[Elizabeth_Zeibig]_Clinical_Parasitology__A_Practi(z-lib.org)

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CHAPTER 2 Specimen Collection and Processing
mixing a small portion of unfixed stool (stool
with no added preservatives) with saline or
iodine and subsequent examination of the resultant
mixture under the microscope, is to detect
the presence of motile protozoan trophozoites.
Trophozoite motility can only be demonstrated
in fresh specimens, especially those of a liquid or
soft consistency. If the specimen is received in the
laboratory in a fixative, this procedure can be
eliminated from the O&P assay. Other parasite
stages that might be observed in a direct wet
preparation include protozoan cysts, oocysts
(Chapter 7), helminth eggs, and larvae. Because
the diagnostic yield of this procedure is low, most
experts agree that technical time is better spent
on the concentration procedure and permanent
stained smear and recommend only performing
the direct wet preparation on fresh specimens.
A direct saline wet preparation is made by
placing a drop of 0.85% saline on a glass slide
(a 3- × 2-inch size is suggested) and mixing with
a small portion of unfixed stool using a wooden
applicator stick or another mixing tool. The
resulting slide should be thin enough for newspaper
print to be read through the smear. A
22-mm square cover slip is placed on the slide
and the preparation is examined microscopically
in a systematic fashion. The entire cover glass
should be scanned using the low power (10×)
objective on the microscope, and the power
should only be increased when a suspicious
object requires further investigation. The use of
oil immersion is not usually recommended on
wet preparations unless the cover slip is sealed
to the slide. A temporary seal can be prepared
using a hot paraffin-petroleum jelly (Vaseline)
mixture around the edges of the cover slip. Performing
this procedure allows for the ability to
observe greater detail using the 100× objective.
A direct iodine wet preparation may be made
to enhance the detail of protozoan cysts. This
type of direct wet preparation is made as
described earlier, using a drop of iodine (Lugol’s
or D’Antoni’s formula) in place of saline. A
suggested recipe for Lugol’s iodine for wet
preparation use is given in Procedure 2-2 at the
end of this chapter. Because iodine kills any
trophozoites present, it is recommended to use
direct saline and direct iodine wet preparations
on each sample that requires this component of
testing. Thus, many laboratories prepare two
direct wet preparations side by side on a large
microscope slide, one preparation with saline
and one with iodine.
Proper adjustment of the microscope is essential
to the successful reading and interpreting of
wet preparations. For example, the light adjustment
of the microscope is critical for the detection
of protozoa, which are often translucent and
colorless. The light should be reduced using the
iris diaphragm to provide contrast between the
cellular elements in the specimen. Lowering
the condenser is often recommended to lower
the light and allow for otherwise transparent
structures to be seen. Screening a slide using
these adjustments typically takes an experienced
laboratory technician approximately 10 minutes.
Quick Quiz! 2-5
The direct wet preparation can be eliminated from
the O&P examination if the specimen is received in a
fixative. (Objective 2-8)
A. True
B. False
Concentration Methods. The next procedure
in an O&P examination is concentration of
the fecal specimen. Concentration techniques
provide the ability to detect small numbers of
parasites that might not be detected using direct
wet preparations. The purpose of concentration
is to aggregate parasites present into a small
volume of the sample and to remove as much
debris as possible that might hinder the laboratory
technician’s ability to see any parasites
present clearly. Concentration techniques can be
performed on fresh or preserved stool specimens.
This portion of the O&P examination allows
the laboratory technician to detect protozoan
cysts, oocysts, helminth eggs, and larvae. Protozoan
trophozoites do not usually survive the
procedure.