The document provides information on stool examination for medical graduates. It discusses the advantages of stool examination in investigating gastrointestinal diseases such as detecting parasites, evaluating chronic diarrhea, dysentery, and identifying causative bacteria. It describes the collection and macroscopic examination of stool samples and preparation of slides for microscopic examination. The microscopic examination may reveal leukocytes, red blood cells, fat, meat fibers, and ova/cysts/trophozoites of parasites. Concentration techniques like sedimentation and floatation are used if parasites are present in low numbers. Common parasites like Entamoeba histolytica and Giardia intestinalis are also described.
2. LEARNING OBJECTIVES
⢠INTRODUCTION
⢠ADVANTAGES OF STOOL EXAMINATION
⢠COLLECTION OF STOOLS SAMPLES
⢠MACROSCOPIC EXAMINATION
⢠PREPARATION OF SLIDES
⢠CONCENTRATION OF STOOL SAMPLES
⢠MICROSCOPIC EXAMINATION
⢠CHEMICAL EXAMINATION
3. INTRODUCTION
STOOL EXAMINATION
⢠It is a neglected part of laboratory test possibly
because it is not very aesthetic.
⢠It is not routinely done, but only in indicated cases.
⢠It can yield useful information in many GIT diseases.
⢠It is non-invasive and the test are simple to do.
4. ⢠INTRODUCTION
⢠ADVANTAGES OF STOOL EXAMINATION
⢠COLLECTION OF STOOL SAMPLES
⢠MACROSCOPIC EXAMINATION
⢠PREPARATION OF SLIDES
⢠CONCENTRATION OF STOOL SAMPLES
⢠MICROSCOPIC EXAMINATION
⢠CHEMICAL EXAMINATION
5. ADVANTAGES OF STOOL EXAMINATION
Examination of stools aids in investigation of GIT
diseases.
⢠Detection of parasites :
â detection of worms (adult worms, tapeworms, larvae, ova)
â detection of protozoa (trophozoites or cysts).
⢠Evaluation of chronic diarrhea
â Random diarrheal sample tested for occult blood, fat, pH,
white blood cells, culture, or microscopy.
â A 48- or 72-hour sample is examined for weight, fat content,
carbohydrate, osmolality or chymotrypsin activity.
6. ADVANTAGES OF STOOL EXAMINATION
Examination of stools aids in investigation of GIT
diseases.
⢠Evaluation of dysentery:
â Identification of causative organism is definitive in amebic V/S bacillary
dysentery.
⢠Bacteriologic examination:
â Infection by bacteria such as Salmonella, Shigella, Vibrio, Yersinia, or
Clostridium difficile can be identified by stool culture.
â Bacterial toxins released by Clostridium botulinum or Clostridium difficile
can also be identified.
7. ADVANTAGES OF STOOL EXAMINATION
Examination of stools aids in investigation of GIT diseases.
⢠Chemical examination:
â to detect occult blood (in ulcerated lesions of gastrointestinal tract,
especially occult carcinoma of colon),
â excess fat excretion (malabsorption syndrome)
â 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):
â Increased numbers of polymorphonuclear neutrophils (identified
by methylene blue stain) are seen in the former
8. ADVANTAGES OF STOOL EXAMINATION
Examination of stools aids in investigation of GIT diseases.
⢠Identification of Rotavirus:
â common cause of diarrhea in infants & young children.
â It can be identified by examination of stool by electron
microscopy, latex agglutination, immunofluorescence or
ELISA.
9. ⢠INTRODUCTION
⢠ADVANTAGES OF STOOL EXAMINATION
⢠COLLECTION OF STOOL SAMPLES
⢠MACROSCOPIC EXAMINATION
⢠PREPARATION OF SLIDES
⢠CONCENTRATION OF STOOL SAMPLES
⢠MICROSCOPIC EXAMINATION
⢠CHEMICAL EXAMINATION
10. 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.
ďź Urine & water destroy trophozoites.
ďź Soil will introduce extraneous organisms & also hinder
proper examination.
11. 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.
â 10% formalin for preservation of eggs, larvae & cysts.
â 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.
â One negative report for ova and parasites does not exclude
the possibility of infection
12. ⢠INTRODUCTION
⢠ADVANTAGES OF STOOL EXAMINATION
⢠COLLECTION OF STOOL SAMPLES
⢠MACROSCOPIC EXAMINATION
⢠PREPARATION OF SLIDES
⢠CONCENTRATION OF STOOL SAMPLES
⢠MICROSCOPIC EXAMINATION
⢠CHEMICAL EXAMINATION
13. MACROSCOPIC EXAMINATION
Findings :-
⢠Consistency
⢠Color
⢠Odor
⢠Presence of blood
⢠Presence of mucus
⢠Presence of adult worms or segments of tapeworms
14. MACROSCOPIC EXAMINATION
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
15. MACROSCOPIC EXAMINATION
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
16. MACROSCOPIC EXAMINATION
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
17. ⢠INTRODUCTION
⢠ADVANTAGES OF STOOL EXAMINATION
⢠COLLECTION OF STOOL SAMPLES
⢠MACROSCOPIC EXAMINATION
⢠PREPARATION OF SLIDES
⢠CONCENTRATION OF STOOL SAMPLES
⢠MICROSCOPIC EXAMINATION
⢠CHEMICAL EXAMINATION
18. 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.
19. 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.
20. ⢠INTRODUCTION
⢠ADVANTAGES OF STOOL EXAMINATION
⢠COLLECTION OF STOOL SAMPLES
⢠MACROSCOPIC EXAMINATION
⢠PREPARATION OF SLIDES
⢠CONCENTRATION OF STOOL SAMPLES
⢠MICROSCOPIC EXAMINATION
⢠CHEMICAL EXAMINATION
21. 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.
22. CONCENTRATION OF STOOL SAMPLES
Concentration techniques are of two main types:
⢠Sedimentation techniques :
â Ova and cysts settle at the bottom.
â Excessive stool debris may make the detection of parasites
difficult.
â e.g. Formolethyl acetate sedimentation procedure.
⢠Floatation techniques :
â Ova and cysts float on surface.
â Some ova and cysts do not float at the top in this procedure.
â e.g. Saturated salt floatation technique, zinc sulphate
concentration technique.
23. CONCENTRATION OF STOOL SAMPLES
⢠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
24. ⢠INTRODUCTION
⢠ADVANTAGES OF STOOL EXAMINATION
⢠COLLECTION OF STOOL SAMPLES
⢠MACROSCOPIC EXAMINATION
⢠PREPARATION OF SLIDES
⢠CONCENTRATION OF STOOL SAMPLES
⢠MICROSCOPIC EXAMINATION
⢠CHEMICAL EXAMINATION
27. MICROSCOPIC EXAMINATION
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.
28. MICROSCOPIC EXAMINATION
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 daysâ stool samples are
examined to rule out parasitic infestations
30. 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
Îź 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).
31. MICROSCOPIC EXAMINATION - PROTOZOA
Entamoeba histolytica
Diagnostic feature of E. histolytica
trophozoites is the presence of
ingested red cells.
Trophozoite of Entamoeba histolytica
showing ingested red cells
(Trichrome stain)
33. MICROSCOPIC EXAMINATION - PROTOZOA
Entamoeba histolytica
Uninucleate, binucleate, trinucleate, &
quadrinucleate cysts of Entamoeba histolytica
⢠Cysts of E. histolytica are spherical &
measure 10-15 Îź in diameter.
⢠Nuclei are 1, 2, 3, or 4 & 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 & central.
⢠Immature cysts may show chromatoid
bodies (aggregates of ribosomes) that
are oblong structures with rounded
ends & glycogen clumps â infective
stage.
35. MICROSCOPIC EXAMINATION - PROTOZOA
Entamoeba histolytica
⢠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.
36. MICROSCOPIC EXAMINATION - PROTOZOA
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
37. MICROSCOPIC EXAMINATION - PROTOZOA
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, non-motile
Absent
Antigen test for E. histolytica Positive Negative
Stool culture Negative Positive for Shigella
38. MICROSCOPIC EXAMINATION - PROTOZOA
Giardia intestinalis (lamblia)
⢠Cysts are more likely to be found
in formed or loose stools.
⢠Cysts of G. lamblia are 8-12 Ο in
diameter, oval and contain 4
nuclei, axonemes, median
bodies and remains of flagella
39. MICROSCOPIC EXAMINATION - PROTOZOA
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 Îź 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
âfalling leafâ.
41. (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.
Ascaris lumbricoides (Roundworm)
42. MICROSCOPIC EXAMINATION - HELMINTHS
Ascaris lumbricoides (Roundworm)
Unfertilized eggs:
⢠Single female worms discharge eggs
which are slightly larger & elliptical in
shape.
⢠Size : 80 Οm x 55 Οm.
⢠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.
43. MICROSCOPIC EXAMINATION - HELMINTHS
Fertilized eggs:
⢠Eggs are round, oval, golden brown in
color & always bile stained.
⢠Size : 70 Ο à 50 Ο 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.
Ascaris lumbricoides (Roundworm)
44. MICROSCOPIC EXAMINATION - HELMINTHS
Ascaris lumbricoides (Roundworm)
Decorticated eggs :
⢠These eggs do not have the unevenly
thick outer albuminous shell &
resemble the hookworm eggs.
45. MICROSCOPIC EXAMINATION - HELMINTHS
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).
Ascaris lumbricoides (Roundworm)
46. MICROSCOPIC EXAMINATION - HELMINTHS
⢠Hookworms are
â Ancylostoma duodenale (old world hookworm)
â 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
47. MICROSCOPIC EXAMINATION - HELMINTHS
Hookworms
⢠Eggs of A. duodenale & N. americanus are 50-75 Ο
in length and 40 Îź 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
48. MICROSCOPIC EXAMINATION - HELMINTHS
Trichuris trichiura (Whipworm)
Diagnosis depends on identification of typical
eggs on stool examination.
Eggs measure 50 Ă 25 Îź 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.
49. MICROSCOPIC EXAMINATION - HELMINTHS
Hymenolepsis nana
⢠Eggs are oval and measures
30-50 Îź 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
50. MICROSCOPIC EXAMINATION - HELMINTHS
Strongyloides stercoralis
Rhabditiform larva of S. Stercoralis in unstained wet mounts of stool.
Note the short buccal canal, the genital primordium (red arrows) & the longer intestinal tract.
51. MICROSCOPIC EXAMINATION - HELMINTHS
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
(âcellophane tape testâ) 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).
⢠Patientâs 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.
52. MICROSCOPIC EXAMINATION - HELMINTHS
Enterobius vermicularis
⢠Eggs of E.vermicularis measure 60 Ο à 30 Ο 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 small childrenâs feces as
infestation is very common in them.
⢠They are white, motile & small in size (male:
0.5 cm; female: 1 cm).
⢠They are planoconvex under the microscope.
⢠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
54. MICROSCOPIC EXAMINATION - HELMINTHS
Taenia solium - Identification of eggs:
⢠Egg measures 30-40 Ο 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.
55. MICROSCOPIC EXAMINATION - HELMINTHS
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.
56. MICROSCOPIC EXAMINATION - HELMINTHS
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 à 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)
57. MICROSCOPIC EXAMINATION - HELMINTHS
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.
58. MICROSCOPIC EXAMINATION - HELMINTHS
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.
59. MICROSCOPIC EXAMINATION - HELMINTHS
Taenia saginata - Identification of gravid segments or
proglottids:
⢠This allows identification of species.
⢠Segments measure 20 mm à 6 mm in size,
and are ivory white.
⢠They contain a central uterine stem > 13
side branches(i.e. 15-20 branches).
60. MICROSCOPIC EXAMINATION - HELMINTHS
Taenia solium - Identification of scolex (head):
⢠Scolex of T. saginata has 4 suckers but no
hooklets.
⢠It measures 2 mm in width.
61. MICROSCOPIC EXAMINATION
Other uncommon parasites found in stool examination
⢠Balantidium coli
⢠Capillaria philippinensis
⢠Sarcocystis species
⢠Fasciola hepatica
⢠Opisthorchis species
⢠Dipylidium caninum
⢠Metagonimus yokogawai
63. 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
64. 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
Pollen grains in stools mistaken for helminth eggs.
65. 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
Plant fibre/Plant cells.
66. 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
Air bubbles
67. ⢠INTRODUCTION
⢠ADVANTAGES OF STOOL EXAMINATION
⢠COLLECTION OF STOOL SAMPLES
⢠MACROSCOPIC EXAMINATION
⢠PREPARATION OF SLIDES
⢠CONCENTRATION OF STOOL SAMPLES
⢠MICROSCOPIC EXAMINATION
⢠CHEMICAL EXAMINATION
68. 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
69. CHEMICAL EXAMINATION
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
70. CHEMICAL EXAMINATION
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.
71. CHEMICAL EXAMINATION
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
72. CHEMICAL EXAMINATION
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.
73. CHEMICAL EXAMINATION
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
â if blood is of neonatal origin it will contain high proportion of hemoglobin F
(Hb F) that is resistant to alkali denaturation.
â On the other hand, maternal blood mostly contains adult hemoglobin or
Hb A that is less resistant.
74. CHEMICAL EXAMINATION
Malabsorption of Fat
⢠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.
75. CHEMICAL EXAMINATION
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,
Whippleâs disease.
76. CHEMICAL EXAMINATION
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).
77. CHEMICAL EXAMINATION
⢠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 ⼠60 fat droplets/HPF
indicates steatorrhea.
Ingestion of mineral or castor oil and use
of rectal suppositories can cause issues in
interpretation. Sudan stain on fecal sample:
(A) Negative; (B) Positive
Microscopic stool examination for fat
78. CHEMICAL EXAMINATION
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:
79. CHEMICAL EXAMINATION
⢠Fecal urobilinogen is determined by Ehrlichâs 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.
⢠Stools become pale or clay-colored if urobilinogen is
reduced or absent.
Test for Urobilinogen in Feces
80. CHEMICAL EXAMINATION
⢠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.
⢠Benedictâs test / ClinitestTM tablet test for reducing sugars is
used to test freshly collected stool sample for lactose.
Test for Reducing Sugars
81. ⢠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
82. 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.