INTRODUCTION

Examination of sample of stool is easily done and is very useful in the evaluation of diarrheal diseases, parasitic infestations, colorectal carcinoma and malabsorption.

Collection

•• Container: Stool sample is collected either in a wide mouthed glass or plastic jars with a screw cap. Container should be clean and dry.

•• Method of collection: The sample is transferred from a clean bed pan or toilet into the container.

•• Amount of sample: Stool required is small and about 2–5 g of the stool sample is adequate.

Precautions in Collection

•• Morning sample is preferred.

•• Sample should be labeled and the time of collection to be mentioned.

•• The container after sample collection should be closed to avoid drying.

•• Contamination with either urine or other substances in the bed pan or toilet should be avoided.

•• Stool must be fresh. Examine within one hour of collection.

•• If blood/mucus/any other abnormal gross features are present in the stool, it should be included in the sample collected.


Preservation

Formal-saline can be used as preservative which preserves morphology of protozoa and helminthic eggs.

STOOL EXAMINATION

Physical Examination

1. Quantity: The quantity of stool varies from 100–250 g/day depending on the type of diet consumed.

2. Consistency and form

•• Normal feces is well-formed.

•• Extensively hard stool is observed during constipation.

•• Large bulky, frothy, pale, foul smelling stool which floats on water is characteristic of steatorrhea (poor fat digestion).

•• Rice water stool is typical of cholera.

•• Watery/semisolid stool in diarrhea, dysentery and following use of a laxative/enema.

3. Color

•• Normal: Golden brown due to stercobilin, a pigment derived from bilirubin metabolism.

•• Black tarry stool: It is usually due to altered blood in stool and known as melena. The source of blood is bleeding from the upper gastrointestinal tract (GIT). But black tarry stool may also be observed following iron administration.

•• Bright red color: It is due to bleeding from the lower GIT like bleeding piles.

•• Clay-colored stools: They are observed in obstructive jaundice.

4. Odor: Normal odor of stool is due to indole and skatole formed by intestinal fermentation and putrefaction. The odor varies according to the pH of the stool.

5. Blood and mucus in stool: This is observed in either amebic dysentery or bacillary dysentery. 

The main differences between these two are given in Table 66.1. 


It may also be seen in ulcerative colitis, neoplasms or tuberculosis of intestine.

6. Parasites: Stool sample may show adult worms/segments of worms (e.g. roundworm, pinworm, whipworm, hookworm or tapeworm).

Chemical Examination

The chemical examination of stool includes:

Reaction and pH

•• Normal stool pH: It ranges from 5.8 to 7.5.

•• Strongly acidic stool: Observed with excess carbohydrate diet or fermentation due to lactose intolerance.

•• Strongly alkaline stool: Observed with excess proteins in diet.

Occult Blood

Small amount of blood in stool cannot be seen on gross examination. Chemical test is necessary to detect occult blood in stool. Presence of blood/hemoglobin in the stool which is detected by a ‘chemical test’ and not by the naked eye is known as ‘occult (hidden) blood’.

Test for Occult Blood

Benzidine test

It is a sensitive test for detecting occult blood.

Principle

The heme compounds derived from hemoglobin molecule in the red cells (from blood in the stool) have peroxidase activity. This peroxidase converts hydrogen peroxide (present in the reagent) to nascent oxygen. The released oxygen oxidizes the benzidine (in the regent) in an acidic pH to colored oxidation products which are blue or green in color.

Procedure

•• The benzidine reagent consists of 4 g benzidine base in 10 mL glacial acetic acid. It is stable for 2–4 months.

•• Emulsify a bit of stool in 5 mL water.

•• Mix 1 mL of emulsion with 1 mL of benzidine reagent in a test tube.

•• Add several drops of 3% hydrogen peroxide.

Interpretation: The appearance of blue color indicates the presence of blood.

Precaution: Benzidine is carcinogenic. 

Guaiac test (fecal occult blood testing)

Occult blood can be detected by chemical (guaiac), hemoporphyrin, or immunologic methods.

Principle

Fecal occult blood test (FOBT) depends on the peroxidase activity of hemoglobin. The heme moiety of hemoglobin in the presence of H2O2 catalyzes the oxidation of the colorless compound guaiaconic acid (hence named guaiac test) to a highly conjugated, blue-colored quinone.

Various commercial tests are available for testing occult blood.

•• Hemoccult is a commonly used slide test.

•• Test using orthotoludine.

•• Test using phenolphthalein.

Interference

These tests indicate the presence of blood in stool which may be found in malignant tumors of colon as well as nonmalignant causes (e.g. hemorrhoids, colitis, diverticulitis, and local trauma to the perineal area.

Causes of False-positive Reactions

•• The positive test is not specific for human hemoglobin. False-positive results may be caused by exogenous factors such as myoglobin and hemoglobin from red meat ingestion (in the diet).

•• Presence of vegetable or fruit peroxidases (e.g. horseradish, bananas, black grapes, pears, plums, melons).

•• Leukocytes and bacteria.

•• Drugs like boric acid, bismuth (present in antacid preparations), bromides, iodine and oxidizing agents.

Causes of False-negative Reactions

•• Use of vitamin C and other oxidants.

Significance

•• Determining the cause of microcytic hypochromic anemia due to chronic blood loss.

Causes of chronic blood loss may be:

–– GIT neoplasms (e.g. colon, stomach cancer)

–– Ulcerative diseases of the GIT

–– Hookworm infection

•• Drugs like aspirin, steroids and indomethacin can be associated with increased gastrointestinal bleeding.

Other Chemical Tests

•• Quantitative fecal fat estimation: Increase in fecal fat (more than 6 gram per day) is found in malabsorption syndromes or diseases of pancreas.

•• Presence of reducing substances like lactose in stool may be found in infants with diarrhea.

Microscopic Examination

A fresh sample of stool without any contamination with disinfectants is used for microscopic examination. On microscopy, examine the stool for:

•• Leukocytes (pus cells), red blood cells, muscle fibers, fat globules, crystals, cysts and yeast cells and are expressed as number seen per high power field.

•• Protozoa, eggs, larvae and cysts of parasites, flagellates and ciliates. 

They are reported as scanty, few, moderate or many.

Methods for the Preparation of Stool for Microscopy

Saline preparation: A small amount of stool sample is picked up with the help of tooth prick and taken on a glass slide. Mix with normal saline to make a thin emulsion so that the fine print can be seen through it. Cover with a cover slip. This is useful for the demonstration of motility especially of E. histolytica.

Iodine preparation: In this, Gram’s iodine is used instead of saline. Iodine imparts brown color to chromatin granules (nuclei) of amoebic cysts and also glycogen vacuole. But iodine kills living parasites thereby preventing identification of motility.

Stool concentration methods: When ova and cysts are few in number and are not detected by routine methods, concentration method will be of help.

•• Floatation method: The stool sample is mixed with either zinc sulfate or magnesium sulfate which has a high specific gravity and causes the parasite to float in the solution. It is used for concentration of cysts, larvae and most of the helminthic eggs.

•• Sedimentation methods: In this method, the parasites sediment and get deposited at the bottom by centrifugation. It can be done by either simple sedimentation method or by formal-saline ether sedimentation method.

–– Simple sedimentation method: A small amount of stool sample is mixed with saline in a tube or bottle and sieved through a strainer. The sieved contents are centrifuged and the supernatant fluid discarded. The deposit is resuspended in more saline, mixed and centrifuged. 

This is repeated till the supernatant fluid appears clear. The deposit is examined directly on a glass slide.

–– Formol-saline ether (or formol-ethyl acetate)

sedimentation method: This yields a good concentration of parasites and is recommended for routine work. But this method cannot be used to concentrate living parasites because the formalin used kills the parasites.

The mode of infection, disease produced and appearances of various trophozoite/egg/ova/larva are shown in Figures 66.1 to 66.4.


STOOL CULTURE AND SENSITIVITY

Collection

Stool sample for culture should be collected in a sterile wide mouthed container.

Culture Media Used

MacConkey’s agar, nutrient agar or selective media depending on the suspected organism.

Procedure

•• Take stool in culture media and incubate at 37°C for 18–20 hours.

•• Suspected colonies are tested by using oxidase test.

•• The causative organisms are identified by using biochemical tests.

•• The organism may be confirmed by agglutination using specific antisera.

Antibiotic sensitivity testing is done for the identified pathogenic organism.