Stool examination - الاتحاد المهني للمختبرات الطبية...

Stool examination

Stool analysis determines the various properties of

the stool for diagnostic purposes. Frequently

ordered tests on faeces includes tests for

leukocytes, blood, fat, parasites, and pathogens.

Bacteria, viruses, intestinal parasites and other

malfunctions can be revealed from stool samples.

Stool cultures are necessary in epidemiology and

public health studies.

Microscopic exam

Fat colorless, neutral, fatty acids, crystals

and soaps.

Undigested food, meat fibers, starch none

Eggs and segment of parasite none

Yeasts none

Leukocytes none

Chemical examination

Water up to 75%

Ph neutral to alkaline

Occult blood None

Minerals variable

Reducing substances <0.25 g/dl

Patient preparation & sample

collection

After patient has been prepared & instructed for stool sampling do:

Collect faeces in dry, clean, urine free container with cover.

Collect entire stool specimen and transfer it in container using tongue depressor, spatula or wooden stick. A sample of 2.5cm or 64.7mg is sufficient.

Warm stool is best for detection of ova &parasites. Do not refrigerate specimen for (o & p).

Techniques used in diagnosis

Wet preparation technique.

Concentration technique :

a/ Flotation technique

b/Sedimentation technique

Staining procedures:

a/ Trichrome

b/ Iron haematoxlin

PCR

Antigen detection

Cultivation or culture

Types of specimens

Ordinary defecated sample

Stool bag sample

Rectal swab sample

Adhesive tape sample

Diagnostic value of stool sample

Detection of ova and parasites

Detection of worms and segments

Culture for bacteria cholera and salmonella

Culture for viruses Rota and Polio

Diagnosis of malabsorption

Detection of occult blood

Macroscopic analysis

Color : Yellow Green Black Red Clay Others

Consistency: Soft Watery Mucoid bloody

Ph : Alkaline or acidic

Odor : varies

Observe and report : Mucus Pus Blood

Segment or Worm.

Microscopic examination

On a clean slide on a drop of a suitable

solution e.g. saline, iodine, methylene blue

eosine, formal saline or Zink sulphate

emulsify a suitable amount of stool and

make a thin homogenous film covered with

cover glass examine on a microscope under

10x power then 40x power and then report.

Macroscopic analysis

Color : Yellow Green Black Red Clay Others

Consistency: Soft Watery Mucoid bloody

Ph : Alkaline or acidic

Odor : varies

Observe and report : Mucus Pus Blood

Segment or Worm.

precautions

Always wear protective gloves

Reject contaminated samples

Reject dry & delayed samples >1hour

Use only covered stool containers

Examine samples within one hour

Do not discard sample till result verification

Stool Analysis What is the stool or feces?

5. Epithelial cells that have been shade

1. Waste residue of indigestible material

(cellulose during the previous 4 days)

2. Bile pigments and salts

3. Intestinal secretions, including mucus

4. Leukocytes that migrate from the

bloodstream

6. Bacteria and Inorphosphatesganic

material(10-20%) chiefly calcium and. Undigested

and unabsorbed food.

Random Collection

1. Universal precaution

2. Collect stool in a dry ,clean container

3. uncontaminated with urine or other body

secretions, such as menstrual blood

4. Collect the stool with a clean tongue blade

or similar object.

5. Deliver immediately after collection

Ova and parasites collection

1. Warm stools are best for detecting ova or

parasites.

Do not refrigerate specimen for ova or

parasites.

2. If the stool should be collect in 10 % formalin

or PVA

fixative, storage temperature is not critical.

3. Because of the cyclic life cycle of parasites,

three

separate random stool specimens are

recommended.

Enteric pathogen collection

1. Some coliform bacilli produce antibiotic substances that

destroy enteric pathogen .Refrigerate specimen

immediately.

2. A diarrheal stool will usually give accurate results.

3. A freshly passed stool is the specimen of choice.

4. Stool specimen should be collected before antibiotic

therapy, or

as early in the course of the disease.

5. If blood or mucous is present, it should be included in the

specimen

Interfering factors

1. Patients receiving tetracycline, anti-diarrheal

drugs, barium, bismuth, oil, iron , or magnesium

may not yield accurate results.

2. Bismuth found in toilet tissue interferes with the

results.

3. Do not collect stool from the toilet bowl. A clean,

dry bedpan is the best.

4. Lifestyle, personal habits, environments may

interfere with proper sample procurement.

Normal values in stool exam

Amount 100-200g/d

Color Brown

Odor varies with ph

Consistency plastic, soft, bulky and small

Size and shape Formed

Gross blood None

Mucus None

Pus None

Parasites None

Normal values in stool analysis

Microscopic examination Normal values

Fat (Colorless, neutral fat (18%)and fatty acid crystals and

soaps)

Undigested food None to small

amount

Meat fibers, Starch, Trypsin None

Eggs and segments of parasites None

Yeasts None

Leukocytes None


Chemical examination Normal values

Water Up to 75 %

pH 6.5-7.5

Occult blood Negative

Urobilinogen 50-300 g/24hr

Porphyrins Coporphyrins:400-1200g/24hr

Uroporphyrins:10-40 mg/24hr

Nitrogen <2.5 g/24hr



Bile Negative in adults :positive in

children

Trypsin 20-950 units/g( positive in small

amounts in adults; present in

greater amounts in normal children.

Osmolarity used 200-250 mOsm with serum

osmolarity to calculate osmotic gap

Sodium 5.8-9.8 mEq / 24hr



Chloride 2.5-3.9 mEq / 24 hr

Potassium 15.7-20.7 mEq /24 hr

Lipids ( fatty acid) 0-6 g / 24 hr

Clinical Implications 1. Fecal consistency may be altered in various disease states

a. Diarrhea mixed with mucous and red blood cells

is associated with

1. Typhus 2. Typhoid 3. Cholera

4. Amoebiasis 5. Large bowel cancer

Clinical Implications

b. Diarrhea mixed with mucus and white blood

cells is associated with

1. Ulcerative colitis 2. Regional enteritis

3. Shigellosis 4. Salmonellosis

5. Intestinal tuberculosis

Clinical Implications

C. ”Pasty” stool is associated with a high fat content in the stool:

1. A significant increase of fat is usually detected on

gross examination

2. With common bile duct obstruction, the fat gives

the stool a putty- like appearance.

3. In cystic fibrosis, the increase of neutral fat gives a

greasy, “butter stool” appearance.

Stool Odor Normal value Varies with pH of stool and diet. Indole

and sketole are the substances that produce normal

odor formed by intestinal bacteria putrefaction

and fermentation.

Clinical implication.

1. A foul odor is caused by degradation of undigested

protein.

2. A foul odor is produced by excessive carbohydrate

ingestion.

3. A sickly sweet odor is produced by volatile fatty

acids and undigested lactose

Stool color Normal value : Brown

Clinical implication:

1. Yellow to yellow-green : severe diarrhea

2. Green : severe diarrhea bile

Black: resulting from bleeding into the upper

gastrointestinal tract (>100 ml blood)

3. Tan or Clay colored : blockage of the common

bile duct.

4. Pale greasy acholic (no bile secretion) stool found

in pancreatic insufficiency.

Stool pH Normal value : Neutral to acid or alkaline

Clinical implication

1. Increased pH ( alkaline)

a. protein break down b. Villous adenoma

c. Colitis d.Antibiotic use

2. Decreased pH ( acid)

a. Carbohydrate malabsorption

b. Fat malabsorption

c. Disaccharidase deficiency

Stool color(con)

4. Maroon-to-red-to-pink : possible result of

bleeding from the lower gastrointestinal tract

(e.g. Tumors, hemorrhoids, fissures, inflammatory

process)

5. Blood streak on the outer surface of usually

indicates hemorrhoids or anal abnormalities.

6. Blood in stool can arise from abnormalities

higher in the colon. In some case the transit time is

rapid blood from stomach or duodenum can appear

as bright or dark red or maroon in stool.

Blood in Stool Normal value : Negative

Clinical Implication :

1. Dark red to tarry black indicates a loss of 0.50 to 0.75 ml of

blood from the upper GI tract.

2. Positive for occult blood may be caused by

a. Carcinoma of colon b. Ulcerative colitis

c. Adenoma d. Diaphramatic hernia

e. Gastric carcinoma f. Diverticulitis

g. Ulcers

Mucous in Stool Normal value : Negative for mucous

Clinical Implication:

1. Translucent gelatinous mucous clinging to the

surface of formed stool occurs in

a. Spastic constipation b. Mucous colitis

c. Emotionally disturbed patients

d. Excessive straining at stool

2. Bloody mucous clinging to the surface suggests

a. Neoplasm b. Inflammation of the rectal canal

Mucous in Stool (con)

3. Mucous with pus and blood is associated with

a. Ulcerative colitis b. Bacilliary dysentery

c. Ulcerating cancer of colon d. Acute

diverticulitis

e. Intestinal tuberculosis

Fat in Stool Normal value : fat in stool will account for up to 20 % of

total solids. Lipids are measured as fatty acids (0-6.0 g/24hr)


1. Increased fat or fatty acids is associated with the

malabsorption syndromes

a. Non tropical sprue b. Crohn’s disease

c. Whipple’s disease d. Cystic fibrosis

e. Enteritis and pancreatic diseases

f. Surgical removal of a section of the intestine

Urobilinogen in Stool Normal value : 125-400 Ehrlich units / 24 hr

75-350 Ehrlich units/100 g


1. Increased values are associated with Hemolytic anemias

2. Decreased values are associated with

a. Complete biliary obstruction

b. Severe liver disease, infectious hepatitis

c. Oral antibiotic therapy that alters intestinal bacteria

flora

d. Infants are negative up to 6 months of age

Bile in Stool Normal value

•Adults –negative

• Children may be positive


1. Bile may be present in diarrheal stools.

2. Increased bile levels occur in Hemolytic

anemia

Trypsin in Stool

Normal value : Positive in small amounts in 95 %

of normal persons.

Clinical Implication : Decreased amounts occur in

a. Pancreatic deficiency

b. Malabsorption syndromes

c. Screen for cystic fibrosis

Leukocytes in Stool

Normal value : Negative Clinical Implication

1. Large amounts of leukocytes

a. Chronic ulcerative colitis b. Chronic bacillary

dysentery

c. Localized abscess

d. Fistulas of sigmoid rectum or anus

2. Mononuclear leukocytes appear in Typhoid

Leukocytes in Stool (con)

3. Polymorphonuclear leukocytes appear in

a. Shigellosis b. Salmonellosis

c. Yersinia d. Invasive Escherichia coli diarrhea

e. Ulcerative colitis

4. Absence of leukocytes is associated with

a. Cholera b. Non specific diarrhea

c. Viral diarrhea d. Amebic colitis

e. Noninvasive E.coli diarrhea

f. Toxigenic bacteria Staphylococci spp., Clostridium

Cholera

g. Parasites-Giardia

Porphyrins in Stool

Normal value : Coproporphyrin 400-1200 g / 24hr

Urophorphyrin 10-40 g / 24 hr.

These values vary from Lab to Lab.


1. Increased fecal coproporphyrin is associated with

a. Coproporphyria (hereditary) b. Porphyria variegata

c. Protoporphyria d. Hemolytic anemia

2. Increased fecal protoporphyrin is associated with

a. Porphyria veriegata b. Protoporphyria

c. Acquired liver disease

Stool Electrolytes

Normal values : Sodium 5.8-9.8 mEq / 24 hr

Chloride 2.5-3.9 mEq / 24 hr

Potassium 15.7-20.7 mEq /24 hr


1. Idiopathic proctocolitis Sodium and Chloride Normal Potassium

2. Cholera Sodium and Chloride

40

• Many of these parasites are causative agents of major public health

problems of the world.

• Recent estimates of prevalence of parasites in the world are:

Ascaris 1.5 billion Hookworms 1.3 billion Whipworms 1 billion Filarial worms 657 million

Malaria 500 million Schistosomes 210 million Amebiasis 50 million Taenia tapeworms 50 million Clonorchis 20 million Chagas’ Disease 15 million

• These parasites cause varying morbidities and even mortalities

Why study Parasitology

41

DIAGNOSIS

DIRECT INDIRECT MOLECULAR

Urine Stool

Sputum Biopsy Blood

Aspirates

PCR DNA probes

IHAT LAT IFAT

ELISA CFT

DEIDT

42

STOOL EXAMINATION

MACROSCOPIC MICROSCOPIC OTHERS

•Consistency •Colour •Composition

•Culture •Cellophane tape

•Baeremann tech. •Ova quantitaion (Stoll & Kato)

Temprory Permanent

Diect saline smear Iodine smear Concentration techniques

Sedimentation Floatation

Saline Formol ether Sat saline Zinc sulphate Sheather’s sugar

43

It is a fast, simple, procedure and provides a quick

answer when positive

It can be used as a safe guard

It provides an estimate of the parasitic burden

It may be more cost effective to delete the direct smear and begin the

stool examination with the concentration procedure.

Results should be confirmed by permanent stained smears

WET MOUNT PREPARATION

• Lugol iodine–acetic acid solution causes the

trophozoite forms to become nonmotile.

• Using a fine Pasteur pipette, allow a drop of

methylene blue solution to run under the coverslip

over the saline preparation (Fig. 7). This will stain the

nuclei of any cells present and distinguish the lobed

nuclei of polymorphs from the large single nuclei of

mucosal cells.

• If a drop of eosin solution is added, the whole field

becomes stained except for the protozoa (particularly

amoebae), which remain colourless and are thus

easily recognized.

45

MACROSCOPIC EXAMINATION

COLOUR CONSISTENCY COMPOSITION Adult PARASITES

Pale=Steatorrhea (G.l)

-Liquid (Troph) -Formed (Cyst) -Semi formed (Cyst)

?? Blood ?? Mucus (dysentry)

*Ascaris worm *E. vermicularis *T. saginata

STOOL EXAMINATION

46

STOOL EXAMINATION

Temporary Saline smear Iodine smear

saline Iodine 1%

Huge number of:

•Eggs

• Protozoal troph. Motility

(Amoeb, flagellates)

Huge number of:

•Cyst morphological details 47

Staining the saline preparation with

methylene blue

Microscopic examination of fecal material

WET MOUNT STAINED SMEAR

Concentration methods generally appear unsuitable for

B.hominis, because they cause disruption of the vacuolar,

multivacuolar and granular forms of the organism.

Concentration procedures

Flotation techniques Zinc sulfate

Sedimentation techniques Formalin ethyl acetate

•It is the method of choice in most

laboratories

•It is ideal for large volume laboratories

•It can be performed on fresh material

and on specimens fixed in most of the

available preservatives

•The sedimentation technique used at

(CDC)

•The preparation is clean.

•Cysts are suitable for inoculation

into culture media

•Contain an excess of fecal debris that

could mask the presence of parasitic

cysts.

•It can only be performed on fresh

material or on specimen preserved in

preservatives containing formalin

STOOL EXAMINATION

Scanty infection

Concentration techniques

Sedimentation Floatation

• Heavy eggs (Ascaris egg)

• Operculated eggs (Trematodes)

• Larvae (Strong sterc.)

• Cysts

• Non Operculated eggs

Trematodes ( S. m.)

Cestode Nematode(Hookworms,Trichoston

g) Cysts

52

STOOL EXAMINATION

Saline sedimentation

10 g stool

Saline

Mesh wire gauze

Conical flask

Sediment

Emulsify

STOOL EXAMINATION

Formol Ether Sed. Conc.

10% Formalin

1 g stool

Sediment

formalin

debris

Ether

Thorough mixing

Ether

• Ether adsorbs fecal debris & floats.

• Formalin fixes & preserves the specimen.

Conical flask centrif. tube

53

STOOL EXAMINATION Floatation concentration

Sat saline Zn sulphate Sheather’s sugar

• Cestode eggs (non op) •Nematode eggs????? •Hookworms??????? •Trichostong؟؟؟؟؟؟؟؟؟؟؟

•Egg of S.m. •Eggs of small tapeworms •Cysts

• Crypto, Iso. oocysts

Tin container

20 min Centrif. 2 min

Seive

Clean light eggs & cysts

54

STOOL EXAMINATION

Kato technique

Mesh screen

Template

Hole

Remove the template

Cellophane soaked by glycerin (clears faeces(

Egg count/ g stool

Egg quant. Of: Ascaris, T. trich., Hookworms, S. mansoni

56

STOOL EXAMINATION

Stoll’s technique

NaOH

4 g Stool

Erlynmeyer flask

56 CC

60 CC

Shake well 0.15 CC

Egg count/ slide

Eggs/1g= Eggs/slideX100

Egg/day=Eggs/1g X stool wt/g in 24 hrs

24 hr stool

Egg quant. Of: Ascaris, T. trich., Hookworms, S. mansoni

STOOL EXAMINATION

Baermann’s technique

Warm water

Stool/soil

seive

Glass funnel

clamp

30 min

25-50 CC

centrifuge

Detec. Of Nematode L. /stool, soil

Cultures are primarily research tools rather than diagnostic

tools.

Xenic in which the parasite is grown in the presence of an undefined

flora.

D. fragilis ,B. coli, E. histolytica and B. hominis

Monoxenic in which the parasite is grown in the presence of a single

additional species E. histolytica and B. coli

Axenic in which the parasite is grown in the absence of any other

metabolizing cells. G. intestinalis, E.histolytica and B.hominis

Cultivation of intestinal protozoa

STOOL EXAMINATION

Cultures for Nematode larvae Filter paper culture

Scanty infection

Larvae of:

• St. stercoralis (A,L)

• Hookworms

• Trichostrong

Water Sealed petri dish

Filter paper Slide

59

INDIRECT IMMUNOLOGICAL

METHODS

• Scanty infection.

• Tissue parasite no portal of exit (Hydatid

dis.)

• Migratory stage (Fasciola)

• Chronic infection fibrosis (Bilharziasis)

60

STOOL EXAMINATION

Permanent Stained smears

Iron haematoxylin stain

Trichrome stain

Modified Ziehl Neelsen stain

(Crptosporidum.)

61

INDIRECT IMMUNOLOGICAL METHODS

Antigen detection Antibody detection

• More specific • More accurate. • Active infection • Early • Quantitative

Ab remain in serum for months even after cure

62

•offer advantages in labor, time, simple to perform, and

batching efficiency that may lead to reduce costs

•offer both increased sensitivity and specificity compared to

conventional staining methods

•These reagents are particularly helpful in an outbreak

•or when screening patients with minimal symptoms

• Can detect only one or two pathogens at one time

• It is costly

Antigen detection

•Many E.histolytica infections were confused with E.dispar

•The only way to distinguish E.dispar from E.histolytica microscopically is

erythrophagocytosis.

•False-positive results due to misidentification of macrophages and

nonpathogenic species of Entamoeba

Low sensitivity of microscopic examination

Antigen –based ELISA

•Some of the assays differentiate E.histolytica from E. dispar

•They have excellent sensitivity and specificity

•They are readily usable by even non experienced laboratory personnel

•Used as large scale screening tools in epidemiological studies


IHAT LAT

+

Sensitized Sheep’s RBC

(O–ve)

Ag

Patient’s serum (?? AB)

Agglutination

+

Agglutination

Ag

Latex particle Patient’s serum (?? AB)

65

Immunofluorescent-antibody test (IFAT)

using monoclonal probes specific for Enterocytozoon bieneusi or

Encephalitozoon intestinalis.

• The sensitivity and specificity of IFAT were 100% Moreover,

• Secies identification by IFAT was more rapid and less expensive

than that by PCR.

• IFAT is a suitable test for detection of microsporidia in

developing countries.


INDIRECT FLUORESCENT ANTIBODY TEST

parasite


Anti human AB

fluorescein

67


ELISA

OPD

OPD

Flat bottom plastic micrititre plate

Ag


Anti human AB

Peroxidase E

AB

68


CFT

Ag


complement

Anti sheep AB

Sheep’s RBC

Tube / microplate

AB

69


Double Electro Immuno Diffusion

Ag Ab

Buffered gel

Electric current

Line of ppt

70

INDIRECT IMMUNOLOGICAL METHODS Immunodiagnostic Strip Test (Dip Stick Test) Ag

Nitrocellulose strip

Monoclonal Ab

Coloured dye

Pt bld (?Ag)

Malaria, Filaria, African tryp.

PCR

PCR was more sensitive, specific, and easier to interpret

Nonhuman pathogens can be distinguished from human pathogens

•It takes longer time

•It is technically complex

•and is costly. Thus it may be not well suited for use in

developing countries.

MOLECULAR BIOLOGICAL TECHNIQUES

Polymerase Chain Reaction (PCR)

Single stranded DNA

Replication

Detection T cruzi, T gondii 73

10 X Objective

74

40 X Objective

75

2 VIAL COLLECTION KITS

5% OR 10%

FORMALIN

CONCENTRATION

GENERAL

MORPHOLOGY

PVA

PERMENANT

STAINED SMEAR

Trichrome or

Iron haematoxylin

PRECISE

MORPHOLOGY

Non-parasitic structures found in faeces:

Care

must be taken not to report as parasites those

structures that can be normally found in faeces such as:

muscle fibres, vegetable fibres, starch cells (stain blue-

black with iodine), pollen grains, fatty acid crystals,

soaps, spores, yeasts, and hairs .

Large numbers of fat globules may be seen in faeces

when there is malabsorption.

Charcot Leyden crystals (breakdown products of

eosinophils) can sometimes be seen in faeces (also in

sputum) in parasitic infections. They appear as slender

crystals with pointed ends, about 30–40m in length 77

Structures found in faeces that required

differentiation from parasites.

Structures found in faeces that required differentiation from

parasites. 78

Image illustrating Yeast Cells in slide preparation

Note similarity to parasitic oocysts.

Image illustrating Vegetable cell in slide

preparation.

Image illustrating Red Blood Cells in slide

preparation. Image illustrating Fat Globules in slide

preparation

79

Image illustrating Vegetable cell in slide

preparation.

Image illustrating a Vegetable Spiral in slide

preparation. Such spirals may appear similar

to proglottids.

Image illustrating Vegetable Spiral in slide

preparation.

80

Image illustrating pollen in slide preparation

using a color filter

Image illustrating pollen in slide preparation

that could be mistaken for a Taenia egg. The

shell is thinner, of non-uniform thickness, and

no hooks are visible.

Image illustrating pollen resembling a

Hymenolepis nana egg. Hooks and polar

filaments are not visible.

Image illustrating geranium pollen cells in

slide preparation. 81

Image illustrating peach hair

in slide preparation. Note the

similarity to Strongyloides

stercoralis.

Image illustrating vegetable

hairs in slide preparation.

82

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