Stool Examination

STOOL EXAMINATION By
Dr. VARUGHESE GEORGE

LEARNING OBJECTIVES
• INTRODUCTION
• ADVANTAGES OF STOOL EXAMINATION
• COLLECTION OF STOOLS SAMPLES
• MACROSCOPIC EXAMINATION
• PREPARATION OF SLIDES
• CONCENTRATION OF STOOL SAMPLES
• MICROSCOPIC EXAMINATION
• CHEMICAL EXAMINATION

INTRODUCTION
• Normal amount of stools in an adult is 100-200 g/day.
• Stools are composed of :
– water (up to 75%),
– indigestible residue
– undigested food
– food which is digested but not absorbed
– bile, epithelial cells, secretions from digestive tract
– inorganic material
– Bacteria
• Stool analysis determines the various properties of the
stool for diagnostic purposes.

• INTRODUCTION
• ADVANTAGES OF STOOL EXAMINATION
• COLLECTION OF STOOLS SAMPLES
• MACROSCOPIC EXAMINATION
• PREPARATION OF SLIDES
• CONCENTRATION OF STOOL SAMPLES
• MICROSCOPIC EXAMINATION
• CHEMICAL EXAMINATION

ADVANTAGES OF STOOL EXAMINATION
Examination of stools aids in investigation of GIT
diseases.
• Detection of parasites :
– detection of worms (adult worms, tapeworms, larvae, ova)
– detection of protozoa (trophozoites or cysts).
• Evaluation of chronic diarrhea
– Random diarrheal sample tested for occult blood, fat, pH,
white blood cells, culture, or microscopy.
– A 48- or 72-hour sample is examined for weight, fat content,
carbohydrate, osmolality or chymotrypsin activity.

Examination of stools aids in investigation of GIT
diseases.
• Evaluation of dysentery:
– Identification of causative organism is definitive in amebic V/S bacillary
dysentery.
• Bacteriologic examination:
– Infection by bacteria such as Salmonella, Shigella, Vibrio, Yersinia, or
Clostridium difficile can be identified by stool culture.
– Bacterial toxins released by Clostridium botulinum or Clostridium difficile
can also be identified.

Examination of stools aids in investigation of GIT diseases.
• Chemical examination:
– to detect occult blood (in ulcerated lesions of gastrointestinal tract,
especially occult carcinoma of colon),
– excess fat excretion (malabsorption syndrome)
– presence or absence of urobilinogen (obstructive jaundice).
• Differentiating infection by invasive bacteria (like
Salmonella or Shigella) from that due to toxin producing
bacteria (like Escherichia coli or Vibrio cholerae):
– Increased numbers of polymorphonuclear neutrophils (identified
by methylene blue stain) are seen in the former

Examination of stools aids in investigation of GIT diseases.
• Identification of Rotavirus:
– common cause of diarrhea in infants & young children.
– It can be identified by examination of stool by electron
microscopy, latex agglutination, immunofluorescence or
ELISA.

COLLECTION OF STOOL SAMPLES
• A random specimen of stool (at least 4 ml or 4 cm3) is
collected in a clean, dry, container with a tightly fitting lid &
transported immediately to the laboratory.
• About 20-40 grams of formed stool or 5-6 tablespoons of
watery stool should be collected.
• Parasites are best detected in warm, freshly passed stools
which should be examined as early as possible (preferably
within 1 hour of collection).
• Stool should not be contaminated with urine, water, soil, or
menstrual blood.
 Urine & water destroy trophozoites.
 Soil will introduce extraneous organisms & also hinder
proper examination.

COLLECTION OF STOOL SAMPLES
• Sample may be refrigerated if delay in examination is
anticipated.
• A fixative may be used if specimen is to be transported to a
distant laboratory.
– 10% formalin for preservation of eggs, larvae & cysts.
– Polyvinyl alcohol for preservation of trophozoites & cysts &
for permanent staining.
• 3 separate samples collected at 3-day intervals are
recommended to detect all parasite infections.
– One negative report for ova and parasites does not exclude
the possibility of infection

MACROSCOPIC EXAMINATION
Findings :-
• Consistency
• Color
• Odor
• Presence of blood
• Presence of mucus
• Presence of adult worms or segments of tapeworms

Consistency of Stool samples
• Cysts are likely to be found in formed stools.
• Trophozoites are most likely to be found in loose or watery
stools or in stools containing blood and mucus.
• Trophozoites die soon after being passed and therefore such
stools should be examined within 1 hour of passing.
• Examination of formed stools can be delayed but should be
completed on the same day.

Consistency of Stool samples
• Dry and hard stools are seen in chronic constipation.
• Pasty stools are due to fat contents
• Common bile obstruction
• Celiac disease
• Ribbon like stools are seen in
• Spastic bowel
• Rectal narrowing
• Stricture
• Partial GIT obstruction

Color/Appearance of Stool samples
Color/Appearance Interpretation
Brown Normal/Stercobilinogen
Black and tarry Bleeding in upper GIT(proximal to cecum)
Drugs (iron salts, bismuth salts,charcoal)
Red Lower GIT bleeding/tumors, inflammatory process
Anal fissure,hemmorhoids,tumors
Undigested tomatoes or beetroot.
Yellow or yellow green Diarrhoea
Clay-colored
(gray-white)
Biliary obstruction
Silvery Carcinoma of ampulla of Vater

Color/Appearance of Stool samples
Color/Appearance Interpretation
Watery Certain strains of Escherichia coli, Rotavirus enteritis,
Cryptosporidiosis
Rice water Cholera
Unformed with blood,
mucus, and pus
Bacillary dysentery, Ulcerative Colitis, Intestinal tuberculosis,
Amoebiasis, Enteritis
Unformed, frothy, foul
smelling, which float on
water
Steatorrhoea
Pale color stool with
greasy appearance
Pancreatic deficiency due to malabsorption

Odour
• Stool odour – Indole and skatole which are formation
by bacterial fermentation and putrefaction.
• Foul odour – Undigested protein & by excessive
intake of carbohydrate.
• Sickly odour – Undigested lactose & fatty acids.

Presence of mucus in stools
• Translucent gelatinous
material clinging to surface
of stool.
• Produced by colonic
mucosa in response to
parasympathetic
stimulation.
• Seen in
– Severe constipation
– Mucous colitis

Presence of mucus and blood in stools
• Seen in
• Bacillary dysentery
• Ulcerative Colitis
• Intestinal tuberculosis
• Amoebiasis
• Enteritis
Mucus with blood clinging to stool is seen in
• Lower GIT malignancy.
• Inflammatory lesions of anal canal

PREPARATION OF SLIDES
• A drop of normal saline is placed near one end of a glass slide and a drop of Lugol
iodine solution is placed near the other end.
• A small amount of feces is mixed with a drop each of saline and iodine using a wire
loop, and a cover slip is placed over each preparation separately.
• If the specimen contains blood or mucus, that portion should be included for
examination (trophozoites are more readily found in mucus).
• If the stools are liquid, select the portion from the surface for examination.

PREPARATION OF SLIDES
• Saline wet mount is used for demonstration of eggs and larvae of helminths, and
trophozoites and cysts of protozoa.
• Saline wet mount can also detect red cells and white cells.
• The iodine wet mount is useful for identification of protozoal cysts as iodine stains
glycogen and nuclei of the cysts.
• Trophozoites become non-motile in iodine mounts.
• A liquid, diarrheal stool can be examined directly without adding saline.

CONCENTRATION OF STOOL SAMPLES
• Useful if very small numbers of parasites are present.
• Indicated in cases of negative wet mount
examination and there is clinical suspicion of
parasitic infection.
• Used for detection of ova, cysts, and larvae of
parasites.
Demerit - Amoebic trophozoites are destroyed in
concentrated specimens and therefore cannot
be detected.

Concentration techniques are of two main types:
• Sedimentation techniques :
– Ova and cysts settle at the bottom.
– Excessive stool debris may make the detection of parasites
difficult.
– e.g. Formolethyl acetate sedimentation procedure.
• Floatation techniques :
– Ova and cysts float on surface.
– Some ova and cysts do not float at the top in this procedure.
– e.g. Saturated salt floatation technique, zinc sulphate
concentration technique.

The most commonly used sedimentation method is
formol-ethyl acetate sedimentation method
because:
i. it can detect eggs and larvae of almost all
helminths, and cysts of protozoa.
ii. it preserves their morphology well.
iii. It is rapid.
iv. risk of infection to the laboratory worker is
minimal because pathogens are killed by
formalin.

• Stool sample suspension is prepared in 10%
formalin (10 ml formalin + 1 gram stool
sample) & passed through a gauze filter till 7
ml of filtered material is obtained.
• Ethyl acetate (3 ml) is added to this filtered
material & the mixture is centrifuged for 1
minute.
• Eggs, larvae & cysts sediment at the bottom
of the centrifuge tube with layers of
formalin, stool debris, and ether above.
• Stool debris is loosened with an applicator
stick & the supernatant is poured off.
• One drop of sediment is placed on one end of
a glass slide & one drop is placed at the other
end.
• One of the drops is stained with iodine, cover
slips are placed, & the preparation is
examined under the microscope.
Formol-ethyl acetate sedimentation method

MICROSCOPIC EXAMINATION
Findings :-
• Leukocytes (WBCs)
• Red Blood Cells (RBCs)
• Macrophages
• Epithelial cells
• Bacteria
• Ova/ Cysts/ Trophozoites of parasites
• Meat/muscle fibres
• Fat

Leukocytes (WBCs)
• Normal stool may contain
occasional (0-1) WBCs.
• To look for WBCs, the smears
should be prepared from areas
of mucous or watery stools.
RBCs
WBCs
Wet mount

Leukocytes (WBCs)
Increased no: of WBCs are stools is associated with
• Bacillary dysentery
• Chronic ulcerative colitis
• Shigellosis
• Salmonella infections
• Invasive E-Coli infections
• Anal/Rectal Fistula
• Localised abscess
• Amoebiasis & typhoid

• Bright red stool is seen in
cases of lower GIT bleeding.
• Black and tarry blood are seen
in cases of
– Upper GIT bleeding.
– Occult bleeding.
• Present in
– Dysentery
– Hemorrhoids
– GIT Malignancies
Red Blood Cells (RBCs)

Macrophages
• seen in
– Bacillary dysentery
– Ulcerative colitis
Wet mount

Epithelial cells
• Seen in inflammatory
conditions of the bowel
Epithelial cell

Bacteria
• Stool cultures are commonly done to identify bacteria
associated with enteric infection.
• Stool samples are examined to detect toxins & to rule out
infection by
– Salmonella, Shigella, Campylobacter & predominating numbers of
Staphylococcus organisms.
– Pseudomonas, Yersinia, Vibrio & Shiga toxin–producing E. coli.
– Clostridium difficile causing antibiotic-associated colitis.
– Yeast.
• At least 3 stool cultures collected on separate days are
recommended if the patient’s clinical picture suggests
bacterial involvement, despite previous negative cultures.

Fat
Present in
• Malabsorption
• Deficiency of pancreatic
digestive enzyme
• Deficiency of bile

Meat/muscle fibres in stools
Their presence show impaired
intraluminal digestion.
• Increased amount of meat
fibres are found in
• Malabsorption syndrome
• Pancreatic functional
defect like cystic fibrosis.

Ova/ Cysts/ Trophozoites of parasites
• Normally there are no parasites/eggs in the stool
sample.
• Multiple stool samples should be examined to rule
out parasitic infestations
• At least 3 consecutive days’ stool samples are
examined to rule out parasitic infestations

MICROSCOPIC EXAMINATION OF STOOL SAMPLES

MICROSCOPIC EXAMINATION - PROTOZOA
Entamoeba histolytica
• Demonstration of trophozoites
of E. histolytica in stool samples
is required for diagnosis of
amoebic dysentery.
• For diagnosis, at least three
fresh stool samples should be
examined to increase sensitivity.
• Trophozoites vary from 15 to 40
μ in diameter.
• In saline wet mounts,
trophozoites show motility in
one direction via pseudopodia,
which form rapidly.
• Cytoplasm shows outer transparent
ectoplasm and inner finely granular
endoplasm.
• Nucleus is visible in the iodine
preparation.
• Fine granules of peripheral nuclear
chromatin are evenly distributed.
Small, single central karyosome.
(Motility is lost in iodine mount).

Diagnostic feature of E. histolytica
trophozoites is the presence of
ingested red cells.
Trophozoite of Entamoeba histolytica
showing ingested red cells
(Trichrome stain)

Entamoeba histolytica trophozoites

Uninucleate, binucleate, trinucleate,
and quadrinucleate cysts of Entamoeba
histolytica
• Cysts of E. histolytica are spherical and
measure 10-15 μ in diameter.
• Nuclei are 1, 2, 3, or 4 and are similar
in morphology to trophozoite nucleus
(mature cyst contains 4 nuclei).
• Nuclear membrane is regular and thin
with finely granular peripheral
chromatin.
• Karyosome is small and central.
• Immature cysts may show chromatoid
bodies (aggregates of ribosomes) that
are oblong structures with rounded
ends and glycogen clumps – infective
stage.

Entamoeba histolytica cyst
Entamoeba histolytica cyst (arrow)

• Plenty of red cells and very few white cells are helpful in
differentiating amebic from bacillary dysentery.
• Detection of antigen of E. histolytica in stools by commercially
available tests based on enzyme immunoassay.
• Detection of DNA specific to E. histolytica is possible by
polymerase chain reaction-based assays.
• Serologic tests like latex agglutination test, indirect
hemagglutination test, enzyme immunoassay, and counter
immunoelectrophoresis detect antibodies to E. histolytica.
• Additionally, endoscopic biopsy of ulcer in the intestine can
demonstrate trophozoites of E. histolytica in 50% ofcases.
Staining with periodic acid Schiff stain facilitates identification
of parasites.

Differences between amoebic and bacillary dysentery
Parameter Amoebic dysentery Bacillary dysentery
Cause Entamoeba histolytica Shigella (most common)
Onset Gradual Acute
Fever/ vomiting Not significant Significant
Toxicity Absent Present
Abdominal tenderness Localized Generalized
Tenesmus Absent Present
Frequency 6-8 per day Over 10 per day
Odor Offensive Nil
Color Dark red Bright red
Nature Feces mixed with blood and
mucus
Blood and mucus with little or
no feces
Consistency Not adherent Adherent to container
Reaction Acid Alkaline

Differences between amoebic and bacillary dysentery
Parameter Amoebic dysentery Bacillary dysentery
Microscopic examination of stool
• Cellular exudates
• Red cells
• Pus cells
• Macrophages
• Eosinophils
• Charcot-Leyden crystals
• Bacteria
• Trophozoites of E. histolytica
Scanty
Clumps
Nil or few
Few
Present
May be present
Many, motile
Motile trophozoites with
ingested red blood cells
Abundant
Discrete
Numerous
Many, some with ingested RBCs
Absent
Absent
Few, nonmotile
Absent
Antigen test for E. histolytica Positive Negative
Stool culture Negative Positive for Shigella

Giardia intestinalis (lamblia)
• Cysts are more likely to be found
in formed or loose stools.
• Cysts of G. lamblia are 8-12 μ in
diameter, oval and contain 4
nuclei, axonemes, median
bodies and remains of flagella

• G. lamblia trophozoites are
found as clusters in fresh liquid
stools, particularly in flakes of
mucus.
• Trophozoites of G. lamblia are
pear-shaped,12-15 μ in
diameter, have 4 pairs of
flagellae, 2 large and oval nuclei,
2 axonemes (axial filaments of
flagella), and 1 or 2 curved
median bodies.
• Motility is likened to that of
“falling leaf”.

Giardia intestinalis (lamblia) trophozoite

• Detection of antigen of G. lamblia in stool sample by
enzyme immunoassay technique with high sensitivity (90-
99%) and specificity (95-100%).
• Direct fluorescent antibody assay is available commercially
in a kit form in which cysts are labeled with
immunofluorescent antibodies and are detected under
fluorescence microscope.

Coccidia
• Demonstration of oocysts of human intestinal coccidia such as
Isospora belli, Cryptosporidium parvum & Cyclospora
cayetanensis.
• These protozoan organisms cause self-limited, mild diarrheal
illness.
• In immunocompromised patients (such as patients with AIDS)
they can induce severe and protracted diarrhea, which may
sometimes be life-threatening.
• Detection of antigen in stool samples by enzyme immunoassay
for detection of specific antigen of C.parvum is available.
• Oocysts of Cryptosporidium labelled with fluorescent antibody
are readily detected under fluorescence microscope.

Coccidia - Isospora belli
Immature oocyst showing
2 sporoblasts
Mature oocyst with 2
sporocysts containing
sporozoites
• Oocysts are seen in direct wet mounts of stools
after formalin-ether concentration technique.
• Oocysts are oval and about 32 × 16 μ in size and
mature outside the human body
• Immature oocysts which are seen in stools
contain 2 sporoblasts.
• Mature oocysts found outside the human body
contains 2 sporocysts, each with 4 sporozoites.
• With modified Ziehl-Neelsen stain (on a stool
smear prepared from the sediment after
formalin-ether concentration), oocysts stain
uniform red-pink.
• Under ultraviolet light, oocysts show
autofluorescence.

.
Under ultraviolet light, oocysts show
autofluorescence.
With modified Ziehl-Neelsen stain (on a stool
smear prepared from the sediment after
formalin-ether concentration), oocysts stain
uniform red-pink
Coccidia - Isospora belli

• Oocysts of C. parvum are difficult to demonstrate in direct stool wet mounts.
• They are demonstrated either by modified Ziehl- Neelsen staining of
concentrated stool smear or by immunofluorescence technique.
• They are 4-6 μ in size, round to oval, and stain pink-red.
Cryptosporidium oocysts appear with a
peripheral green fluorescence
Cryptosporidium oocysts in stools
by modified Ziehl- Neelsen staining
Coccidia - Cryptosporidium parvum

• In direct wet mounts of feces,oocysts measure 8-10 μ in
diameter and contain a cluster of refractile globules (morula-like
appearance).
With modified Ziehl-Neelsen stain, they
appear similar to C. parvum, but are
larger.
Under ultraviolet light (365 nm), oocysts show
intense blue autofluorescence.
Coccidia - Cyclospora cayetanensis

• Microsporidia are obligate intracellular protozoa, which cause opportunistic
infection in immunocompromised patients leading to persistent diarrhea and
weight loss.
• Common species causing infection in humans are Enterocytozoon bieneusi,
Encephalitozoon intestinalis, and Encephalitozoon hellem.
• Spores are very small (1-5 μ), stain red on modified trichrome stain and may
show a transverse band.
Microsporidia (arrows) in stool stained with modified acid-fast trichrome
Microsporidia

MICROSCOPIC EXAMINATION - HELMINTHS
• Diagnosis of A. lumbricoides infection is made by demonstration
of eggs on stool examination.
• Eggs can be demonstrated in direct wet mount of stools in
moderate to heavy infections.
• The recommended procedure is formol-ethyl acetate
sedimentation technique for concentration of eggs.
• In stools, the types of eggs are found:
– fertilized (double-shelled or decorticated)
– unfertilised (double-shelled or decorticated)
Ascaris lumbricoides (Roundworm)

(A) Adult female and male worms
(B) Anterior end of worm. Head-on view, showing one dorsal and two ventral lips with
papillae
(C) Posterior end of female, showing anal opening, a little above the conical tip
(D) Posterior end of male, showing two protruding copulatory spicules(s)
(E) Specimen showing Ascaris lumbricoides, male and female.

Unfertilized eggs:
• Single female worms discharge eggs
which are slightly larger & elliptical in
shape.
• Size : 80 μm x 55 μm.
• The egg has a thinner shell with an
irregular coating of albumin.
• The egg is filled with a mass of large
disorganized highly refractile granules
of various sizes (atrophied ovum).
• Eggs do not float in salt solution.

Fertilized eggs:
• Eggs are round, oval, golden brown in
color & always bile stained.
• Size : 70 μ × 50 μ in size.
• Surrounded by thick smooth translucent
shell with an outer coarsely
mammillated albuminous coat, a thick
transparent middle layer & the inner
lipoidal vitelline membrane.
• The egg contains a single central
granular mass (fertilized ovum).
• Eggs float in saturated solution of
common salt.

Decorticated eggs :
• These eggs do not have the unevenly
thick outer albuminous shell &
resemble the hookworm eggs.

Identification of adult worms
• Occasionally adult worms are passed spontaneously in the feces
and brought to the laboratory for identification.
• Adult ascaris worms are cylindrical or round, pinkish, and
measure about 15 cm (male) or 30 cm (female) in length.
• 0.5 cm in diameter
• Tail is either curved (male) or straight (female).
• There are three lips at the anterior end (mouth).

• Hookworms are
– Ancylostoma duodenale (old world hookworm)
– Necator americanus (new world hookworm).
• Diagnosis is based on identification of hookworm eggs on stool
• examination.
• Technique of formol-ethyl acetate sedimentation is preferred.
• Alternatively, direct wet mount of stool sample can demonstrate
eggs in moderate to heavy infections.
• Eggs of A. duodenale & N. americanus are morphologically similar.
Hookworms

Hookworms
• Eggs of A. duodenale & N. americanus are 50-75 μ
in length and 40 μ in width, oval, colourless, and
have a thin shell.
• In fresh stools, eggs show 4-8 gray, granular cells.
• If stool > 12 hrs old, eggs will show a rhabditiform
larva folded upon itself , which are called
embryonated eggs.
• If feces > 24 hrs old, free rhabditiform larvae will
be seen.
• Buccal cavity of hookworm larva is longer which distinguishes itself from
• larvae of Strongyloides stercoralis Test for occult blood in stools is positive.
• Additional findings in blood -
• Blood examination often shows eosinophilia.
• Microcytic hypochromic anemia develops due to chronic blood loss.
Hookworm egg in fresh stools.
Egg in oval shape with a thin shell, has a
clear space between wall and developing
cleavage, and contains granular cells

Trichuris trichiura (Whipworm)
Diagnosis depends on identification of typical
eggs on stool examination.
Eggs measure 50 × 25 μ in size, are yellow-
brown and barrel-shaped.
A rounded, transparent plug is present at both
poles
Eggs contain central, uniformly granular mass.
Eggs are often quantitated to assess the
severity of infection.

Hymenolepsis nana
• Eggs are oval and measures
30-50 μ in size.
• The inner membrane has 2
poles, from which 4-8 polar
filaments spread out
between the two
membranes.
• The oncosphere has 6
hooklets

Strongyloides stercoralis
Diagnosis of S. stercoralis infection depends on demonstration of
rhabditiform larvae in fresh stool specimens.
Eggs of S.stercoralis are rarely seen in stool samples because they
hatch and release rhabditiform larvae in the intestine.
Rhabditiform larvae are 200-300 μ in length and 15 μ in width and
are actively motile.
They have two esophageal swellings, a prominent genital
primordium and a short buccal cavity.
In old stool samples, rhabditiform larvae of hookworms are seen
which resemble those of S. stercoralis; the differentiating feature
is the longer buccal cavity of the former.

Strongyloides stercoralis
• Excretion of larvae is often irregular and their number may be
few  stool examination may yield negative result.
• Concentration technique (formol-ethyl acetate) is helpful in
suspected cases.
• Duodenal fluid can be aspirated for detection of larvae or
Entero-test (String test) can be performed.
• In disseminated infection, larvae may be detected in sputum.
• Enzyme immunoassay test that detects IgG antibodies to S.
stercoralis is available &
• indicated if organism is not detected in feces, duodenal
aspirate, or string test and clinical suspicion is strong.
• cannot differentiate between recent and past infection.

Enterobius vermicularis
Special technique for collection and demonstration of pinworm eggs :
• Adult female pinworm migrates at night from the intestine (cecum) to the
perianal skin folds and deposits eggs which are often not found on routine
stool examination.
• Pinworm eggs can be collected either by a transparent adhesive tape
(“cellophane tape test”) or by anal swab.
• Specimen should preferably be collected late into the night or early morning
before patient passes urine, feces or takes a bath.
• A transparent adhesive tape is folded over the end of a glass slide, spoon
handle or a wooden tongue depressor (sticky surface outwards).
• Patient’s buttocks are separated & the slide or spoon handle covered with
tape is pressed over perianal skin at many sites.
• The tape is then spread over a glass slide with adhesive side down & pressed
flat onto the slide surface.
• Slide covered with tape is then examined under the microscope.

• Eggs of E.vermicularis measure 60 μ × 30
μ in size, are oval & flattened on one side.
• They are colorless, transparent with a
double-lined smooth shell & contain a
small granular mass or a larva.
• Adult pinworms may be recovered from
perianal skin folds (by adhesive tape) or
may be found in children’s feces.
• They are white, motile & small in size
(male: 0.5 cm; female: 1 cm).
• Diagnosis depends on demonstration of
eggs in samples collected from perianal
skin or demonstration of adult worms.
Enterobius egg
Enterobius eggs collected by
transparent tape method

Enterobius vermicularis egg

Taenia solium - Identification of eggs:
• Egg measures 30-40 μ in diameter, is
round to oval, and has a thick, brown
wall with transverse lines.
• The egg contains an embryo, which is a
round granular mass containing 3 pairs
of hooklets and surrounded by a fine
membrane.

Taenia solium - Identification of eggs:
• Occasionally, the egg is
enclosed in a clear sac.
• Eggs are discharged
intermittently by the
tapeworm and therefore may
not be detected easily.
• Repeated stool examinations and formol-ether concentration
technique are often required for their demonstration.

Taenia solium - Identification of gravid segments or
proglottids:
• This allows identification of species.
• The segment is flattened between two
glass slides and examined under a
magnifying glass.
• Gravid segment is 13 mm × 8 mm in size,
translucent, and pale blue.
• It has a central uterine stem with 8-13
lateralbranches.
• (Uterine branches are >13 in T. saginata)

Taenia solium - Identification of scolex (head):
• Scolex of a tapeworm is very small
(pinhead size) and is rarely seen.
• When examined with a magnifying glass,
scolex of T. solium shows 4 suckers and a
crown of hooklets.

Taenia saginata - Identification of eggs:
• Eggs of T. saginata can be identified in
feces and perianal skin.
• They are morphologically similar to those
of T. solium.

Taenia saginata - Identification of gravid segments or
proglottids:
• This allows identification of species.
• Segments measure 20 mm × 6 mm in size,
and are ivory white.
• They contain a central uterine stem > 13
side branches(i.e. 15-20 branches).

Taenia solium - Identification of scolex (head):
• Scolex of T. saginata has 4 suckers but no
hooklets.
• It measures 2 mm in width.

MICROSCOPIC EXAMINATION - ARTEFACTS
It is also important to identify artefacts during microscopic
examination of stool samples which could be confused with ova
& cysts of various protozoa & helminths
Yeast & fungal elements in stool

Pollen grains in stools mistaken for helminth eggs.

Plant fibre/Plant cells.

Air bubbles

CHEMICAL EXAMINATION
Chemical examination of feces is usually carried out for the
following tests :
• Occult blood
• Excess fat excretion (malabsorption)
• Urobilinogen
• Reducing sugars
• stool osmotic gap
• stool pH

Test for Occult Blood in Stools
• Presence of blood in feces which is not apparent on gross
inspection and which can be detected only by chemical tests is
called as occult blood.
• Causes of occult blood in stools are:
i. Intestinal diseases: hookworms, amebiasis,typhoid fever, ulcerative
colitis, intussusception,adenoma, cancer of colon or rectum.
ii. Gastric and esophageal diseases: peptic ulcer,gastritis, esophageal
varices, hiatus hernia.
iii. Systemic disorders: bleeding diathesis, uremia.
iv. Long distance runners.
• Recommended as a screening procedure for detection of
asymptomatic colorectal cancer

Test for Occult Blood in Stools - Peroxidase-like activity of Hb
• Benzidine and orthotolidine are carcinogenic and are no longer
used.
• Benzidine test is also highly sensitive and false-positive reactions
are common.
• Since bleeding from the lesion may be intermittent, repeated
testing may be required.
Principle:
• Hemoglobin has peroxidase-like activity & releases oxygen
from hydrogen peroxide.
• Oxygen molecule then oxidizes the chemical reagent
(benzidine, orthotolidine, aminophenazone, or guaiac) to
produce a colored reaction product.

Causes of False +ve Tests
1. Ingestion of peroxidase-
containing foods like red meat,
fish, poultry, turnips,
horseradish, cauliflower,
spinach, or cucumber. Diet
should be free from peroxidase-
containing foods for at least 3
days prior to testing.
2. Drugs like aspirin and other anti-
inflammatory drugs, which
increase blood loss from GITin
normal persons.
Causes of False -ve Tests
1. Foods containing large amounts
of vitamin C.
2. Conversion of all hemoglobin to
acid hematin (which has no
peroxidase-like activity) during
passage through the
gastrointestinal tract.
Test for Occult Blood in Stools - Peroxidase-like activity of
Hb

Test for Occult Blood in Stools - Immunochemical Tests
• These tests specifically detect human hemoglobin.
• There is no interference from animal hemoglobin or myoglobin
(e.g. meat) or peroxidase-containing vegetables in the diet.
• The test consists of mixing the sample with latex particles coated
with anti-human haemoglobin antibody & if agglutination occurs,
test is positive.
• This test can detect 0.6 ml of blood per 100 grams of feces.

Test for Occult Blood in Stools - Apt test
• This test devised by Dr. Apt is done to decide whether blood in
the vomitus or in the feces of a neonate represents swallowed
maternal blood or is the result of bleeding in the GIT.
• The baby swallows blood during delivery or during breastfeeding
if nipples are cracked.
• Apt test is based on the principle that
– if blood is of neonatal origin it will contain high proportion of hemoglobin F
(Hb F) that is resistant to alkali denaturation.
– On the other hand, maternal blood mostly contains adult hemoglobin or
Hb A that is less resistant.

Malabsorption of Fat
• Dietary fat is absorbed in the small intestine with the help of bile
salts and pancreatic lipase.
• Fecal fat mainly consists of neutral fats (unsplit fats), fatty acids,
and soaps (fatty acid salts).
• Normally very little fat is excreted in feces (<7 grams/day in
adults).
• Excess excretion of fecal fat indicates malabsorption and is known
as steatorrhea.
• It manifests as bulky, frothy, and foul-smelling stools,which float
on the surface of water.

Causes of Malabsorption of Fat
i. Deficiency of pancreatic lipase (insufficient lipolysis): chronic
pancreatitis, cystic fibrosis.
ii. Deficiency of bile salts (insufficient emulsification of fat): biliary
obstruction, severe liver disease, bile salt deconjugation due to
bacterial overgrowth in the small intestine.
iii. Diseases of small intestine: tropical sprue, celiac disease,
Whipple’s disease.

Test for Malabsorption of Fat
Tests for fecal fat are
• qualitative (i.e. direct microscopic examination after fat
staining)
• quantitative (i.e. estimation of fat by gravimetric or titrimetric
analysis).

• A random specimen of stool is
collected after putting the patient
on a diet of > 80 gm fat per day.
• Stool sample is stained with a fat
stain (oil red O, Sudan III or Sudan
IV) and observed under the
microscope for
fat globules.
• Presence of ≥ 60 fat droplets/HPF
indicates steatorrhea. Ingestion of
mineral or castor oil and use of
rectal suppositories can cause
problems in interpretation. Sudan stain on fecal sample:
(A) Negative; (B) Positive
Microscopic stool examination for fat

Patient should be on a diet of 70-100 gm of fat per day for 6
days before the test.
Stool samples are collected over 72 hours and stored in a
refrigerator during the collection period.
Samples should not be contaminated with urine.
Fat quantitation can be done by gravimetric or titrimetric
method.
In gravimetric method, an accurately weighed sample of
feces is emulsified, acidified, and fat is extracted in a
solvent; after evaporation of solvent, fat is weighed as a
pure compound.
Quantitative estimation of fecal fat:

• Titrimetric analysis is the most widely used method.
• An accurately weighed stool sample is treated with
alcoholic potassium hydroxide to convert fat into
soaps.
• Soaps are then converted to fatty acids by the
addition of hydrochloric acid.
• Fatty acids are extracted in a solvent and the solvent
is evaporated.
• The solution of fat made in neutral alcohol is then
titrated against sodium hydroxide.

• Fatty acids comprise about 80% of fecal fat.
• Values >7 grams/day are usually abnormal.
• Values >14 grams/day are specific for diseases
causing fat malabsorption.

• Fecal urobilinogen is determined by Ehrlich’s aldehyde test
• Sample should be fresh and kept protected from light.
• Normal amount of urobilinogen excreted in feces is 50-300 mg
per day.
• Increased fecal excretion of urobilinogen is seen in hemolytic
anemia.
• Urobilinogen is decreased in biliary tract obstruction, severe
liver disease, oral antibiotic therapy (disturbance of intestinal
bacterial flora) & aplastic anemia (low hemoglobin turnover).
• Stools become pale or clay-colored if urobilinogen is reduced
or absent.
Test for Urobilinogen in Feces

• Deficiency of intestinal enzyme lactase which converts lactose
(in milk) to glucose and galactose, is a common cause of
malabsorption.
• If lactase is deficient, lactose is converted to lactic acid with
production of gas which leads to diarrhea, vomiting, and
failure to thrive in infants.
• Benedict’s test / ClinitestTM tablet test for reducing sugars is
used to test freshly collected stool sample for lactose.
Test for Reducing Sugars

Trypsin in Stools & Fecal Chymotrypsin
• Trypsin is a proteolytic enzyme formed in the small intestine &
it is destroyed by bacteria in older children and adults.
• Inadequate trypsin secretion can lead to malabsorption &
abdominal discomfort.
• No trypsin activity is detectable in constipated stools owing to
prolonged exposure to intestinal bacteria, which inactivates
trypsin.
• Chymotrypsin, an intestinal proteolytic enzyme secreted by
the pancreas, can be used to assess pancreatic function.
• Fecal chymotrypsin is a more reliable measurement of
pancreatic function than trypsin.

Stool Electrolytes: Sodium, Chloride, Potassium &
Osmolality
• Stool electrolyte tests are used to assess electrolyte imbalance in
patients with diarrhea.
• Stool electrolytes must be evaluated along with the serum and
urine electrolytes as well as clinical findings in the
• patient.
• Stool osmolality is used in conjunction with blood serum
osmolality to calculate the osmotic gap and to diagnose intestinal
disaccharide deficiency.

Fecal Osmotic Gap
• Fecal osmotic gap is calculated from concentration of
electrolytes in stool water by the formula 290-2([Na+] + [K+]).
(290 is the assumed plasma osmolality).
• In osmotic diarrhea, osmotic gap >150 mOsm/kg.
• In secretory diarrhea, osmotic gap < 50 mOsm/kg.
Fecal pH
• Stool pH below 5.6 is characteristic of carbohydrate
malabsorption.

EVALUATION OF CHRONIC DIARRHEA

• Bulk: 100-200 grams/day
• Color: Brown
• Water: Up to 75%
• pH: 7.0-7.5
• Red blood cells: Absent
• White blood cells: Few
• Epithelial cells: Present
• Crystals: Calcium oxalate, triple phosphate
REFERENCE RANGES IN
STOOL EXAMINATION

REFERENCE RANGES IN
STOOL EXAMINATION
• Fat (Adults): <7 grams/day (gravimetric method), <6
• grams/day (titrimetric method)
• Fat droplets: Average 2.5 per high power field in random
• sample
• Urobilinogen: 50-300 mg/24 hours
• Parasites: Nil
• Ova, cysts, trophozoites: Nil
Critical Values
• Stool culture positive for Salmonella, Shigella, Campylobacter,
Vibrio, or Yersinia.

Stool Examination

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