EXPERIMENTAL PARASITOLOGY 54, 135-144 (1982)

Tritrichomonas foetus: Ultrastructural Localization of Basic Proteins

and Carbohydrates

MARLENE BENCHIMOL, CEZAR A. ELIAS, AND WANDERLEY DE SOUZA

Institute de Biojkca, Universidade Federal do Rio de Janerio, Cidade Universitdria, Ilha do FundBo,
21.910, Rio de Janeiro, RJ, Brasil

(Accepted for publication 30 May 1980)

BENCHIMOL, M., ELIAS, C. A., AND DE SOUZA, W. 1982. Tritrichomonas foetus: Ul-
trastructural localization of basic proteins and carbohydrates. Experimental Parasitology
54, 135- 144. The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic
acid (E-PTA) techniques were used to localize basic proteins at the ultrastructural level in
the protozoa, Tritrichomonas foetus. The periodic acid-thiosemicarbazide-silver protein-
ate technique was used to localize carbohydrates. With the AS technique reaction product
was seen only in the hydrogenosomes. With the E-PTA technique, reaction product was
seen in the microtubules that form the basal bodies, flagella, pelta, and axostyle, in the costa,
in the hydrogenosomes, and at the region of the recurrent flagellums adhesion to the cell
body. Previous acetylation of the cells under conditions that block most free amino groups
abolished (AS technique) or greatly reduced (E-PTA technique) staining. Carbohydrates
were localized on the cell surface; in the membranes of the hydrogenosomes, Golgi complex,
and cytoplasmic vesicles; and in the glycogen particles. The specificity of the AS and E-PTA
techniques to detect basic proteins is based on observations made with T. foetus as well as
with other cell types.
INDEX DESCRIPTORS: Tritrichomonas foetus; Protozoa, parasitic; Basic proteins; Carbo-
hydrates: Ultrastructure; Cytochemistry

INTRODUCTION pathogenic protozoa. Previously we pre-

Two cytochemical methods associated
with electron microscopy have been used to
localize basic polypeptides: one uses phos-
photungstic acid in alcoholic solutions
(E-PTA) and the other uses ammoniacal
silver (AS).
Few papers have been published on
cytochemical localization of basic polypep-
tides in protozoa. These studies would be of
interest since basic proteins are involved in
the control of cellular differentiation and
regulation of gene function in eucaryotic
cells. Moreover, recent studies show that
they may play a role in the process of pen-
etration of parasitic protozoa inside host
cells (Kilejian 1976; de Souza and Souto-
Padron 1978). We thought, therefore, that it
would be of interest to investigate the lo-
calization of basic proteins in some
sented results related to some trypanoso-
matids (Souto-Padron and de Souza 1978,
1979) and in the sporozoon Toxoplasma
gondii (de Souza and Souto-Padron 1978).
In the present report we describe results
obtained by using the AS and E-PTA meth-
ods to locate basic proteins in Tritricho-
monas foetus, a pathogenic protozoon of
the urogenital tract of cattle. In addition
the specificity of both techniques is dis-
cussed in relation to controls performed
and results obtained in other cell types. In
order to analyze better the E-PTA method,
carbohydrates were also detected using the
periodic acid - thiosemicarbazide - silver
proteinate method (Thiery 1967).
MATERIALS AND METHODS
Tritrichomonas foetus was isolated by Dr. H. Guida
(EMBRAPA, Rio de Janeiro) from the urogenital tract

13.5
0014-4894/82/050135-10$02.0010
Copyright 0 1982 by Academic Press, Inc.
All rights of reproduction in any form reserved
136 BENCHIMOL,ELIAS,
of a bull in the state of Rio de Janeiro, Brasil, and has
been maintained by weekly transfers at room temper-
ature (about 23 C) in a medium with the following
composition (g/liter): liver hydrolysate, 25.0; D(f)-
glucose, 5.0; sodium chloride, 6.5; agar-agar, 0.5; and
10% inactivated and filter-sterilized fresh bovine
serum. Cells were grown in 150 x 15-mm tubes con-
taining 20 ml of medium. The inoculum consisted of 1
ml of 24-hr culture at 36.5 C. About 4.5 x lo6 cells/ml
were inoculated per tube. The cells were cultivated for
24-28 hr at 36.5 C. A layer (about 1.5 cm) of paraffin
oil was added to the culture tubes to keep the diffusion
of oxygen low.
Parasite cells were centrifuged at 15OOg for 10 min
and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate
buffer, pH 7.2, for 2 hr at room temperature. After
fixation they were washed in buffer, postfixed in 1%
0~0, in cacodylate buffer for 2 hr at room tempera-
ture, dehydrated in either acetone or ethanol, and em-
bedded in Epon. Ultrathin sections were obtained with
an LKB Ultratome III ultramicrotome and examined
unstained or after staining with uranyl acetate or lead
citrate or both, in an AEI EM6-B electron microscope.
For detection of carbohydrates, ultrathin sections of
cells fixed in glutaraldehyde and OsOl were collected
on gold grids and treated with periodic acid (l%, 20
min at room temperature) to oxidize adjacent hydroxyl
or o-amino alcohol groups into aldehydes. After rins-
ing in distilled water, aldehydes were condensed with
thiosemicarbazide (1% thiosemicarbazide in 10% ace-
tic acid for 48 hr at room temperature) to yield the
corresponding thiosemicarbazones which are powerful
reducing agents. Thus, after rinsing sequentially with
10, 5, and 1% acetic acid and with distilled water, the
sections were exposed to silver proteinate (1%) for 30
min in the dark at room temperature. Sections were
observed unstained (Thiery 1967). Controls were done
by omission of one of the steps in the method.
For detection of basic proteins, the phosphotungstic
acid method (E-PTA) was employed as previously de-
scribed (Gordon and Bensch 1968; Souto-Padron and
de Souza 1978). After glutaraldehyde fixation the cells
were dehydrated (without postlixation with 0~0~) in
ethanol and incubated for 2 hr at room temperature in
2% PTA in absolute ethanol after which they were
washed in ethanol and embedded in Epon. The am-
moniacal silver (AS) method was used as described by
MacRae and Meetz (1970). After fixation, the cells
were thoroughly washed in distilled water and incu-
bated for 5 min at room temperature in the AS solu-
tion. This solution was prepared just before use by
gradual addition of 10% silver nitrate solution to con-
centrated ammonium hydroxide until a slight turbidity
appeared and persisted. After incubation in this solu-
tion, the cells were washed in distilled water and
placed for 5 min in a 3% formaldehyde solution, where
a brown coloration appeared. After washing in distilled
water they were postfixed in OsO,, dehydrated in
AND DE SOUZA
ethanol, and embedded in Epon. Some glutaraldehyde-
fixed cells were acetylated by reaction with 10% acetic
anhydride in pyridine for 90 min at 37 C before either
the E-PTA or the AS treatment. Control cells were in-
cubated under the same conditions in pyridine only.
RESULTS
The general structure of Tritrichomonas
foetus has been described in detail by
Honigberg et al. (1971). Figure 1 shows
some of the structures such as basal body,
nucleus, costa, hydrogenosome, cytoplas-
mic vacuoles, and recurrent flagellum.
The periodic acid-thiosemicarbazide-
silver proteinate method (Thiery 1967) was
used to detect carbohydrates in thin sec-
tions of Epon-embedded cells. In controls
where either the periodic acid oxidation
step or the thiosemicarbazide incubation
were omitted there was no reaction prod-
uct. Reaction product is seen mainly in
many particles preferentially localized near
the axostyle and which correspond to
glycogen particles (Fig. 2). They have a di-
ameter of 27 nm and are composed of 3-nm
subunits. The glycogen particles react even
with a short incubation time (30 min) in
thiosemicarbazide. With longer incubation
periods (24-48 hr) reaction product is as-
sociated with the plasma membrane which
surrounds the whole body of the parasite as
well as the flagella. The membranes of vesi-
cles, food vacuoles, and the Golgi complex
also show reaction product. The membrane
of the hydrogenosomes shows a weak, but
positive, reaction. The matrix of the hydro-
genosomes shows a weak, but positive,
reaction. The matrix of the hydro-
genosomes and the microtubules which form
the basal bodies, flagella, axostyle, and
pelta do not react. The costa also does not
show any reaction product (Fig. 2).
For detection of basic proteins, the
treatment of the cells with ethanolic phos-
photungstic acid (E-PTA) gives a homoge-
neous electron-dense reaction product with
some structures of T. foetus. After appli-
cation of this technique, the cells appear
well preserved. However, since they are
 Tritrichomonasfoetus: CARBOHYDRATES AND BASIC PROTEINS 137

FIG. 1. General aspect of Tritrichomonas foe&s. Ultrathin section stained with uranyl acetate and
lead citrate. BB, basal body; C, costa; N, nucleus; RF, recurrent flagellum. The small arrow indicates
the lateral arcuate system which surrounds the costa. x 12,000. Bar = 1 pm.

not postfixed with osmium tetroxide there
is no good contrast in the cellular struc-
tures, except in those which react with E-
PTA. We have observed that when thin
sections of E-PTA-treated cells are stained
with lead citrate there is a precipitation of
lead on structures which react with PTA.
Since this precipitation improves the con-
trast, we observed either nonstained or lead-
stained sections.
All hydrogenosomes show an intense
reaction (Fig. 4). In some of them, the
reaction is less intense in the central por-
tion, probably reflecting difficulties in the
penetration of the PTA. The cytoplasmic
ribosomes show a weak reaction. The mi-
crotubules which form the flagella, basal
bodies, pelta, and axostyle show an intense
reaction (Figs. 3, 4). The costa also reacts
strongly showing the presence of a very
dense band intercalated by a less dense one
in the center of which there is a dense line.
The lateral arcuate system which sur-
rounds the costa does not react. The
clockwise curving rootlet filaments as-
sociated with the basal bodies also react
(Figs. 3, 4). Filaments radiating from the
peripheral doublet microtubules of flagellum
number 2 (following the numeration found
in the diagram presented by Honigberg et
al. (1971) are seen. Electron-transparent
areas are seen in the cytoplasm, mainly
near the axostyle, and may represent sites
in which glycogen particles are located
(Fig. 4).
Reaction product is seen at the region of
138 BENCHIMOL, ELIAS, AND DE SOUZA

FIG. 2. Tritrichomonas foetus submitted to the periodic acid-thiosemicarbazide-silver proteinate

technique for detection of carbohydrates. Ultrathin section unstained. Reaction product is seen on the
cell surface, in the membranes of the Golgi complex (G), in the membrane of cytoplasmic vacuoles
(star), in the membrane of hydrogenosomes (H) and in the glycogen particles (Cl). The microtubules
which form the axostyle (arrowhead) and costa (C) did not show reaction. ~37,000. Bar = 0.5 pm.

adherence of the recurrent flagellum to the
cell body. The reaction is restricted to a
layer localized at the inner face of the cell
body plasma membrane, any reaction ap-
pearing in the flagellum (except the
axonemal microtubules). It appears as a very
dense line from which some projections
radiate perpendicularly toward the cell,
with a periodicity of about 16 nm. There is
also a second plate which does not show
reaction product (Fig. 4).
Exposure of the cell suspension to 10%
acetic anhydride in pyridine for 90 min
greatly reduced the E-PTA staining (Fig. 5).
 FIGS. 3, 4. Trirrichomonas foetus submitted to the ethanolic phosphotungstic acid (E-PTA) tech-
nique. Reaction product is seen in the microtubules which form the basal bodies (BB), flagella (F),
axostyle, and pelta. The costa (C) and the hydrogenosomes (H) react strongly. Reaction product was
also seen in filaments radiating from the peripheral doublet microtubules of flagellum number 2 (curved
arrow in Fig. 3) and the cytoplasmic portion of the recurrent flagellum (RF)-cell body adhesion zone
(curved arrow in Fig. 4). Ultrathin sections stained only with lead citrate (2 min). Fig. 3, X20,000; Fig.
4, ~15,000. Bar = 1 pm.
139
140 BENCHIMOL, ELIAS,
Pyridine alone did not suppress the stain-
ing. After application of the postformalin
ammoniacal silver technique, the cell sus-
pension has a brown to black color which
can also be seen by light microscopy,
mainly when the amplitude contrast method
associated with Nomarski optics is used.
The reaction product is seen mainly in
spherical structures with a mean diameter
of 0.7 ,um. Treatment of the cells with the
ammoniacal silver and formalin solutions
interferes with the preservation of cell
structure. Some empty cytoplasmic areas
were occasionally seen. In the electron mi-
croscope the reaction product appears in
the form of electron-opaque particles with
diameters of about 20 nm. They are found
either isolated or in contact with each
other. The silver particles are found mainly
in the hydrogenosomes (Fig. 6). Not all hy-
drogenosomes, however, showed silver
particles. Other large dense membrane-
bounded structures also do not react.
After exposure of T. foetus cells to 10%
acetic anhydride in pyridine for 90 min be-
fore AS treatment, no silver particles are
seen in the hydrogenosomes. Pyridine
alone does not suppress the reaction.
DISCUSSION
Protozoa of the family Trichomonadidae,
which includes agents of important human
and veterinary diseases, have a large
number of filamentous and tubular struc-
tures which form the mastigont, a group of
structures which has been studied by elec-
tron microscopy by using either thin sec-
tions or negative staining technique. In ad-
dition they have a spherical structure which
has been designated as paracostal granules,
paraxostylar granules, chromatic granules,
or hydrogenosomes (Honigberg et al. 1971;
Nielsen and Diemer 1976; Lindmark and
Muller 1973).
The results obtained by us confirm previ-
ous morphological studies on Tritricho-
monas foetus (Inoki et al. 1961; Simpson
and White 1964; Honigberg et al. 1971). We
AND DE SOUZA
will discuss here only aspects related to the
structures which result in reaction product
when the periodic acid - thiosemicarbazide-
silver proteinate, the ammoniacal silver or
the ethanolic phosphotungstic acid tech-
niques are used.
The postformalin ammoniacal silver
technique was fust introduced in light mi-
croscopy by Black and Ansley (1964), who
used it for investigations related to the
biological functions of histones (Black and
Ansley 1964, 1966). Experimental studies
using isolated histones suggest that when a
yellow color appears after interaction of the
histone with AS a lysin-rich histone is indi-
cated, whereas the black staining is as-
sociated with arginine-rich histones (Black
and Ansley 1966). However, the authors
pointed out that differences in the AS
staining may reflect differences in reactivity
of amino and guanidino groups rather than
differences in the relative content of lysine
and arginine residues in the histone.
MacRae and Meetz (1970) introduced the
AS technique to electron microscopy ap-
plying it to localize histones in the eryth-
ropoietic cells of the chick. They show
that stem cells and early erythroblasts ex-
hibit little reaction while more differ-
entiated cells such as polychromatophilic
erythrocytes and reticulocytes exhibit more
silver granules. More recently this tech-
nique has been applied to localize basic
proteins in erythroid precursors from pa-
tients with anemia (Kass and Gray 1975), in
polymorphonuclear leukocytes (MacRae
and Spitznagel 1975; Brown and Wood
1978), in eosinophils (Pimenta et al. 1979),
and in two developmental stages of Try-
panosoma cruzi (Souto-Padron and de
Souza 1978). In all these studies the reac-
tion product appears as electron-dense par-
ticles. In T. foetus, silver particles are ob-
served only in hydrogenosomes. A few
granules are seen in the cytoplasm. How-
ever, we could not associate them with any
particular structure of the cell. T. foetus
submitted to the AS method after acetyla-
 FIG. 5. Tritrichomonas foetus submitted to the ethanolic phosphotungstic acid technique after previ-
ous acetylation under conditions which block most free amino groups. A weak reaction is seen in the
microtubules which form the axostyle (M). No reaction product is seen in the hydrogenosomes (H).
~30,000. Bar = 0.5 pm.
FIG. 6. Tritrichomonas foetus submitted to the postformal in ammoniacal silver method. Silver
particles are seen only in the hydrogenosomes (H). ~22,000. Bar = 0.5 pm.
141
142 BENCHIMOL, ELIAS,
tion did not show silver particles, thus con-
firming its specificity.
Phosphotungstic acid is an anionic stain
introduced to electron microscopy by Hall
et al. (1945) and widely used in negative
staining (Haschmeyer and Myers 1972). In
cytochemistry PTA has been used to detect
polysaccharides (Pease 1970) and proteins.
Bloom and Aghajanian (1968) suggest that
E-PTA binds principally to proteins rich in
lysine, arginine, and histidine which are lo-
calized at synapses. Sheridan and Barrnett
(1969) use it to locate nuclear proteins, pre-
sumably histones, in the meiotic chromo-
somes of the prophase nuclei of lily micro-
sporocytes. Reaction product is observed in
the nuclear chromatin, in the nucleoli, in
material found in the nuclear membrane
pore, in the annulate lamellae, and in the
lateral element of the synaptinemal com-
plex. Silverman and Glick (1969), on the
basis of histochemical experiments, suggest
that PTA selectively stains proteins. Pease
(1966) has used PTA to detect polysac-
charides associated with the exterior sur-
face (glycocalix) of epithelial cells. How-
ever, the results we have recently obtained
show that E-PTA, under the conditions de-
scribed under Materials and Methods, does
not detect carbohydrates. In Trypanosoma
cruzi, E-PTA reaction product is seen in the
kinetoplast, in the nucleus, and in some
vesicles; all of them are structures which do
not show any reaction product when the
PA-TSC-SP technique is used (Souto-
Padron and de Souza 1978). A similar result
is obtained when the same technique is
applied to other trypanosomatids where
reaction product is seen also at the region of
adhesion between the cell body and the
flagellum (Souto-Padron and de Souza
1979). In addition, reaction product is as-
sociated with the conoid, rhoptries, and
micronemes of the sporozoa Toxoplasma
go&ii, structures which do not have car-
bohydrates (de Souza and Souto-Padron
1978). Biochemical studies suggest that the
rhoptries of the sporozoa Plasmodium
AND DE SOUZA
lophurae contain polypeptides rich in his-
tidine which may be involved in the process
of penetration of this protozoa inside a host
cell (Kilejian 1976). The results obtained
with T. foetus also support the idea that
E-PTA does not detect polysaccharides. In
addition, previous acetylation of the cells
under conditions which block most free
amino groups (Pearse 1968) greatly reduced
the E-PTA staining. When we used the
PA-TSC-SP technique, reaction product
was associated with structures such as the
plasma membrane, the membrane of some
intracellular structures such as the Golgi
complex, pinocytotic vesicles, hydro-
genosomes, and mainly with glycogen par-
ticles. These structures do not react with
E-PTA. E-PTA reaction product is seen to
be associated with the hydrogenosomes, in
the costa, in the microtubules which form the
basal bodies, flagella, axostyle, and pelta,
in the clockwise curving rootlet filaments
associated with the basal bodies, in the fi-
laments which radiate from the peripheral
microtubules of flagellum number 2, and the
region of adherence of the recurrent flagel-
lum to the cell body (undulate membrane).
All the microtubules show the same intense
reaction, a result which has not been ob-
tained in other cells. Gordon and Bensch
(1968) found a strong staining in the
peripheral doublet microtubules and a less
intense staining in the central pair of mi-
crotubules of the flagellum of the guinea pig
sperm. In trypanosomatids less intense or
no staining is observed in the central mi-
crotubules (Souto-Padron and de Souza
1978, 1979)
The zone of adherence of the recurrent
flagellum to the cell body appears to be
specialized as previously observed in try-
panosomatids (de Souza et al. 1978, 1979).
A plate which shows an intense PTA stain-
ing is seen below the inner face of the cell
body plasma membrane.
The hydrogenosome is an organelle
which contains enzymes which participate
in the metabolism of pyruvate formed in
 Tritrichomonas foetus: CARBOHYDRATES
glycolysis (Lindmark and Muller 1973;
Muller 1975). This organelle appears to be
rich in basic proteins since it gives intense
reaction with either the E-PTA or the AS
technique. Its membrane, however, shows
reaction product when the PA-TSC-SP
method, which detects carbohydrates, is
used.
The absence of reaction product, indica-
tive of the presence of carbohydrates, in
the costa of cells submitted to the PA-
TSC-SP method was not expected in
view of the results recently described by
Sledge et al. (1978). Analysis of purified
costa of Tritrichomonas foetus is shown to
be composed of about 95% carbohydrate
(mainly glucose) and 5% protein. Our re-
sults agree with those recently reported for
some trichomonad species (Amos et al.
1979). Using a histochemical method these
authors could not detect PAS-stained mate-
rial in the costa. Biochemical analysis
(SDS-gel electrophoresis) showed the
presence of three main classes of proteins.
As suggested by Amos et al. (1979) it is
possible that the preparations of Sledge et
al. (1978) contained glycogen which is
abundant in the surrounding cytoplasm.
Our results suggest that the E-PTA and
AS techniques detect different proteins.
Reaction product is seen with both tech-
niques only in the hydrogenosomes, sug-
gesting that the AS technique is more spe-
cific. Results recently obtained with other
cell types support this assertion. Although
an intense staining is seen in the rhoptries
and micronemes of E-PTA-treated T. gon-
dii cells, no silver particles are seen in these
structures (de Souza and Souto-Padr6n
1978; de Souza, unpublished observations).
In eosinophils and mast cells similar results
have been obtained (Pimenta et al., 1979).
However, we cannot at the present time
know which basic amino acids have more
affinity to either E-PTA or AS. Studies are
being carried out in different cell types as
well as in isolated polypeptides with a de-
fined composition of amino acids in order to
AND BASIC PROTEINS 143
clarify the specificity of these two tech-
niques .
ACKNOWLEDGMENTS
We thank Mr. A. L. de Oliveira for help with pho-
tography and Mrs. Zelia de Freitas for secretarial as-
sistance. The technical assistance of Miss Silvia R. V.
Sales was very much appreciated. This work has been
supported by Brasils Conselho National de Desen-
volvimento Cientifico e Tecnoldgico (CNPq), by Con-
selho de Ensino e Pesquisa da UFRJ, and by Finan-
ciadora de Estudos e Projetos (FINEP).
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