JOURNAL OF CLINICAL MICROBIOLOGY, July 1998, p. 1974–1976 Vol. 36, No.

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0095-1137/98/$04.0010
Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Evaluation of Intestinal Protozoan Morphology in Human Fecal

Specimens Preserved in EcoFix: Comparison of
Wheatley’s Trichrome Stain and EcoStain
LYNNE S. GARCIA* AND ROBYN Y. SHIMIZU
Department of Pathology and Laboratory Medicine, Clinical Microbiology, University of California
at Los Angeles Medical Center, Los Angeles, California 90095-1713
Received 6 November 1997/Returned for modification 24 February 1998/Accepted 15 April 1998

As a result of disposal problems related to the use of mercury compounds, many laboratories have switched
from mercuric chloride-based Schaudinn’s and polyvinyl alcohol (PVA) stool preservatives to other, non-
mercury-based preservatives. A comparison of organism recoveries and morphologies of the intestinal protozoa
was undertaken with PVA containing the EcoFix zinc-based Schaudinn’s preservative (Meridian Diagnostics,
Inc.); both Wheatley’s modification of Gomori’s trichrome stain (WT) and EcoStain (ES) were used to stain 51
human fecal specimens. Morphology, clarity of nuclear and cytoplasmic detail, overall color differences, and
the ease or difficulty in detecting intestinal protozoa in fecal debris were assessed for the two permanent
stained smears. Overall, organism morphology of the intestinal protozoa stained with WT and that of protozoa
stained with ES were not equal in nuclear and cytoplasmic detail or range of color. However, the same or-
ganisms were identified in stained fecal smears with either WT or ES, with the exception of situations in which
organism numbers were characterized as rare. Included were 67 protozoan challenges (number of organisms):
Entamoeba histolytica-Entamoeba dispar (5), Entamoeba coli (9), Entamoeba hartmanni (6), Endolimax nana (12),
Iodamoeba bütschlii (8), Blastocystis hominis (19), Giardia lamblia (6), Dientamoeba fragilis (2), yeast (2), and
leukocytes (2). Five specimens were negative for parasites but contained fecal debris that was compared for
morphologic detail and color range. The ES produces a more gray-green monotone with very little pink or red
tone; contrast among the various colors is less than that seen with WT. Stain intensity for all organisms was
acceptable, and there were no problems with stain deposition. The quality of the protozoan morphology with
ES was often comparable to that with WT (36 of 67 [53.7%]) and, in some cases, better (24 of 67 [35.8%]).
Organisms on the WT-stained smear exhibited better morphology in a few instances (4 of 67 [6%]), and in
three instances, there were discrepant organism numbers.

For many years, Schaudinn and polyvinyl alcohol (PVA)
fixatives with a mercuric chloride (HgCl2) base have been used
to preserve stool specimens for the recovery and identification
of intestinal parasites (1, 5, 8, 9). The concentration technique
for either 5 or 10% formalin- or PVA-preserved specimens has
been used for the recovery of helminth eggs and larvae and
protozoan cysts. The permanently stained smear prepared from
Schaudinn- or PVA-fixed material is used primarily for the
identification of intestinal protozoa and is considered to be the
most important technique for this purpose (2–4, 8, 10, 12).
During the past few years, the issue of mercury disposal has
become more important for clinical laboratories. Many facili-
ties do not have the ability to dispose of small quantities of
materials contaminated with mercury compounds. It is becom-
ing almost impossible to find companies that will accept mer-
cury-containing waste, and even if an appropriate company can
be found, the cost is prohibitive. The use of Schaudinn’s fixa-
tive prepared with a compound other than mercuric chloride
(HgCl2) would be advantageous. Several studies using the sub-
stitute copper sulfate (CuSO4) indicated that this compound
did not provide consistent fixation for adequate protozoan
morphology (5, 7). Some laboratories have switched to the use
of sodium acetate-acetic acid-formalin (SAF) fixative coupled
with the iron hematoxylin stain for the permanently stained
* Corresponding author. Mailing address: Department of Pathology
and Laboratory Medicine, UCLA Medical Center (171315), 10833 Le
Conte Ave., Los Angeles, CA 90095-1713. Phone: (310) 794-2752. Fax:
(310) 794-2765. E-mail: lgarcia1@ucla.edu.
fecal smears (11, 13, 15). Although this approach provides a
good alternative, other laboratories want to maintain use of
the trichrome stain. The combination of SAF fixative and tri-
chrome stain may not always provide the same quality of re-
sults as those seen with the SAF-iron hematoxylin combina-
tion. Manufacturers have also begun investigating the possibility
of providing non-mercury-based fixatives coupled with a tri-
chrome-based stain that can be used as a combination ap-
proach, very similar to the combination of SAF and iron he-
matoxylin. The main objective of this study was to determine
whether the same organisms preserved in EcoFix preservative,
regardless of morphologic differences or numbers other than
rare (see Materials and Methods), could be identified with
either the EcoStain (ES) or Wheatley’s trichrome stain (WT)
(14).
MATERIALS AND METHODS
Human fecal specimens (51 total) were collected in EcoFix preservative (Me-
ridian Diagnostics, Inc., Cincinnati, Ohio). Of the 51 vials, 43 were positive for
intestinal protozoa, 3 were positive for human cells and yeast, and five contained
only fecal debris.
Two permanent stained smears were prepared from each vial, one smear being
stained with ES according to the manufacturer’s directions (Meridian Diagnos-
tics, Inc.) and the second smear being stained with WT (1, 3, 8, 10, 14). Both
smears were examined by reviewing approximately 300 oil immersion fields
(magnification, 31,000), and the results were recorded by technologists other
than those preparing the smears. This approach is generally more consistent than
reading each smear for a set amount of time, since different individuals scan
smears at different speeds (3, 10). Although the smears were identified as to stain
used, without examination of the smears by microscopy, they could not be
identified by gross appearance as being stained with ES or WT. Numbers of
organisms and cells, clarity of nuclear and cytoplasmic detail, overall staining

1974
VOL. 36, 1998 EVALUATION OF INTESTINAL PROTOZOAN MORPHOLOGY 1975

TABLE 1. Protozoa identified on permanent stained smears and in a few cases, the WT-stained smears were better (4 of 67
(ES or WT) from human fecal specimens preserved in EcoFix [6%]). The overall color of ES-stained fecal smears is a gray-
ES WT No.
green or gray-blue monotone with very little pink tone, and the
Sp. or cell type No. of contrast among the range of colors is less than that seen with
compa-
and form challenges
No. Result No. Result rablea WT. Stain intensity among both ES- and WT-stained smears
was acceptable, and there were no problems with stain depo-
Entamoeba histolytica-
Entamoeba dispar sition.
Cyst 2 1 Missed 1
Trophozoite 3 1 Better 1 Better
DISCUSSION
1 Missed
Although HgCl2 has been used in the preparation of
Entamoeba coli
Schaudinn’s and PVA fixatives, other compounds have been
Cyst 7 1 Fewer organisms 6
Trophozoite 2 1 Better 1 used as substitutes in order to eliminate the many problems
with disposal and cost inherent in the use of mercury com-
Entamoeba hartmanni pounds (5–7, 13, 15). The use of ZnSO4 as a mercury substitute
Cyst 2 2 Better is gaining popularity. Many companies are manufacturing stool
Trophozoite 4 3 Better 1 fixatives with ZnSO4, in addition to other chemicals; these
formulations tend to be proprietary (6). Recently, interest has
Endolimax nana
Cyst 8 3 Better 1 Better 4
also been expressed in developing stains that are formulated to
Trophozoite 4 2 Better 2 be used with very specific fixatives for a combination approach
to fixation and permanent staining.
Iodamoeba bütschlii Overall differences between WT and ES are related to the
Cyst 6 3 Better 3 range of organism color, rather than stain intensity. Although
Trophozoite 2 2 Better there were differences in color and some differences in proto-
Blastocystis hominis 19 4 Better 1 Better 14
zoan morphology, the majority of the smears stained with
central body form either ES or WT showed no significant differences that would
influence the ability to recognize the organism. Studies have
Giardia lamblia shown that when permanent WT-stained smears from fecal
Cyst 5 2 Better 3 specimens preserved in HgCl2 and ZnSO4 were examined, the
Trophozoite 1 1 overall differences in recovery and morphology were minimal.
However, protozoan morphology was superior in specimens
Dientamoeba fragilis 2 2 Better
trophozoite preserved in HgCl2, with clear, well-defined nuclear and cyto-
plasmic detail (6). Although the range of colors was present
Leukocyte with WT (pink, red, purple, blue, and green), stained smears
PMNb 1 1 prepared from ZnSO4-fixed material were more green and
Eosinophil 1 1 HgCl2 smears were more uniformly blue with better differen-
tial colors (pink, red, and purple). However, organisms were
Yeast cells 2 1 Better 1
detectable on both types of smears (6).
Total number 71 24 Better 5 Better 39 Definitive identification of intestinal protozoa frequently de-
2 Missed 1 Fewer organisms pends on the permanent stained smear, and it is important that
a
this procedure be performed as a routine part of the ova and
Although “comparable” means that the organisms were identified in both
permanent stained smears (ES and WT), there were differences in color ranges,
parasite examination (2–4, 8–10, 12). The ability to identify the
with ES-stained smears exhibiting a more monotonous color and WT-stained organisms after staining depends on obtaining the best fixation
smears exhibiting a wider range of color, including more pink, red, and purple as quickly after specimen passage as possible.
tones.
b
The first matched set of fixative and stain, EcoFix stool
PMN, polymorphonuclear leukocyte.
preservative and the ES permanent fecal stain, has been de-
veloped. Data from this study has confirmed that these two
reagents used in combination provide an acceptable, and in
differences, and ability to detect organisms in fecal debris were assessed from the some cases better, alternative to WT with fecal specimens
two permanent stained smears for both trophozoite and cyst stages and human
cells. Although there were morphologic and color differences, stained smears
preserved in EcoFix.
were considered equivalent if the same organisms were identified in both spec- Some discrepancies are inevitable; sampling errors and or-
imens and the levels of nuclear and cytoplasmic detail were comparable. In cases ganism shedding cycles contribute to the failure to always iden-
where the number of organisms per smear was rare (no organisms per 10 oil tify all intestinal protozoa in the stool sample, particularly
immersion fields at a magnification of 31,000 but at least one organism in the
smear), we anticipated that there might be situations where one smear would be
when organism numbers are quite low. However, the labora-
positive and the other smear would be negative. No discrepant results were tory community accepts these limitations as normal factors
anticipated with organism recovery and identification when organisms were char- related to diagnostic test results in parasitology.
acterized as more than rare. With more laboratories switching from mercury-based fixa-
tives to alternatives, it continues to be important to match
RESULTS these fixatives with stains that provide the best possible mor-
phology. Until present disposal and cost limitations on the use
A wide range of intestinal protozoa (67 protozoan chal- of mercury-based fixatives are modified, it is important to re-
lenges and cells were identified from the 46 positive specimens. member that any permanent stain will certainly increase the
In most cases, the comparative morphologies of the intestinal chances for protozoan recovery over that with concentration
protozoa from ES- and WT-stained smears were equal (36 of sediment alone (4). With the substitute fixatives and methods
67 [53.7%]) (Table 1). In some cases, the ES-stained smears re- available at this time, the combination of EcoFix and ES pro-
vealed better overall protozoan morphology (24 of 67 [35.8%]), vides a better alternative than that seen with EcoFix and WT
1976 GARCIA AND SHIMIZU
formula. This study demonstrates the importance of develop-
ing matched stool preservative and stain combinations. As
more fixative-stain matched sets become available, organism
morphology may approach that of results seen with the “gold
standard” combinations of mercury-based fixatives with either
WT or iron hematoxylin stain. It is also interesting to note that
proficiency testing fecal specimens used in the United States
for diagnostic parasitology testing are all preserved in mercury-
based fixatives. Currently, there are no plans to send profi-
ciency testing fecal specimens preserved in any of the nonmer-
cury fixatives to participants for testing in their laboratories.
Until substitutes for mercury-based fixatives can provide the
same day-to-day consistency in organism fixation and subse-
quent excellent morphology after staining, laboratories will
continue to explore other reagent options.
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