Indications

 To identify the parasitic causes of blood and mucus in

faeces and differentiate amoebic dysentery from bacterial
dysentery.
 To identify intestinal parasitic infections that require
treatment
 To identify chronic significant infections that if not treated
can lead to serious complications developing later in life,
e.g. intestinal schistosomiasis leading to portal
hypertension
 To detect serious hookworm infection in patients
with severe iron deficiency anemia, especially in a
pregnant woman or child.
 To assist in the surveillance and control of local
parasitic infections caused by geohelminths (soil
transmitted nematodes), and helminths
transmitted by the ingestion of infected meat,
freshwater fish, crabs, or shell-fish.
Collection of faecal specimen for
parasitic examination
 For clinical purposes, a fresh faecal specimen is required.
 It should be uncontaminated with urine and collected into
a suitable size, clean, dry, leak-proof container.
 The container need not be sterile but must be free of all
traces of antiseptics and disinfectants.
 A large teaspoon amount of faeces is adequate or about 10
ml of a fluid specimen.
 Several specimens collected on alternate days may be
required to detect parasites that are excreted
intermittently, e.g. Giardia
 In the case of Infants, collect from the diaper.
 Caution:
Faecal specimens, like other specimens received in the
laboratory, must be handled with care to avoid acquiring
infection from infectious parasites, bacteria, or viruses.
Stool preservatives
1. Preservatives for the wet preparation are:
 1. 10% formol-saline for the wet preparation. This is best
preservatives as it kills the bacteria and preserves the
protozoa and helminths.
 2. Sodium acetate formalin.
 3. Methionate iodine formalin. This is a good preservative for
the field collection of the stool.

2. For staining use Polyvinyl alcohol.

3. Avoid preservatives for the culture of stool.

 Usually three parts of the preservatives and one part of the
stool.
 The stool is examined;
1. Grossly
2. Microscopically
3. Chemically
Gross Examination
 Gross Stool examination includes:
1. Color.
2. Consistency.
3. Quantity.
4. Odor.
5. Mucous.
6. Helminths.
7. Concretions (gallbladder stone rarely may be found).
Chemical Examination
 The chemical examination includes:
1. Stool pH.
2. Reducing substances.
3. For occult blood.
4. Presence of fat, carbohydrate, and proteins.
Microscopic Examination
 Microscopic examination includes:
1. Presence of leukocytes (pus cells).
2. Presence of Red Blood Cells.
3. Ova and parasites.
4. Presence of meat fibers and muscle fibers.
5. Presence of fat.
6. Yeast and molds.
Consistency
 Normal is soft and formed.
 Loosely formed stools.
 watery stools.
 Thin stools.
 Dry or hard stools found in constipated patients.
 small round hard stool is due to habitual constipation.
 Cystic fibrosis due to pancreatic involvement and are
greasy.
 Diarrheal stools are watery.
 Constipated stools are firm and may see spherical masses.
 The very hard stool is due to excessive absorption of water
due to prolonged contact with colonic mucosa.
Color
 Normal color is due to the presence of stercobilinogen.
 Yellow or yellow-green color is seen in diarrhea.
 Black and tarry (related with consistency) stools are due to
bleeding of upper GI tract from tumors.
 Maroon or pink color is from lower GI tract due to tumors,
hemorrhoids, fissure, or inflammatory process.
 Clay-colored stools are due to biliary tract obstruction.
 Mucous in the stool indicate constipation, colitis or
malignancy.
 Pale color with greasy appearance is due to pancreatic
deficiency leading to malabsorption.
 1. Brown, dark brown or yellow-brown
 Normal color is due to oxidation of bile pigments.
 2. Gray color
 Ingestion of chocolate or Cocoa. steatorrhea.
 3. Green color
 Ingestion of spinach, and chlorophyl vegetables, administration of calomel.
 4. Black (Taary black)
 Iron or bismuth ingestion, bleeding from the upper GI tract.
 5. Very dark brown
 Diet high in meat.
 6. Red color
 Diet high in beats, laxatives of vegetable origin, Bleeding from lower GI tract.
 7. Green or yellow-green
 Diet high in spinach, green vegetables.
 8. Red streaks of blood on feces
 Bleeding from the hemorrhoids, fissure, ulcerative lesion or carcinoma of rectum or anus.
Mucous
1. Mucous is produced by the mucosa of the colon in response to parasympathetic stimulation
2. Pure mucous is translucent gelatinous material clinging to the surface of the stool. This may be seen in:
 1. Severe constipation.
 2. Mucous colitis.
 3. Excessive straining of the stool.
 4. emotionally unstable patient.
3. Mucous in diarrhea with microscopically present with RBCs and WBCs is seen in:
 1. Bacillary dysentery.
 2. Ulcerative colitis.
 3. Intestinal tuberculosis.
 4. amoebiasis.
 5. Enteritis.
 6. Acute diverticulitis.
 7. ulcerating malignancy of the colon.
4. Mucus with blood which is clinging to stool is seen in:
 1. Malignancies of the colon.
 2. Inflammatory lesion of rectal canal.
5. An excessive amount of mucus seen in:
 1. Villous adenoma of the colon.
 2. This depends upon the dietary intake.
pH
 Normally stool is slightly acidic or alkaline or neutral.
 pH is 7.0 to 7.5 depending on the diet.
 Newborn pH = 5.0 to 7.5.
 pH of the stool depends upon the diet and bacterial fermentation in the small intestine.
 Carbohydrate changes the pH to acidic while the protein breakdown changes to alkaline.
 1. Breastfed infants pH has a slightly acidic stool.
 2. Bottle fed infants have a slightly alkaline stool.
 pH stool test helps to evaluate carbohydrate and fat malabsorption.
 pH stool also helps to know disaccharidase deficiency.
 Alkaline (Increased pH) stool is seen in:
 1. Colitis.
 2. Villous adenoma.
 3. Diarrhea.
 4. Antibiotic therapy.
 5. Excess intake of proteins.
 Acidic (Decreased pH) stool seen in:
 Fat malabsorption.
 Disaccharidase deficiency.
 Carbohydrate malabsorption.
 Excess intake of carbohydrates.
Reducing Substances
 Sample:
 This is done on the stool of infants or adult.
 A small amount of stool is needed, just 5 grams are enough.
 Precaution
 Stool should be delivered to the laboratory as soon as
possible, preferably within 1 hour.
 Because lactose (or other sugars) in the stool will normally
be broken down by chemical processes within 2 to 4 hours.
 Avoid contamination with urine or other material like
water or toilet paper.
 Bacterial fermentation may give the false low result
 Indication:
 To diagnose the intolerance to disaccharides.
 Pathophysiology
 1. Normally sugars are rapidly absorbed in the upper small
intestine.
 2. If sugars are not absorbed then these produce diarrhea
due to the osmotic pressure produced by these unabsorbed
sugars.
 3. These unabsorbed sugars draw fluid and electrolytes into
the intestine.
 4. These unabsorbed sugars are measured as reducing
substances.
How to test reducing substances:
 Mostly there are commercial devices available e.g Clinitest
(Benedict's solution) and etc.
 A yellow-brown color indicates reducing substances.
 This color indicates ++ sugars (lactose).
 Glucose oxidase reagent strip.
 Copper reduction reagent tablet.
 Also, check the pH of the stool.
 Another method is:
 Give a load of lactose.
 In case of deficiency of lactase, there will be no increase in
the glucose level.
 Increased reducing substances are seen in:
 Disaccharidase enzyme deficiency in the intestine.
 Short bowel syndrome.
 Idiopathic lactase deficiency leads to lactose
intolerance.
 Carbohydrate malabsorption saw in :
 Sprue.
 Viral gastroenteritis.
 Celiac disease.
Stool for Occult Blood, OB
(Fecal occult blood)
 Sample:
 This test is done on the stool.
 The random sample can be taken and 3 mL quantity is
enough.
 Avoid the outer portion and take a sample from the central
area of the formed stool.
 Three consecutive stools samples are needed.
 Collect the stool in dry, sterilized wide mouth container.
 Instruct the patient to stop vit.C, iron-containing drugs,
meat and vegetable for at least three days prior to the test.
 Fresh stool testing is recommended.
Indication:
 This is a screening test for colorectal carcinoma.
 This can be used for the bleeding ulcer in the
gastrointestinal tract.
 This test can be included in the periodic medical
checkup.
 This test is advised in older people after the age of 40
which is asymptomatic to rule out GI malignancy
 There is a presence of hemoglobin in the stool, evidenced
by the chemical test but not seen by the naked eye.
 Occult blood is hidden and it requires a chemical test for
its detection.
 Occult blood will be positive when the tumors grow in the
lumen of the intestine and if it ulcerate and give rise to
bleeding.
 Bleeding in the upper GI tract produces a black tarry stool.
 While bleeding from the lower GI tract produces just blood
in the stool.
 Heme has a peroxidase-like activity which is detected by
the Guaiac dye test.
Procedure
 Chemical method:
 This is a modification of the benzidine and 0-tolidine test because these are
carcinogenic.
 1. Take 95% alcohol 15 mL.
 2. Dissolve 4- aminophenazone 0.4 grams.
 3. Add Acetic acid 10% 1 mL.
 4. H O (Hydrogen peroxide)1 volume and 10 ml of water.
 1. Take 10 to 15 mL of D.water and emulsify the stool (10 mm in diameter).
 2. Now centrifuge and take the clear emulsified fluid.
 3. Take three tubes and label those as a patient, Negative control, and positive control.
 4. Add 5 mL of emulsified stool material to the patient tube.
 5. Add D.water to the negative control.
 6. Add one drop blood to the positive control.
 3. Now layer 5 mL of the mixed aminophenazone prepared reagent above the
suspension and don't mix. Just layer it over the suspension.
 4. Now add 10 drops of Hydrogen peroxide and don't mix
 5. Check the result as follows.
 Every time you have to make a fresh sample to run this
test.
 The false positive test is seen if the patient stool has a
peroxidase-like substance.
 The false negative reaction is seen in case of ascorbic acid
excess in the stool.
 In case of suspected case repeat the test for two more times.
False positive OB test is seen in:
1. Ingested meat.
2. Peroxidase rich vegetables like turnip, horseradish,
mushroom, broccoli, beans, sprouts, cauliflower,
oranges, bananas, cantaloupe, and grapes.
3. Drugs may lead to bleeding like anticoagulants,
aspirin, iron preparation, nonsteroidal antiarthritic
drugs, and steroids.
4. White cells and bacteria also cause a false positive test.
 False negative OB test is seen in:
 1. Vit C may cause a false negative.
 2. Oxidants also cause a false negative result.
Causes of positive OB test
 Gastrointestinal tumors.
 Rectal carcinoma.
 Gastric carcinoma.
 Inflammatory bowel disease.
 Diverticulosis.
 Varices.
 Ischemic bowel disease.
 Arteriovenous malformations of GI tract.
 Hemorrhoids.
 Blood swallowed from the oral cavity or nasopharynx.
 Adenoma.
 Peptic ulcer.
 Gastritis.
 Amyloidosis.
 Kaposi’s sarcoma.
 We examine stool by following methods:
 Direct Examination
 Faecal Concentration Techniques
 Formol ether concentration technique
 Zinc sulphate floatation technique
 Saturated sodium chloride floatation technique
DIRECT EXAMINATION
 Routinely faecal specimens are examined in district
laboratories by direct technique. This involves:
 Reporting the appearance of the specimen and
identifying any parasitic worms or tapeworm
segments.
 Examining the specimen microscopically for:
 – motile parasites such as the larvae of S. stercoralis and
trophozoites of E. histolytica, G. lamblia, and more
rarely, B. coli
 – helminth eggs,
 – cysts and oocysts of intestinal protozoa.
FAECAL CONCENTRATION
TECHNIQUES
 The number of parasitic forms of both protozoan and/or
helminthic parasites) in fecal specimens is often too low to
be observed microscopically in direct wet mounts or in
stained smear preparation.
 In such cases, the use of concentration techniques
increases the chances of detecting parasitic organisms,
thus increasing the sensitivity of copromicroscopic
techniques.
 The two most commonly used stool concentration
techniques are sedimentation and flotation.
 Sedimentation techniques are preformed commonly in
general diagnostic laboratories because they are easier to
perform and less prone to technical errors.
Formal Ether Sedimentation
Technique
 Sedimentation techniques use solutions of lower specific
gravity than the parasitic organisms, thus concentrating
the latter in the sediment.

 It takes advantage of the high specific gravity of protozoan

cysts and helminth eggs compared to water. Their natural
tendency to settle out in aqueous solutions can be
accelerated by light centrifugation.

 Formalin fixes the eggs, larvae, oocysts, and spores, so that

they are no longer infectious, as well as preserves their
morphology. Fecal debris is extracted into the ethyl acetate
phase of the solution. Parasitic elements are sedimented at
the bottom.
 Materials required
 Glass container
 Guaze
 Funnel
 Centrifuge tube (15ml capacity)
 Centrifuge
 Physiological saline (0.85% w/v Nacl)
 10% buffered formalin
 Ether (ethyl acetate)
 Test tubes with stopper
 Glass rod
 Iodine
 Microscope
Procedure for Formal Ether
Sedimenation Technique
 Wear gloves when handling stool specimens.
 In a suitable container, thoroughly mix a portion of stool
specimen about the size of a walnut into 10mL of saline solution.
Mix thoroughly.
 Filter the emulsion through fine mesh gauze into a conical
centrifuge tube.
 Centrifuge the suspension at relative centrifugal force (RCF) of
600 g (about 2000 rpm) for no less than 10 minutes. The
suspension should yield about 0.75mL of sediment for fresh
specimens and 0.5 mL for formalinized feces.
 Decant the supernatant and wash the sediment with 10 mL of
saline solution. Centrifuge again and repeat washing until
supernatant is clear.
 After the last wash, decant the supernatant and add 10 mL
of 10% formalin to the sediment. Mix and let stand for 5
minutes to effect fixation.
 Add 1 to 2 mL of ethyl acetate, Stopper the tube and shake
vigorously.
 Centrifuge at 450 g RCF (about 1500 rpm) for 10 minutes.
Four layers should result as follows
 a top layer of ethyl acetate;
 plug of debris;
 layer of formalin; and
 sediment
 Free the plug of debris from the side of the tube by
ringing with an applicator stick. Carefully decant the
top three layers.
 With a pipette, mix the remaining sediment with the
small amount or remaining fluid and transfer one drop
each to a drop of saline and iodine on a glass slide.
Cover with a coverslip and examine microscopically for
the presence of parasitic forms.
Observation and Results
 Systematically examine the entire surface of each
coverslip with the 10x objective or, if needed for
identification, higher power objectives of the
microscope in a systematic manner so that the entire
coverslip area is observed. When organisms or
suspicious objects are seen, switch to higher
magnification (40X) to see more detailed morphology
of the object in question.
Zinc sulphate floatation technique
 A zinc sulphate solution is used which has a specific
gravity (relative density) of 1.180–1.200. Faeces are
emulsified in the solution and the suspension is left
undisturbed for the eggs and cysts to float to the
surface. They are collected on a cover glass.
 Saturated sodium chloride
floatation technique
 The saturated sodium chloride technique is a useful and
inexpensive method of concentrating hookworm or Ascaris eggs,
e.g. in field surveys.
 Stir sodium chloride (e.g. table salt) into hot clean distilled or
boiled filtered water until no more can be dissolved. Add a few
more grams of the salt so that a layer of the undissolved salt
remains in the bottom of the stock container.
 Mix well and leave the undissolved salt to sediment. When cool,
filter some of the solution and use a hydrometer to check that
the specific gravity is 1.200.
 Immediately before use, filter the amount of solution required
from the stock bottle and recheck the specific gravity.
Leukocytes
 Normally there are no WBCs.
 WBCs only appear in infection or inflammation.
 Their presence is important in case of diarrhea or dysentery.
 >3 WBCs /high field are seen in ulcerative colitis and bacterial infection.
 Greater numbers of WBCs indicate invasive pathogens.
 Virus and parasites don’t cause WBCs in the stool.
 Increased number of WBCs in the stool.
 Bacillary dysentery.
 chronic ulcerative colitis.
 Shigellosis.
 salmonella infection.
 Yersinia infection.
 Invasive E.coli diarrhea.
 Fistula of anus or rectum.
 Localized abscess.
 Typhoid
 Few WBCs are seen in amoebiasis.
 The absence of WBCs seen in some of the diarrhoeal
conditions alike:
 Cholera.
 Viral diarrhea.
 Drug-induced diarrhea.
 Amoebic colitis.
 Non-invasive E.coli diarrhoea.
 Parasitic infestation.
 Toxigenic bacterial infection.
Red Blood Cells
 Blood in the stool can be:
 Bright red from the bleeding in the lower GI tract.
 Maroon in color.
 Black and tarry from bleeding from the upper GI tract.
 Occult blood (not visible to the naked eye).
 Causes of blood in stool:
 Hemorrhoids.
 Cancer.
 Dysentery.
Ova and parasites
 Normally there are no parasites or eggs in the stool sample.
 Multiple stool sample is needed to rule out the parasitic infestation, at
least three consecutive days.
 An abnormal result means parasites or eggs are present in the stool.
Such infections include:
 Roundworms: Ascaris lumbricoides.
 Hookworms: Necator americanus.
 Pinworms: Enterobius vermicularis.
 Whipworm: Trichuris trichiura.
 Tapeworms: Diphyllobothrium latum, Taenia saginata, and Taenia
solium
 Protozoa: Entamoeba histolytica (an amoeba), and Giardia lamblia (a
flagellate)
 Strongyloidiasis.
Fat
 The fat in the stool shows the possibility of:
 Malabsorption.
 Deficiency of pancreatic digestive enzyme.
 Deficiency of Bile.