Stool Examination:- Part 1 – Stool Analysis ,

Complete Stool studies, Example of ova and

parasite,
Sample
1. A random stool sample can be taken.
1. More than 2 grams of the stool is needed, ideally 2 to 5 grams and
sometimes referred to as pigeon’s egg.
2. To rule out worm infestation three consecutive stools are tested.
1. Collect three stools in the span of 10 days.
2. Two samples on alternate days.
1. In the hospitalized patient can take a stool sample every day.
2. Multiple samples are needed to rule out the parasitic infestation.
3. One sample after purgation.
3. Collect the sample in a clean, water-tight dry urine free container with a tight lid.
4. In the case of Infants, collect from the diaper.
5. Or cellophane tape method:
1. This is also known as Scotch tape preparation.
2. This is the best method in infants and children to diagnose pinworms
(Enterobius vermicularis).
3. The female at night when the child is resting comes out of the rectum and
lays eggs in the perianal area.
4. Put the tape at night and collect the tape in the morning.
5. Procedure:
1. Take 10 cms of the transparent adhesive tape.
2. Fold it on tongue depressor with the adhesive side out.
3. Now press the adhesive side of tape over the perianal area and
cover the maximum area. This tape should be applied at night.
4. In the morning remove the tape and put the adhesive side on the
slide.
5. See under the microscope.

Method to prepare the stool smears:

Saline and iodine Direct wet preparations:
Saline wet preparation:

1. Take one drop of 0.85% saline.

2. Take a small amount of stool and mix well.
3. The smear should be thin so that you can see the newsprint under the slide.
4. Put cover glass and see under the microscope 100x and 400x objective.
1. This is best to see helminth eggs, larva, and trophozoites.

Iodine wet preparation:

1. Take a drop of Lugol’s iodine solution.

 2. Take a small amount of stool and mix it well.
3. Make a thin smear.
4. Put the cover glass on it and gently press it to get an evenly thin smear.
5. See under 100x and 400x objective lenses.
1. Too weak iodine solution, in that case, organisms will not stain properly.
2. Too strong iodine solution will clump the stool.

Stool wet preparation

Concentration method
The main aim of the concentration method is to remove the debris.  Also when the
parasite is low in number.
There are three methods used for the concentration of stool:

1. Formalin-ethyl-acetate concentration method.

2. Zinc-floatation method.
3. Sheather sugar floatation method.

The formalin-ethyl-acetate concentration method is most commonly used.

This method recovers the helminth eggs and larvae. To a lesser extent trophozoites.
Principle: This is based on specific gravity. After centrifugation, the parasites present in
the stool are heavier and settles down at the bottom as sediments. Debris is lighter and
rises to the upper layers.
Advantages are:
 1. It is easy to prepare the solution.
2. This is inexpensive.
3. The procedure is easy to perform.
4. There is a rare distortion of parasites forms (eggs).

The procedure of formalin-ethyl-acetate concentration:

1. The stool should be fixed in formalin for at least 30 minutes.

1. Take 2 to 5 grams of the stool and mix thoroughly in the 10% formalin.
2. Filter the above stool in the formalin.
1. This can be done by two layers of gauze or a wire screen and collect
around 3 mL.
3. add 10 to 12  mL of 0.85% saline and mix it well.
4. Centrifuge for 2 minutes at 2000 RPM ( or 2500 RPM).
5. Discard the supernatant and leave 1 to 1.5 mL of the sediment.
1. If the supernatant is cloudy then repeat the above steps of saline.
6. Add 9 mL of 10% formalin to the sediment.
7. Now add 3 mL of ethyl acetate.
8. Cap the test tube and shake well for 30 seconds.
9. Centrifuge the tubes for 1 minute at 2000 RPM.
10. Four layers will form.  The bottom is the sediment which is needed to prepare the
smear.

Stool formalin-ethyl acetate concentration method

11. Remove the debris with a wooden applicator stick. Decant the upper three layers
carefully and leave the sediments in the test tube.
12. Clean the sides of the test tube with a swab.
1. Giardia cyst may stick to the side of the test tube.
13. Add a few drops of the formalin and mix the sediment thoroughly. This will
preserve the sediment.
14. Concentrated wet preparation: Now make the smears saline and iodine wet
preparation.
15. Examine under the microscope.
Precautions and Handling of the Stool samples
1. Advise patients for the following things for at least 48 hours before the collection
of the stool:
1. Avoid mineral oils.
2. Do not take bismuth.
3. Don’t take antibiotics like tetracyclines.
4. Don’t take anti-diarrheal drugs which are non-absorbent.
5. Avoid anti-malarial drugs.
6. The patient should not have a barium swallow examination before the
stool examination.
7. For occult blood stop iron-containing drugs, meat, and fish at least 48
hours before the collection.
1. In the case of antibiotics or contrast media either take a sample
before or after one week.
2. These substances produce unknown objects or mask the
parasites.
2. Warm stools are better for the ova and parasites.
3. Don’t refrigerate the stool for ova and parasites.
4. Stool for ova and parasites can be collected in formalin and polyvinyl alcohol.
These are used as a fixative.
5. If there is blood or mucus, that should be included in the stool. Because most of
the pathogens are found in this substance.
6. Exam the stool before giving antibiotics or other drugs.
7. The semi-formed stool should be examined within 60 minutes of collection.
1. The liquid stool should be examined within the first 30 minutes.
2. The solid stool should be examined within the first hour of collection.
8. Trophozoites degenerate in liquid stool rapidly, so exam the stool within 30
minutes.
9. In the case of constipated cases, use non-residual purgative on the night before
the collection of the stool.
10. Avoid urine contamination, because some of the parasites are destroyed by the
urine.
11. Water destroys some of the parasites like Schistosome eggs and amoebic
trophozoites.
12. Toilet paper in the stool makes it difficult to exam the stool and may mask the
parasites.

Stool preservatives are:

In routine stool preservatives used are:

1. Formalin
1. 5% is ideal for protozoan cysts.
2. 10% preserves eggs and cysts.
3. Advantages are:
1. It is easy to prepare.
2. It can preserve the stool for several years.
3. It has a long shelf life.
4. Disadvantages are:
 1. It is not good for a permanent smear.
2. Details of eggs and cysts fade away.
3. Trophozoites can not be recovered.
2. Polyvinyl alcohol
1. This is an effective parasitic fixative.
2. This can be used along with Scaudinn’s solution.
3. Advantages are:
1. This is easy to with stool.
2. This helps in gluing the sample on the slide.
3. It has a long shelf life when stored at room temperature.
4. Smears can stain with trichrome and iron, hemotoxylin.
4. Disadvantages are:
1. There is a large amount of mercury present in the solution which is
hazardous for health.
2. It is not easy to prepare in the lab.
5. The formula of Polyvinyl alcohol:

Stool polyvinyl alcohol formula

1.
1.
1. Procedure: Add glycerol to PVA in a large beaker.
2. Blend it with a glass rod by stirring.
3. Gradually add water and keep on mixing.
4. Leave it overnight before the use.

3. Sodium-acetate formalin mixture (SAF)

1. There is increased interest in the use of this fixative.
2. Advantages are:
1. This is good for intestinal protozoa and coccidia like bodies.
2. This fixative eliminates the use of mercury compounds.
3. This is an inexpensive fixative.
4. This is easy to prepare in the lab.
5. It has a buffering effect to decrease the distortion of the protozoa.
6. This can be used in a concentration of the stool smear.
7. It has good results when used with iron and hematoxylin
permanent stain.
3. Disadvantages are:
1. This does not have adhesive properties and you may need
albumin for this purpose.
2. It is diluted with water.
 4. The formula of Sodium acetate formalin:

Stool SAF formula

4. If the sample needs delay then must use stool preservatives, otherwise reject the
sample.
1. Send sample in two vials:
1. One containing 5% or 10% formalin.
2. The second vial contains either polyvinyl or sodium acetate
formalin.

Stool preservatives

5. Preservatives for the wet preparation are:

1. 10% formol-saline for the wet preparation. This is the best preservatives
as it kills the bacteria and preserves the protozoa and helminths.
2. Another preservative is Sodium acetate formalin (SAF).
3. Methionate iodine formalin. This is a good preservative for the field
collection of the stool.
6. For staining use Polyvinyl alcohol.
7. Avoid preservatives for the culture of stool.
1. Usually, three parts of the preservatives and one part of the stool is used.
The permanent stain of the stool smears:
The sample of the choice for stains is a thinly prepared slide from the preservative of
PVA (polyvinyl alcohol).
There are three methods for the permanent stain:

1. Wheatley trichrome.
2. Iron hematoxylin.
3. Modified acid-fast stain.
4. The most commonly used is the Wheatley trichrome.

Wheatley trichrome procedure:

1. Take the following Coplin jar and put the chemicals in those jars:

2. 1 2 3 4 5 6 7

Reagents Iodine 70% 70% Trichrome 90% 95% 95% alco

+70% alcohol alcohol stain acidified alcohol
alcohol alcohol

Purpose remove remove Rinse, Stain Destain Stop Dehydrat

of the the HgCl iodine and hydration staining
reagents and hydration
hydration

Time for 10 minutes 5 minutes 5 minutes 7 minutes 5 to 10 Quick 5 minutes

each jar seconds rinse

7. Now seal the with the fixative and keep it for some time.
8. Examine the slide under the oil immersion lens.

Indications
1. To evaluate the function and integrity of the GI tract.
2. To rule out the presence of WBCs and RBCs.
3. To find ova or parasites.
4. To see the presence of fat for malabsorption syndrome.
5. For screening of colon cancer.
6. For asymptomatic ulceration of GI tract.
7. Evaluate diseases in the presence of diarrhea and constipation.
 8. Summary of stool studies are done to evaluate:
1. Intestinal bleeding.
2. Infestation.
3. Inflammatory diseases.
4. Malabsorption.
5. Different causes of diarrhea.

Pathophysiology
1. The stool examination includes:
1. Grossly.
2. microscopically.
3. Chemically.
2. Gross Stool examination includes:
1. Color.
2. Consistency.
3. Quantity.
4. Odor.
5. Mucous.
6. Helminths.
7. Concretions (gallbladder stone rarely may be found).
3. The 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.
7. Bacteria.
4. The chemical examination includes:
1.
1. Stool pH.
2. Reducing substances.
3. For occult blood.
4. Presence of fat, carbohydrate, and proteins.

Normal components of the stool:

1. Undigested food particles like:
1. Vegetable cells.
1. Vegetable fibers.
2. Plant hairs.
3. Amorphous vegetable material.
 Stool plant cells and hairs

1.
1.
4. Pollen grains are regular in size and often present in large
numbers.

Stool pollen grain and Starch

2. Muscle fibers.  These are usually light brown in color. Sometime it may see
striations.
1. Meat fibers. and muscle fibers are seen in the stool. Their presence
shows defective indigestion.
1. The increased amount of meat fibers are found in:
1. Malabsorption syndrome.
2. A pancreatic functional defect like cystic fibrosis.
 Stool muscle fibers

3. Starch granules may be spherical if undigested then these are having concentric
layers of white, homogenous material.

Stool starch

Stool potato granules

4. Fishbones.
5. Water.
6. Bacteria.  On the stool smear do the gram stain and can see the bacteria.
Normally there are bacteria in the stool.
 Stool gram-negative bacteria

7. Desquamated epithelial cells. This depends upon the size of nuclei in relation to
the cytoplasm.

Stool squamous epithelial cells

8. Polymorphonuclear leucocytes have four separate spheres with peripheral

chromatin.

Stool polymorphonuclear leucocyte

9. Macrophagic cells have numerous inclusions and these need to differentiate

from the intestinal amoebae. There are large particles in the cytoplasm. The
nucleus has no karyosomes.
2. Degenerated macrophagic cells lost their nucleus and have ingested
material.
 Stool macrophagic cells

10. Digestive tracts products like:

1. Enzymes.
2. Mucus.
3. Bile pigments products.
4. Digested but not absorbed food.
5. Products produced by the decomposition of the stool are:

1. Skatole.
2. Indole.
3. Various gases like H2S, CO2, and nitrogen.
4. Presence of Fat. The fat in the stool shows the possibility of :

1.
1. Malabsorption.
2. Deficiency of pancreatic digestive enzyme.
3. Deficiency of Bile.

Stool normal findings

Macroscopic or gross appearance of the stool:
The gross findings of the stool are important. Note the consistency and the color of the
stool.
Try best to examine the stool as received in the lab, in case of delay use the
preservatives.
The mushy or liquid stool may suggest the presence of protozoan trophozoites. In this
case, if the stool is examined one-half hours gives the best result.
Helminth eggs and larvae are found in the liquid or formed stool. Grossly you may see
proglottids or adult tapeworms.

The Consistency Of The Stool May Be:

1. Normal is soft and formed.

2. Loosely formed stools.
3. watery stools.
4. Thin stools.
5. Pellet-like stools.
6. Dry or hard stools found in constipated patients.
7. Puttylike stools.
8. The small round hard stool is due to habitual constipation.
9. Pasty stools are due to high-fat contents and seen in:
1. common bile duct obstruction.
2. In Celiac disease, the stool looks like aluminum paint.
3. Cystic fibrosis due to pancreatic involvement and are greasy.
10. Diarrheal stools are watery.
11. Steatorrhea stool is:
1. Large in amount.
2. Frothy.
3. Foul-smelling.
12. Constipated stools are firm and may see spherical masses.
13. Ribbon-like stool suggests the spastic bowel, rectal narrowing, stricture, or
partial obstruction.
14. The very hard stool is due to excessive absorption of water due to prolonged
contact with colonic mucosa.

Color

1. Normal color is due to the presence of stercobilinogen and is brown.

2. Yellow or yellow-green color is seen in diarrhea.
3. Black and tarry (related with consistency) stools are due to bleeding of the upper
GI tract from tumors.
4. The maroon or pink color is from the lower GI tract due to tumors, hemorrhoids,
fissure, or inflammatory process.
5. Clay-colored stools are due to biliary tract obstruction.
6. Mucous in the stool indicate constipation, colitis, or malignancy.
7. Pale color with greasy appearance is due to pancreatic deficiency leading to
malabsorption.
The color of the stool  Causes

1. Brown, dark brown, or yellow-brown Normal color is due to the oxidation of bile pigments.

2. Gray color Ingestion of chocolate or Cocoa. steatorrhea.

3. Green color Ingestion of spinach, and chlorophyll vegetables, administ

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 f

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, o
anus.

Quantity

1. Normally there is 100 to 200 G/day.

1. With a vegetable diet maybe 250 g/day.
2. Many disorders cause large, bulky stools even in people who don’t eat a lot.
1. Like malabsorption syndrome, and carbohydrate indigestion.
3. The size of your stools has more to do with how well you digest your foods than
how much you eat.
4. Some types of foods produce larger stools because they don’t break down
completely.
5. Some gastrointestinal disorders also cause poor food breakdown and
absorption, which leads to large, bulky stools.

Odor

1. The foul odor is caused by the undigested protein and by excessive intake of
carbohydrates.
1. Stool odor is caused by indole and skatole which are formed by bacterial
fermentation and putrefaction.
2. A bad odor which is sickly produced by undigested lactose and fatty acids.
3. The odor is increased due to excess intake of proteins.
 4. The putrid odor is due to severe diarrhea of malignancy or gangrenous
dysentery.

Mucus

1. Mucus 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. Mucus in diarrhea with microscopically present with RBCs and WBCs are 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 the rectal canal.
5. An excessive amount of mucus seen in:
1. Villous adenoma of the colon.
2. This depends upon the dietary intake.

PH

1. Normally stool is slightly acidic or alkaline or neutral.

1. pH is 7.0 to 7.5 depending on the diet.
2. Newborn pH = 5.0 to 7.5.
2. The pH of the stool depends upon the diet and bacterial fermentation in the
small intestine.
3. Carbohydrate changes the pH to acidic while the protein breakdown changes to
alkaline.
1. The breastfed infant’s pH has a slightly acidic stool.
2. Bottle-fed infants have a slightly alkaline stool.
4. pH stool test helps to evaluate carbohydrate and fat malabsorption.
5. pH stool also helps to know disaccharidase deficiency.
6. Alkaline (Increased pH) stool is seen in:
1. Colitis.
2. Villous adenoma.
3. Diarrhea.
4. Antibiotic therapy.
5. Excess intake of proteins.
7. Acidic (Decreased pH) stool seen in:
1. Fat malabsorption.
 2. Disaccharidase deficiency.
3. Carbohydrate malabsorption.
4. Excess intake of carbohydrates.
8. Precautions for pH estimation:
1. Barium intake and laxatives change the pH.
2. If the specimen is contaminated with the urine, will need to discard the
sample.

Reducing Substances

● Please see the details of the Reducing substances in the stool on this link.

Microscopic Examination
● This is the preliminary examination to find the causes of diarrhea.

1. Presence of Leukocytes Normally there are no WBCs.

1. WBCs only appear in infection or inflammation.
2. Their presence is important in case of diarrhea or dysentery.
3. >3 WBCs /high fields are seen in ulcerative colitis and bacterial infection.
4. Greater numbers of WBCs indicate invasive pathogens.

Stool with the presence of WBCs and RBCs

2. Viruses and parasites don’t cause WBCs in the stool.

3. Increased number of WBCs in the stool.
1. Bacillary dysentery.
2. chronic ulcerative colitis.
3. Shigellosis.
4. salmonella infection.
5. Yersinia infection.
6. Invasive E.coli diarrhea.
7. Fistula of anus or rectum.
8. Localized abscess.
4. Few WBCs are seen in amoebiasis.
1. Also, WBCs are seen in typhoid.
5. The absence of WBCs seen in some of the diarrhoeal conditions alike:
1. Cholera.
 2. Viral diarrhea.
3. Drug-induced diarrhea.
4. Amoebic colitis.
5. Non-invasive E.coli diarrhoea.
6. Parasitic infestation.
7. Toxigenic bacterial infection.
6. Presence of Red Blood Cells in the stool. Blood in the stool can be:
1. Bright red from the bleeding in the lower GI tract.
2. Maroon in color.
3. Black and tarry from bleeding from the upper GI tract.
4. Occult blood (not visible to the naked eye).
5. Causes of blood in stool:
1. Hemorrhoids.
2. Cancer.
3. Dysentery.
7. Make smear from the mucus area or from the blood-colored area from the
watery or semiformed stool.

Stool gross presence of the  blood

Stool physical character and possible causes:

Stool findings (Physical features) Possible Causes

1. Diarrhea mixed with blood and mucous Typhoid, Amoebiasis, and large colon carcinoma

2. Diarrhea mixed with Pus and mucous Ulcerative colitis, Salmonellosis, Intestinal tuberculosis, S
acute diverticulitis

3. Patty stool with high-fat contents Cystic fibrosis and CBD – obstruction
4. Formed stool with attached mucous Constipation, Mucous colitis, and excessive straining

5. Small, hard dark balls like Constipation

6. Clay-colored,  pasty, and little odor Bile duct obstruction, and barium ingestion.

7. Black, tarry, sticky, watery, voluminous Upper GI tract bleeding, Noninvasive infections like Cho
poisoning, and Toxigenic E. Coli and Disaccharidase defi

8. Ova and parasites. Normally there are no parasites or eggs in the stool sample.
1. Multiple stool sample is needed to rule out the parasitic infestation, at
least three consecutive days.
2. An abnormal result means parasites or eggs are present in the stool. Such
infections include:
3. Roundworms: Ascaris lumbricoides.
4. Hookworms: Necator americanus.
5. Pinworms: Enterobius vermicularis.
6. Whipworm: Trichuris trichiura.
7. Tapeworms: Diphyllobothrium latum, Taenia saginata, and Taenia solium.
8. Protozoa: Entamoeba histolytica (an amoeba), and Giardia lamblia (a
flagellate)
9. Strongyloidiasis.

Examples of some of the parasites

Tinea Saginiata egg with Hooklets Hymenolepis Nana Egg showing hooklets
Entamoeba Histolytica Trophozoite

Entamoeba Histolytica Cyst

Ascaris lumbricoides egg

 Hookworm egg

Important facts:

1. The intestinal protozoan is usually found in the soft and liquid stool.
2. Cysts are rarely found in liquid stool.
3. Cysts are found in the formed stool.
4. Helminth eggs are found in liquid or formed stool.
5. Liquid stools are diluted, so difficult to finds these parasites.
6. Examined the surface of the unpreserved stool for macroscopic parasites.
7. Pinworms are seen at the surface and tapeworms in the interior of the stool.
8. The freshly passed stool is essential for the detection of amoebae or the
flagellate.
9. All liquid or soft stool should be examined within 30 minutes of the collection.
10. Formed stool immediate examination is not critical, can wait for 3 to 4 hours