Acanthamoeba sp. is a free-living amoeba
known to cause chronic central nervous system infection
or eye infection in humans. Many cases remain unde-
tected for want of a good detection system. We report
for the ®rst time a rapid staining method to facilitate the
identi®cation of Acanthamoeba sp. using the modi®ed
Field's staining technique. A. castellanii, which was used
in the present experiment, is maintained in our labora-
tory in mycological peptone medium (Gibco). The cul-
tures were pooled together and smears were made on
glass slides for staining purposes. Di€erent types of
stains such as Field's stain, modi®ed Field's stain,
Wright's stain, Giemsa stain, Ziehl-Neelsen stain, and
trichrome stain were used to determine the best stain for
the identi®cation of this amoeba. The concentration of
various stains and the duration of staining were varied
to provide the best color and contrast for each stain.
Acanthamoeba was also obtained from the brain of ex-
perimentally infected mice and was stained with various
stains as mentioned above to determine the best stain for
use in identifying the presence of this parasite in exper-
imentally infected animals. The modi®ed Field's stain
gives a very good color contrast as compared with other
stains. Furthermore, it takes only 20 s to be carried out
using the least number of reagents, making it suitable for
both laboratory and ®eld use.
Introduction
Acanthamoeba sp. is a free-living amoeba known to
cause granulomatous amebic encephalitis and Acanth-
amoeba keratitis in humans (Martinez 1985). It has been
isolated from the human throat, nasal swabs, and ab-
M. Pirehma á K. Suresh á S. Sivanandam á A.K. Anuar
K. Ramakrishnan á G. Suresh Kumar (&)
Department of Parasitology, Faculty of Medicine,
University of Malaya, 50603 Kuala Lumpur, Malaysia
Ó Springer-Verlag 1999
scesses and is sometimes found as a contaminant in cell
cultures (Casemore 1969). Acanthamoeba keratitis has
been reported from Europe, Australia, India, Taiwan,
Japan, Africa, Israel, and South and North America
(Govinda and Jeanette 1990), but the parasite's true
incidence is unknown. Recently, the ®rst recognized case
of Acanthamoeba keratitis was reported in Malaysia
(Mohamed Kamel and Norazah, 1995).
It is highly likely that many cases remain undetected
for want of a good detection system. Currently, corneal
scrapings from infected eyes are cultured in agar plates
coated with a lawn of Escherichia coli and then subcul-
tured in peptone medium (Visvesvara et al. 1975).
Generally it takes about 5±7 days before the parasite is
detected. Staining of the parasite can facilitate early
detection of Acanthamoeba sp. from corneal scrapings.
We report for the ®rst time a rapid staining method
for the identi®cation of Acanthamoeba using the modi-
®ed Field's staining technique (Field et al. 1963), which
is simpler to perform and yields better results as com-
pared with other recommended staining techniques such
as Giemsa stain, Wright's stain (Martinez 1985), and
trichrome stain (Visvesvara et al. 1975). Modi®ed
Field's stain also proved to be the best stain for identi-
®cation of Acanthamoeba among the brain cells of mice
that had been experimentally infected intranasally with
trophozoites of Acanthamoeba.
Materials and methods
Acanthamoeba castellanii, previously obtained from a corneal
scraping from an infected patient at the Department of Microbi-
ology, National University of Singapore, is the isolate currently
maintained in our laboratory. The parasites were maintained in
10-ml screw-capped tubes containing mycological peptone medium
(Gibco) at room temperature and were subcultured once every 7th
day. The cultures were pooled together in 10-ml screw-capped
tubes and then spun at 2000 rpm using a Kubota 2010 centrifuge.
Thin smears were subsequently made on a clean glass slide from the
sediment containing the parasites. The smears were air-dried and
then stained with modi®ed Field's stain prepared according to the
method of Field et al. (1963), the modi®cation being the use of
792

Fig. 1 Fig. 2

Fig. 3 Fig. 4

Figs. 1±5 Trophozoites of Acanthamoeba spp. stained with modi®ed

Field's stain and examined under oil immersion. Fig. 1 Note the
distinct nucleolus (n) stained dark blue, the nucleus (N) stained pink,
and the cytoplasm stained purplish blue. Fig. 2 Acanthapodia are
demonstrated clearly with this stain (arrow). Fig. 3 Note the dividing
nucleus within the cell (arrow). Fig. 4 Scrapings of Acanthamoeba spp.
from the Escherichia coli lawned agar plates. Note the de®ned outline
of the parasite (arrow) among the lawn of E. coli bacteria (E). Fig. 5
Brain smears from infected laboratory-bred mice. The parasite has
stained purplish blue (arrow) and can easily be distinguished from the
pink-stained brain cells (B). Note that water and food vacuoles are
Fig. 5 visible in the cytoplasm of the parasite

methanolic eosin as the ``B'' stain instead of the original ``B'' stain,
which contains bu€ered eosin.
The glass slides with thin smears were then ®xed with ®ve to ten
drops of a 0.2% solution of methanolic eosin followed by the same
number of drops of Field's stain ``A'' solution. The duration of the
staining was varied from 5 to 30 s. The slides were subsequently air-
dried and the stained slides were examined under oil immersion
(´100).
Experimental infection of three laboratory-bred mice was car-
ried out by intranosal inoculation of Acanthamoeba trophozoites
into the animals for the assessment of pathological changes. The
infected mice were dissected at the end of the month. Thin brain
smears were made on clean glass slides. The slides were air-dried and
stained by the modi®ed Field's staining technique according to the
method described above for identi®cation of the presence of tro-
phozoites or cysts of Acanthamoeba in the brain of infected mice.
Staining was also done by other techniques such as Field's stain
originally used for staining of thick blood ®lms for malaria para-
sites (Field 1941), Wright's stain and Giemsa stain (Field et al.
1963), Ziehl-Neelsen stain (Cheesbrough 1991), and trichrome stain
(Wheatley 1951). The concentration of various stains and the du-
ration of staining were varied to provide the best color and contrast
for each stain.
Results and discussion
The trophozoites stained a distinct purplish blue,
whereas the nucleus and the nucleolus stained pink and
dark blue, respectively, when stained with modi®ed
Field's stain (Fig. 1). The staining also demonstrated the
presence of acanthapodia, the typical feature of the
parasite (Page 1967), and rendered the slender, spine-like
processes more obvious than did the other stains
(Fig. 2). The color contrast appeared to be sharper and
clearer than that provided by other stains such as Gi-
emsa stain, Wright's stain, trichrome stain, Ziehl-Neel-
sen stain, or the original Field's stain and clearly
distinguished the parasite from debris and contami-
nants. Furthermore, the cell membrane under modi®ed
Fields staining was more de®nite and clear. The modi-
®ed Field's stain also clearly showed the division of the
nucleus (Fig. 3), and this will prove especially useful in
elucidating in detail the life-cycle stages of the parasite.
When stained with Giemsa stain, the cell body of the
trophozoites and the nucleus of Acanthamoeba were
light and dark purple in color, respectively, but the
contrast was not as good as that produced by the
modi®ed Field's stain. The nucleolus and other internal
structures were not obvious. When stained with Wright's
stain, trophozoites showed the same contrast as did
Giemsa stain, whereby the nucleus stained dark blue and
the cytoplasm stained light purplish blue. When tri-
chrome stain was used, in most cases the parasites had
the whole cell stained green, with only a few showing the
nucleolus stained red. The whole cell was stained pink
following staining with the Ziehl-Neelsen stain, which
showed no color contrast between the nucleus and the
cytoplasm. The original Field's stain colored the nucleus
and cytoplasm dark and light purple, respectively, but
the internal structures and other details were not ob-
vious. The trophozoites could not be distinguished from
debris or other contaminants.
793
Since agar plates lawned with Escherichia coli were
used to isolate Acanthamoeba spp. from corneal scrap-
ings, we assessed the e€ectiveness of the stain on the
parasite obtained from E. coli-lawned agar plates. A
scraping was taken from the E. coli lawn, smeared on a
glass slide, and subsequently stained by the modi®ed
Field's staining technique. The parasites showed a good
color contrast between the nucleus and the cytoplasm,
which was similar to that seen on parasites obtained
from the axenic culture (Fig. 4). The brain smears ob-
tained from the experimentally infected mice, which
were stained with modi®ed Field's stain, also demon-
strated a color contrast similar to that achieved with the
axenic culture of Acanthamoeba. The parasite stained
purplish blue and the nucleus stained pink, whereas the
brain cells stained pink (Fig. 5).
The results obtained in this study show that staining
of Acanthamoeba trophozoites with modi®ed Field's
stain gives a very good color contrast and takes only
20 s making this technique suitable for both laboratory
and ®eld use. The other staining techniques require the
use of more reagents, are comparatively time-consum-
ing, and involve complex staining procedures. The rec-
ommended stain also proved to be useful in
di€erentiating the parasites from the brain cells of the
infected laboratory-bred mice, thus aiding in the eluci-
dation of the pathogenicity of Acanthamoeba. We
strongly recommend that this rapid staining method be
used by clinicians and medical laboratory workers for
the rapid diagnosis of Acanthamoeba spp.
Acknowledgements The authors are grateful for the axenic cultures
of Acanthamoeba obtained from the Department of Medical Mi-
crobiology, Faculty of Medicine, National University of Singapore.
The study was funded by IRPA grant 06-02-03-0486.
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