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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?
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
5. Epithelial cells that have been shade
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:400-1200g/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
Why study Parasitology
• 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


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DIAGNOSIS
DIRECT INDIRECT MOLECULAR
Urine IHAT
Stool LAT
Sputum IFAT PCR
Biopsy ELISA DNA probes
Blood CFT
Aspirates DEIDT

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STOOL EXAMINATION

MACROSCOPIC MICROSCOPIC OTHERS

•Culture
•Consistency •Cellophane tape
•Colour Permanent Temprory •Baeremann tech.
•Composition •Ova quantitaion (Stoll & Kato)

Diect saline smear Iodine smear Concentration techniques

Floatation
Sedimentation

Saline Formol ether Sat saline Zinc sulphate Sheather’s sugar

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WET MOUNT PREPARATION

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


answer when positive
 It provides an estimate of the parasitic burden

 It can be used as a safe guard

 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


• 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
STOOL EXAMINATION
MACROSCOPIC EXAMINATION

COLOUR CONSISTENCY COMPOSITION Adult PARASITES

-Liquid (Troph) *Ascaris worm


Pale=Steatorrhea ?? Blood ?? Mucus
-Formed (Cyst) *E. vermicularis
(G.l) (dysentry)
-Semi formed (Cyst) *T. saginata

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STOOL EXAMINATION
Temporary
Saline smear Iodine smear
saline Iodine 1%

Huge number of:


•Eggs Huge number of:

• Protozoal troph. Motility •Cyst morphological details


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(Amoeb, flagellates)
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 Sedimentation techniques


Zinc sulfate Formalin ethyl acetate

•The preparation is clean. •It is the method of choice in most


•Cysts are suitable for inoculation laboratories
into culture media
•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)

•It can only be performed on fresh •Contain an excess of fecal debris that
material or on specimen preserved in could mask the presence of parasitic
preservatives containing formalin cysts.
STOOL EXAMINATION
Scanty infection
Concentration techniques

Sedimentation Floatation
• Non Operculated eggs
• Heavy eggs (Ascaris
egg) Trematodes ( S. m.)

• Operculated eggs Cestode


(Trematodes)
Nematode(Hookworms,Trichoston
• Larvae (Strong sterc.) g) Cysts
STOOL EXAMINATION
Saline sedimentation

Mesh wire gauze

Saline Emulsify
Conical flask

10 g stool
Sediment

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STOOL EXAMINATION
Formol Ether Sed. Conc.

Ether
Ether debris

10% Formalin formalin

1 g stool
Sediment
Thorough mixing Conical flask centrif. tube

• Ether adsorbs fecal debris & floats.


• Formalin fixes & preserves the
specimen. 53
Floatation concentration
STOOL EXAMINATION
Clean light eggs &
cysts

Sat saline Zn sulphate Sheather’s sugar

Tin container
• Cestode eggs (non op) Seive
•Egg of S.m.
•Nematode eggs?????
•Eggs of small tapeworms • Crypto, Iso. oocysts
•Hookworms???????
•Cysts
•Trichostong‫؟؟؟؟؟؟؟؟؟؟؟‬
20 min Centrif. 2 min

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STOOL EXAMINATION
Kato technique
Mesh screen

Hole
Template
Remove the
template

Cellophane soaked by
glycerin (clears faeces(

Egg count/ g stool

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


STOOL EXAMINATION
Stoll’s technique
Egg quant. Of: Ascaris, T. trich., Hookworms, S. mansoni

24 hr stool
60 CC
4 g Stool
56 CC
Shake well 0.15 CC

NaOH
Egg count/ slide

Eggs/1g= Eggs/slideX
Erlynmeyer flask Egg/day=Eggs/1g
56 X stool
STOOL EXAMINATION
Baermann’s technique
Stool/soil
seive
25-50 CC
Warm water
Glass funnel 30 min centrifuge

clamp

Detec. Of Nematode L. /stool, soil


Cultivation of intestinal protozoa

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

Cultures are primarily research tools rather than diagnostic


tools.
STOOL EXAMINATION
Cultures for Nematode larvae
Filter paper culture

Filter paper
Slide
Sealed petri dish
Water

Scanty infection
Larvae of:
• St. stercoralis (A,L)
• Hookworms

59 • Trichostrong
INDIRECT IMMUNOLOGICAL
METHODS
• Scanty infection.
• Tissue parasite no portal of exit (Hydatid
dis.)
• Migratory stage (Fasciola)
• Chronic infection fibrosis (Bilharziasis)

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STOOL EXAMINATION
Permanent Stained smears

Iron haematoxylin stain


Trichrome stain
 Modified Ziehl Neelsen stain
(Crptosporidum.)

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INDIRECT IMMUNOLOGICAL METHODS

Antigen detection Antibody detection

ore specific
ore accurate.
ctive infection
Ab remain in serum for
arly
months even after cure
uantitative

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Antigen detection

•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
Low sensitivity of microscopic examination

•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

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
Ag
Ag +
+
Latex particlePatient’s serum
Patient’s serum (?? AB)
Sensitized
heep’s RBC (?? AB)
(O–ve)

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Agglutination Agglutination
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

fluorescein
Anti human AB

Patient’s serum
(?? AB)

parasite

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INDIRECT IMMUNOLOGICAL METHODS
ELISA

OPD

Peroxidase E OPD
Anti human AB
atient’s serum
(?? AB) AB
Ag
Flat bottom plastic micrititre plate
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INDIRECT IMMUNOLOGICAL METHODS
CFT

Sheep’s
Anti sheepRBC
AB
AB

comple
ment
Patient’s serum
(?? AB)
Ag
Tube /
microplate 69
INDIRECT IMMUNOLOGICAL METHODS
Double Electro Immuno Diffusion
Line of
ppt
Electric current

Ag A
b

Buffere
d gel
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INDIRECT IMMUNOLOGICAL METHODS
Immunodiagnostic Strip Test (Dip Stick Test) Ag

Pt bld (?Ag)

Coloured dye

Monoclonal Ab
trocellulose strip 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

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Detection
T cruzi, T gondii
10 X Objective

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40 X Objective

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2 VIAL COLLECTION KITS

5% OR 10%
FORMALIN PVA

PERMENANT
STAINED SMEAR
ONCENTRATION
Trichrome or
Iron haematoxylin

GENERAL PRECISE
MORPHOLOGY 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
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Structures found in faeces that required
differentiation from parasites.

Structures found in faeces that 78required differentiation from


parasites.
Image illustrating Red Blood Cells in slide
preparation. Image illustrating Fat Globules in slide
preparation

Image illustrating Yeast Cells in slide preparation79 Image illustrating Vegetable cell in slide
Note similarity to parasitic oocysts. preparation.
Image illustrating Vegetable Spiral in slide
preparation.

Image illustrating a Vegetable Spiral in slide


Image illustrating Vegetable cell in slide 80 preparation. Such spirals may appear similar
preparation. to proglottids.
Image illustrating pollen in slide preparation Image illustrating pollen resembling a
that could be mistaken for a Taenia egg. The Hymenolepis nana egg. Hooks and polar
shell is thinner, of non-uniform thickness, and filaments are not visible.
no hooks are visible.

Image illustrating pollen in slide preparation Image illustrating geranium pollen cells in
81 slide preparation.
using a color filter
Image illustrating peach hair Image illustrating vegetable
in slide preparation. Note the hairs in slide preparation.
similarity to Strongyloides
stercoralis. 82