This document discusses stool examination, including the composition of stool, collection and examination of stool samples, and various macroscopic, chemical, and microscopic tests that can be performed on stool samples. It provides details on normal findings and what various abnormalities may indicate. The tests described allow examination of stool volume, color, consistency, odor, presence of blood or mucus, pH, fat, nitrogen, and occult blood levels. Microscopic evaluation includes wet mount preparations, staining, and concentration methods to detect parasites, eggs, cysts, trophozoites, and other elements.

1. Stool Examination
Dr. Nishith A. Vachhani
(Ph.D. Medical Micro.)
nv2805@gmail.com

2. Introduction
• Human feces is called as STOOL.
• Faeces / Feces is plural of Latin term faex meaning RESIDUE.
• Scatology or Caprology is the study of feces.
• Composition of Stool
– ¾ Water, ¼ Solid
– Undigested and Unabsorbed food
– Intestinal secretions, Mucous
– Bile pigments and Salts
– Bacteria and Inorganic material
– Epithelial cells, Leukocytes
Dr. Nishith A. Vachhani

3. Collection of Stool Sample
• Follow Universal Precautions.
• Always a fresh sample should be tested.
• Stool should be collected in a dry, sterilized, wide mouthed
container.
• It should be uncontaminated with Urine or any other body
secretions.
• Container should be properly named.
Dr. Nishith A. Vachhani

4. Dr. Nishith A. Vachhani

5. Macroscopic Examination
• Volume : Normal <200 grams/day
• Colour:
– Normal: yellowish brown (due to combination of bile and bilirubin)
– Abnormal: Yellow, Green, Blood streak, Bright Red, Black, White
• Consistency
– Normal: Soft and Formed
– Soft, mushy, liquid and voluminous- diarrhoea, intake of purgatives
– Small numerous, largely mucus and blood with small amount of stool-
dysenteries
– Rice watery without fecal matter- Cholera
Dr. Nishith A. Vachhani

6. Macroscopic Examination
• Odour
– Normal: Aromatic (due to indole and sketole)
– Increased: excessive protein ingestion
– Sour rancid: fatty acid in milk indigestion (in children and adults), normal
in infants
– Putrid: severe diarrhoea of malignancy, gangrenous dysentery.
• Blood: Normally absent
• Mucous:
– Small quantity of mucin is normal
– Small quantity – faeces from small gut
– Excessive quantity – infection of intestine
– Entirely mucus with little or no faeces and streaks of blood- dysentery,
ileo colitis, intussusception.
• Parts of Parasite and Adult Parasite
Dr. Nishith A. Vachhani

7. Chemical Examination
• Reaction (pH)
– Normal is neutral (6.9 to 7.2)
– pH is dependent on bacterial fermentation and putrefaction in the bowel.
– Alkaline: Excess protein ingestion, Antibiotic use, colitis.
– Acidic: Excess carbohydrate ingestion, Fat malabsorption.
• Fats:
– Normally up to 20% of total solids
– Increase in pancreatic diseases. Malabsorption syndrome and enteritis.
• Nitrogen
– Normal is 1 to 1.5 gm /day
– Increase in azotorrhoea, pancreatic achylia, pancreatogenous fatty
diarrhoea, idiopathic steatorrhoea.
Dr. Nishith A. Vachhani

8. Chemical Examination
• Stercobilinogen
– Fecal urobilinogen.
– It is a chemical created by bacteria in the gut.
– It is made of broken-down hemoglobin.
– It is further processed to become the chemical that gives feces its brown
color
– Normal is 40 to 280 mg/day.
– Average is 150 mg/day.
– Dependent on amount of bilirubin passing to intestine (jaundice).
• Coproporphyrin
– Normal is 300 to 1100 mg/ day.
– Abnormally increased in congenital porphyria.
– Abnormally decreased in liver disease like cirrochis, hepatitis, passive
venous congestion, metastatic carcinoma in liver.
Dr. Nishith A. Vachhani

9. Chemical Examination
• Occult Blood
– Detect blood which is present in amount or form not visible
macroscopically
– Normal: Nil
– Abnormal presence in condition of occult haemorrhage in the GI tract.
– Methods:
• Benzidine Test
• Guaiac Test
• Orthotolidine Test
– Found positive in…
• Ulcers,
• Diverticulitis,
• Ulcerative Colitis,
• Diaphragmatic Hernia,
• Adenoma, CA Colon
Dr. Nishith A. Vachhani

10. Chemical Examination
Benzidine Test:
• Principle: Perioxidase action of hemoglobin in blood converts hydrogen
peroxide to water and nascent oxygen. This oxygen oxidizes benzidine in acid
medium to form green to blue coloured complex.
• Interpretation:
– Trace- faint blue colour (after 1 minute)
– + : Definite blue green (slowly)
– + + : Green blue (rapidly)
– + + + : Blue (almost immediately)
– + + + + : dark blue (immediately)
Dr. Nishith A. Vachhani

11. Chemical Examination
• Reducing Substance
– Routinely carried out in infants with chronic diarrhoea.
– To diagnose lactose intolerance.
– Sample of 5 gm stool needs to be delivered within 1 hour, because lactose
in the stool will normally be broken down by chemical processes within
2-4 hrs after the specimen is produced.
– It also positive in case of Rota virus infection in infants.
Dr. Nishith A. Vachhani

12. Microscopic Examination
• Direct Wet Mount Preparation:
– Saline Preparation:
– Lugol’s Iodine Preparation:
– Buffered Methylene Blue Preparation:
– Eosin solution Preparation:
• Staining Preparations:
– Trichrome Staining
– Giemsa’s Staining
– Modified ZN Staining
– Calcofluor White Staining Procedure
• Concentration Methods:
– Floatation method: Saturated Sodium Chloride, Zink Sulphate centrifugal
floatation, Sheather’s Sugar Centrifugal Flotation technique.
– Sedimentation method: Formal Ether method
Dr. Nishith A. Vachhani

13. Microscopic Examination
Saline Preparation :
• Used to demonstrate worm’s eggs, larva and protozoan trophozoites and
cysts.
• Small amount of faeces is mixed with drops of normal saline and examined.
• Large amounts of leukocytes is suggestive of chronic ulcerative colitis,
chronic bacillary dysentery and localized abscess.
• Increased amount of RBCs are found in ulcero-inflammatory conditions of
bowel and in bleeding from colon and rectum.
• Epithelial cells in increased number are associated with catarrhal
inflammation of bowel and colon.
• Crystal: Charcot leyden crystals (colorless, diamond or needle shaped,
hexagonal) are found in amoebic dysentery.
• Fat globules, starch granules, muscle fibers, vegetable cells are also seen.
Dr. Nishith A. Vachhani

14. Microscopic Examination
Iodine Preparation:
• Small amount of faeces is mixed with drops of normal saline and Lugol’s
iodine solution.
– Iodine crystals (powdered): 5 gm
– Potassium iodide :10 gm
– Distilled water : 100 ml
• Iodine kills the organisms, therefore motility is lost.
• Used mainly to stain nuclei and glycogen mass if present.
• Flagella becomes recognizable.
• Cyst can usually be specifically identified in this method.
Dr. Nishith A. Vachhani

15. Microscopic Examination
Buffered Methylene Blue Preparation :
• Stains only trophozoites of amoeba.
• It does not stain amoebic cyst or trophozoites and cyst of flagellates.
• Nucleus and the inclusions such as RBC or yeast cells stain dark blue.
• Cytoplasm stains light blue.
Eosin Wet Preparation :
• Detection of trophozoites and cyst.
• They can be much more easily detected against the pink- red background of
eosin preparation.
Dr. Nishith A. Vachhani

16. Microscopic Examination
Concentration Method
• Mainly performed to differentiate parasitic eggs from debris.
• It makes eggs more visible by removing organic and inorganic elements of
stool.
Floatation Method
• It is easy to perform.
• These method use the high specific gravity of a solution to float the lighter
ova and cyst.
Sedimentation Method
• Sedimentation process enable the concentration of parasitic eggs in sediment
of procedure.
Dr. Nishith A. Vachhani

17. Microscopic Examination
Saturated Sodium Chloride Solution Method:
• Boil granular sodium chloride in excess in water to produce a saturated
solution which when cooled has a specific gravity of 1.18 - 1.2.
• Half fill a wide- mounted flat bottomed container with the saturated salt
solution.
• Emulsify 1gm of feces in the solution and strain it to remove the debris from
the surface.
• Pour the filtrate into meniscus and fill it to the top with saturated salt
solution.
• Lay a glass slide over the top, avoiding any bubbles being trapped.
• Leave for 20 min before quickly inverting the slide.
• Scan for ova using the 10x objectives.
Dr. Nishith A. Vachhani

18. Microscopic Examination
Zink Sulphate Centrifugal Floatation Technique
• Fill a 15 ml centrifuge tube with ZnSO4 solution (1.18 specific gravity) and
pour into a glass dish.
• Add the feces (2 to 3 grams) into the ZnSO4 solution in the dish.
• Using a funnel, pour the ZnSO4-fecal mixture back into the centrifuge tube.
• Centrifuge for 2 min at high speed (1500 - 2000 rpm).
• Using a headed-rod or loop, remove a sample from the surface of the solution
and place on a microscope slide.
• Add a drop of iodine (to stain the cysts and ova) and a coverslip.
• Examine at 10x.
Dr. Nishith A. Vachhani

19. Microscopic Examination
Formal ether sedimentation technique.
• Thoroughly mix a portion of stool specimen into 10mL of saline solution. Mix
thoroughly.
• Filter the emulsion through fine mesh gauze into a conical centrifuge tube.
• Centrifuge the suspension at about 2000 rpm for 10 minutes.
• 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.
Dr. Nishith A. Vachhani

20. Microscopic Examination
Formal ether sedimentation technique.
• Centrifuge at 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 with an applicator stick.
Carefully decant the top three layers.
• Mix the remaining sediment.
• Transfer one drop each to a drop of saline and iodine on a glass slide.
• Cover with a coverslip and examine microscopically.
Dr. Nishith A. Vachhani

21. Microscopic Examination
Staining Methods
 Trichrome Staining: The permanent stained smear facilitates detection and
identification of cysts and trophozoites.
 Giemsa’s Staining: For Cryptosporidium and Isospora Spp.
 Modified ZN Staining: This technique is useful for the identification of
oocysts of the coccidian species (Cryptospora, Isospora, and Cyclospora).
 Calcofluor White Staining Procedure: This chemofluorescent technique is
useful for the detection of Microsporidia, Acanthamoeba spp., Pneumocystis
jiroveci, and Dirofilaria spp.
Dr. Nishith A. Vachhani