Soil Ameba Infection
Specific Indirect Immunoenzymatic (Peroxidase)
Staining of Formalin-fixed Paraffin Sections
C L Y D E G. C U L B E R T S O N ,
M.D.
ABSTRACT
Culbertson, Clyde G.: Soil ameba infection. Specific indirect immunoenzymatic (peroxidase) staining of formalin-fixed paraffin sections. Am J Clin
Pathol 63: 475-482, 1975. This report describes the use of the immunoenzymatic (peroxidase) method to identify the species and to stain distinctively
the amebas in formalin-fixed paraffin-mounted sections. This permits the
use of hematoxylin and eosin counterstaining. T h e method, now well
developed by others for many purposes, is an alternative to immunofluorescence and seems to offer a number of advantages and a lesser number of
disadvantages. (Key words: Immunoenzyme staining; Peroxidase-labeled
antiglobulin; Soil ameba infection; Naegleria; Hartmannella; Acanthamoeba;
Amebic meningoencephalitis.)
S O I L AMEBA I N F E C T I O N has
sometimes
escaped recognition clinically because the
amebas have been overlooked in the cerebrospinal fluid, since they resemble leukocytes. Similarly, the amebas in the brain
and other tissue sections have not been
identified using ordinary methods since
the stained amebas may be mistaken for
leukocytes. 1 Inasmuch as the infections
mimic bacterial infections clinically, the
condition is usually not suspected at the
time of autopsy and therefore the tissue is
placed in formalin. This, of course, precludes culture studies that could detect
and identify the amebas. 4 The general
assumption that no further opportunity
exists to identify the amebas and to
determine their species has inhibited atReceived August 26, 1974; received revised manuscript November 8, 1974; accepted for publication
November 8, 1974.
Presented at the Fall Meeting of the American
Society of Clinical Pathologists, Washington, D . C ,
October 9, 1974.
Address reprint requests to Dr. Culbertson. Lilly
Laboratory for Clinical Research, Marion County
General Hospital, 960 Locke Street, Indianapolis,
Indiana 46202.
475
tempts to do immunologic tests for specific identification upon the amebas in
formalin-fixed paraffin sections. Recent
reports support the view that formalinfixed antigens may be at least partially
stable and retain specific reaction sites in a
number of instances where different antigen and antibody reactions are concerned. 6-8'16-21 The need of the diagnostic histologist is not only for distinctive
staining of infectious agents in the
tissues—in this case soil amebas—but also
for clear staining of the tissue elements.
Immunofluorescence has been of great
value in research and in routine diagnostic work in the demonstration of infectious agents, and has been employed successfully to identify soil amebas in infected tissue. 8,16 However, this technic has
some limitations, such as fading, lack of
permanent mounting and of clear, definitive staining of the tissue elements, and
the need for special equipment. This
report outlines our study of the already
well known and widely used immunoperoxidase method as an alternative
476
CULBERTSON
to immunofluorescence in the recognition
of soil ameba infection in tissue sections. 14 ' 15
We also believe this infection is a suitable model for further study of this procedure, since ameba antigens appear
stable and strong in formalin-fixed tissue.
The amebas are of large size, permitting
easy histologic observations of the results.
Also, the species can often be confirmed
by other methods such as culture and
observation of morphologic features,
which include the presence of distinctive
cysts in the case of hartmannellid amebas,
and by the size and appearance of the
trophozoites. Experimental infections can
be achieved with known species and
known pathogenic isolates and with individual pathogenic soil amebas, thus
permitting experimentation upon the
m e t h o d itself. T h e i m m u n o e n z y m e
• technic will doubtless be of value for still
other infectious agents where these experimental studies cannot as easily be
made—as, for instance, in viral infections.
T h e general availability of the immunologic reagents now makes it possible
to use this procedure in the medical
laboratory.
Materials and Methods
1. Histologic Material
a. We had available formalin-fixed
paraffin blocks that had been prepared
over the past 10 years from experimentally infected animals using both
Hartmannella and Naegleria. 5 We also
made fresh cryostat and fixed sections on
fresh material from experimental animals.
b. Human and animal tissues sent to us
by pathologists as fixed tissue or in
paraffin blocks or stained slides were also
used after experiments with animal tissues
were successful.8,9,17'2°
A.J.C.P.—Vol.
63
intravenous injections of the live amebas
produced during the period from 1965 to
1970, and stored since in glass-sealed
ampoules at - 2 0 C. T h e complementfixing titers of these sera ranged from 200
to 1,000 at the time of preparation. For
the present studies, a range of dilutions
from 1:10 and 1:2,000 was tested for each
serum against the appropriate amebas
dried on multiple spot glass slides by the
method of Goldman 7 for E. histolytica, and
those sera producing titers of 500 to
1,000 were considered satisfactory for
use.
3. Dilution of Serum Used for
Immunoperoxidase Staining of Sections
The end titers usually ran from 500 to
1,000, after which the staining diminished
in intensity in the above-described test. In
order to insure adequate staining of the
amebas in tissue sections, since they usually stained less readily than the culture
amebas, the sera were diluted to 1:100.
4. Goat Anti-rabbit
Peroxidase-labeled Globulin
Most of the tests were done using a
single lot of peroxidase-labeled globulin
obtained from Bioware, Inc. Another
similar preparation from Microbiological
Associates was also found satisfactory.
The shelf life of these reagents is unknown to us, but it is our impression the
conjugate can be used for about a month
if stored at 4 C after reconstitution.
5. Saline Solution
Phosphate-buffered saline solution
(P.B.S.), pH 7.2, was freshly made from
vials of buffer salt (Bioware) in deionized
water.
6. Benzidine Histochemical Reagent
2. Antiamebic Serum
These studies were done utilizing
anti-Hartmannella (Al) and anti-Naegleria
(HBl) rabbit serum made by multiple
Two forms of 3-3 diaminobenzidine
(D.A.B.) were available, and generally
both gave satisfactory results. The D.A.B.
was dissolved in 10 ml. fresh P.B.S. and
April 1975
IMMUNOPEROXIDASE STAINING OF AMEBAS
10 jul. 30% H 2 0 2 were added with a
micropipette. Seven milligrams of the
more insoluble D.A.B. base was used, or 4
mg. when the D.A.B. tetrahydrochloride
was employed. The D.A.B.-H 2 0 2 reagent
was made immediately before it was
needed. 18 T h e D.A.B. base preparation
had to be filtered. After trying both, we
preferred the D.A.B. tetrahydrochloride
substrate. Plastic disposable tubes and
pipettes were found best to insure against
chemical contamination. T h e solution
needed to be clear and colorless when
applied, and only very slowly did it
change to a dark reddish-brown color.
Rapid color development meant dirty
glassware, and such solutions were unsatisfactory.
7. Technical Procedure
A. Tissue preparation10
1. Fix representative tissue blocks in
10% formalin.
2. Dehydrate and imbed in usual
manner.
3. Cut sections at 5 m/u,.
4. Float upon water bath containing
gelatin (1 Gm. U.S.P. gelatin in
1,500 ml. H 2 0).
5. Mount sections on slides, avoiding air bubbles, and carefully
flatten tissue.
6. Place rack of slides in closed
staining dish into which has been
placed 2 - 3 ml. of full-strength
formalin, for 2 - 3 hours or overnight. (Do not immerse slide in
formalin.)
7. Transfer slides to incubator or
dryer at 50 C. and leave until
thoroughly dry.
B. Slide preparation or pretreatment11
1. Deparaffinize in xylol.
2. Remove xylol in graded alcohols
to water.
3. Place in fresh 1% a q u e o u s
NH 4 OH for 3 - 5 minutes.
477
4. Wash with P.B.S. or tap water
1-2 minutes.
5. Immerse in fresh 3% aqueous
Tween 80 3 - 5 minutes.
6. Wash as above 3 - 5 minutes.
7. Place flat on level staining rack.
C. Immunologic procedure (indirect)2
1. Label three identical slides,
treated as above, "Naegleria,"
"H-A group," and "P.B.S."
2. Cover tissue sections with corresponding antiserum in appropriate dilution and P.B.S. (control)
and incubate at room temperature (23 C.) for 10 minutes.
3. Wash in running tap water or
three changes of P.B.S. for 5
minutes. Drain or blot.
4. Cover all three tissue sections
with a p p r o p r i a t e dilution of
peroxidase-conjugated anti-rabbit
globulin (goat or horse); incubate
as above 10 minutes.
5. Wash, drain, or blot as in 3.
D. Histochemical procedure19
1. Immediately before use prepare
H 2 0 2 -D.A.B. as follows: Weigh
out 4 mg. D.A.B. HC1. (Take care
to prevent ingestion of this substance; it may be carcinogenic!)
Dissolve in 10 ml. fresh P.B.S. in
a clean tube. Add 10 pi. 30%
H 2 0 2 with clean micropipette—
(Caution: this material is
corrosive!)—or 0.1 ml. of a 1-10
dilution of 30% H 2 0 2 in P.B.S.
These quantities should be determined as accurately as possible.
2. Cover tissue sections with this
fresh reagent and leave for
exactly 4 minutes.
3. Rinse with P.B.S. and proceed
immediately to step E.
E. Hematoxylin and eosin counterstain
1. Immerse rinsed sections in Harris hematoxylin (without acetic
478
CULBERTSON
2.
3.
4.
5.
6.
acid) for time required for satisfactory staining—usually 10-20
minutes. If only a few sections
are to be stained, this procedure
may be carried out on the staining rack.
Sections may be "blued" by rinsing or immersing in P.B.S. (pH
7.0) for 5 - 1 0 minutes or by the
conventional acid alcohol ( 1 %
HC1 in 70% alcohol), followed by
tap-water wash with NH 4 OH, or
by the use of lithium carbonate
solution, if preferred.
Immerse or cover with 5% eosin
Y in equal parts absolute alcohol
and distilled water for V4 — 1 minute. Do not wash in H 2 0 , as this
will remove most of the eosin, but
place sections directly in absolute
or 95% alcohol.
Pass sections t h r o u g h t h r e e
changes of absolute alcohol and
two changes of xylol.
Mount in usual resin mounting
material.
Examine preparations at X100
and x400 for yellowish-brownstaining amebas. We used a
# 8 0 A Photar blue filter, and
sometimes a didymium filter with
a 60-watt halogen bulb light
source. A positive staining reaction must be interpreted. If the
tissue cells are stained similarly,
obviously the staining of the
ameba is not specific, and something is wrong. Positive-reacting
amebas can be regarded as indicating specific identification of
species only when suitable controls are negative.
8. Control Tests
Sera against the two species of interest
showed practically no immunologic
cross-reactivity, so that the use of both
sera upon unknown specimens, together
with known positive and negative control
tissue specimens, served to make rea-
A.J.C.P.—Vol.
63
sonably certain that the staining was
truly dependent upon specific antigenantibody reactions. In a few sections,
where anti-Hartmannella serum was used
as a negative control on Naegleriainfected tissue, a rare pale yellowishbrown ameba appeared among many that
stained only with hematoxylin. However,
the vast majority of the amebas did not
stain yellowish-brown in the control, and
an equally overwhelming number did so
stain with the homologous serum. While
this inconsistency did not hamper the
interpretation of the results, it was
studied. Of the several possible explanations it was found that when the antiHartmannella serum (anti-Al) was incubated with Naegleria amebas (HB1), as
described in Section 9, this unexpected
staining was abolished. It thus appears
that for some unknown reason a low titer
of anti-Naegleria antibody exists in this
sample of anti-Hartmannella serum.
Other investigations were also carried
out with sera from which antibody had
been removed by absorption with amebas
prepared as indicated below. In order to
control various other possibilities for
error, known positive sections were tested,
substituting (1) P.B.S. for antiamebic
serum to prove that the conjugate alone
would not react with amebas; (2) P.B.S.
for the conjugate to prove that a positive
reaction was not due to residual peroxidase in the amebas. Finally, blocking
of the peroxidase staining with unlabeled globulin containing anti-rabbit antibody was used to determine further that
the immunologic evidence was valid.12
Results of these tests are shown in Table 1.
9. Absorption of Antibody from
Antiamebic Serum
Live amebas of both Naegleria and
Hartmannella species were each grown in
plastic culture bottles, 20 ml. medium per
bottle. After two days, when the growth
covered the bottom, the amebas were
loosened by shaking, pipetted into centrifuge tubes, centrifuged, and washed
April 1975
479
IMMUNOPEROXIDASE STAINING OF AMEBAS
Table 1. Results of Confirmatory Tests For Immunologic Specificity of Indirect
Peroxidase Globulin Staining
Antiserum
Used*
H-AI
N-HB1
N-HB1
H-Al
H-AI
N-HB1
0
0
H-AI
H-AI
N-HB1
N-HB1
0
0
Unconjugated
Goat
Anti-rabbit
Globulin
0
0
0
0
0
0
0
0
+
0
+
0
0
0
Normal Goat
Immune
Globulin
Peroxidaseconjugated
Goat
Anti-rabbit
Globulin
Species of
Ameba
in Section
0
0
0
0
0
0
0
0
0
+
0
+
0
0
+
+
+
+
0
0
+
+
+
+
+
+
0
0
H-AI
H-AI
N-HBI
N-HB1
H-AI
N-HBI
H-AI
N-HBI
H-AI
H-AI
N-HBI
N-HBI
H-AI
N-HBI
Peroxidase
Staining
+
+
-t
-
±t
+
±t
+
—
* ll-Al Auli-liarliiiannella Acanlhamoeba (AI-/Y. nilbntsmii): N-HBI Anli-Naegleria (rlBI-M foideri [or aerobia]).
t See text, ilem S (Control Tests) under Materials and Methods.
t See text, paragraph 3, under "blocking," in Results.
three times in 0.85 sterile NaCl solution.
The amebas were suspended in the original volume of saline solution, counted in a
hemocytometer and adjusted to approximately 1,000,000 per ml. Two tubes containing 10 ml. of this suspension were
centrifuged and all possible supernatant
fluid removed. The serum to be absorbed
was inactivated at 56 C. for 30 minutes,
diluted 1:10 with saline solution, and each
serum was added to two tubes—one
containing hartmanneilid amebas, the
other Naegleria. These mixtures were
agitated on a roller drum at 37 C. for 30
minutes and placed at 4 C. for another 30
minutes. T h e homologous mixture
showed strong agglutination of the
amebas. T h e tubes were centrifuged and
the serum removed and treated again in
similar tubes with identical amebic pellets
and washed in the same manner. After
centrifugation the sera were stored at 4 C.
and tested against sections of tissue containing known species of amebas. When
this was done at a dilution of 1:100 of the
absorbed serum, the staining reaction was
completely inhibited. A single absorption
diminished the staining, but did not obliterate it—thus the above-described double
procedure. T h e heterologous absorption
resulted in no noticeable change in the
staining reaction of the appropriate
amebas in section, proving that the
washed, live amebas did not by enzymatic
or other non-immunologic means inactivate the antibody protein.
Results
The first efforts made without any
counterstaining gave yellowish sections
with brownish amebas, and the contrast
was poor. The use of a hematoxylin
counterstain fortunately and unexpectedly gave much better contrast, making
possible picking out the yellowish-brown
amebas at 100X magnification. Observation of a considerable number of stains of
human sections of both Naegleria and
Hartmannella and also of experimental
animal tissue suggested that the peroxidase staining inhibits the hematoxylin and eosin staining of the positively
reacting amebas. T h e mechanism of this
was not determined. Not all amebas in a
section stained, and not all stained uniformly. This variability has also been
observed in the immunofluorescence procedure, and one explanation advanced
480
CULBERTSON
was based upon the different ages or
phases of the amebas in the reproductive
cycle.3 Figures 1-9 show the microscopic
appearance.
Some of the older material, of which
only stained slides were available, was
studied by removing the coverslips and
destaining for a short time in 1% HC1 in
70% alcohol, followed by the method
outlined above. This was found to give
excellent results in most instances. On two
occasions—in one older and in one of the
more recent human cases—the amebas,
which resembled hartmannellid amebas in
morphology, failed to stain distinctively
with either of the two sera, either from
paraffin blocks or old stained slides. The
reason for the failure has not been determined, but it is possible the causative
ameba was of a different species, yet
unknown as a pathogen, or the fixation or
processing of the tissue varies in some
unknown manner.
Experiments designed to show a blocking effect indicated a rather marked dim-
A.J.C.P.—Vol.
63
inution of the staining when the standard incubation times were used. As
others have reported with reference to
the immunofluorescence method, 12 the
preliminary treatment of the sections already incubated with specific antiserum
by nonconjugated anti-rabbit globulin did
not inhibit all staining seen when the
sections were incubated with normal goat
immunoglobulins. There was, however, a
marked reduction in the staining after use
of the "blocking" serum.
Discussion
The immunoenzymatic staining method
of infectious agents may become the most
useful of the several methods for those
microbiologic agents for which antisera
can be prepared and the antigens of
which are at least partially stable to fixation. There is need to study further
particularly those infections in which the
chemical stains cannot be used, such a
trichomoniasis or even viral infections.
Formalin fixation, according to our ex-
FIG. 1. (upper left). Monkey cerebellum. Animal infected intraspinally with Naegleria (fowleri, aerobia, HB1),
then sacrificed and the cerebellum perfused intravenously with 10% formol saline. Section stained using
anti-Hartmannella (Acanthamoeba) (H-Al) serum plus peroxidase-labeled goat anti-rabbit serum, followed by
D . A . B . - H 2 0 2 reagent, and hematoxylin and eosin stain. Note clump of amebas that stain only with
hematoxylin and eosin, above and to left of Purkinje cell. x320.
FIG. 2 (upper center). Same as Figure 1 except that anti-Naegleria (anti N-HB1) serum was used. Note
ring of yellowish-brown amebas around small blood vessel. X320.
FIG. 3 (upper right). Same as Figure 2 except that amebas are diffusely scattered and are more deeply
colored in spite of a heavier hematoxylin stain. Note contrasting erythrocytes, which retain a bright red
color with eosin. x320.
FIG. 4 (middle left). Nasal mucosa of mouse. Animal infected intranasally with Naegleria (HB1). Formalin
fixation-formic
acid decalcification. Section stained with anti-H-Al serum plus peroxidase conjugate, plus
D . A . B . - H 2 0 2 reagent, and hematoxylin and eosin. Note that amebas take normal hematoxylin stain. X320.
FIG. 5 (middle center). Tissue from the same block as in Figure 4, stained identically except that antiN-HB1 serum was used. Note yellowish-brown color of amebas present in epithelium and also in nasal
cavity exudate.
FIG. 6 (middle right). Human brain (case of Jager and Stamm), stained with anti-H-Al serum plus
peroxidase conjugate and D . A . B . - H 2 0 2 followed by hematoxylin and eosin. Note yellowish-brown amebas
with reddish blue background of glial fibers. x320.
FIG. 7. (lower left). Human brain (case of Patras and Andujar). Perivascular exudate in cerebral tissue stained
by anti-H-Al serum plus peroxidase conjugate and D . A . B . - H 2 0 2 followed by hematoxylin and eosin. Note
that amebas take hematoxylin stain only. X320.
FIG. 8 (lower center). Tissue from same block as in Figure 7, stained with anti-N-HBl serum and treated
as in Figure 7. Note yellowish-brown color of amebas. x320.
FIG. 9 (lower right). Same tissue as in Figure 6, just above, but stained with anti-N-HBl serum plus
peroxidase conjugate and D.A.B., and H 2 0 2 plus hematoxylin and eosin. Note that the amebas stain with
hematoxylin and eosin; therefore the ameba belongs to the hartmannellid group. x320.
April 1975
481
IMMUNOPEROXIDASE STAINING OF AMEBAS
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*
*
*
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482
CULBERTSON
perience as well as a considerable number
of reports in the literature, 13 does not
destroy the immunologic specificity and
reactivity always or completely. As in the
case of the soil amebas, there is sufficient
residual antigenicity for practical identification by immunologic methods. The
chemical nature of the antigens resistant
to formalin, etc., has not been determined
in the case of the soil amebas, but most
evidence would seem to suggest that they
are polysaccharide in nature.
T h e application to diagnosis of soil
amebas may also be helpful in demonstrating the presence of amebas when
they are not numerous in a particular
slide. T h e use of this method to restudy
undiagnosed granulomas, etc., seen in the
past, as well as the effects of other
fixatives, may yield some interesting information.
Finally, it should be stressed that this is
a rather involved procedure, depending
upon many factors. As will be noted, we
found much shorter times of incubation
could be used than are usually specified.2
Actually the shorter periods seemed to
improve the staining. Some study and
practice may be needed to achieve satisfactory results. However, since the
method may give information unobtainable by any other means, the rather tedious
and somewhat expensive process is, in our
judgment, worth the trouble. We have
found that, after some experience, the
procedure is not as formidable as it first
appeared to be!
Acknowledgments. Willis Overton, Lilly Research
Laboratories, supplied many of the sections, and
Paul Ensminger, Lilly Research Laboratories, had
previously prepared most of the antisera used in
the study. Doctors Patras and Andujar; Robert and
Rorke; Kernohan, Magath, and Schloss; Jager and
Stamm permitted us to use human tissue from their
reported cases.
References
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2. Cawley L: Personal communication, February
1974
3. Cerva L, Kramar J: Antigenic relationships
A.J.C.P.—Vol. 63
among several Limax amoebae isolates assessed
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1973
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of acute, fatal amebic meningoencephalitis.
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21. Taylor CR, Burns J: T h e demonstration of
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