Stool Examination

STOOL
EXAMINATION
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
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.
• INTRODUCTION
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
• INTRODUCTION
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 Biliary obstruction

(gray-white)
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, Bacillary dysentery, Ulcerative Colitis, Intestinal tuberculosis,

mucus, and pus Amoebiasis, Enteritis
Unformed, frothy, foul Steatorrhoea

smelling, which float on
water
Pale color stool with Pancreatic deficiency due to malabsorption
greasy appearance
• Odor
• Stool odor – Indole and skatole which are formation
by bacterial fermentation and putrefaction.
• Foul odor – Undigested protein & by excessive
intake of carbohydrate.
• Sickly odor – 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
• INTRODUCTION
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.
• INTRODUCTION
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.
Formol-ethyl acetate sedimentation method
• 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.
• INTRODUCTION
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
Red Blood Cells (RBCs)

• 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
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
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 • Cytoplasm shows outer transparent
ectoplasm and inner finely granular
examined to increase sensitivity. endoplasm.
• Trophozoites vary from 15 to 40 • Nucleus is visible in the iodine
preparation.
μ in diameter. • Fine granules of peripheral nuclear
• In saline wet mounts, chromatin are evenly distributed.
Small, single central karyosome.
trophozoites show motility in (Motility is lost in iodine mount).
one direction via pseudopodia,
which form rapidly.
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
• 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
Uninucleate, binucleate, trinucleate,
are oblong structures with rounded and quadrinucleate cysts of Entamoeba
ends and glycogen clumps – infective histolytica
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 Blood and mucus with little or
mucus 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 Scanty Abundant
• Red cells Clumps Discrete
• Pus cells Nil or few Numerous
• Macrophages Few Many, some with ingested RBCs
• Eosinophils Present Absent
• Charcot-Leyden crystals May be present Absent
• Bacteria Many, motile Few, nonmotile
• Trophozoites of E. histolytica Motile trophozoites with Absent
ingested red blood cells
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
Giardia intestinalis (lamblia)
• 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”.
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
• 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 Immature oocyst showing
contain 2 sporoblasts. 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 Mature oocyst with 2
autofluorescence. sporocysts containing
sporozoites
Coccidia - Isospora belli
With modified Ziehl-Neelsen stain (on a stool Under ultraviolet light, oocysts show
smear prepared from the sediment after autofluorescence.
formalin-ether concentration), oocysts stain
uniform red-pink
Coccidia - Cryptosporidium parvum
• 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 in stools Cryptosporidium oocysts appear with a

by modified Ziehl- Neelsen staining peripheral green fluorescence
Coccidia - Cyclospora cayetanensis
• 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 Under ultraviolet light (365 nm), oocysts show
appear similar to C. parvum, but are intense blue autofluorescence.
larger.
Microsporidia
• 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

Ascaris lumbricoides (Roundworm)
• 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.
MICROSCOPIC EXAMINATION - HELMINTHS
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
• 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
• 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 Hookworm egg in fresh stools.
Egg in oval shape with a thin shell, has a
be seen. clear space between wall and developing
cleavage, and contains granular cells
• 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.
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.
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
• INTRODUCTION
CHEMICAL EXAMINATION
Chemical examination of feces is usually carried out for the

following tests :
• Occult blood • Reducing sugars
• Excess fat excretion (malabsorption) • stool osmotic gap
• Urobilinogen • 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
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.
• 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.
Test for Occult Blood in Stools - Peroxidase-like activity of Hb

Causes of False +ve Tests Causes of False -ve Tests
1. Ingestion of peroxidase- 1. Foods containing large amounts

containing foods like red meat, of vitamin C.
fish, poultry, turnips, 2. Conversion of all hemoglobin to
horseradish, cauliflower, acid hematin (which has no
spinach, or cucumber. Diet peroxidase-like activity) during
should be free from peroxidase- passage through the
containing foods for at least 3 gastrointestinal tract.
days prior to testing.
2. Drugs like aspirin and other anti-
inflammatory drugs, which
increase blood loss from GITin
normal persons.
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).
• Microscopic stool examination for fat
• 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
Quantitative estimation of fecal 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.
• 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.
Test for Urobilinogen in Feces
• 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 Reducing Sugars
• 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.
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.
REFERENCE RANGES IN STOOL EXAMINATION
• 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
• 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.
THANKS

Stool Examination

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Stool Examination

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STOOL

• Normal amount of stools in an adult is 100-200 g/day.

Examination of stools aids in investigation of GIT

Examination of stools aids in investigation of GIT

Examination of stools aids in investigation of GIT diseases.

Examination of stools aids in investigation of GIT diseases.

• Sample may be refrigerated if delay in examination is

• Cysts are likely to be found in formed stools.

Black and tarry Bleeding in upper GIT(proximal to cecum)

Clay-colored Biliary obstruction

Watery Certain strains of Escherichia coli, Rotavirus enteritis,

Unformed with blood, Bacillary dysentery, Ulcerative Colitis, Intestinal tuberculosis,

Unformed, frothy, foul Steatorrhoea

Presence of mucus in stools

Presence of mucus and blood in stools

Mucus with blood clinging to stool is seen in

• Useful if very small numbers of parasites are present.

Concentration techniques are of two main types:

The most commonly used sedimentation method is

• Normal stool may contain

Increased no: of WBCs are stools is associated with

Red Blood Cells (RBCs)

• Black and tarry blood are seen

Their presence show impaired

• Increased amount of meat

• Normally there are no parasites/eggs in the stool

Diagnostic feature of E. histolytica

Trophozoite of Entamoeba histolytica

Entamoeba histolytica cyst (arrow)

Cause Entamoeba histolytica Shigella (most common)

Parameter Amoebic dysentery Bacillary dysentery

Microscopic examination of stool

• Cysts are more likely to be found

• Cysts of G. lamblia are 8-12 μ in

Cryptosporidium oocysts in stools Cryptosporidium oocysts appear with a

Microsporidia (arrows) in stool stained with modified acid-fast trichrome

(A) Adult female and male worms

• These eggs do not have the unevenly

• Diagnosis is based on identification of hookworm eggs on stool

• Buccal cavity of hookworm larva is longer which distinguishes itself from

• Additional findings in blood -

Yeast & fungal elements in stool

Pollen grains in stools mistaken for helminth eggs.

Plant fibre/Plant cells.

Chemical examination of feces is usually carried out for the

• Benzidine and orthotolidine are carcinogenic and are no longer

Test for Occult Blood in Stools - Peroxidase-like activity of Hb

1. Ingestion of peroxidase- 1. Foods containing large amounts

i. Deficiency of pancreatic lipase (insufficient lipolysis): chronic

Tests for fecal fat are

Patient should be on a diet of 70-100 gm of fat per day for 6

Quantitative estimation of fecal fat:

• Fatty acids comprise about 80% of fecal fat.

• Bulk: 100-200 grams/day

• Fat (Adults): <7 grams/day (gravimetric method), <6

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