Cleanse finger, ear lobe, heel, or toe (of children) thoroughly with spirit and allow to dry. Prick skin deeply to cause a few drops of blood to flow freely. On one end of
motility, local tissue and organ dysfunction
Urine: Direct smear for eggs, sedimentation
and hatching of miracidia in diluted urine
for S. haematobium, preferably in last
portion of urine passed. Feces: Direct smear;
concentration for S. mansoni or japonicum
eggs. Rectal biopsy and sigmoidoscopy (S.
haematobium). Blood: Precipitin (circumoval
Localized inflammation of jejunum or duodenum, followed
often by ulceration at sites of worm attachment. Diarrhea
with foul-smelling stools; abdominal pain. In severe
infections eosinophilia, ascites, anorexia, nausea, vomiting,
Feces: Direct smear, sedimentation
Worm migrations cause tissue necrosis and fibrosis.
Bile duct damage resembles that for Clonorchis. Picture
like any biliary derangement (gallbladder, choledochus
involvement). Eosinophilia. Infection runs a chronic course
Feces: Direct smear, sedimentation. Duodenal
drainage; complement fixation, skin tests
Similar hepatic involvement, large numbers of
parasites cause diarrhea, jaundice, cachexia, eosinophilia.
Proliferation and desquamation of biliary epithelium,
dilatation, and thickening of the wall occur, severe
symptoms of liver dysfunction, recurring jaundice with
hepatomegaly may follow. Long continued chronic course
Direct smear, sedimentation; eggs in biliary
Lung: Parasites are embedded, usually in pairs, in
subpleural cysts (eggs act as foreign bodies) with
inflammation, eosinophilia and fibrous capsule
formation. Chronic cough with fever, brown sputum,
hemoptysis, severe chest pain, bronchial pneumonia or
pleural fluid common. May enter any organ and produce
local symptoms, e.g. abdominal pain, diarrhea, CNS
involvement. Similar lesions in other tissues.
Sputum: Direct smear. Feces: Direct smear,
sedimentation; complement fixation, etc.
Fasciola hepatica (The Sheep Liver Fluke)
Recapitulation—Parasitology at a Glance
The Amebae of the Intestinal Canal
The Body Fluid and Tissue Flagellates (Causing Leishmaniasis and Trypanosomiasis)
Malaria Species Identification in the Mosquito—Pigment in Oocysts
188 Concise Book of Medical Laboratory Technology: Methods and Interpretations Nematoda
190 Concise Book of Medical Laboratory Technology: Methods and Interpretations Contd...
Recapitulation Pathogenesis and Pathology of Worm Infections
192 Concise Book of Medical Laboratory Technology: Methods and Interpretations Contd...
194 Concise Book of Medical Laboratory Technology: Methods and Interpretations Contd...
Local Effects of Worm Infections
196 Concise Book of Medical Laboratory Technology: Methods and Interpretations Contd...
LABORATORY EXAMINATION FOR PARASITES
Preservation and Shipment of Specimens to be
Examined for Trophic and Encysted Protozoa
(PVA) Polyvinyl Alcohol Method of
Excellent for ameba, especially if permanent slides are
required. For collection of field specimens and for mass
surveys, the Merthiolate-Iodine-Formalin (MIF) method
1. Reagent—Modified Schaudinn’s solution is prepared
by mixing 5 mL of glacial acetic acid, 1.5 mL of glycerol
and 93.5 mL of Schaudinn’s solution (2 parts saturated
aqueous mercuric chloride and 1 part 95% ethyl
Heat the above solution to 75oC; while stirring,
slowly add 5 g polyvinyl alcohol (PVA) powder. This
final solution should be clear and free of lumps after
cooling. It is used at room temperature, and lasts
a. One specimen should be sent without above
fixative to be used for detection of protozoan
cysts and helminthic ova. This specimen can be
used for temporary or permanent smears or for
To prepare slides, a small amount of this fecal
mixture (recent or months old) is spread thinly
over about one-third of the slide. After drying
for 3 or more hours at 37oC or overnight at room
temperature, the smear is stained by the iron
hematoxylin method. This procedure is particularly suitable for the preservation and staining
of the trophic forms of intestinal protozoa. To
obtain satisfactory stained fecal smears containing cystic stages, the excess clear PVA solution
is decanted from the vial and a small amount of
the remaining fixed fecal material is placed on a
piece of facial tissue or toilet paper. The excess
PVA solution is allowed to absorb for 5–10 minutes, leaving a moist fecal residue. Gently scrape
up a small amount of the residue with the sharp
edge of a broken applicator stick and smear with
gentle brushing strokes on a slide. Drop smear
immediately into 70% alcohol to which iodine
has been added to produce a portwine color, and
stain by the iron hematoxylin method. To ensure
satisfactory preparation of both types smears are
Merthiolate-Iodine-Formalin (MIF)
This is now the standard method for mass surveys,
collection of bulk material, or parasitologic field studies.
Fecal specimens are fixed and stained immediately, and
can be examined at any time within several months of
collection. This method is particularly good for protozoa.
1. Reagents: MIF stock solution is made from 250 mL
distilled water, 200 mL of tincture of merthiolate, 25
mL of formaldehyde, 5 mL of glycerol. Store in brown
bottle. Lugol’s solution (5% iodine in 10% potassium
iodide in distilled water), not over 1 week old, forms
2. Preparation of specimens: For each specimen
to be collected, have ready 2.35 mL MIF stock
solution in a Kahn test tube with cork stopper and
0.15 mL Lugol’s solution in a second Kahn tube
with a rubber stopper. Combine the 2 solutions
just before adding the fecal specimen. Break up
about 0.25 g feces into the combined solution, mix
thoroughly, and stopper well (may be examined
immediately on a slide, 1 drop fecal preparation and
1 drop distilled water; or stored in a well-stoppered
tube, where the stain will be retained for several
Routine Stool Examination and Concentration
Methods have been Dealt with Elsewhere (Previous
Negative Stain Direct Fecal Smear Examination
Prepare normal fecal smear in saline to which 1–2 drops
of 1% isotonic eosin are added. Background material and
dead parasites turn uniformly pink. Stain will not penetrate
living trophozoites, causing them to stand out markedly as
clear, translucent organisms against a pink background. A
Isotonic eosin, 1%, and brilliant cresyl blue, 0.2%,
makes a good vital stain, causing living material to appear
as shiny pale blue-green objects on the pink background.
Kato Cellophane Thick Smear Technique (Kato and
This method permits rapid examination of a large number
of samples (up to 70/h) for eggs of the common helminths.
It is not suitable for protozoa or minute helminths or for
highly fibrous or gaseous samples.
202 Concise Book of Medical Laboratory Technology: Methods and Interpretations Materials
1. Wettable cellophane of medium thickness (40–50 µm),
2. Glycerin malachite green solution: 100 mL pure
glycerin, 100 mL water, 1 mL 3% aqueous malachite
The cellophane strips should be soaked in the glycerin
mixture for at least 24 hours before use.
1. Place 50 to 60 mg feces (4 mm cube) on a clean slide.
2. Cover with a glycerin-soaked cellophane strip, press to
spread feces in an even layer. Feces need not be spread
to all areas of cellophane; a circumference equal to the
width of the strip is sufficient.
3. Allow to stand at room temperature for 1 hour (or
20–30 minutes at 40oC in a dry incubator). This dries
and clears the specimen. (Do not over dry, as gas
bubbles will form and air cells will surround the eggs).
4. Examine the entire film under low poor magnification.
Heidenhain’s Iron Hematoxylin Staining Method for
Iron hematoxylin is generally accepted as the most
reliable stain for nuclear detail in amebae and for accurate
laboratory diagnosis. It also provides a permanent stain
handicap in routine handling of specimens by clinical
laboratories. The trichrome stain has therefore, superseded
iron hematoxylin staining for routine diagnosis, though for
critical definition, iron hematoxylin is still unsurpassed.
Both iron hematoxylin and trichrome can be used for
either PVA-fixed or nonpreserved material.
1. Schaudinn’s fixative—described earlier.
2. Hematoxylin stain (stock solution)—dissolve 100 g
hematoxylin powder in 100 mL absolute ethanol. Let
stand several weeks for maturation. For use, 5 mL of
ripened hematoxylin is added to 95 mL distilled water.
3. Mordant—dissolve 5 g ferric ammonium sulfate in
10 mL distilled water and filter.
1. Make thin fecal smear on a clean glass slide with
toothpick, applicator, or stiff haired paste brush.
2. Before drying occurs, immerse slide in Schaudinn’s
fluid with acetic acid added, heated to 45oC. Fix for
5–15 minutes at this temperature or for 30 minutes
at room temperature (omit this step for PVA-fixed
aqueous iron-alum Usually 3–5 minutes
Tap water (running) 15–20 minutes
Isopropyl alcohol 2 changes of 5
Xylol (2 changes) 5 minutes each
Mount in xylol-balsam, xylol-Damar or DPX mountant
with a cover glass of No. l thickness.
Preferred permanent stain for routine diagnosis.
To 100 mL distilled water, add 0.6 g chromotrope 2 R,
0.3 g light green SF, 0.7 g phosphotungstic acid, and 1 mL
Prepare slides as for hematoxylin staining. (Omit steps 1
and 2 for PVA-fixed specimens).
Schaudinn’s fixative room temperature
2. 70% alcohol (wash) 15 minutes
3. 70% iodine alcohol 10 minutes
4. 70% alcohol (wash) 2 changes of
6. Acidified 90% alcohol 2 dips
10. Mount immediately as for hematoxylin preparations.
Cultivation of Intestinal Protozoa
Numerous types of special media have been developed
for the cultivation of the intestinal amebae and flagellates
as well as for the ciliate Balantidium coli. Among these
are Boeck-Drbohlav-Locke-egg serum medium, Nelson’s
egg yolk infusion-liver extract medium, and Dobelle’s
medium which contains serum, egg albumin and starch.
The parasite (E. histolytica) grows in 24 to 36 hours at 37oC.
The morphological character of the colony is quite typical
The cultivation of free-living juveniles of Ancylostoma
duodenale and Strongyloides stercoralis is done on
sterilized sand or charcoal paste. Samples of soil or feces
containing ova are mixed with equal quantity of sterilized
fine sand or animal charcoal and water to make a thick
paste. The paste is kept on a filter paper in a petri dish and
covered with lid. It is kept at 25 to 30oC for several days. The
free-living juveniles collect in the water of condensation.
Collect urine in a clean, dry container, avoiding fecal
contamination. Study centrifuged sediment. Trophozoites
of Trichomonas vaginalis, unhatched eggs of Schistosoma
haematobium, and intact scolices or hooklets of
Echinococcus granulosus may appear in the urine. Viable
S. haematobium eggs will hatch only after dilution of urine.
Examine for parasites as a direct smear under a cover glass.
If the sputum is thick, bloody, or pus laden, mix with equal
volume 1–2% sodium hydroxide. Stir, let settle, and study
sediment. Larvae of Strongyloides stercoralis, scolices
of Echinococcus granulosus, eggs of P. westermani, or
migrating nematode larvae may be present in the sputum.
Charcot-Leyden crystals and eosinophils can also be
observed in wet mounts or after wet fixation and staining.
For night-swallowed sputum will often yield Paragonimus
eggs or migrating nematode larvae better than will sputum
or feces (owing to less detritus).
These are useful for nematode eggs and are particularly
helpful for eggs of Clonorchis and other bile dwelling
Examine centrifugate directly under the microscope. Make
smears, stain with Giemsa’s stain and examine. Culture
some of the sediment, as for blood. Inoculate guinea pigs
Wet preparation may be examined directly for Trichomonas
vaginalis. Cultures can also be made.
Graham Cellulose Tape Technique for Diagnosis of
With the help of a tongue depressor, press the adhesive
side of a small strip or loop of cellulose tape (e.g. Scotch
tape) over the anal and perianal surfaces, preferably at
night. Then place tape with adhesive side down in a drop
of toluene on a microscopic slide. Examine for eggs or
worms which have adhered to the tape.
Cleanse finger, ear lobe, heel, or toe (of children)
thoroughly with spirit and allow to dry. Prick skin deeply
to cause a few drops of blood to flow freely. On one end of
a meticulously clean slide free of fingerprints or oil film,
make a thin smear as for a blood count. For the preparation
of the thick film, deposit a large drop of blood at the other
end of the slide and spread it out evenly with the corner
of another slide to a diameter of about 20 mm. The film
should not be too thick since it may crack and peel when
dry. Dry the slide in a flat position so that the distribution
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