[Elizabeth_Zeibig]_Clinical_Parasitology__A_Practi(z-lib.org)

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CHAPTER 2 Specimen Collection and Processing
procedure allows laboratory technicians to
observe detailed features of protozoa by staining
intracellular organelles. Although some protozoa
may be recognized in the direct or concentrated
wet preparation, the identification is considered
tentative until confirmed with the permanent
stained smear. In addition, there are some protozoa
that only possess a trophozoite stage and will
not be detected in the concentrated wet mount
preparation. Dientamoeba fragilis (Chapter 4) is
one example and, if a permanent stained smear
is not performed, this parasite will likely be
missed. The permanent stained smear is not the
method of choice for the identification of helminth
eggs or larvae because these parasites often
stain too dark or appear distorted. Helminth
eggs or larvae are best detected and identified
using a concentration technique.
The sample of choice for such stains is a
thinly prepared slide of see-through thickness
made from a PVA-preserved sample. Specimens
fixed with SAF may also be used, but the choice
of stain is limited to iron hematoxylin. The slide
should be allowed to air-dry thoroughly before
staining. Slides can also be prepared from a
fresh stool specimen but must not be allowed to
dry and should be immediately placed into a
fixative, such as the Shaudinn fixative. On completion
of staining, the slides can be sealed with
a permanent mounting sealant and can be kept
for years, serving as an effective teaching tool.
The slides are reviewed under oil immersion
(100×); 300 fields are reviewed before the slide
can be considered negative. Because the slides
being examined are permanently stained, an
increased light source is recommended for
achieving optimal results. This may be done by
adjusting the microscope iris and raising the
microscope condenser.
Two common stains used for routine O&P
testing include trichrome (Wheatley modification)
and iron hematoxylin. Specialized stains are
also available for specific groups of parasites.
These are not part of the routine O&P examination
and must be specifically requested. These
specialized stains include the modified acid-fast
and modified trichrome stains.
TABLE 2-3
Appearance of Select
Protozoan Structures and
Background Material on
Trichrome Stain
Structure or Material
Cytoplasm of Entamoeba histolytica
trophozoites and cysts
Cytoplasm of Entamoeba coli cysts
Nuclear karyosomes
Degenerated parasites
Background
Appearance
Light pink or
blue-green
Purple tint
Bright red to
red-purple
Light green
Green
Wheatley Trichrome. The most widely used
permanent stain is the Wheatley trichrome stain.
Laboratory technicians choose this stain because
it uses reagents with a relatively long shelf life
and the procedure is easy to perform. There are
distinct color differences among the cytoplasmic
and nuclear structures of select parasitic forms,
as well as background material, as noted in Table
2-3. Some technicians find that the distinct color
differences between the parasites and background
material make this stain easier for review
of patient slides. Others think that the contrasting
colors are more stressful to their eyes, which
is a matter of personal opinion. A suggested procedure
for trichrome staining of a slide made
from a PVA-fixed specimen may be found in
Procedure 2-6.
Iron Hematoxylin. The iron hematoxylin
stain may be used instead of the trichrome technique.
Historically, this procedure was considered
to be time-consuming. However, a shorter
technique using this stain, described in Procedure
2-7, is now available. The iron hematoxylin stain
reveals excellent morphology of the intestinal
protozoa. In some cases, the nuclear detail of
these organisms is considered to be stained
clearer and sharper than when stained with trichrome.
The color variations among specific
parasitic structures and background material are
not as distinct as with the hematoxylin stain,
described in Table 2-4.