(c) Procedure. If the specimen has not been previously fixed in MIF,
mix one portion with 10 portions of MIF solution in a disposable container.
STEP 1: Suspend the fecal material and strain through two layers of wet gauze into a
STEP 2: If needed, add MIF to make 12 milliliters.
STEP 3: Add three milliliters of ethyl acetate, stopper, invert the tube, and shake
vigorously until thoroughly mixed (about 30 seconds). Release the stopper
with care to avoid spraying the area or self with the specimen.
STEP 4: Centrifuge for two minutes at 2,500 rpm. Four layers will result in the tube
after centrifugation. From top to bottom, the layers are: ethyl acetate, debris
plug, MIF, and the sediment containing the parasites (if present).
STEP 5: Free the debris plug with an applicator stick and carefully decant the top three
layers, leaving the sediment undisturbed. Use a cotton swab to clean the
remnants of ethyl acetate from inside of the tube.
STEP 6: Remix the sediment with an applicator stick, transfer a drop to a clean slide,
and observe microscopically for parasites.
c. Permanent Stains. Because of the difficulty in recognizing protozoan
organisms in wet preparations, a parasitology laboratory should have a procedure for
permanent stain. In fact, no trophozoite should be reported without first being positively
identified in a stained preparation under the oil immersion objective. Another advantage
of permanent mounts is that they can be kept as a permanent record and/or for training.
(1) Iron hematoxylin stain. The phosphotungstic/iron hematoxylin stain for
staining protozoans in fecal smears, proposed by Tompkins and Miller, is one of the best
techniques for demonstrating nuclear details and cytoplasmic inclusions. It is a short
procedure and does not have to be adjusted with microscopic observation as does
1 Iodine alcohol.
a Stock solution. Dissolve sufficient iodine crystals in 70
percent ethanol to make a dark brown concentrated solution. This solution is stable for
two to three months.
b Working solution--solution A. Dilute the stock solution with
enough 70 percent ethanol to make it the color of strong tea. This solution should be
made just prior to use.