[Elizabeth_Zeibig]_Clinical_Parasitology__A_Practi(z-lib.org)

CHAPTER 2 Specimen Collection and Processing
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PROCEDURE 2-3
FORMALIN–ETHYL ACETATE CONCENTRATION—cont’d
2 and 3. When the supernatant is basically clear,
proceed to step 5.
5. Add 9 mL of 10% formalin to the sediment and mix
thoroughly.
6. Add 3 mL of ethyl acetate, stopper the tube, and shake
the mixture vigorously in an inverted position for at
least 30 seconds.
7. Centrifuge the tube for 10 minutes at 500 × g
(1500 rpm). Four layers will form in the tube. From top
to bottom they are as follows:
Layer of ethyl acetate
Plug of specimen debris
Layer of formalin
Sediment
8. Remove the stopper and, with an applicator stick,
gently rim the plug of debris to loosen it from the sides
of the tube. Carefully decant the top three layers.
9. With a cotton-tipped swab, wipe down the sides of
the tube, absorbing any remaining ethyl acetate.
Excess ethyl acetate may appear as bubbles in the
microscopic preparation and can dissolve the plastic in
the tube.
10. Make side by side saline and iodine wet preps from
the sediment on a large glass slide. These are called
concentrated wet preps.
11. Examine each concentrated wet prep under the microscope,
as described in text.
NOTE: Specimen should be formalin-fixed for a minimum of 30 minutes prior to beginning this procedure.
PROCEDURE 2-4
ZINC SULFATE FLOTATION
1. Strain the specimen through a filter containing a singlelayer
thickness of gauze into a conical centrifuge tube.
2. Fill the tube with saline and centrifuge for 10 minutes
at 500 × g (1500 rpm). Decant the supernatant. If the
supernate is cloudy, repeat this step for a second wash.
3. Resuspend the sediment with 1-2 mL of zinc sulfate
solution. Fill the tube with additional zinc sulfate to
within 2-3 mm of the rim. (The zinc sulfate must have
a specific gravity of 1.18-1.20.)
4. Centrifuge for 2 minutes at 500 × g (1500 rpm). Allow
the centrifuge to come to a complete stop.
5. While the tube is in the centrifuge, remove one or two
drops of the top film using a Pasteur pipette or a bent
wire loop and place on a slide.
6. Add a cover slip and examine microscopically. Iodine
can also be added.
NOTE: Specimen should be formalin-fixed prior to beginning this suggested procedure.
PROCEDURE 2-5
PREPARING SMEARS FOR PERMANENT STAINING
Fresh Stool
1. Using applicator sticks, spread a thin film of stool on a
microscope slide and place immediately into Schaudinn
fixative. Allow to fix for 30 minutes to overnight. Prior
to staining, the slide must be placed into 70% alcohol
to remove fixative.
2. Liquid specimen—add three or four drops of PVA to a
slide and mix with several drops of stool. Spread the
mixture to allow to dry overnight at room temperature
or for 2-3 hours at 35° C.
PVA-Preserved Stool
1. Mix the vial of the PVA-preserved specimen well and
pour some of the material onto several layers of paper
towels. Let stand for 3 minutes to absorb excess PVA.
2. Using applicator sticks, apply the material to the slide,
ensuring that it covers the slide to the edges.
3. Dry the slide overnight at room temperature or for 2-3
hours at 35° C.
SAF-Preserved Stool
1. Mix SAF-preserved specimen well and strain through
gauze into a conical centrifuge tube.
2. Centrifuge for 1 minute at 500 × g (1500 rpm) and
decant the supernatant.
3. Prepare the slide by adding two drops of albumin to the
slide and mixing with one drop of fecal sediment.
4. Allow to dry and place into 70% alcohol to begin the
staining procedure.