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Routine examination of stool

This document provides an overview of stool examination. It defines stool and describes its normal composition as 3/4 water and 1/4 solid matter. It outlines precautions for stool collection such as avoiding certain foods/medications prior. Collection details like using a clean container and preserving samples are provided. The types of examination covered are physical, chemical, and microscopic. Physical examines properties like color, consistency, and contents. Chemical analyzes pH, fats, nitrogen, and occult blood. Microscopic looks for parasites, bacteria, epithelial cells, and other components under stained and unstained preparations with concentration techniques.

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STOOL EXAMINATION Presented by: Dr. Priyanka Buragohain Guided by: Dr. Hemen Kalita Deptt. Of Roga Nidan. Govt. Ayurvedic College
DEFINITION • Human feces is called as stool. • faeces / feces is plural of latin term faex meaning RESIDUE. • It is the waste residue of indigestible materials of an animal’s digestive tract expelled through the anus during defecation. • Meconium is newborn’s first feces. • SCATOLGY or CAPROLOGY is the study of feces. Stool examination by Dr. Priyanka Buragohain 2
COMPOSITION • ¾ water, ¼ solid • Undigested and unabsorbed food • Intestinal secretions, mucous • Bile pigments and salts • Decomposed products • Bacteria and inorganic material • Epithelial cells, leukocytes. Stool examination by Dr. Priyanka Buragohain 3
PRECAUTION BEFORE COLLECTION • Patient should avoid the following things for at least 48 hours before collection of stool: • Mineral oils, bismuth, non absorbable anti diarrhoeal drugs, antimalarial drugs, antibiotics, etc • Pt. Should not have barium swallow examination before stool R/E • Avoid iron containing drugs, meat, fish etc for atleast 48 hours before stool for occult blood. • In constipated patients use only non residual purgative Stool examination by Dr. Priyanka Buragohain 4
COLLECTION • Universal precautions • Pt. is asked to pass stool in a clean container. • Stool should be collected in a steralized, wide mouthed container. • Loose/last/portion containing mucus, blood etc is to be collected in a wide mouthed bottle. • Should be uncontaminated with urine or any other body secretions. • >2gm is required. • Properly named and always a fresh sample should be tested. • Liquid stool to be examined within ½ hour • Solid stool to be examined within 1 hour. • If delayed store in a refrigerator. Stool examination by Dr. Priyanka Buragohain 5
COLLECTION CONTD... • 3 samples of stool within 10 days to exclude false negatives. • 2 samples to be examined on alternate days after normal defaecation and 1 sample after a purgative for certain worms. • Formalin is the best preservative. It kills the bacteria but ptreserves the protozoa and helminthes. • For culture no preservatives to be used Stool examination by Dr. Priyanka Buragohain 6
7 Stool examination by Dr. Priyanka Buragohain
TYPES OF EXAMINATION • PHYSICAL EXAMINATION: colour, volume, consistency, odour, mucus, pus, concreations, helminths. • CHEMICAL EXAMINATION: reactions, occult blood, fat, carbohydrate, protein, etc • MICROSCOPIC EXAMINATION: remnants of food, pus cells, macrophages, RBCs, crystals, bacteria, yeasts, molds, protozoa, helminths. • STOOL CULTURE: Stool examination by Dr. Priyanka Buragohain 8
Physical examination • AMOUNT • CONSISTENCY • COLOUR • ODOUR • REACTION • MUCUS • CONCRETION • BLOOD • PUS • FOOD REMNANT • UNDIGESTED TABLETS Stool examination by Dr. Priyanka Buragohain 9
MACROSCOPIC EXAMINATION AMOUNT • Normal is 150 g to 200 g/day • Increased in steatorrhoea, diarrhoea, indigestion of carbohydrate. Stool examination by Dr. Priyanka Buragohain 10
CONSISTENCY OR FORM • Normal is soft but formed • Excessively hard/scybala- habitual constipation • Flattened or ribbon like-intake of excess of mineral oil, carcinoma of rectum, stricture of rectum • 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 Stool examination by Dr. Priyanka Buragohain 11
Stool examination by Dr. Priyanka Buragohain 12
Colour • Dark Grey- excessive cocoa or chocolate ingestion • Reddish or blackish brown- large amount of fruits • Green – ingestion of green leafy vegetables, administration of calomel due to biliverdin • Red – Beat ingestion fresh blood • Yellow – rhubarb or senna ingestion, normal stool Stool examination by Dr. Priyanka Buragohain 13
• Clay – obstructive jaundice, barium meal x-ray • Tarry black – haemorrhage in stomach/upper intestine • Dark brown to bright red – bleeding in rectum or sigmoid colon • Red streaks of blood on the surface of faeces- haemorrhoids, fissures, carcinoma ,ulcerative colitis Stool examination by Dr. Priyanka Buragohain 14
Odour • Normal odour is aromatic due to indole and skatole • Increased- excessive protein ingestion • Sour rancid- fatty acid in milk indigestion (in children and adults), normal in infants • Putrid- severe diarrhoea of malignancy, gangrenous dysentry Stool examination by Dr. Priyanka Buragohain 15
Reaction • Normal is neutral • Ph varies from 6.9 to 7.2 • pH is dependent on bacterial fermentation and putrefaction in the bowel. • Alkaline – excess protein ingestion • Acidic – excess carbohydrate ingestion Stool examination by Dr. Priyanka Buragohain 16
Mucus • 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 Stool examination by Dr. Priyanka Buragohain 17
Concretion • In infants whitish curds may be found • Gall bladder stones may be rarely found Stool examination by Dr. Priyanka Buragohain 18
Blood • Absent in normal faeces • Formed stool with streaks of blood – lesion in sigmoid colon, rectum or anal canal • Liquid stool with bright red blood, pus and mucus- bacillary dysentery, ulcerative colitis • Semi formed stool with deep tarry black blood- melena • Loose stool with deep cherry red blood- melena Stool examination by Dr. Priyanka Buragohain 19
Pus • Normally absent • Pus with blooded mucus- ulcerative colitis, bacillary dysentery, ulcerative carcinoma Stool examination by Dr. Priyanka Buragohain 20
• GROSS FOOD REMNANTS MAY IN NORMAL STOOL • UNDIGESTED TABLETS MAY BE FOUND Stool examination by Dr. Priyanka Buragohain 21
CHEMICAL EXAMINATION Stool examination by Dr. Priyanka Buragohain 22
Chemical Examination Of Stool • Acidity/basicity • Fats • Nitrogen • Stercobilinogen • Coproporphyrin • Occult blood • Reducing substances • N. B : most commonly used chemical examination of stool is pH, occult blood and reducing substances Stool examination by Dr. Priyanka Buragohain 23
FATS • Normally upto 20% of total solids • Lipids are measured as fatty acids:2-5 gm/24 hrs • Known dietary intake and timed stool collection. • Take diet containing 100 gm of fat daily, 3 day stool collection. • >6 gm/day is abnormal • Quantitative or semiquantitative methods:  Gravimetric method  Isotopic techniques(radio-isotopes)  Electrical capacitance method  Titrimetric method of Van de Kamer Stool examination by Dr. Priyanka Buragohain 24
Titrimetric method • Boil with alcoholic potassium hydroxide to convert fats and fatty acid into soap • cool • HCl is added to convert soap to fatty acid • Fatty acid extracted with petroleum ether • Aliquot is evaporated, taken up in neutral alcohol • Ttitrate with sodium hydroxide • Fatts are calculated as fatty acids Stool examination by Dr. Priyanka Buragohain 25
Electrical capacitance methods • Fecal suspension is extracted with solvent specially with chlorinated benzene • Extract is filtered • Electrical capacitance is measured and compared with standard of triolein simillarly treated Stool examination by Dr. Priyanka Buragohain 26
Interpretation • Fecal fat increase in • enteritis and pancreatic disease (lack of lipase) • Surgical removal of a section of intestine • Mal absorption syndrome • Chronic pancreatic disease(> 10 gm / 24 hr) • Neutral fat increase in • Use of rectal suppositories • Ingestion of castor oil or mineral oil • Ingestion of dietetic low calories mayonnaise • Tropical sprue Stool examination by Dr. Priyanka Buragohain 27
Nitrogen • Varies with the amount and nature of diet • Normal is 1 to 1.5 gm /day • Increase in azotorrhoea, pancreatic achylia, pancreatogenous fatty diarrhoea, idiopathic steatorrhoea Stool examination by Dr. Priyanka Buragohain 28
Stercobilinogen • Normal is 40 to 280 mg/day • Average is 150 mg/day • Dependent on amount of bilirubin passing to intestine(jaundice) Stool examination by Dr. Priyanka Buragohain 29
Coproporphyrin • Normal is 300 to 1100 mg/ day • Type 1 – 70% • Type 3 – 10 to 30% • Abnormally increased in congenetial porphyria • Abnormallly decreased i9n liver disease like cirrochis, hepattitis, passive4 venous congestion, metastatic carcinoma in liver Stool examination by Dr. Priyanka Buragohain 30
Occult blood • Detect blood which is present in amount or form not visible macroscopically • Normally nil • Abnormal presence in condition of occult haemorrhage in the GI tract • BENZIDINE TEST • GUAIAC TEST • ORTHOTOLIDINE TEST • Most commonly used test is benzidine test Stool examination by Dr. Priyanka Buragohain 31
BENZIDINE TEST • 4 gm benzidine in 100 ml of glacial acetic acid • Emulsify pea sized bit of faeces in 5 ml of water. • Mix 1 ml emulsion and 1 ml of reagent in test tube • Add several drops of 35 H2O2 • Blue colour indicates positive reaction Stool examination by Dr. Priyanka Buragohain 32
• Trace- faint blue colour after 1 min • 1+ - definite blue green slowly • 2+ - green blue rapidly • 3+ - blue almost immediately • 4+ - dark blue immediately Stool examination by Dr. Priyanka Buragohain 33
GUAIAC TEST • Less sensitive • With loss of 20 to 30 ml of blood all test will be positive • Guaiac reagent consist of 1 gm Guaiac in 5 ml of 95% ethanol. • Make a small smear of feces on a filter paper • Add 2 to 3 drops of gum guaiac solution + 2 to 3 drops of glacial acetic acid + 2 to 3 drops of 3% H2O2 Stool examination by Dr. Priyanka Buragohain 34
• Trace- faint blue green in 1 min • 1+ light blue slowly • 2+ clear blue rapidly • 3 + deep blue almost immediately • 4+ deep blue immediately Stool examination by Dr. Priyanka Buragohain 35
ORTHOTOLIDINE TEST • Intermediate sinsitivity • Smear the stool on a filter paper with an applicator • Pipette a few drops of the reagent on to the filter paper(orthotolidine barium peroxide 200 mg+ glacial acetic acid 5 ml) • After 30 sec examine for a blue colour • Blue green colour within 30 sec means positive test Stool examination by Dr. Priyanka Buragohain 36
Interpretation • Gastric disease eg chronic ulcer and malignancy • Intestinal diseases eg dysentery, typhoid fever, carcinoma • Haemorrhoids • During instrumentation Stool examination by Dr. Priyanka Buragohain 37
Faecal reducing substance test • To diagnose lactose intolerance • Sample of 5 gm stool is needed • Sample needs to be delivered to the laboratory as soon as possible, preferably within 1 hr , cause lactose in the stool will normally be broken down by chemical processes within 2-4 hrs after the specimen is produced. Stool examination by Dr. Priyanka Buragohain 38
Interpretation • Negative/ trace- < 0.25 g/dl • Suspicious(grade 1) – 0.25-0.5 g/dl • Abnormal(grade 2-4)->0.5 g/dl • Found in Carbohydrate malabsorption • Tropical sprue Stool examination by Dr. Priyanka Buragohain 39
MICROSCOPIC EXAMINATION Stool examination by Dr. Priyanka Buragohain 40
NEED FOR MICROSCOPIC EXAMINATION • For the diagnosis of microscopic elements. • Trophozoites and its movements are better seen in unstained preparation of a fresh material. • Cystic forms &Nuclear character are better seen in stained preparation(iodine) • Gycogen mass- stained with iodine • Chromatoid bars- unstained preparation • N.B – Both stained and unstained materials are to be prepared Stool examination by Dr. Priyanka Buragohain 41
Stool examination by Dr. Priyanka Buragohain 42
Need of concentration technique • To see whether treatment of parasite is successful • To find ova of S. Mansoni or Taenia if few or other ova and cyst are not seen in routine examination • To examine stool specimens from patients who do not come from an area where a particular parasite is found Stool examination by Dr. Priyanka Buragohain 43
Stool examination by Dr. Priyanka Buragohain 44
FLOATATION TECHNIQUE • Use solutions which have highier specific gravity(zinc sulphate or Sheather’s sugar) than the organisms to be floated so that the organisms rise to the top and the debries sink to the bottom. • Advantage – produce a cleaner material than the sedimentation technique • Disadvantage – walls of eggs and cyst will often collapse, hindering identification. • Some parasite eggs do not float. Stool examination by Dr. Priyanka Buragohain 45
SEDIMETATION TECHNIQUE • Use solutions of lower specific gravity than the parasitic organisms(formalin ethyl acetate technique) • Recommended for general diagnostic laboratories due to easy to perform and less prone to technical error. Stool examination by Dr. Priyanka Buragohain 46
Sedimentation techniques • Mix a small piece of stool with 10 ml of water or saline in a tube/ bottle • Sieve the suspension into a beaker through a strainer with small holes. • Pour the contents into a centrifuge tube • Centrifuge at 2000-3000/rpm for 1 min • Pour off the supernatant part • Resuspend the deposit in clean water and add enough water to fill the tube. • Mix well and recentrifuge • Pour off the supernatant part • Resuspend in zinc sulphate solution, fill the tube with the solution • Centrifuge at high speed for 1 min • Transfer the contents from the surface of the tube to a slide, using a bacteriological wire loop • Add small drops of saline and mix • Cover with a cover slip • Examine under 10x and 40x objectives Stool examination by Dr. Priyanka Buragohain 47
Stool examination by Dr. Priyanka Buragohain 48
Stainning methods Wet mount normal saline Iodine solution Buffered methylene blue solution Eosin solution Stainning for permanent preparation Schaudinn’s fluid Heidenhain’s Haematoxilin method Trichome stain Stool examination by Dr. Priyanka Buragohain 49
Microscopic examination of wet mount Stool examination by Dr. Priyanka Buragohain 50
Saline wet mount Stool examination by Dr. Priyanka Buragohain 51
Iodine wet mount • Iodine kills the organisms, therefore motility is lost. • Used mainly to stain nuclei and glycogen mass if present. • Flagella becomes recognisable. • Cyst can usually be specifically identified in this method. • Lugol’s iodine solution is used. Stool examination by Dr. Priyanka Buragohain 52
Lugol’s iodine • Its very strong • Must be diluted about 5 times with distilled water • Stain deteriorates quickly hence to be prepared every 2 weeks • Contains: Iodine crystals(powdered): 5 g Potassium iodide :10 g Distilled water : 10 • Potassium iodide is dissolved in distilled water and iodine crystals are slowly added. Solution is filtered and kept in a stoppered bottle of amber colour Stool examination by Dr. Priyanka Buragohain 53
Buffered methylene blue wet mount • 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 Stool examination by Dr. Priyanka Buragohain 54
Eosin wet mount • Detection of trophozoites and cyst • They can be much more easily detected against the pink- red background of eosin preparation Stool examination by Dr. Priyanka Buragohain 55
Stool examination by Dr. Priyanka Buragohain 56
Stool examination by Dr. Priyanka Buragohain 57
Stool examination by Dr. Priyanka Buragohain 58
Stool examination by Dr. Priyanka Buragohain 59 Cover with a cover slip
Stool examination by Dr. Priyanka Buragohain 60
Stool examination by Dr. Priyanka Buragohain 61
microscopic examination findings • REMNANTS OF FOOD • vegetable cells • Muscle fibres • Starch granules • Fat globules • Connective tissue/ elastic fibres • Mineral oil or castor oil globules • CELLS • Epithelial cells • Pus cells • Macrophages • Ghost cells • Pyknotic bodies • Eosinophills • RBC • Crystals • Yeasts and molds • Protozoa • Helminthic parasites Stool examination by Dr. Priyanka Buragohain 62
Vegetable cell • Sometimes causes confusion with ova, eggs, cyst or cell bodies • IRREGULAR OUTER MARGIN Excess quantity is seen in excess intake of vegetables or indigestion Stool examination by Dr. Priyanka Buragohain 63
Muscle fibres • May confuse with Tinea segments • Excess protein intake or indigestion • Its excess excretion is called Creatorrhoea(flesh-flow) Stool examination by Dr. Priyanka Buragohain 64
Starch granules • Variable in size, round to polygonal in shape, colourless, circular or Y shaped dot in the centre • Confused with ova of helminths • Found in carbohydrate dyspepsia • Better seen in iodine preparation Stool examination by Dr. Priyanka Buragohain 65
Fat globules • Appear similar to parasitic cyst or cell bodies • Emulsifying agents are used to eliminate confusion • Confused with ova of helminths • Found in fat dyspepsia Stool examination by Dr. Priyanka Buragohain 66
Connective tissue/elastic fibres • Confused with tinea segments • Signify indigestion Stool examination by Dr. Priyanka Buragohain 67
Mineral oil/ castor oil globules • When taken as purgative • May be confused with ova of helminthes Stool examination by Dr. Priyanka Buragohain 68
Epithelial cells • Excess presence due to inflammatory conditions of colon, rectum, anal canal Stool examination by Dr. Priyanka Buragohain 69
Pus cells • Commonly found in normal stool, help to ease the passage of stool • Normally not visible to human eye. • If visible indicates disease • Bacillary dysentery, UC, acute Amoebic dysentery, malignancy of rectum, drug induced enterocilitis Stool examination by Dr. Priyanka Buragohain 70
Macrophages • Large mononuclear cells with vesicular nucleus and ingested materials including RBC • Confused with E. Histolytica cyst or E. Coli cyst • Excess in Amoebic or bacillary dysentery Stool examination by Dr. Priyanka Buragohain 71
Ghost cells • Degerative form of macrophages, epithelial cells • Its an enlarged/swollen eosinophilic epithelial cell with only eosinophilic cytoplasmic outline but without a nucleus • Characteristic of bacillary dysentery Stool examination by Dr. Priyanka Buragohain 72
Pyknotic bodies • Nuclear remains of tissue cells and leucocytes • Characteristic of acute amoebic dysentery Stool examination by Dr. Priyanka Buragohain 73
Eosinophils • In intestinal allergy • Diluting fluid used- Randolph’s diluting fluid,Pilot’s stain • Carbol chromotrope technique • A measured quantity of the deposit is taken and diluted with the diluting fluid 1:10 or 1: 20 according to the concentration of the residue and counted in haemocytometer. • Increased in allergic conditions, parasitic infestation and drug allergy, ulcerative colitis Stool examination by Dr. Priyanka Buragohain 74
RBCs • seen in cases of ulcrative lesions of gut • in bacillary dysentery – yellowish discrete • Amoebic dysentery – greenish and in clumps Stool examination by Dr. Priyanka Buragohain 75
Crystals • Fatty acid crystals • Calcium oxalate crystals • Triple phosphate crystals • Charcot Leyden crystals • Haemotoidin crystals • Crystals of drugs Stool examination by Dr. Priyanka Buragohain 76
• Charcot Leyden crystal: • Slender and pointed at both ends, Hexagonal bipyramidal structures localised in the primary granues of cytoplasm of eosinophils and basophils • Evidence of parasitic infiltrate eg amoeba, ascaris, hookworm, fasciola • diamond shaped or whetstone shaped crystals • Normally colourless, stained purplish- red by trichome • Vary in size and may be as large as 50 µm in length • Found in UC, dysentery, malignant ulcers, schistosomiasis etc Stool examination by Dr. Priyanka Buragohain 77
• Haematoidin crystals: • Ironless pigment derived from haemoglobin and formed within tissues(reticuloendothelial cells) but found extracellularly after 5-7 days in foci of previous haemorrhage. • Occurs as refractile, yellow- brown and orange-red granules • Characteristically as rhomboid plates arranged in a radial pattern, so called hematoidin burrs. Stool examination by Dr. Priyanka Buragohain 78
Yeast and molds • Yeast are normally present • Excess in cases of AIDS • Molds are rare but may be seen in immunodeficiency conditions Stool examination by Dr. Priyanka Buragohain 79
Parasites Stool examination by Dr. Priyanka Buragohain 80
Stool examination by Dr. Priyanka Buragohain 81
Stool examination by Dr. Priyanka Buragohain 82
Stool examination by Dr. Priyanka Buragohain 83 protozoa
Entamoeba histolytica TROPHOZOITE STAGE • Identified by motility and presence of ingested RBC • Shape : constantly changing position • Size :ranges from 18 to 40 µm ,average being 20 to 20 µm • Cytoplasm :divisible in two portion • Nucleus :spherical in shape Stool examination by Dr. Priyanka Buragohain 84
Stool examination by Dr. Priyanka Buragohain 85 RBC appear yellowish green inside the endoplasm Nucleus is not visible but a faint outline may be detected Endoplasm shows bluish or ground glass appearance Eccentric nucleus with karyosome (a small dot at the centre surrounded by a clear halo), nuclear membrane, Linen network having a spoke like radial arrangement
PRE CYSTIC STAGE • Size :small in size,10 to 20 µm • Shape :round or slightly ovoid with blunt pseudopodium • Free from ingested RBC and other materials • Nucleus : large nucleus • Retains the characteristics of trophozoite. Stool examination by Dr. Priyanka Buragohain 86
CYSTIC STAGE • Size : 6 to 9 µm / 12 to 15 µm • Shape : round, surrounded by a highly refractile membrane called cyst wall • Nucleus :quadrinucleate • Clear and hyaline cytoplasm • Nuclear structure retainning the character of trophozoite Stool examination by Dr. Priyanka Buragohain 87
Stool examination by Dr. Priyanka Buragohain 88 •Iodine mounting •Body of the parasite stains yellow to light brown •Nucleus is clearly seen with a karyosome •Cytoplasm is smooth and hyaline appearance •Glycogen mass stains brown Saline mounting •Chromatid bodies are seen as round refracile bars •Cyst wall smooth and thin •Glycogen bar not visible •Outlines of nuclei may be visible Iron haematoxillin stain •Chromatid body and nucleus stain jet black •Cytoplasm stains bluish or greyish •Glycogen mass gets dissolved in the process of stainning and remains as a vacuole
Entamoeba coli TROPHOZOITE STAGE • Largest amoeba 20-40 µm in diameter • Sluggishly motile • Cytoplasm not clearly defined • Opaque endoplasm packed with food vacuoles with bacteria and others but no RBC • Nucleus visible in unstainned preparation • In stainned prep nucleus shows large eccentric karyosome surrounded by broader halo and coarse chromatin ranules linning nuclear membrane Stool examination by Dr. Priyanka Buragohain 89
• CYST • 15- 20 µm in diameter • Rounded body • Octanucleate • Largen glycogen mass in binucleate stage • Chromatoid body if present are in slender filaments or pointed threads • Glycogen mass and chromatoid bodies are absent in mature cyst Stool examination by Dr. Priyanka Buragohain 90
Endolimax nana • Commensal in large intestine of man • Trophozoites are smaller in size(8-9 µm in diameter) • Sluggish in motility • Cytoplasmic inclusions contain bacteria and food particles but no RBC • Nucleus has irregular karyosome, eccentric and in contact with nuclear membrane Stool examination by Dr. Priyanka Buragohain 91
CYST • Cyst are oval • Same size as the trophozoites • Number nuclei are 1-4 • Mature cyst are quadrinucleate • Chromatoid bodies and glycpgen mass are not seen Stool examination by Dr. Priyanka Buragohain 92
Difference between different trophozoites Stool examination by Dr. Priyanka Buragohain 93
Difference between different cyst Stool examination by Dr. Priyanka Buragohain 94
Stool examination by Dr. Priyanka Buragohain 95 OTHER NON PATHOGENIC AMOEBIDA SPECIES Trophozoite of E. hartmani Trophozoite of Iodamoeba butschlii Cyst of blastocystis hominis
Stool examination by Dr. Priyanka Buragohain 96 Balantidium coli •Largest protozoal parasite •Pig is the common reservoir •Two stages •Trophozoite and encysted stage TROPHOZOITE •Oval body •60-70 µm in length and 40-50 µm breadth •Body is covered with a delicate pellicle showing longitudinal striations •Cilia are short and delicate, of uniform length, on mouth are longer called adoral cilia •Thin layer of ectoplasm and granular endoplasm •Groove at the anterior end(peristome) leading to a mouth(cytostome) terminating in a short funnel shaped gullet(cytopharynx) extending upto 1/3rd of the body •At the posterior part permanent anus called cytophage is situated •2 nuclei: kidney shaped macronucleus, round micronucleus in the concavity of micronucleus •2 contractile vacuole, many food vacuole
CYST • Smaller than trophic, 50-60 µm in diameter • Cytoplasm is granular contains the macronucleus, micronucleus, refractile body • Contractile vacuole • Thick transparent double layered wall Stool examination by Dr. Priyanka Buragohain 97
Giardia lambia • TROPHOZOITE • Flat view: a tennis or badminton racket • Side view: longitudinally split pear • Dorsal surface is convex and the ventral surface in concave with a sucking disc • Size is 14 × 7 µm • Anterior end is broad and rounded, posterior end tapers to a sharp point • Bilaterally symmetrical and all organs are paired. • 2 axostyles, 2 nuclei, 4 pairs of flagella Stool examination by Dr. Priyanka Buragohain 98 Exist in 2 phase: trophozoite and cyst
CYST • Oval in shape • Size is 12 × 7 µm • Axostyle lie diagonally like dividing wall within the cyst wall • 4 nuclei lie clustered at one end, lie in pairs at opposite poles • Remains of flagella and sucking disc may be seen in cytoplasm • Acid causes the parasite to encyst Stool examination by Dr. Priyanka Buragohain 99
Stool examination by Dr. Priyanka Buragohain 100
What we can see in microscopic examination of stool ? Stool examination by Dr. Priyanka Buragohain 101 ADULT WORM LARVA OVA CYST
Stool examination by Dr. Priyanka Buragohain 102 HELMINTHES PLATYHELMINTHES NEMATHELMINTHES CESTODE TREMATODE NEMATODE TAPEWORM Taenia solium Taenia saginata ECHINOCOCCUS G. Hominis F. Buski F. Hepatica C. Sinensis P. westermani • A. Lumbricoids • T. Trichiura • A. Duodenale • E. Vermicularis •S. Stercoralis
ADULT WORM • TAENIA SAGINATA/SOLIUM • DIPHYLLOBOTHRIUM • HYMENOLEPIS • DYPILIDIUM LARVAL STAGE • ECHINOCOCCUS: Hydatid cyst • SPIROMETRA • HYMENOLEPIS • TAENIA SOLIUM • MULTICEPS: Stool examination by Dr. Priyanka Buragohain 103
Taenia solium • Segments of Tape worm or single segment may be found • White in colour, semi transparent. • May be 3 – 10 m long/ 1-3 cm segments maybe upto 24 m • Variable length(1000-2000 proglottids) • When stool is allowed to dry up the pieces of segments will roll upand appear as round worm, moistening the segments will restore the shape • Head is quadrate in outline, has 4 circular suckers • Head is absent of rostellum/hooklets Stool examination by Dr. Priyanka Buragohain 104
Eggs of T. saginata • Spherical and brown in colour • 31-43µm in diameter • Thin outer transparent shell(remnants of yolk mass), causes egg to clump together • Inner embryophore is brown, thick walled, radially striated • Contains an oncosphere(14- 20µm), with 3 pairs of hooklets • Doesnot float in saturated solution of common salt Stool examination by Dr. Priyanka Buragohain 105
Taenia solium • 2-3 metres long(800-900 proglottides) • Scolex is 1mm in diameter, globular in outline, 4 circular suckers, • Head with rostellum armed with a double row of alternating large and small hooklets, shaped like daggers or Arabian poniards • Segments are shed in chains of 5-6 at a time, not single. Stool examination by Dr. Priyanka Buragohain 106
• EGGS • Same as T. Solium • 30x40 µm sized egg • Pale yellow • Thick radially striated embryophore with 6 hooklets inside Stool examination by Dr. Priyanka Buragohain 107
Echinococcus granulosus • Commonly called dog tapeworm/ hydatid worm • Man harbours the larval form, not the adult • Larva found wiyhin the hydatid cyst, scolex of the future adult worm remains invaginated within a vesicular body. Stool examination by Dr. Priyanka Buragohain 108
Stool examination by Dr. Priyanka Buragohain 109 Hymenolepis diminuta
Gastrodiscoides hominis • Prevalent in Assam and Bengal • Pyriform in shape • Measures 5-10 mm × 4-6 mm • Body has 2 parts: anterior conical and posterior hemispherical portion which is hollowed out ventrally to form a concave disc • Acetabulum is postero terminal, situated ventrally • Notch at posterior end • Eggs are ovoid, operculated, 130×60 µm, immature when oviposited Stool examination by Dr. Priyanka Buragohain 110
Fasciola hepatica • Eggs • Large, operculated, ovoid , brownish yellow(bile stained) • Size is 140× 80 µm • Contains a large unsegmentad ovum in a mass of yolk cells • Excreted with bile into duodenum and then passed out along with the faeces • Does not float in saturated common salt sol. • Can develop only in water Stool examination by Dr. Priyanka Buragohain 111
Fasciola buski • Reported in Assam, bengal, china, thailand and other oriental regions • Largest trematode(2-7.5 length, 8-20 mm bredth, 0.5-3 mm thickness) • Elongated and oval in shape • Resembles F. Hepatica but does not possess any cephalic cone • Each worm lay 25,000 eggs per day • Eggs are indistinguishable from F. hepatica Stool examination by Dr. Priyanka Buragohain 112
Clonorchis sinensis EGGS • Yellowish brown • Flask shaped • Operculated • Possess a terminal hook like spine(resembling an electric bulb) • Small in size(35×20µm) • Ciliated embryo (oviposited stage) • Do not float in saturated solution of common salt Stool examination by Dr. Priyanka Buragohain 113
Paragonimus westermani • Golden brown in colour • Oval in shape with flattened opercula • 80- 55 µm • Each egg contains an unsegmented ovum surrounded by yolk cells • Prevalent in Assam • Found in sputum and faeces Stool examination by Dr. Priyanka Buragohain 114
Ascaris lumbricoids • Large round worms may be males and females or both • Pinkish in colour • 0.3-0.4 cm in thickness • 15-25 cm long • Males are shorter than females • Have curved tapering tail Stool examination by Dr. Priyanka Buragohain 115
• UNFERTILISED EGGS • Do not float on floatation method • Size- 40×70 µm • Yellow in colour • Elongated • Mammilated thin shell, ovum containing refractile yolk globules occupying the whole inside space • May be confused with veg cell • FERTILISED EGGS • Float on floatation method • Size- 40×70 µm • Yellow in colour • Oval or round • Thick mammillated coat and single celled ovum inside Stool examination by Dr. Priyanka Buragohain 116
Enterobius vermicularis • Small round worm or thread like worm or pin worm(spindle shaped) • White coloured • 0.5-1 cm long • Tail pointed • Males smaller than females and posterior body is curved and sharply truncated(found only after purgation) Stool examination by Dr. Priyanka Buragohain 117
• EGGS • Colourless(not bile stained) • 20×50 µm • Assymetrical, Oval planoconvex, • thin transparent shelled, • contain coiled tadpole likelarva inside • Floats in saturated salt solution Stool examination by Dr. Priyanka Buragohain 118
Ancylostoma duodenale • Hook worm • Small greyish white or pink coloured cylindrical • 1-1.5 cm long • One end is curved like a hook • 6 teeth, 4 hook like on ventral surface and 2 knob like on the dorsal surface Stool examination by Dr. Priyanka Buragohain 119
EGGS • Oval or elliptical in shape • 40x60 µm sized egg • Colourless(not bile stained) • Surrounded by a transparent hyaline shelled membrane • Contains 4 segmented ovum inside. • Floats on saturated solution of salt Stool examination by Dr. Priyanka Buragohain 120
Trichuris trichiura • Whip worm • Looks like a tiny whip with a handle and a lash • 3-5 cm in length • White coloured Stool examination by Dr. Priyanka Buragohain 121
• 25x50 µm sized egg • Brown coloured • Double shelled, outer one is bile stained • Thick shelled, barrel shaped with mucus plug at both pole • Single ovum • floats in saturated solution of common salt Stool examination by Dr. Priyanka Buragohain 122
Strongyloids stercoralis • Adult worm: • Females are readily discovered than males • 2.5mm ×40-50 µm (females) • Posterior extrimity is pointed • Males are shorter and broader than females Stool examination by Dr. Priyanka Buragohain 123
• Eggs • Eggs are conspicuous within the body in a single line • 55-30µm • Thin shelled • Transparent, oval • Contain larva ready to hatch. It is the larva not the eggs are found in stool. Stool examination by Dr. Priyanka Buragohain 124
• Rhabditiform larvae • Develop directly from gravid females • Short mouth, double- bulb oesophagus • Filariform larvae • Longer and slender • Short mouth and cylindrical oesophagus Stool examination by Dr. Priyanka Buragohain 125
Stool examination by Dr. Priyanka Buragohain 126
Helminthes that float/do not float on saturated solution of saline Eggs that float • A. Duodenale • N. Americanas • E. Vermicularis • H. Nana • A. Lumbricoids • T. Trichuria • H. Diminuta Eggs that do not float • A. Lumbricoids • T. solium • T. Saginata • Trematodes • F. Buski • F. Hepatica • C. Sinensis Stool examination by Dr. Priyanka Buragohain 127
Stool examination by Dr. Priyanka Buragohain 128
Stool examination by Dr. Priyanka Buragohain 129
Stool examination by Dr. Priyanka Buragohain 130
Stool examination by Dr. Priyanka Buragohain 131 Plant hairs can be confused for larvae oh hookworm or Strongyloides stercoralisPlant hair resembling S. strongiloids
Stool examination by Dr. Priyanka Buragohain 132 Parasite of earth worm Pollen grain resemblening to fertile egg of A. lumbricoids Bee pollen resembling t. Trichuria egg
Stool examination by Dr. Priyanka Buragohain 133
STOOL CULTURE • Used to detect the presence of disease causing(pathogenic )bacteria • Help to diagnose an infection of digestive system • Used in conjunction with stool test • Reference range for stool culture is negative Stool examination by Dr. Priyanka Buragohain 134
Most common bacteria for culture of stool • Campylobacter species • Salmonella species • Shigella species • Yersinia species • Vibrio species(travel history) • Some bacteria cause illness by producing toxins(PCR, Antigen test are to be done with stool test) • Escherichia coli • Clostridium difficile Stool examination by Dr. Priyanka Buragohain 135
Collection • Specimen collected via rectal swab(in infants) • Sterile collection container not required • No detergent or preservative should be present in the container • Specimen should be immediately transported to the laboratory • If transport is delayed by longer than 2 hours, transport media(eg Cary- Blair) is recommended • Samples must be sent in a sealed, leak-proof container marked with a biohazard sticker Stool examination by Dr. Priyanka Buragohain 136
Medias used commonly • MacConkey agar- Salmonella species, shigella species • Eosin methylene blue agar • Triple sugar iron(TSI)- differentiate salmonella and shigella • Sabouraud agar • Hekteon enteric agar • Selenite broth Stool examination by Dr. Priyanka Buragohain 137
Summary • Definition of stool • Composition of stool • Precaution and collection of sample • Physical examination • Chemical examination • Microscopic examination • Artifacts • Stool culture Stool examination by Dr. Priyanka Buragohain 138
THANK YOU !!!Stool examination by Dr. Priyanka Buragohain 139

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Routine examination of stool

  • 1.
    STOOL EXAMINATION Presented by: Dr.Priyanka Buragohain Guided by: Dr. Hemen Kalita Deptt. Of Roga Nidan. Govt. Ayurvedic College
  • 2.
    DEFINITION • Human fecesis called as stool. • faeces / feces is plural of latin term faex meaning RESIDUE. • It is the waste residue of indigestible materials of an animal’s digestive tract expelled through the anus during defecation. • Meconium is newborn’s first feces. • SCATOLGY or CAPROLOGY is the study of feces. Stool examination by Dr. Priyanka Buragohain 2
  • 3.
    COMPOSITION • ¾ water,¼ solid • Undigested and unabsorbed food • Intestinal secretions, mucous • Bile pigments and salts • Decomposed products • Bacteria and inorganic material • Epithelial cells, leukocytes. Stool examination by Dr. Priyanka Buragohain 3
  • 4.
    PRECAUTION BEFORE COLLECTION •Patient should avoid the following things for at least 48 hours before collection of stool: • Mineral oils, bismuth, non absorbable anti diarrhoeal drugs, antimalarial drugs, antibiotics, etc • Pt. Should not have barium swallow examination before stool R/E • Avoid iron containing drugs, meat, fish etc for atleast 48 hours before stool for occult blood. • In constipated patients use only non residual purgative Stool examination by Dr. Priyanka Buragohain 4
  • 5.
    COLLECTION • Universal precautions •Pt. is asked to pass stool in a clean container. • Stool should be collected in a steralized, wide mouthed container. • Loose/last/portion containing mucus, blood etc is to be collected in a wide mouthed bottle. • Should be uncontaminated with urine or any other body secretions. • >2gm is required. • Properly named and always a fresh sample should be tested. • Liquid stool to be examined within ½ hour • Solid stool to be examined within 1 hour. • If delayed store in a refrigerator. Stool examination by Dr. Priyanka Buragohain 5
  • 6.
    COLLECTION CONTD... • 3samples of stool within 10 days to exclude false negatives. • 2 samples to be examined on alternate days after normal defaecation and 1 sample after a purgative for certain worms. • Formalin is the best preservative. It kills the bacteria but ptreserves the protozoa and helminthes. • For culture no preservatives to be used Stool examination by Dr. Priyanka Buragohain 6
  • 7.
    7 Stool examination byDr. Priyanka Buragohain
  • 8.
    TYPES OF EXAMINATION •PHYSICAL EXAMINATION: colour, volume, consistency, odour, mucus, pus, concreations, helminths. • CHEMICAL EXAMINATION: reactions, occult blood, fat, carbohydrate, protein, etc • MICROSCOPIC EXAMINATION: remnants of food, pus cells, macrophages, RBCs, crystals, bacteria, yeasts, molds, protozoa, helminths. • STOOL CULTURE: Stool examination by Dr. Priyanka Buragohain 8
  • 9.
    Physical examination • AMOUNT •CONSISTENCY • COLOUR • ODOUR • REACTION • MUCUS • CONCRETION • BLOOD • PUS • FOOD REMNANT • UNDIGESTED TABLETS Stool examination by Dr. Priyanka Buragohain 9
  • 10.
    MACROSCOPIC EXAMINATION AMOUNT • Normalis 150 g to 200 g/day • Increased in steatorrhoea, diarrhoea, indigestion of carbohydrate. Stool examination by Dr. Priyanka Buragohain 10
  • 11.
    CONSISTENCY OR FORM •Normal is soft but formed • Excessively hard/scybala- habitual constipation • Flattened or ribbon like-intake of excess of mineral oil, carcinoma of rectum, stricture of rectum • 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 Stool examination by Dr. Priyanka Buragohain 11
  • 12.
    Stool examination byDr. Priyanka Buragohain 12
  • 13.
    Colour • Dark Grey-excessive cocoa or chocolate ingestion • Reddish or blackish brown- large amount of fruits • Green – ingestion of green leafy vegetables, administration of calomel due to biliverdin • Red – Beat ingestion fresh blood • Yellow – rhubarb or senna ingestion, normal stool Stool examination by Dr. Priyanka Buragohain 13
  • 14.
    • Clay –obstructive jaundice, barium meal x-ray • Tarry black – haemorrhage in stomach/upper intestine • Dark brown to bright red – bleeding in rectum or sigmoid colon • Red streaks of blood on the surface of faeces- haemorrhoids, fissures, carcinoma ,ulcerative colitis Stool examination by Dr. Priyanka Buragohain 14
  • 15.
    Odour • Normal odouris aromatic due to indole and skatole • Increased- excessive protein ingestion • Sour rancid- fatty acid in milk indigestion (in children and adults), normal in infants • Putrid- severe diarrhoea of malignancy, gangrenous dysentry Stool examination by Dr. Priyanka Buragohain 15
  • 16.
    Reaction • Normal isneutral • Ph varies from 6.9 to 7.2 • pH is dependent on bacterial fermentation and putrefaction in the bowel. • Alkaline – excess protein ingestion • Acidic – excess carbohydrate ingestion Stool examination by Dr. Priyanka Buragohain 16
  • 17.
    Mucus • Small quantityof 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 Stool examination by Dr. Priyanka Buragohain 17
  • 18.
    Concretion • In infantswhitish curds may be found • Gall bladder stones may be rarely found Stool examination by Dr. Priyanka Buragohain 18
  • 19.
    Blood • Absent innormal faeces • Formed stool with streaks of blood – lesion in sigmoid colon, rectum or anal canal • Liquid stool with bright red blood, pus and mucus- bacillary dysentery, ulcerative colitis • Semi formed stool with deep tarry black blood- melena • Loose stool with deep cherry red blood- melena Stool examination by Dr. Priyanka Buragohain 19
  • 20.
    Pus • Normally absent •Pus with blooded mucus- ulcerative colitis, bacillary dysentery, ulcerative carcinoma Stool examination by Dr. Priyanka Buragohain 20
  • 21.
    • GROSS FOODREMNANTS MAY IN NORMAL STOOL • UNDIGESTED TABLETS MAY BE FOUND Stool examination by Dr. Priyanka Buragohain 21
  • 22.
    CHEMICAL EXAMINATION Stool examinationby Dr. Priyanka Buragohain 22
  • 23.
    Chemical Examination OfStool • Acidity/basicity • Fats • Nitrogen • Stercobilinogen • Coproporphyrin • Occult blood • Reducing substances • N. B : most commonly used chemical examination of stool is pH, occult blood and reducing substances Stool examination by Dr. Priyanka Buragohain 23
  • 24.
    FATS • Normally upto20% of total solids • Lipids are measured as fatty acids:2-5 gm/24 hrs • Known dietary intake and timed stool collection. • Take diet containing 100 gm of fat daily, 3 day stool collection. • >6 gm/day is abnormal • Quantitative or semiquantitative methods:  Gravimetric method  Isotopic techniques(radio-isotopes)  Electrical capacitance method  Titrimetric method of Van de Kamer Stool examination by Dr. Priyanka Buragohain 24
  • 25.
    Titrimetric method • Boilwith alcoholic potassium hydroxide to convert fats and fatty acid into soap • cool • HCl is added to convert soap to fatty acid • Fatty acid extracted with petroleum ether • Aliquot is evaporated, taken up in neutral alcohol • Ttitrate with sodium hydroxide • Fatts are calculated as fatty acids Stool examination by Dr. Priyanka Buragohain 25
  • 26.
    Electrical capacitance methods •Fecal suspension is extracted with solvent specially with chlorinated benzene • Extract is filtered • Electrical capacitance is measured and compared with standard of triolein simillarly treated Stool examination by Dr. Priyanka Buragohain 26
  • 27.
    Interpretation • Fecal fatincrease in • enteritis and pancreatic disease (lack of lipase) • Surgical removal of a section of intestine • Mal absorption syndrome • Chronic pancreatic disease(> 10 gm / 24 hr) • Neutral fat increase in • Use of rectal suppositories • Ingestion of castor oil or mineral oil • Ingestion of dietetic low calories mayonnaise • Tropical sprue Stool examination by Dr. Priyanka Buragohain 27
  • 28.
    Nitrogen • Varies withthe amount and nature of diet • Normal is 1 to 1.5 gm /day • Increase in azotorrhoea, pancreatic achylia, pancreatogenous fatty diarrhoea, idiopathic steatorrhoea Stool examination by Dr. Priyanka Buragohain 28
  • 29.
    Stercobilinogen • Normal is40 to 280 mg/day • Average is 150 mg/day • Dependent on amount of bilirubin passing to intestine(jaundice) Stool examination by Dr. Priyanka Buragohain 29
  • 30.
    Coproporphyrin • Normal is300 to 1100 mg/ day • Type 1 – 70% • Type 3 – 10 to 30% • Abnormally increased in congenetial porphyria • Abnormallly decreased i9n liver disease like cirrochis, hepattitis, passive4 venous congestion, metastatic carcinoma in liver Stool examination by Dr. Priyanka Buragohain 30
  • 31.
    Occult blood • Detectblood which is present in amount or form not visible macroscopically • Normally nil • Abnormal presence in condition of occult haemorrhage in the GI tract • BENZIDINE TEST • GUAIAC TEST • ORTHOTOLIDINE TEST • Most commonly used test is benzidine test Stool examination by Dr. Priyanka Buragohain 31
  • 32.
    BENZIDINE TEST • 4gm benzidine in 100 ml of glacial acetic acid • Emulsify pea sized bit of faeces in 5 ml of water. • Mix 1 ml emulsion and 1 ml of reagent in test tube • Add several drops of 35 H2O2 • Blue colour indicates positive reaction Stool examination by Dr. Priyanka Buragohain 32
  • 33.
    • Trace- faintblue colour after 1 min • 1+ - definite blue green slowly • 2+ - green blue rapidly • 3+ - blue almost immediately • 4+ - dark blue immediately Stool examination by Dr. Priyanka Buragohain 33
  • 34.
    GUAIAC TEST • Lesssensitive • With loss of 20 to 30 ml of blood all test will be positive • Guaiac reagent consist of 1 gm Guaiac in 5 ml of 95% ethanol. • Make a small smear of feces on a filter paper • Add 2 to 3 drops of gum guaiac solution + 2 to 3 drops of glacial acetic acid + 2 to 3 drops of 3% H2O2 Stool examination by Dr. Priyanka Buragohain 34
  • 35.
    • Trace- faintblue green in 1 min • 1+ light blue slowly • 2+ clear blue rapidly • 3 + deep blue almost immediately • 4+ deep blue immediately Stool examination by Dr. Priyanka Buragohain 35
  • 36.
    ORTHOTOLIDINE TEST • Intermediatesinsitivity • Smear the stool on a filter paper with an applicator • Pipette a few drops of the reagent on to the filter paper(orthotolidine barium peroxide 200 mg+ glacial acetic acid 5 ml) • After 30 sec examine for a blue colour • Blue green colour within 30 sec means positive test Stool examination by Dr. Priyanka Buragohain 36
  • 37.
    Interpretation • Gastric diseaseeg chronic ulcer and malignancy • Intestinal diseases eg dysentery, typhoid fever, carcinoma • Haemorrhoids • During instrumentation Stool examination by Dr. Priyanka Buragohain 37
  • 38.
    Faecal reducing substancetest • To diagnose lactose intolerance • Sample of 5 gm stool is needed • Sample needs to be delivered to the laboratory as soon as possible, preferably within 1 hr , cause lactose in the stool will normally be broken down by chemical processes within 2-4 hrs after the specimen is produced. Stool examination by Dr. Priyanka Buragohain 38
  • 39.
    Interpretation • Negative/ trace-< 0.25 g/dl • Suspicious(grade 1) – 0.25-0.5 g/dl • Abnormal(grade 2-4)->0.5 g/dl • Found in Carbohydrate malabsorption • Tropical sprue Stool examination by Dr. Priyanka Buragohain 39
  • 40.
  • 41.
    NEED FOR MICROSCOPICEXAMINATION • For the diagnosis of microscopic elements. • Trophozoites and its movements are better seen in unstained preparation of a fresh material. • Cystic forms &Nuclear character are better seen in stained preparation(iodine) • Gycogen mass- stained with iodine • Chromatoid bars- unstained preparation • N.B – Both stained and unstained materials are to be prepared Stool examination by Dr. Priyanka Buragohain 41
  • 42.
    Stool examination byDr. Priyanka Buragohain 42
  • 43.
    Need of concentrationtechnique • To see whether treatment of parasite is successful • To find ova of S. Mansoni or Taenia if few or other ova and cyst are not seen in routine examination • To examine stool specimens from patients who do not come from an area where a particular parasite is found Stool examination by Dr. Priyanka Buragohain 43
  • 44.
    Stool examination byDr. Priyanka Buragohain 44
  • 45.
    FLOATATION TECHNIQUE • Usesolutions which have highier specific gravity(zinc sulphate or Sheather’s sugar) than the organisms to be floated so that the organisms rise to the top and the debries sink to the bottom. • Advantage – produce a cleaner material than the sedimentation technique • Disadvantage – walls of eggs and cyst will often collapse, hindering identification. • Some parasite eggs do not float. Stool examination by Dr. Priyanka Buragohain 45
  • 46.
    SEDIMETATION TECHNIQUE • Usesolutions of lower specific gravity than the parasitic organisms(formalin ethyl acetate technique) • Recommended for general diagnostic laboratories due to easy to perform and less prone to technical error. Stool examination by Dr. Priyanka Buragohain 46
  • 47.
    Sedimentation techniques • Mixa small piece of stool with 10 ml of water or saline in a tube/ bottle • Sieve the suspension into a beaker through a strainer with small holes. • Pour the contents into a centrifuge tube • Centrifuge at 2000-3000/rpm for 1 min • Pour off the supernatant part • Resuspend the deposit in clean water and add enough water to fill the tube. • Mix well and recentrifuge • Pour off the supernatant part • Resuspend in zinc sulphate solution, fill the tube with the solution • Centrifuge at high speed for 1 min • Transfer the contents from the surface of the tube to a slide, using a bacteriological wire loop • Add small drops of saline and mix • Cover with a cover slip • Examine under 10x and 40x objectives Stool examination by Dr. Priyanka Buragohain 47
  • 48.
    Stool examination byDr. Priyanka Buragohain 48
  • 49.
    Stainning methods Wet mount normalsaline Iodine solution Buffered methylene blue solution Eosin solution Stainning for permanent preparation Schaudinn’s fluid Heidenhain’s Haematoxilin method Trichome stain Stool examination by Dr. Priyanka Buragohain 49
  • 50.
    Microscopic examination ofwet mount Stool examination by Dr. Priyanka Buragohain 50
  • 51.
    Saline wet mount Stoolexamination by Dr. Priyanka Buragohain 51
  • 52.
    Iodine wet mount •Iodine kills the organisms, therefore motility is lost. • Used mainly to stain nuclei and glycogen mass if present. • Flagella becomes recognisable. • Cyst can usually be specifically identified in this method. • Lugol’s iodine solution is used. Stool examination by Dr. Priyanka Buragohain 52
  • 53.
    Lugol’s iodine • Itsvery strong • Must be diluted about 5 times with distilled water • Stain deteriorates quickly hence to be prepared every 2 weeks • Contains: Iodine crystals(powdered): 5 g Potassium iodide :10 g Distilled water : 10 • Potassium iodide is dissolved in distilled water and iodine crystals are slowly added. Solution is filtered and kept in a stoppered bottle of amber colour Stool examination by Dr. Priyanka Buragohain 53
  • 54.
    Buffered methylene bluewet mount • 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 Stool examination by Dr. Priyanka Buragohain 54
  • 55.
    Eosin wet mount •Detection of trophozoites and cyst • They can be much more easily detected against the pink- red background of eosin preparation Stool examination by Dr. Priyanka Buragohain 55
  • 56.
    Stool examination byDr. Priyanka Buragohain 56
  • 57.
    Stool examination byDr. Priyanka Buragohain 57
  • 58.
    Stool examination byDr. Priyanka Buragohain 58
  • 59.
    Stool examination byDr. Priyanka Buragohain 59 Cover with a cover slip
  • 60.
    Stool examination byDr. Priyanka Buragohain 60
  • 61.
    Stool examination byDr. Priyanka Buragohain 61
  • 62.
    microscopic examination findings •REMNANTS OF FOOD • vegetable cells • Muscle fibres • Starch granules • Fat globules • Connective tissue/ elastic fibres • Mineral oil or castor oil globules • CELLS • Epithelial cells • Pus cells • Macrophages • Ghost cells • Pyknotic bodies • Eosinophills • RBC • Crystals • Yeasts and molds • Protozoa • Helminthic parasites Stool examination by Dr. Priyanka Buragohain 62
  • 63.
    Vegetable cell • Sometimescauses confusion with ova, eggs, cyst or cell bodies • IRREGULAR OUTER MARGIN Excess quantity is seen in excess intake of vegetables or indigestion Stool examination by Dr. Priyanka Buragohain 63
  • 64.
    Muscle fibres • Mayconfuse with Tinea segments • Excess protein intake or indigestion • Its excess excretion is called Creatorrhoea(flesh-flow) Stool examination by Dr. Priyanka Buragohain 64
  • 65.
    Starch granules • Variablein size, round to polygonal in shape, colourless, circular or Y shaped dot in the centre • Confused with ova of helminths • Found in carbohydrate dyspepsia • Better seen in iodine preparation Stool examination by Dr. Priyanka Buragohain 65
  • 66.
    Fat globules • Appearsimilar to parasitic cyst or cell bodies • Emulsifying agents are used to eliminate confusion • Confused with ova of helminths • Found in fat dyspepsia Stool examination by Dr. Priyanka Buragohain 66
  • 67.
    Connective tissue/elastic fibres •Confused with tinea segments • Signify indigestion Stool examination by Dr. Priyanka Buragohain 67
  • 68.
    Mineral oil/ castoroil globules • When taken as purgative • May be confused with ova of helminthes Stool examination by Dr. Priyanka Buragohain 68
  • 69.
    Epithelial cells • Excesspresence due to inflammatory conditions of colon, rectum, anal canal Stool examination by Dr. Priyanka Buragohain 69
  • 70.
    Pus cells • Commonlyfound in normal stool, help to ease the passage of stool • Normally not visible to human eye. • If visible indicates disease • Bacillary dysentery, UC, acute Amoebic dysentery, malignancy of rectum, drug induced enterocilitis Stool examination by Dr. Priyanka Buragohain 70
  • 71.
    Macrophages • Large mononuclearcells with vesicular nucleus and ingested materials including RBC • Confused with E. Histolytica cyst or E. Coli cyst • Excess in Amoebic or bacillary dysentery Stool examination by Dr. Priyanka Buragohain 71
  • 72.
    Ghost cells • Degerativeform of macrophages, epithelial cells • Its an enlarged/swollen eosinophilic epithelial cell with only eosinophilic cytoplasmic outline but without a nucleus • Characteristic of bacillary dysentery Stool examination by Dr. Priyanka Buragohain 72
  • 73.
    Pyknotic bodies • Nuclearremains of tissue cells and leucocytes • Characteristic of acute amoebic dysentery Stool examination by Dr. Priyanka Buragohain 73
  • 74.
    Eosinophils • In intestinalallergy • Diluting fluid used- Randolph’s diluting fluid,Pilot’s stain • Carbol chromotrope technique • A measured quantity of the deposit is taken and diluted with the diluting fluid 1:10 or 1: 20 according to the concentration of the residue and counted in haemocytometer. • Increased in allergic conditions, parasitic infestation and drug allergy, ulcerative colitis Stool examination by Dr. Priyanka Buragohain 74
  • 75.
    RBCs • seen incases of ulcrative lesions of gut • in bacillary dysentery – yellowish discrete • Amoebic dysentery – greenish and in clumps Stool examination by Dr. Priyanka Buragohain 75
  • 76.
    Crystals • Fatty acidcrystals • Calcium oxalate crystals • Triple phosphate crystals • Charcot Leyden crystals • Haemotoidin crystals • Crystals of drugs Stool examination by Dr. Priyanka Buragohain 76
  • 77.
    • Charcot Leydencrystal: • Slender and pointed at both ends, Hexagonal bipyramidal structures localised in the primary granues of cytoplasm of eosinophils and basophils • Evidence of parasitic infiltrate eg amoeba, ascaris, hookworm, fasciola • diamond shaped or whetstone shaped crystals • Normally colourless, stained purplish- red by trichome • Vary in size and may be as large as 50 µm in length • Found in UC, dysentery, malignant ulcers, schistosomiasis etc Stool examination by Dr. Priyanka Buragohain 77
  • 78.
    • Haematoidin crystals: •Ironless pigment derived from haemoglobin and formed within tissues(reticuloendothelial cells) but found extracellularly after 5-7 days in foci of previous haemorrhage. • Occurs as refractile, yellow- brown and orange-red granules • Characteristically as rhomboid plates arranged in a radial pattern, so called hematoidin burrs. Stool examination by Dr. Priyanka Buragohain 78
  • 79.
    Yeast and molds •Yeast are normally present • Excess in cases of AIDS • Molds are rare but may be seen in immunodeficiency conditions Stool examination by Dr. Priyanka Buragohain 79
  • 80.
    Parasites Stool examination byDr. Priyanka Buragohain 80
  • 81.
    Stool examination byDr. Priyanka Buragohain 81
  • 82.
    Stool examination byDr. Priyanka Buragohain 82
  • 83.
    Stool examination byDr. Priyanka Buragohain 83 protozoa
  • 84.
    Entamoeba histolytica TROPHOZOITE STAGE •Identified by motility and presence of ingested RBC • Shape : constantly changing position • Size :ranges from 18 to 40 µm ,average being 20 to 20 µm • Cytoplasm :divisible in two portion • Nucleus :spherical in shape Stool examination by Dr. Priyanka Buragohain 84
  • 85.
    Stool examination byDr. Priyanka Buragohain 85 RBC appear yellowish green inside the endoplasm Nucleus is not visible but a faint outline may be detected Endoplasm shows bluish or ground glass appearance Eccentric nucleus with karyosome (a small dot at the centre surrounded by a clear halo), nuclear membrane, Linen network having a spoke like radial arrangement
  • 86.
    PRE CYSTIC STAGE •Size :small in size,10 to 20 µm • Shape :round or slightly ovoid with blunt pseudopodium • Free from ingested RBC and other materials • Nucleus : large nucleus • Retains the characteristics of trophozoite. Stool examination by Dr. Priyanka Buragohain 86
  • 87.
    CYSTIC STAGE • Size: 6 to 9 µm / 12 to 15 µm • Shape : round, surrounded by a highly refractile membrane called cyst wall • Nucleus :quadrinucleate • Clear and hyaline cytoplasm • Nuclear structure retainning the character of trophozoite Stool examination by Dr. Priyanka Buragohain 87
  • 88.
    Stool examination byDr. Priyanka Buragohain 88 •Iodine mounting •Body of the parasite stains yellow to light brown •Nucleus is clearly seen with a karyosome •Cytoplasm is smooth and hyaline appearance •Glycogen mass stains brown Saline mounting •Chromatid bodies are seen as round refracile bars •Cyst wall smooth and thin •Glycogen bar not visible •Outlines of nuclei may be visible Iron haematoxillin stain •Chromatid body and nucleus stain jet black •Cytoplasm stains bluish or greyish •Glycogen mass gets dissolved in the process of stainning and remains as a vacuole
  • 89.
    Entamoeba coli TROPHOZOITE STAGE •Largest amoeba 20-40 µm in diameter • Sluggishly motile • Cytoplasm not clearly defined • Opaque endoplasm packed with food vacuoles with bacteria and others but no RBC • Nucleus visible in unstainned preparation • In stainned prep nucleus shows large eccentric karyosome surrounded by broader halo and coarse chromatin ranules linning nuclear membrane Stool examination by Dr. Priyanka Buragohain 89
  • 90.
    • CYST • 15-20 µm in diameter • Rounded body • Octanucleate • Largen glycogen mass in binucleate stage • Chromatoid body if present are in slender filaments or pointed threads • Glycogen mass and chromatoid bodies are absent in mature cyst Stool examination by Dr. Priyanka Buragohain 90
  • 91.
    Endolimax nana • Commensalin large intestine of man • Trophozoites are smaller in size(8-9 µm in diameter) • Sluggish in motility • Cytoplasmic inclusions contain bacteria and food particles but no RBC • Nucleus has irregular karyosome, eccentric and in contact with nuclear membrane Stool examination by Dr. Priyanka Buragohain 91
  • 92.
    CYST • Cyst areoval • Same size as the trophozoites • Number nuclei are 1-4 • Mature cyst are quadrinucleate • Chromatoid bodies and glycpgen mass are not seen Stool examination by Dr. Priyanka Buragohain 92
  • 93.
    Difference between differenttrophozoites Stool examination by Dr. Priyanka Buragohain 93
  • 94.
    Difference between differentcyst Stool examination by Dr. Priyanka Buragohain 94
  • 95.
    Stool examination byDr. Priyanka Buragohain 95 OTHER NON PATHOGENIC AMOEBIDA SPECIES Trophozoite of E. hartmani Trophozoite of Iodamoeba butschlii Cyst of blastocystis hominis
  • 96.
    Stool examination byDr. Priyanka Buragohain 96 Balantidium coli •Largest protozoal parasite •Pig is the common reservoir •Two stages •Trophozoite and encysted stage TROPHOZOITE •Oval body •60-70 µm in length and 40-50 µm breadth •Body is covered with a delicate pellicle showing longitudinal striations •Cilia are short and delicate, of uniform length, on mouth are longer called adoral cilia •Thin layer of ectoplasm and granular endoplasm •Groove at the anterior end(peristome) leading to a mouth(cytostome) terminating in a short funnel shaped gullet(cytopharynx) extending upto 1/3rd of the body •At the posterior part permanent anus called cytophage is situated •2 nuclei: kidney shaped macronucleus, round micronucleus in the concavity of micronucleus •2 contractile vacuole, many food vacuole
  • 97.
    CYST • Smaller thantrophic, 50-60 µm in diameter • Cytoplasm is granular contains the macronucleus, micronucleus, refractile body • Contractile vacuole • Thick transparent double layered wall Stool examination by Dr. Priyanka Buragohain 97
  • 98.
    Giardia lambia • TROPHOZOITE •Flat view: a tennis or badminton racket • Side view: longitudinally split pear • Dorsal surface is convex and the ventral surface in concave with a sucking disc • Size is 14 × 7 µm • Anterior end is broad and rounded, posterior end tapers to a sharp point • Bilaterally symmetrical and all organs are paired. • 2 axostyles, 2 nuclei, 4 pairs of flagella Stool examination by Dr. Priyanka Buragohain 98 Exist in 2 phase: trophozoite and cyst
  • 99.
    CYST • Oval inshape • Size is 12 × 7 µm • Axostyle lie diagonally like dividing wall within the cyst wall • 4 nuclei lie clustered at one end, lie in pairs at opposite poles • Remains of flagella and sucking disc may be seen in cytoplasm • Acid causes the parasite to encyst Stool examination by Dr. Priyanka Buragohain 99
  • 100.
    Stool examination byDr. Priyanka Buragohain 100
  • 101.
    What we cansee in microscopic examination of stool ? Stool examination by Dr. Priyanka Buragohain 101 ADULT WORM LARVA OVA CYST
  • 102.
    Stool examination byDr. Priyanka Buragohain 102 HELMINTHES PLATYHELMINTHES NEMATHELMINTHES CESTODE TREMATODE NEMATODE TAPEWORM Taenia solium Taenia saginata ECHINOCOCCUS G. Hominis F. Buski F. Hepatica C. Sinensis P. westermani • A. Lumbricoids • T. Trichiura • A. Duodenale • E. Vermicularis •S. Stercoralis
  • 103.
    ADULT WORM • TAENIASAGINATA/SOLIUM • DIPHYLLOBOTHRIUM • HYMENOLEPIS • DYPILIDIUM LARVAL STAGE • ECHINOCOCCUS: Hydatid cyst • SPIROMETRA • HYMENOLEPIS • TAENIA SOLIUM • MULTICEPS: Stool examination by Dr. Priyanka Buragohain 103
  • 104.
    Taenia solium • Segmentsof Tape worm or single segment may be found • White in colour, semi transparent. • May be 3 – 10 m long/ 1-3 cm segments maybe upto 24 m • Variable length(1000-2000 proglottids) • When stool is allowed to dry up the pieces of segments will roll upand appear as round worm, moistening the segments will restore the shape • Head is quadrate in outline, has 4 circular suckers • Head is absent of rostellum/hooklets Stool examination by Dr. Priyanka Buragohain 104
  • 105.
    Eggs of T.saginata • Spherical and brown in colour • 31-43µm in diameter • Thin outer transparent shell(remnants of yolk mass), causes egg to clump together • Inner embryophore is brown, thick walled, radially striated • Contains an oncosphere(14- 20µm), with 3 pairs of hooklets • Doesnot float in saturated solution of common salt Stool examination by Dr. Priyanka Buragohain 105
  • 106.
    Taenia solium • 2-3metres long(800-900 proglottides) • Scolex is 1mm in diameter, globular in outline, 4 circular suckers, • Head with rostellum armed with a double row of alternating large and small hooklets, shaped like daggers or Arabian poniards • Segments are shed in chains of 5-6 at a time, not single. Stool examination by Dr. Priyanka Buragohain 106
  • 107.
    • EGGS • Sameas T. Solium • 30x40 µm sized egg • Pale yellow • Thick radially striated embryophore with 6 hooklets inside Stool examination by Dr. Priyanka Buragohain 107
  • 108.
    Echinococcus granulosus • Commonlycalled dog tapeworm/ hydatid worm • Man harbours the larval form, not the adult • Larva found wiyhin the hydatid cyst, scolex of the future adult worm remains invaginated within a vesicular body. Stool examination by Dr. Priyanka Buragohain 108
  • 109.
    Stool examination byDr. Priyanka Buragohain 109 Hymenolepis diminuta
  • 110.
    Gastrodiscoides hominis • Prevalentin Assam and Bengal • Pyriform in shape • Measures 5-10 mm × 4-6 mm • Body has 2 parts: anterior conical and posterior hemispherical portion which is hollowed out ventrally to form a concave disc • Acetabulum is postero terminal, situated ventrally • Notch at posterior end • Eggs are ovoid, operculated, 130×60 µm, immature when oviposited Stool examination by Dr. Priyanka Buragohain 110
  • 111.
    Fasciola hepatica • Eggs •Large, operculated, ovoid , brownish yellow(bile stained) • Size is 140× 80 µm • Contains a large unsegmentad ovum in a mass of yolk cells • Excreted with bile into duodenum and then passed out along with the faeces • Does not float in saturated common salt sol. • Can develop only in water Stool examination by Dr. Priyanka Buragohain 111
  • 112.
    Fasciola buski • Reportedin Assam, bengal, china, thailand and other oriental regions • Largest trematode(2-7.5 length, 8-20 mm bredth, 0.5-3 mm thickness) • Elongated and oval in shape • Resembles F. Hepatica but does not possess any cephalic cone • Each worm lay 25,000 eggs per day • Eggs are indistinguishable from F. hepatica Stool examination by Dr. Priyanka Buragohain 112
  • 113.
    Clonorchis sinensis EGGS • Yellowishbrown • Flask shaped • Operculated • Possess a terminal hook like spine(resembling an electric bulb) • Small in size(35×20µm) • Ciliated embryo (oviposited stage) • Do not float in saturated solution of common salt Stool examination by Dr. Priyanka Buragohain 113
  • 114.
    Paragonimus westermani • Goldenbrown in colour • Oval in shape with flattened opercula • 80- 55 µm • Each egg contains an unsegmented ovum surrounded by yolk cells • Prevalent in Assam • Found in sputum and faeces Stool examination by Dr. Priyanka Buragohain 114
  • 115.
    Ascaris lumbricoids • Largeround worms may be males and females or both • Pinkish in colour • 0.3-0.4 cm in thickness • 15-25 cm long • Males are shorter than females • Have curved tapering tail Stool examination by Dr. Priyanka Buragohain 115
  • 116.
    • UNFERTILISED EGGS •Do not float on floatation method • Size- 40×70 µm • Yellow in colour • Elongated • Mammilated thin shell, ovum containing refractile yolk globules occupying the whole inside space • May be confused with veg cell • FERTILISED EGGS • Float on floatation method • Size- 40×70 µm • Yellow in colour • Oval or round • Thick mammillated coat and single celled ovum inside Stool examination by Dr. Priyanka Buragohain 116
  • 117.
    Enterobius vermicularis • Smallround worm or thread like worm or pin worm(spindle shaped) • White coloured • 0.5-1 cm long • Tail pointed • Males smaller than females and posterior body is curved and sharply truncated(found only after purgation) Stool examination by Dr. Priyanka Buragohain 117
  • 118.
    • EGGS • Colourless(notbile stained) • 20×50 µm • Assymetrical, Oval planoconvex, • thin transparent shelled, • contain coiled tadpole likelarva inside • Floats in saturated salt solution Stool examination by Dr. Priyanka Buragohain 118
  • 119.
    Ancylostoma duodenale • Hookworm • Small greyish white or pink coloured cylindrical • 1-1.5 cm long • One end is curved like a hook • 6 teeth, 4 hook like on ventral surface and 2 knob like on the dorsal surface Stool examination by Dr. Priyanka Buragohain 119
  • 120.
    EGGS • Oval orelliptical in shape • 40x60 µm sized egg • Colourless(not bile stained) • Surrounded by a transparent hyaline shelled membrane • Contains 4 segmented ovum inside. • Floats on saturated solution of salt Stool examination by Dr. Priyanka Buragohain 120
  • 121.
    Trichuris trichiura • Whipworm • Looks like a tiny whip with a handle and a lash • 3-5 cm in length • White coloured Stool examination by Dr. Priyanka Buragohain 121
  • 122.
    • 25x50 µmsized egg • Brown coloured • Double shelled, outer one is bile stained • Thick shelled, barrel shaped with mucus plug at both pole • Single ovum • floats in saturated solution of common salt Stool examination by Dr. Priyanka Buragohain 122
  • 123.
    Strongyloids stercoralis • Adultworm: • Females are readily discovered than males • 2.5mm ×40-50 µm (females) • Posterior extrimity is pointed • Males are shorter and broader than females Stool examination by Dr. Priyanka Buragohain 123
  • 124.
    • Eggs • Eggsare conspicuous within the body in a single line • 55-30µm • Thin shelled • Transparent, oval • Contain larva ready to hatch. It is the larva not the eggs are found in stool. Stool examination by Dr. Priyanka Buragohain 124
  • 125.
    • Rhabditiform larvae •Develop directly from gravid females • Short mouth, double- bulb oesophagus • Filariform larvae • Longer and slender • Short mouth and cylindrical oesophagus Stool examination by Dr. Priyanka Buragohain 125
  • 126.
    Stool examination byDr. Priyanka Buragohain 126
  • 127.
    Helminthes that float/donot float on saturated solution of saline Eggs that float • A. Duodenale • N. Americanas • E. Vermicularis • H. Nana • A. Lumbricoids • T. Trichuria • H. Diminuta Eggs that do not float • A. Lumbricoids • T. solium • T. Saginata • Trematodes • F. Buski • F. Hepatica • C. Sinensis Stool examination by Dr. Priyanka Buragohain 127
  • 128.
    Stool examination byDr. Priyanka Buragohain 128
  • 129.
    Stool examination byDr. Priyanka Buragohain 129
  • 130.
    Stool examination byDr. Priyanka Buragohain 130
  • 131.
    Stool examination byDr. Priyanka Buragohain 131 Plant hairs can be confused for larvae oh hookworm or Strongyloides stercoralisPlant hair resembling S. strongiloids
  • 132.
    Stool examination byDr. Priyanka Buragohain 132 Parasite of earth worm Pollen grain resemblening to fertile egg of A. lumbricoids Bee pollen resembling t. Trichuria egg
  • 133.
    Stool examination byDr. Priyanka Buragohain 133
  • 134.
    STOOL CULTURE • Usedto detect the presence of disease causing(pathogenic )bacteria • Help to diagnose an infection of digestive system • Used in conjunction with stool test • Reference range for stool culture is negative Stool examination by Dr. Priyanka Buragohain 134
  • 135.
    Most common bacteriafor culture of stool • Campylobacter species • Salmonella species • Shigella species • Yersinia species • Vibrio species(travel history) • Some bacteria cause illness by producing toxins(PCR, Antigen test are to be done with stool test) • Escherichia coli • Clostridium difficile Stool examination by Dr. Priyanka Buragohain 135
  • 136.
    Collection • Specimen collectedvia rectal swab(in infants) • Sterile collection container not required • No detergent or preservative should be present in the container • Specimen should be immediately transported to the laboratory • If transport is delayed by longer than 2 hours, transport media(eg Cary- Blair) is recommended • Samples must be sent in a sealed, leak-proof container marked with a biohazard sticker Stool examination by Dr. Priyanka Buragohain 136
  • 137.
    Medias used commonly •MacConkey agar- Salmonella species, shigella species • Eosin methylene blue agar • Triple sugar iron(TSI)- differentiate salmonella and shigella • Sabouraud agar • Hekteon enteric agar • Selenite broth Stool examination by Dr. Priyanka Buragohain 137
  • 138.
    Summary • Definition ofstool • Composition of stool • Precaution and collection of sample • Physical examination • Chemical examination • Microscopic examination • Artifacts • Stool culture Stool examination by Dr. Priyanka Buragohain 138
  • 139.
    THANK YOU !!!Stoolexamination by Dr. Priyanka Buragohain 139