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 ¶sites. 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
Normal values in stool analysis
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
Normal values in stool analysis
Chemical examination Normal values
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
osmol- arity to calculate osmotic gap
Sodium 5.8-9.8 mEq / 24hr
Normal values in stool analysis
Chemical examination Normal values
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)
Clinical Implication :
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
Clinical Implication:
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
Clinical Implication:
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.
Clinical Implication:
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
Clinical Implication :
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
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
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
INDIRECT IMMUNOLOGICAL METHODS
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 IMMUNOLOGICAL METHODS
INDIRECT FLUORESCENT ANTIBODY TEST
parasite
Patient’s serum (?? AB)
Anti human AB
fluorescein
67
INDIRECT IMMUNOLOGICAL METHODS
ELISA
OPD
OPD
Flat bottom plastic micrititre plate
Ag
Patient’s serum (?? AB)
Anti human AB
Peroxidase E
AB
68
INDIRECT IMMUNOLOGICAL METHODS
CFT
Ag
Patient’s serum (?? AB)
complement
Anti sheep AB
Sheep’s RBC
Tube / microplate
AB
69
INDIRECT IMMUNOLOGICAL METHODS
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
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