or, more commonly secondary to liver abscess. Peritonitis—bacterial with usual manifestations. Ulcerations and abscesses of other organs or tissues, manifestations depending upon site infected. (Incubation period: acute,
for human hemoglobin in feces. Nonetheless, as with
any in vitro diagnostic test, occasional false positive
2. False negatives may occur due to improper feces
suspension preparation or the lesion did not bleed or
bleed sufficiently to produce a positive result.
3. Blood secondary to aspirin use or use of other nonsteroidal anti-inflammatory agents may cause GI
bleeding and show false positive results.
4. Stool samples collected during menstrual bleeding,
constipation induced bleeding, bleeding hemorrhoids
and rectal medication may also cause false positive
¾ Enteritis and pancreatic diseases when there is lack of
¾ Surgical removal of a section of intestine
¾ In chronic pancreatic disease, fat is more than
¾ A stool specimen high in fat content will have a pasty
appearance and can be detected by gross examination.
1. Increased neutral fat may occur under the following
• With use of rectal suppositories
• With ingestion of castor oil or mineral oil
• With ingestionof dietetic low calories mayonnaise.
2. Barium interferes with test results.
Other Methods of Assessing Malabsorption
The GTT curve becomes flat since adequate amounts of
orally given glucose are not absorbed, whereas in the same
patient if glucose is given parenterally, a normal curve is
Protein loss estimation is not necessary for diagnosing
Proteins within the intestine are reduced enzymatically
to their component amino acids, which are then reabsorbed.
If mucosal abnormalities prevent reabsorption or if protein
leakage exceeds reabsorptive capacity, hypoproteinemia
may result. The fecal protein excretion can be documented
by administering isotopically labeled albumin or povidone
(PVP) rather than by chemical analysis of feces.
Microscopic Examination of Stool Specimens
Stool specimens should be fresh and must not be
contaminated with detergents or disinfectants, etc.
Having described the gross appearance, proceed on for
microscopic examination for cells and parasites as follows:
1. Place a small piece of stool on a slide and mix with
saline until smooth. Cover with a coverslip. If the
specimen contains mucus, examine preferably without
saline. The mucus is put on the slide and covered with
2. Examine under 10X and 40X objectives, with a reduced
• Large numbers of pus cells or muscle fibers
• Free living amebae, flagellates or ciliates.
Parasitic amebae, flagellates, ciliates, eggs, larvae and
cysts are usually reported as the number seen in the entire
¾ Moderate number: (2+) 10 to 20
Cells are usually reported as the number seen per high
power field as in urine deposit.
Normal (0.85%) saline is used for routine examination of
stool specimens, as it is isotonic with living organisms. Use
Iodine is used to examine the nuclear structure of cysts,
the preparation is made in the same way as for saline.
Using an iodine solution, the chromatin granules and
karyosome of nuclei stain brown. The glycogen vacuole
stains brown and the chromatid bars remain unstained.
The solution used is called Dobells’s iodine.
Iodine should not replace saline for routine use, as it kills
living material, and would therefore, make it impossible to
detect motility of amebae, flagellates, ciliates and larvae. In
addition, iodine makes the chromatid bars of E. histolytica
This provides a pink background against which the cysts
and amebae stand out as clear unstained objects.
This is used to stain the chromatid bars of cysts, and is of
value especially for E. histolytica.
The nuclear structure stains pale green, the chromatid
concentration method, because the use of ether appears
to be necessary for the staining reaction.
Where heavy infestation is present, this method is not
Concentration Methods may be Used
1. To see whether treatment of the parasites has been
2. To find ova of S. mansoni or Taenia if few or for other
ova and cysts if they have not been seen on routine
examination (being very few) and are suspected to be
3. To examine stool specimens from patients who do not
come from an area where a particular parasite is found.
Flotation Concentration Methods
In flotation methods, the stool is mixed with a solution,
e.g. zinc or magnesium sulfate, which has a high specific
gravity so that the parasitic contents float to the surface.
Zinc Sulfate Concentration Method
This method can be used to concentrate cysts, larvae
and most helminth eggs, except those of P. westermani,
F. buski, C. sinensis and D. latum and other operculated
eggs and also Schistosoma eggs which do not float.
Zinc sulfate solution of specific gravity 1.180 is needed.
Prepared by dissolving 33 g of chemical in 100 mL of
1. Mix a small piece of stool with about 10 mL of water
or saline, in a bottle or tube.
2. Sieve the suspension into a beaker, through a strainer
3. Pour the contents of the beaker into a centrifuge tube.
4. Centrifuge at 2000–3000 rpm/min for 1 minute.
5. Pour off the supernatant fluid.
6. Resuspend the deposit in clean water and add enough
8. Pour off the supernatant fluid.
9. Resuspend in zinc sulfate solution, fill the tube with
10. Centrifuge at high speed for 1 minute.
11. Transfer the contents from the surface of the tube to
a slide, using a bacteriological wire loop. This surface
film must be removed immediately.
12. Add small drops of saline and mix.
14. Examine under 10X and 40X objectives.
Sedimentation Concentration Methods
In sedimentation methods, the parasites are not floated
but deposited, usually by centrifuging.
A small piece of stool is mixed with saline in a tube or
bottle and sieved through a strainer. The sieved contents
are centrifuged and the supernatant fluid poured off.
The deposit is resuspended in more saline, mixed, and
centrifuged. This is repeated until the supernatant fluid is
clear. The deposit is examined directly on a slide.
By this simple method, parasitic cysts, eggs, and free
living parasites can be concentrated.
Formol-saline Ether Sedimentation Method
This method gives a good concentration of parasitic
contents and is recommended for routine work. This
method, however, cannot be used to concentrate free
living forms as formalin kills the parasites.
Concentrate formaldehyde solution 50 mL (40% w/v)
Add the formaldehyde solution to the saline and mix well.
1. Mix a small piece of stool in about 10 mL of 10% formol
2. Sieve the suspension into a beaker through a strainer
3. Pour about 6 mL of the sieved suspension into a
5. Mix well and immediately centrifuge at 3,000 rpm/min
6. Four layers are seen (Fig. 7.5)
b. Middle layer of stool particles
c. A lower layer of formol saline
d. The deposit in which parasitic contents will be
7. Using an applicator stick, separate the middle layer
from the sides of the tube and pour this away together
with the ether and formol saline.
8. Resuspend the deposit by tapping the bottom of the
9. Transfer the deposit to a slide using a pasteur pipette.
10. Cover with a coverslip and examine under 10X and
40X objectives with a reduced condenser aperture. For
the identification of cysts; iodine, eosin, or Sargeaunt’s
FIG. 7.5: Various layers as seen after centrifugation
Parasitism is a category of association of living things in
which one partner (the parasite) maintains itself at the
expense of the energy of the other (the host). By definition,
medical parasitology, then, would include even viruses
and bacteria, but it is restricted to those animal parasites,
chiefly protozoa and helminths, that produce a state of
disease in man or are closely related to others that do.
Microbial human parasites are included, for convenience,
in the field of microbiology (discussed elsewhere), and with
the specialized subspecialties of virology, bacteriology,
Importance of Morphologic Identification
Recognition and differentiation of the animal parasites of
man, often involving separation of pathogenic from very
similar nonpathogenic (harmless) forms, need precise
knowledge of their morphology. Proficiency in this
part of laboratory work is gained by extensive practical
experience with properly collected and processed clinical
specimens. The diagnosis and differentiation of protozoal
diseases, whether intestinal or systemic, often present
unusual problems. Differentiation of intestinal amebae
very similar larger form (E. histolytica) that is responsible
The following pages present medical parasitology in
Infections worldwide prevalence depends on sanitation
level and degree of natural or acquired resistance.
Entamoeba histolytica is found often in Indian rural and
urban population. The intestinal protozoans present a
serious threat in tropical rather than temperate climates,
but usually much less common than asymptomatic
infection. Incidence of Giardia lamblia varies with age,
most common in children, relatively rare in adults.
Balantidium coli is comparatively rare but may be
common where sanitary conditions are very poor. Only
these three commonly accepted forms are considered in
Tables 8.1A and 8.1B. Pathogenicity of other species is rare
Nonpathogenic protozoa are commonly found in
the feces of man and should be differentiated from the
recognized pathogenic forms. The commoner organisms:
amebae—Entamoeba hartmanni, E. coli, Endolimax
main importance is that they are a sign of environmental
pollution with fresh feces and may elicit unnecessary
treatment or inaccurate diagnosis.
Trichomonas vaginalis, known only in the trophozoite
stage, inhabits the human vagina and urethra of male
and female. Produces vaginitis with severe itching and
mucopurulent discharge in small proportion of cases.
T. vaginalis is worldwide in distribution, occurring in
TABLE 8.1A: Tabulated life cycle of common human intestinal pathogenic protozoans
Life cycle in man Exit Reservoir
-do- -do- -do- Duodeuum and bile
Balantidium coli -do- -do- -do- Many invade mucosa of
TABLE 8.1B: Intestinal protozoal diseases of man
Possible clinical features Laboratory diagnosis
Amebiasis Flask-shaped ulcers in mucosa of large intestine which appear as pinpoint dots
on surface with mild inflammation but may produce extensive undermining below
surface. Localized in cecum and whole large intestine, especially on flexures and
Blood: Leukocytosis, anemia. Eosinophilia rare in uncomplicated protozoal
Abscess and ulcer material (as for
E. histolytica Symptoms: Dysentery, bloody diarrhea, sometimes followed by constipation;
abdominal pain, gas distension; poor appetite, weight loss, headache, nervous
manifestations, local tenderness
Complications: Liver abscess (single or multiple) with liver enlargement and
congestion. Pain, swelling, leukocytosis, anemia, fever. Lung abscess primary
or, more commonly secondary to liver abscess. Peritonitis—bacterial with
usual manifestations. Ulcerations and abscesses of other organs or tissues,
manifestations depending upon site infected. (Incubation period: acute,
8–10 days, chronic, 2 months to years.)
Limited to large intestine where parasites localize, with pathology and symptoms
that may resemble amebic dysentery. Most cases asymptomatic with high natural
resistance; acute or chronic disease. Epidemic outbreaks may occur with cases of
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