UNIVERSITY OF CALIFORNIA PUBLICATIONS
~ j ' " Vin
ZOOLOGY
Vol. 20, No. 15, pp. 365-371 February 1, 1923
A COMPARISON
OF THE CYSTS OF ENDAMOEBA COLI AND
COUNCILMANIA LAFLEURI IN CONGO RED
BY
NATHANIEL BERCOV1TZ, M.D.
: i 'Tu 'to,
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UNIVERSITY OF CALIFORNIA PRESS
BERKELEY, CALIFORNIA
1923
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UNIVERSITY OF CALIFORNIA PUBLICATIONS
IN
ZOOLOGY
Vol. 20, No. 15, pp. 365-371 February 1, 1923
A COMPARISON
OF THE CYSTS OF ENDAMOEBA COLI AND
COUNCILMANIA LAFLEURI IN CONGO RED
BY
NATHANIEL BERCOVITZ, M.D.
Early in January, 1922, I conducted a series of experiments in
the protozoological laboratory at the University of California to
determine the permeability of the cysts of intestinal protozoan para-
sites by a number of anilin stains. During the course of the experi-
ments it was noticed that there was no staining of the cysts by aqueous
solutions of the stains until one day a number of cysts of Council-
mania lafleuri Kofoid and Swezy were seen to appear red after being
exposed to Congo red. The next specimen contained cysts of Enda-
moeba coli, and exposure to Congo red showed no reaction in the
cysts which appeared clear in the,red background. A third specimen
contained cysts of Councilmania and these also took the stain when
exposed to Congo red. This seemed to be unusual, so it was decided
to make an intensive study of this phenomenon. At first it was
thought that this might be a quick way to differentiate between these
two types of amoeba, but as more cases were studied it was found
that the phenomenon was not constant. There were instances where
the Congo red had no effect on the cysts of Councilmania, and there
were instances where the Congo red stained cysts of Endamoeba coli
deeply.
In order to arrive at some more accurate conclusion a series of
fifty cases was studied. Fresh solutions of Congo red, 1 per cent in
normal salt, were used throughout. The effect of this stain on every
type of cyst of human intestinal parasites available was studied, but
in the cysts of Councilmania and Endamoeba coli only was there any
366 University of California Publications in Zoology [Vol. 20
staining. Some twenty-five of the more common anilin stains in
1 per cent normal salt solution were also used on these cysts, which
included those of Endamoeba dysenteriae, large and small races;
Endamoeba nana, small race; Iodamoeba buetschlii; Chilomastix
davainei, and Ciardia enterica, in addition to Councilmania and Enda-
moeba coli. In no cyst, and with no stain, was there any staining
of live cysts except with the Congo red on Councilmania and Enda-
moeba coli and, as already stated, this stain acted differentially on
the whole between these two.
There were a number of variables to be considered. First of all
there Avas the matter of determining whether the cysts were alive or
dead, as the dead cysts stained intensively with any of the dyes used.
It was soon found that in Congo red dead cysts stained a deep orange
red with nuclei showing very dark; it also becamp evident that in
live cysts the dye did not penetrate the cyst wall, therefore did not
stain either the cytoplasm or the nuclei.
This fact was therefore established early, that in live cysts the
dye did not stain the cytoplasm or nuclei of the cyst, but only the
cyst wall. It was- also found that there were degrees of intensity of
the staining of the cyst wall, and for this reason it was decided to
designate three degrees of staining as follows: the intense shade
as '' stained,'' the light shade '' pinkish,'' and where no staining was
apparent as ''unstained." It was found in many of the cysts which
were ordinarily regarded as ''unstained," that in a powerful light a
faint pinkish shade might be seen. This might have been due, how-
ever, to the film of staining fluid around the cyst.
The other variables were the amounts of material used and the
dye used. Obviously both of these could not be kept absolutely con-
stant, but continuous practice reduced to a minimum error due to
these factors.
After a few trials it was found that if a dye was going to act at
all on a cyst it acted immediately, and that the cyst did not stain
more intensively if exposed for a long time, even as long as twTo days
in the dye. So there was no error due to the time element. Variations
in room temperature did not make any difference in the results.
It is obvious that in a study like this the personal element enters
in a large way, since what would be considered stained by one might
be considered pinkish by another, and even with the same person,
there would be variations of judgment as to the intensity of the color,
especially with different densities of the surrounding field of material
1923] Bercovitz: Endamoeba coli and Councilmania lafleuri -367
and stain. For this reason this study is not absolute, but the findings
are recorded as a general result and are liable to the variations of
personal and other elements.
Early in the study it was noticed that in stools in which Council-
mania were found the cysts seemed to be more numerous than in those
containing Endamoeba coli. This feature was also studied in connec-
tion with the action of stains.
The results of the findings in the first fifty cases are recorded in
tables 1, 2, and 3. In general it was found that the cysts of Council-
mania, when present are abundant in the stool, while on the other
hand those of Endamoeba coli are usually very scarce, at times not
more than one or two cysts being found on a slide. It was also found
that the tendency of the cyst wall of Councilmania to take the Congo
red was much more marked than in the case of Endamoeba coli.
Furthermore, the cysts of Councilmania seem to be ovoidal or
ellipsoidal oftener than the cysts of Endamoeba coli.
In the first series of 50 cases there were 17 cases, or 34 per cent,
with E. coli infection; 29 cases, or 58 per cent, with Councilmania
infection, and 4 cases, or 8 per cent, with infection of both E. coli and
Councilmania.
TABLE 1
Series I. Relative Abundance of Cysts of E. coli and Councilmania in Slides
Few
Moderate
Many
Endamoeba coli
64.7%
17.2% •
23.6%
10.4%
11.8%
72.4%
Councilmania
This table indicates that Councilmania cysts are more abundant
in stools than are the cysts of Endamoeba coli.
TABLE 2
Series I. Reaction of Cysts of E. coli and Councilmania to Congo Bed
Type of Cyst
No. of
Cases
Unstained
Pinkish
Stained
Pink and
Unstained in
same
Specimen
Total No.
Showing
Color
No.
%
No.
%
No.
%
No.
%
No.
%
E. coli
17
29
9
6
53.1
20.7
6
3
35.4
10.3
2
9
11.8
31.0
0
11
0
37.9
8
23
46.9
79.3
Councilmania..
368 University of California Publications in Zoology [Vol. 20
This table shows that the cyst wall of Councilmania takes the
Congo red stain more readily than that of E. coli.
In order to place these conclusions on a more accurate basis, a
second series of fifty stools was examined in which the following
factors were noted: cysts dead or alive in stool; stool formed or
liquid; actual count of cysts on slides to determine more accurately
the proportion staining, as well as abundance of cysts in these cases.
Congo red itself being a very delicate indicator, it was noted
whether the stool became dark in the Congo red, and this was used
as the test for acidity.
Where cysts were abundant one hundred were counted, and the
results with the stain noted. But in no case were more than two slides
of each specimen examined, as in many cases it would have required
examination of ten to fifteen slides to find one hundred cysts. Where
one hundred cysts were found on one slide it was considered that
the cysts were abundant. Where less than one hundred cysts were
found on one slide, but more than fifty cysts on two slides, the cysts
were considered to be moderately abundant. Where there were less
than fifty cysts on two slides the number was considered few, and
where less than ten cysts on two slides, very few.
The results of the second series are recorded in tables 3 and 4.
In this series 22 cases, or 44 per cent, were infected with Endamoeba
coli, and 28 cases, or 56 per cent, were infected with Councilmania.
No mixed infections were included in this series.
TABLE 3
Series II. Eelative Abundance of Cysts of E. coli and Councilmania Cysts
Few
Many
(including very
few)
(including moderately
abundant)
E. coli
86.4%
21.5%
13.6%
78.5%
Councilmania
The contrast between abundance of cysts in infections by Enda-
moeba coli as compared with infections by Councilmania is very strik-
ing, and is of value because the cysts were actually counted. The
three groups of table 1 have been changed to two large groups as being
more accurate. Wherever there were less than fifty cysts on two slides
it was recorded as few, and where more than fifty cysts were found
on two slides the case was recorded as many.
1923] Bercovitz: Endamoeba coli and Councilmania lafleuri 369
TABLE 4
Series II. Reaction of Cysts of Endamoeba coli and Councilmania to
Congo Red
Type of Cyst
No.
of
Cases
Unstained
Stained
(showing
any color
at all)
Unstained
Stained
Many
Few
Many
Few
No.
%
No.
%
No.
%
No.
%
No.
%
No.
%
E. coli
22
28
8
3
36.3
10.7
14
25
63.7
89.3
1
2
12.5
66.6
7
1
87.5
33.3
2
20
14.5
80.0
12
5
85
20
Councilmania
In this series eleven cases, or 22 per cent, showed no coloring of
any of the cysts. In the first series fifteen cases, or 30 per cent,
showed no coloring. In the second series the contrast between the
staining in the two types is marked. The relative difference between
percentage of cysts of each type stained is practically the same for
the two series, the second series showing an actual higher percentage
of all cysts which are stained.
In both series the diagnosis between E. coli and Councilmania was
made after examination of slides stained with iron haematoxylin.
In the first series there were four cases where Councilmania and
E. coli were found in the same specimen. An attempt w7as made to
record the location of cysts which took the Congo red and of those
which did not; the slides in these cases were stained in haematoxylin,
in order later to find the marked cysts and thus to determine whether
they were E. coli or Councilmania. Inasmuch as in these four cases
both stained and unstained cysts were found, it would have been of
the greatest value if it could have been determined whether all of the
stained cysts were Councilmania and the unstained cysts E. coli, or
vice versa. However, another very curious phenomenon was observed
in these cases, in that after the Congo red had come in contact with
the cyst wall the haematoxylin did not seem to have free access to
the cyst; thus the cytoplasm and nuclei were not stained, and the
cysts could not be differentiated.
Repeated trials of this were made, and in every case the result was
the same. A large number of the cysts were distorted, and some
seemed to be more or less collapsed. The haematoxylin seemed to
cling to the cyst wall with great tenacity, as the cysts were exceed-
ingly difficult to destain. An investigation of this phenomenon is
to be reported later.
The results of the second series of fifty cases are in the main
similar to the results recorded in the first series. The incidence of
370 University of California Publications in Zoology [Vol. 20
Endamoeba coli infection is somewhat higher than in the first series
of fifty, but still considerably lower than the number of Councilmania
infections. As regards abundance of cysts, there are strikingly few
cysts in the great percentage of cases infected with Endamoeba coli,
while nearly 80 per cent of the cases infected with Councilmania show
abundance of cysts. In cases of E. coli infection, a smaller number
show absolutely no cysts staining at all. In many cases, however,
perhaps only two or three cysts showed any color, yet these cases were
classed as specimens showing color! In this second series Council-
mania again showed a preponderance of specimens in which cysts
take the Congo red stain.
TABLE 5
Series I and II. Eelative Abundance or Cysts of E. coli and Councilmania
in One Hundred Cases
Cysts
Many and Moderate
Few
No.
%
No.
%
E. coli
5
14.7'
34
85.3
Councilmania
46
80.7
11
19.3
Four mixed cases of first series not recorded.
In the total series of one hundred cases there were thirty-nine
cases showing E. coli infection; fifty-seven cases showing Council-
mania infection, and four cases with both E. coli and Councilmania.
Table 6
Action of Congo Eed on E. coli and Councilmania in Ninety-six Pure
Infections
E. coli
Councilmania
No. of Cases
%
No. of Cases
%
Unstained
17
43.6
9
15.7
Stained
22
56.4
48
84.3
Acknowledgments are made to the California State Board of
Health for the supply of material used, to the University of California
for assistance from the grant made in aid of research on human
intestinal protozoa, and for the use of the facilities of the proto-
zoological laboratory, to the China Medical Board for a fellowship
permitting this study, and to Professor Kofoid and Dr. Swezy for
counsel.
1923] Bercovitz: Endamoeba coli and Councilmania lafleuri 371
SUMMARY
1. In infections by Endamoeba coli the cysts are present as a gen-
eral rule in small numbers; while in infections by Councilmania the
cysts are abundant.
2. In general the cysts of Councilmania take the Congo red stain
whereas the cysts of E. coli either do not take the stain, or else in most
cases take it very faintly.
3. Of all cysts of intestinal protozoa tested with some twenty-five
anilin stains, only cysts of E. coli and Councilmania took any of the
stains at all, and these two took only the Congo red. Furthermore,
only the cyst wall was stained, the dye apparently not passing through
the wall and thus not staining either cytoplasm or nuclei.
4. The cysts of the common intestinal protozoa when dead are
quickly penetrated by anilin dyes and the protoplasm is deeply
stained. Living cysts resist penetration by these dyes.
5. The shape of cysts of Councilmania in many cases is ovoidal,
or ellipsoidal, whereas cysts of Endamoeba coli are as a rule spherical,
and also slightly smaller than Councilmania cysts (see Kofoid and
Swezy, 1921, a, b).
6. The condition of the stool, i.e., whether formed or unformed,
whether acid or alkaline, had no effect either upon the abundance of
the cysts or the way in which they reacted to the Congo red stain.
There was no evidence that in the more fluid stools the cysts were
less protected than in the formed stools.
Presbyterian Hospital,
Katchek, Hainan, China.
Transmitted, December 8, 1922.
LITERATURE CITED
Kofoid, C. A., and Swezy, O.
1921a. On the free, encysted, and budding stages of Councilmania lafleuri, a
parasitic amoeba of the human intestine. Univ. Calif. Publ. Zool.,
20, 170-189, pis. 18-22, 3 figs, in text.
1921b. Councilmania lafleuri, a new amoeba of the human intestine. Proc.
Soc. Exper. Biol. Med., 18, 310-312.
UNIVERSITY OF CALIFORNIA PUBLICATIONS—(Continued)
11. The Life Cycle of EcHnostoma revolutum (Froelich), by John C. Johnson.
Pp. 338-388, plates 19-25, 1 figure in text. May, 1920 ... .60
12. On Some New Myriopods Collected in India in 1916 "by O. A. Kofoid, by
Ralph V, Chamberlin. Pp. 389-402, plates 26-28. August, 1920 ....... ...._ .20
IS. Demonstration of the Function of the Neuromotor Apparatus in Euplotes
by the Method of Microdissection, by Charles V. Taylor. Pp. 403-470,
plates 29-33, 2 figures in text. October, 1920 ... ....... .85
Index, pp. 471-480.
Vol.20. 1. Studies on the Parasites of the Termites. L On Streblomastix strix, a Poly-
mastigote Flagellate with a Linear Plasmddial Phase, by Charles Atwood
Kofoid and Olive Swezy. Pp. 1-20, plates 1-2,1 figure in text. July, 1919 .25
2. Studies on the Parasites of the Termites. H. On Trichomitus terrnitidis, a
Polymastigote Flagellate with a Highly Developed Neuromotor System,
by Charles Atwood Kofoid and Olive Swezy. Pp. 21-40, plates 3-4, 2
figures in text. July, 1919 ...... _.1............„......„ ........ 25
3. Studies on the Parasites of the Termites, HI. On Trichonympha campanula
sp. nov., by Charles Atwood Kofoid and Olive Swezy. Pp. 41-98, plates
5-12, 4 figures in text. July, 1919 —..... .............. .75
4. Studies on the Parasites of the Termites. TV. On Leidyopsis sphaerica gen.
nov., sp. nov., by Charles Atwood Kofoid and Olive Swezy. Pp. 99-116,
plates 13-14, 1 figure in text. July, 1919 ...... < .25
5. On the Morphology and Mitosis of Chilomastix mesnili (Wenyon), a Common
Flagellate of the Human Intestine, by Charles A. Kofoid and Olive Swezy.
Pp. 117-144, plates 15-17, 2 figures in text. April, 1920 ...... .85
6. A Critical Review of the Nomenclature of Human Intestinal Flagellates,
Ccrcomonas, Chilomastix, Trichomonas, and Giardia, by Charles A. Kofoid.
Pp. 145-168, 9 figures in text. June, 1920 ................. .85
7. On the Free, Encysted, and Budding Stages of Councilmania lafleuri, a Para-
sitic Amoeba of the Human Intestine, by Charles Atwood Kofoid and
Olive Swezy. Pp. 169-198, plates 18-22, 3 figures in text. June, 1921 .60
8. Mitosis and Fission in the Active and Encysted Phases of Giardia enterica
(Grassi) of Man, with a Discussion of the Method of Origin of Bilateral
Symmetry in the Polymastigote Flagellates, by Charles A. Kofoid and
Olive Swezy. Pp. 199-234, plates 23-26, 11 figures in text. March,
1922 .........................„...„.™ .50
9. The Micro-Injection of Paramaecium, by Chas. Wm. Rees. Pp. 235-242.
April, 1922.. .". . ............ .20
10. On Balantidium coli (Malmsten) and Balantidium suis (sp. nov.), with
an account of their neuromotor apparatus, by J. Daley McDonald,
Pp. 243-300, plates 27, 28, 15 figures in text. May, 1922 1.00
11. Mitosis in Endamoeba dysenteriae in the Bone Marrow in Arthritis de-
formans, by Charles Atwood Kofoid and Olive Swezy. Pp. 301-307,
7 figures in text.
12. Endamoeba dysenteriae in the Lymph Glands of Man in Hodgkin's Disease,
by Charles A. Kofoid, Luther M. Boyers, M.D., and Olive Swezy. Pp. 300-
312, 4 figures in text.
Nos. 11 and 12 in one cover. April, 1922 ...... .25
13. Mitosis in the Encysted Stages of Endamoeba coli (Loesch), by Olive
Swezy. Pp. 313-332, plates 29-31. May, 1922 .30
14. The Neuromotor Apparatus of Paramaecium, by Charles William Rees.
Pp. 333-364; plates 32-36, 5 figures in text. November, 1922.. .45
15. A Comparison of the Cysts of Endamoeba coli and Councilmania lafleuri in
Congo Red, by Nathaniel Bercovitz, M.D. Pp. 365-371. February, 1923. .25
Vol. 21. 1. A Revision of the Microtus californicus Group of Meadow Mice, by Reming-
ton Kellogg. Pp. 1-42, 1 figure in text. December, 1918 .50
2. Five New Five-toed Kangaroo Rats from California, by Joseph Grinnell.
Pp. 43-47. March, 1919 ... .. ....... ...... ,05
3. Notes on the Natural History of the Bushy-tailed Wood Rats of California,
by Joseph Dixon. Pp. 49-74, plates 1-3, 3 figures in text, December, 1919 .25
4. Revision of the Avian Genu3 Passerella, with Special Reference to the Dis-
tribution and Migration of the Races in California, by H. S. Swarth. Pp.
75-224, plates 4-7, 80 figures in text. September, 1920 ..... 81.75
UNIVERSITY OF CALIFORNIA PUBLICATIONS—(Continued)
5. A Study of the California Jumping Mice of the Genua Zapus, by A. Brazier
HowelL Pp. 225-238, 1 figure in text. May, 1920 .IS
6. Two New Rodents (Genera Thomomys and Marmota) from the Eastern
Border of California, by Joseph GrinnelL Pp. 239-244, 6 figures in text.
November, 1921 15
7. A Study of the Californian Forms of the Miorotus montanus Group of
Meadow Mice, by Remington Kellogg. Pp. 245-274, 25 figures in text.
8. A Synopsis of the Microtus mordax Group of Meadow Mice in California,
by Remington Kellogg. Pp. 275-302, plate 8, 29 figures in text.
Nos. 7 and 8 in one cover. April 1922 76
9. Behavior of the Leaf-nosed Snake, Phyllorhynchus decurtatus, by Sarah
Rogers Atsatt. Pp. 303-312. February, 1923 25
10. A Systematic List of the Mammals of California, by Joseph Grinnell.
Pp. 313-324. January, 1923 . 25
VoL 22.1. A Quantitative and Statistical Study of the Plankton of the San Joaquin
River and Its Tributaries in and near Stockton, California, in 1913, by
Winfred Emory Allen. Pp. 1-292, plates 1-12, 1 figure in text. June, 1920. $3.00
2. Variations in the Shell of Teredo navalis in San Francisco Bay, by Robert
Cunningham Miller. Pp. 293-328, plates 13-17, 6 figures in text. Novem-
ber, 1922 50
S. Quantitative Studies on Marine Phytoplankton at La Jolla in 1919, by
Winfred Emory Allen. Pp. 329-347, 2 figures in text. November, 1922 .30
4. On the Effect of Low Salinity on Teredo navalis, by Harold Francis Blum.
Pp. 349-368, 4 figures in text. December, 1922 35
5. Quantitative Studies on Inshore Marine Diatoms and Dinofiagellates of
Southern California in 1920, by Winfred Emory Allen. Pp. 369-378,
1 figure in text.
6. Stylarioides papillosa, sp. nov., a New Annelid from the San Diego Region,
by Christine E. Essenberg. Pp. 379-381, 8 figures in text.
Nos. 5 and 6 in one cover. December, 1922 25
7. The Digestion of Wood by Teredo navalis, by Walter H. Dore and Robert
C. Miller. Pp. 383-400, plate 18. February, 1923 25
8. Variations in the Pallets of Teredo navalis in San Francisco Bay, by Robert
Cunningham Miller. Pp. 401-414, plates 19-20. February, 1923 25
VoL 23. The Marine Decapod Crustacea of California, by Waldo L. Schmitt. Pp.
1-470, plates 1-50, 165 figures in text. May, 1921 5.00
Vol.24. 1. A Geographical Study of the Kangaroo Rats of California, by Joseph
Grinnell. Pp. 1-124, plates 1-7, 24 figures in text. June, 1922 1.75
2. Birds and Mammals of the Stikine River Region of Northern British
Columbia and Southeastern Alaska, by H. S. Swarth. Pp. 125-314, plate
8, 34 figures in text. June, 1922 2.50