Vol. 48, No. 5
Printed in U.S.A.
THE AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Copyright © 1967 by The Williams & Wilkins Co.
PROTOZOAN CHARACTERISTICS OF BLASTOCYSTS
HOM1NJS
CHARLES H. Z1ERDT, PH.D., WILLADENE S. RUDE, AND BRIAN S. BULL, M.D.
Department of Clinical Pathology, Clinical Center, National Institvtes of Health,
U. S. Department of Health, Education and Welfare, Bethesda, Maryland £001b
Blastocyslis hominis, an inhabitant of the
lower intestinal tract of man, was described
and named by Brumpt in 1912.6 Since that
time it has attracted the attention of several investigators, notably Prowazek,12 who
believed it to be the cyst form of a flagellate,
and Alexeieff,1'2 who considered it to be a
vegetable organism allied to Blastomyces.
More recently, after reexamining the cultural characteristics of the organism,
Lynch, 8 ' 9 Aragao,3 and Knowles and Das
Gupta 7 concluded that it was a yeast of the
genus Schizosaccharomyces. Because so
many investigators studying the same organism have come to such strikingly different conclusions, it seems reasonable to agree
with Bach and Keifer" that the true nature
of B. hominis is still an enigma. The present
study was undertaken in the hope that a reexamination of the morphology of this
organism at the ultrastructural level, combined with further study of its cultural characteristics, might provide more definitive
information and aid in the problem of classification.
MATERIAL AND METHODS
Source of B. hominis. Stools Avhich were
otherwise normal but contained large numbers of Blastocysts were selected during
routine examination for ova and parasites.
Media. Small portions of stool were emulsified in tubes and layered on Petri plates.
They were then incubated aerobically,
anaerobically, and in an atmosphere of 5%
CO.. Boeck and Drbohlav (B-D), s an inspissated egg medium, and Nelson and
Jones (N-J)11 which is composed of Hank's
solution with added horse serum, sodium
bicarbonate, and rice powder were employed. The solid B-D medium was covered
Received December 27, 1966.
Dr. Bull's present address is Department of
Hematology, Postgraduate Medical School of
London, Ducane Road, London, W.12, England.
with a liquid overlay of Locke's solution
with 10 % horse serum and rice powder.
Cultural methods. Cultures were transferred by adding inoculum to fresh medium
in a ratio of 1:10. Old or deteriorating cultures were renewed by inoculating fresh
medium with aliquots from several of the
deteriorating cultures.
In addition to the two media described
above, attempts were also made to grow
Blastocystis on media commonly employed
for the selection of fungi. Sabouraud's
agar, Sabouraud's agar with chloramphenicol,
Sabouraud's agar with chloramphenicol and
cycloheximide, and brain heart infusion agar
with 5 % sheep blood and chloramphenicol
were inoculated with 5 strains of Blastocystis
and incubated aerobically and anaerobically
at room temperature and at 37 C. Cultural
attempts were also made on high serum media commonly used for Mycoplasma cultivation, with isolation technics, so that bacterial
and Blastocystis colonies might grow side by
side.
For short-term microscopic observation of
living organisms and for photography,
slide cultures were used. These consisted of
a drop of culture on a 1- by 3-in. slide covered
with a 22-mm. glass coverslip and ringed
with immersion oil. The preparation was
incubated at 37 C. by means of a stage
heater. The strain selected for further study
by both light and electron microscopy was
an isolation from patient M. H. This strain
has been successfully maintained in culture
for more than 9 months.
For electron microscopy, culture samples
were concentrated by centrifligation at f>0 X
g for 10 min. The sediment was fixed for 10
sec. in 4 % osmium, transferred to 2% glutaraldehyde for 30 min., and then placed in
a sucrose cacodylate buffer overnight. The
specimens were postfixed in 2% osmium,
dehydrated, and embedded in Epon. Sections were cut on an LKB ultramicrotome
495
496
Z1EEDT ET AL.
with glass knives. They were examined in an
RCA EMU 3G microscope at 50 kv.
RESULTS AND DISCUSSION
There was no growth during a 1-month
period of observation of any of the five
strains of B. hominis on the fungal or on
Mycoplasma media. On the B-D and N-J
media, growth was optimal at 37 C. Growth
was generally unsuccessful at 30 C. and none
of the strains could be grown at room temperature. In feces, B. hominis died rapidly
at4C.
B-D and N-J media were originally devised to permit in vitro cultivation of Entamoeba histolytica. Both media permit the
long-term in vilro cultivation of B. hominis,
but only if a mixed bacterial flora is also
present. Growth was much faster on B-D
medium, with maximal growth often occurring within 3 days. N-J medium required
about 10 to 15 days for maximal growth.
The dependence of B. hominis on the presence of the mixed bacterial flora probably
accounts for unpredictable changes in its
growth rate. A change to fresh medium or to
different conditions of aerobiosis would often
successfully rejuvenate a culture that was
growing poorly. The bacterial flora of the
initial culture was that of the source stool.
This evolved slowly into a triad or tetrad
culture consisting of a lactose-fermenting
Gram-negative bacillus, usually Escherichia
coli, a Bacteroides species, a Streptococcus
species, and a Micrococcus species. The only
strict anaerobe was the Bacteroides species.
B. hominis did not reproduce well at a pH
of less than 6.0. Growth was most rapid between pH 7.0 and 8.0. In cultures in which lactose-fermenting Gram-negative bacteria had
lowered the pH it was necessary to add 7 %
NaHCOa, with phenol red used as an indicator to restore neutrality. Phenol red incorporated in the medium was not injurious
to B. hominis.
B. hominis in culture is extremely polymorphous. The majority of the organisms
could, however, be divided into three large
groups.
1. Vacuolated form (Figs. 1 to 5). These
predominated in fecal specimens and recent
isolates. The organisms were about 8 to 10 n
Vol. 48
in diameter. Nucleus and cytoplasm were
compressed at the periphery of the cell by a
large central vacuole, the contents of which
did not stain for lipid, starch, cellulose, or
glycogen. Lipid granules formed in older
cells (Fig. 5). This form reproduced rapidly
by binary fission, division occurring as often
as every 4 hr. in young cultures (Fig. 4).
A slime capsule was present around some
B. hominis of the vacuolated type. In some
cultures this capsule was very prominent,
having a thickness almost one half that of
the cell diameter (Figs. 1, 3, and 4). The
capsule formed a clear halo around the cell
that could not be altered by changing the
level of focus. The outer limits of this capsule could be distinguished by the bacteria
which outlined it, much as carbon particles
outline the capsule of Pneumococcus.
2. Ameba-like form. These organisms occurred in stool specimens along with the
vacuolated forms but were not as numerous
as the latter. As the cultures aged, however,
and growth of the organisms slowed, the
amebiform organisms became increasingly
prominent (Figs. 6 to 8). Cultures maintained in N-J medium for more than 1 month consisted almost exclusively of small, irregularly
shaped, amebiform organisms that often
contained one or two lipid inclusions. Other
amebiform organisms maintained under
more optimal cultural conditions were
larger, sometimes containing indistinct
granular inclusions. The nucleus, when
visible, showed peripheral clumping of the
nuclear chromatin (Figs. 6 and 7). Some of
the larger amebiform cells appeared to reproduce by developing one to three smaller
cells at different points on the cytoplasmic
membrane (Fig. S). These separated after a
period of hours and were indistinguishable
from smaller amebiform organisms present
elsewhere in the culture. Active extension
and retraction of pseudopods were seen
occasionally, but this activity did not result
in cell progression.
8. Granule-filled form. This large spherical
form was most frequently found in old cultures maintained on B-D medium. Often it
was the only form present. The cells measured 10 to 60 M in diameter and were filled
with smaller granules (Figs. 9 to 11). The
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FIG. 1 (upper, left). Typical Blastocysiis hominis cells of the vacuolated form, one with a distinct slime
capsule.
FIG. 2 (upper, center). Vacuolated form cell with distinct peripheral cytoplasm containing organelles
and lipid granules.
FIG. 3 (upper, right). Vacuolated form showing thick capsule
FIG. 4 (second row, left). Vacuolated form. Dividing cell
FIG. 5 (second row, center). Vacuolated form. Lipid granules stained with Sudan III
FIG. 6 (second row, right). Ameba form showing nucleus
FIG. 7 (third row, left). Ameba form showing nucleus
FIG. 8 (third row, center). Ameba form reproducing
FIG. 9 (third row, right). Granule-filled form. Early development
FIG. 10 (lower, left). Granule-filled form. Intermediate stage of development
FIG. 11 (lower, center). Granule-filled form. Mature
FIG. 12 (lower, right). Granule-filled form. Internal granules developed into daughter cells of ameba
form, leaving ruptured parent cell. FIGS. 1 to 12, phase light. X 480.
497
49S
ZIERDT ET AL.
Vol. 48
FIG. 13 (upper left). Various stages in the formation of micropinocytotic vesicles may be seen around
the periphery of the cell. The central vacuole contains amorphous flocculent material. X 11,000.
FIG. 14 (upper right). The Golgi complex of vesicles formed from smooth-surfaced membranes arranged in a pile of flattened sacs is closely apposed to the nulcear membrane. The nuclear
membrane is loose, except for several regions of thickening corresponding to the nuclear pores.
These thickenings are more clearly demonstrated in Figure 11, bottom. This is an oblique section and
the central vacuole is not visible. X 11,000.
FIG. 15 (lower left). The cytoplasm contains a lipid inclusion, two mitochondria, and a large number
of both rough- and smooth-surfaced vesicles. The slime layer external to the plasma membrane shows
characteristic thickening at the site of incipient micropinocytotic vesicle formation. X 12,000.
FIG. 16 (lower right). There is a prominent perinuclear space, except at the site of nuclear pores. A
Golgi complex occupies the thickening in the perinuclear cytoplasmic shell. The central vacuole contains large numbers of small vesicles. X 11,000.
Key to symbols, FIGS. 13 to 17: g, Golgi apparatus; I, lipid inclusion; m, mitochondria; mpv, micropinocytotic vesicles; n, nucleolus; A'", nucleus; p, nuclear pores; rer, rough endoplasmic reticulum; s,
slime layer; ser, smooth endoplasmic reticulum.
Nov. 1967
BLASTOCYSTS HOMINIS
499
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Fia. 17. A typical mature vacuolated form of B. hominis. The plasma membrane is coated with a thin
layer of amorphous slime. The cytoplasm contains mitochondria and endoplasmic reticulum of both the
rough and smooth types, as well as free polyribosomes. There is a prominent perinuclear space and a
nucleolus is demonstrable. X 20,000.
granules were of two types. Those located
centrally after rupture of the cyst developed
into the typical B. hominis forms described
above (Fig. 12). The granules located in the
periphery of the cell did not develop further
and were interpreted as having a metabolic
function.
There was suggestive evidence that in
addition to the three modes of reproduction
described above, a reproductive form that
could not be visually differentiated from
bacteria occurred occasionally. During continuous observation of slide culture preparations, typical B. hominis appeared in places
where only bacteria had been identifiable at
the beginning of the observation period.
Cells with ingested bacteria were seen in
fresh stool preparations and in cultures.
Needle-shaped crystals were present in some
cultures, both within B. hominis and lying
free in the medium. The crystals contained
within organisms were occasionally large
500
Vol. 48
ZIERDT ET AL.
enough to distend the cell and appeared to
have been phagocytized. The process of
crystal phagocytosis was never actually observed, however, and the possibility remains
that the B. hominis were excreting the crystals.
Smears from fresh stool specimens were
successfully fixed in Schaudinn's solution
and stained with Mallory's phosphotungstic
acid hematoxylin, Giemsa, and periodic acidSchiff reagents. Acidophilic bodies were
evident in the nucleus and cytoplasm. The
nucleus of the ameba-like form was well
demonstrated
and
showed
peripheral
clumping of chromatin. PAS stains were
uniformly negative.
Morphology {Ultrastructure)
Although the ultrastructure of B. hominis
has been previously reported,10 our findings
(Figs. 13 to 17) differed in that there were
clearly defined structures identifiable as mitochondria, a Golgi complex, and endoplasmic reticulum.
The cytoplasm, which formed a hollow
sphere, was delimited on the outside by a
plasma membrane and a slime layer. This
amorphous layer of slime was thicker in
regions where micropinocytotic vesicles were
forming and where it lined such vesicles
(Fig. 13). The cytoplasmic shell was separated from the central vacuole by a convoluted membrane. Mitochondria were present
in the cytoplasm, as was smooth and rough
endoplasmic reticulum. The ribosomes lining
the rough endoplasmic reticulum were unusually large and prominent. Similar polyribosomes covered the loose nuclear membrane. The nucleus contained a nucleolus
and was surrounded by a perinuclear space
except where the nuclear membrane was
closely apposed at the site of a nuclear pore.
A Golgi complex was present near the nucleus (Figs. 14 and 16).
The contents of the central vacuole varied
considerably from organism to organism.
Sometimes flocculent, amorphous precipitates were present; sometimes very little
electron-dense material of any sort was preserved by fixation. The most frequent
finding, particularly in the larger and presumably older forms, was a large number of
tiny spherical bodies (Figs. 16 and 17).
TABLE 1
COMPARATIVE CHARACTERISTICS OP
BLASTOCYSTS HOMINIS AND OF
YEAST
of a Typical
Characteristics of B. hominis Characteristics
Yeast
No cell wall, organism
surrounded only by a
thin membrane capable of forming micropinocytotic vesicles
Growth in vitro only in
mixed culture with
bacteria
No growth on fungal
media
Preference for neutral or
slightly alkaline pH
No evidence of budding
or mycelial formation
in vivo or in vitro
Reproduction by binary
fission or sporulation
Pseudopod extension
and retraction
Ingestion of bacteria
and other particulate
matter
Optimal temperature
for growth 37 C. No
growth at room temperature
Killed at 4 C, and in
mildly hypotonic or
hypertonic environment
Cell wall present, usually heavy, nutrients
transferred by diffusion
Growth in vitro without bacteria
Growth on fungal media
Preference for acid pH
and resistant to
quite a low pH
Budding usual in vivo
and production of
mycelia common in
vitro
Reproduction by budding, fragmentation,
or sporulation
No pseudopod formation
No ingestion of particulate matter
Optimal temperature
usually less than 37
C. Good growth at
room tempei-ature
Usually resists these
conditions
CONCLUSIONS
The reason that previous investigators
failed to reach a consensus on B. hominis is
understandable. The organism is capable of
assuming a bewildering variety of forms. I t
reproduces in at least two different ways and
has possibly as many as four modes of reproduction. I t certainly cannot be neatly
and unequivocally classified as either a
yeast or a protozoan, but the latter classification seems to fit the present data best.
Very few of its characteristics are those that
would be expected of a yeast, as indicated
in Table 1.
Nov. 1967
BLASTOCYSTS HOMINIS
SUMMARY
Blastocyslis hominis, an organism long regarded as an intestinal yeast, was studied by
means of light and electron microscopy; its
cultural characteristics were also investigated.
B. hominis in culture occurs in three major
forms: 1. Vacuolated. This cell, S to 10 ^ in
diameter, has a large central vacuole and
multiplies by binary fission. 2. Ameba-like
form. A polymorphous cell with a large size
range. I t manifests pseudopodal and phagocytic activity and multiplies by asymmetrical fission. 3. Granule-filled form. A spherical
cell found predominantly in older cultures
containing reproductive granules. This is
probably a cyst form of B. hominis.
B. hominis grows only at 37 C. in the
presence of a mixed bacterial flora, fails to
grow on fungal media, and shows a preference for neutral or slightly alkaline pH. In
culture it manifests pseudopod extension
and retraction and phagocytic activity. It
is rapidly destroyed by environmental
changes in tonicity, shows no evidence of
mycelial formation, and fails to stain with
periodic acid-Schiff reagent. The ultrastructure shows absence of a cell wall, presence
of a well-defined nucleus and nucleolus,
mitochondria, ribosomes, Golgi apparatus,
and smooth and rough endoplasmic reticulum. The majority of these characteristics
seem to ally the organism more with the
protozoa than with the fungi.
501
ology Department, Medical College of Virginia,
who observed the unyeastlike behavior of B.
hominis and stimulated us to undertake research
into its true nature. The valuable technical assistance of Darlcne C. Brindley, who prepared the
sections for electron microscopy, is also acknowledged.
REFERENCES
1. AlcxcielT, A.: Sur la nature des formations
dites "Kystcs de Trichomonas inteslinalis."
C. R. Soc. Biol., 71: 296-298, 1911.
2. Alexeieff, A.: Sur la cycle dvolutif et les affinites do Blastocyslis enlerocola. Arch. Zool.
Exp., 56: 113,_1917.
3. Aragao, IT.: Etudes sur les Blastocyslis.
Mem. Inst. Oswaldo Cruz XV, Fas. /: 143151, 1922.
4. Bach, F. W., and Keifer, K. H.: Untersuchungen iiber Blastocyslis. Cent. Bkt. 1,
Abt. Orig. LXXXIX, No. 4-5, 72-98, 1922.
5. Boeck, W. C, and Drbohlav, J.: The cultivation of Endamoeba histolytica. Am. J.
Hyg., 5: 271-407, 1925.
6. Brumpt, E.: Blastocyslis hominis n. sp. et
formes voisiiies- Bull. Soc. Path. Exot., 5:
725-730, 1912.
7. Knowles, R., and Das Gupta, B. M.: On the
Nature of Blastocyslis hominis. Indian J.
M. Res., IS: 31-38, 1924.
8. Lynch, K. M.: Blastocyslis Species in culture. Am. J. Trop. Med., 8: 215-221, 1922.
9. Lynch, K. M.: Cultivation of Blastocysts and
determination of species. Am. J. Trop.
Med., 2: 539-549, 1922.
10. Magaudda-Borzi, L., and Pennisi, L-: The
morphology of Blastocyslis hominis under
the electron microscope. Atti Soc. Peloritana Sci. Fis., Matemat. Natur., Messina.
(Suppl.), 7: 575-582, 1961.
11. Nelson, E. C , and Jones, M. M.: Cultivation
of Entamoeba histolytica in carbon dioxidebicarbonate buffer system media. Am. J.
Trop. Med. Hyg., 13: 667-673, 1964.
12.
Prowazek,
S.: Untersuchungen iiber oblige
Acknowledgments. We wish to acknowledge the
parasitische Flagellaten. Arb. K. Gesundcontribution of Muriel M. Jones, of the Bacteriheitsamte, XXI: 1-41, 1904.