Topics
1. Introduction
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•
Sample Collection and Preservation Methods
Wet mount Preparation
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

Normal saline 0.85%
Iodine
BMB
2. Artifacts
3. Concentration Techniques
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•
Modified Formal- Ether Sedimentation technique
Acid- Ether Sedimentation technique
4. Flotation Techniques
•
•
By using Sheather’s solution
By using Sodium Chloride solution
5. Staining of parasites
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Topics (cont.)
6. Detecting of Blood Parasites
•
Thick and thin Blood smear
7. Counting of Helminthes Eggs in Feces
8. Chemical Tests
•
•
•
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Fecal PH test
Testing feces for Occult Blood
Fecal fat test
Stool reducing sugar test
9. Medical Entomology
Raed Z. Ahmed, Medical Parasitology Lab.,2012
‫‪Grades‬‬
‫‪10%‬ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ‪Quizzes:‬‬
‫‪10%‬ــــــــــــــــــــــــ ‪Assignment & Participation:‬‬
‫‪30%‬ــــــــــــــــــــــــــــــــــــ ‪Midterm examination:‬‬
‫‪50%‬ــــــــــــــــــــــــــــــــــــــــــ ‪Final examination:‬‬
‫‪20%‬ـــــــــــــــــــــــــــــــــــ ‪– Practical exam:‬‬
‫‪30%‬ـــــــــــــــــــــــــــــــــــــ ‪– Written exam:‬‬
‫‪Raed Z. Ahmed, Medical Parasitology Lab.,2012‬‬
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General Lab Objectives
1. To familiarizes the student with the most widely
used techniques for detection of parasites.
2. To be able to identify the parasite stages.
3. To learn the student, how to deal with risk samples.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
What is the stool or feces?
 Waste residue of indigestible material (cellulose during
the previous 4 days),
 Bile pigments and electrolyte,
 Intestinal secretions, including mucus,
 Leukocytes that migrate from the bloodstream,
 Epithelial cells that have been shade,
 Bacteria and Inorganic material(10-20%) chiefly calcium
and phosphates. Undigested and unabsorbed food
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Fecal Specimen
• Fecal specimen are examined for protozoa, helminthes
larvae or eggs.
• The stages of protozoa found in stool samples are
trophozoites and cysts or oocysts.
• The stages of helminthes usually found in the stool
samples are eggs and larvae, through whole adult worms
or segment of worms may also be seen.
• Adult worms and segment of tape worms are usually
visible to naked eye, but eggs, larvae, cyst, oocyst and
trophozoites can be seen only with the microscope.
• In order to see these structure, the fecal material must be
properly collected and examined.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Parasites
Protozoa
Cyst
Helminths
Platyhelminths
Nematoda
Trophozoite
Oocyst
Cestoda
Trematoda
Eggs
Adult
Eggs
Eggs
Adult
Adult
Segments
Cercariae
Larva
Number of Specimens and Collection Time
• No technique is 100% successful in detecting parasites by
single stool examination, and at least three serial stools
must be examined before a patient can be considered free
from infections in which stages of parasites would be
expected to be free in the faeces.
• Because of the intermittent passage of certain parasites, the
possibility of finding organisms is increased by examining
multiple specimens.
• It is suggested that 3 specimens, collected at 2 to 3 day
intervals, should be examined both pretreatment and post
treatment (to ensure eradication of documented pathogenic
protozoa).
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Collection of Fecal Specimen
• Because of fragile nature of many intestinal
parasites, and the need to maintain their morphology for
accurate identification.
• Reliable microscopic diagnosis can not made unless the
stool is collected properly.
• The stool specimen must be enough for satisfactory
examination of fresh feces uncontaminated by urine, dirt*,
water or other body secretion such as menstrual blood.
• If the sample is too small or contaminated with urine, it
should not be accepted. Ask the patient to pass another
specimen.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Collection of Fecal Specimen (cont.)
• Collect the specimen in a clean dry screw-capped top
container
• Collect the stool with a clean tongue blade or similar
object.
• The container should be free from antiseptics and
disinfectant.
• Random specimen: suitable for qualitative testing for
blood and microscopic examination.
• Timed specimen: for quantitative fecal testing such as
fecal fat testing, because of the variability of bowel habit
and the transit time required for food to pass through the
digestive tract, so the most representative sample is a-3
day collection. Raed Z. Ahmed, Medical Parasitology Lab.,2012
Collection of Fecal Specimen (cont.)
• The container with the specimen should be clearly labeled
with the following:
o Patient’s name or number.
o Date and time of collection.
• All samples should be accompanied by a requisition form
from the physician giving relevant clinical details and
recent travel history.
• Samples and forms from patient with a confirmed or
suspected diagnosis of certain infectious diseases such as
AIDS or hepatitis should be clearly labeled with
“Biohazard”
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Collection of Fecal Specimen (cont.)
• Most viable parasites are susceptible to desiccation or
temperature variation.
• If time lapse between collection and observation is
considerable, i.e. more than 4 days, it may be necessary to
add some form of preservative to faeces specimen to retain
morphology.
• Formed samples can be kept in a refrigerator at 4 C° for a
short time, but not in incubator.
• Any whole worms or segments passed should be placed in
a separate container
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Preservation methods for fecal specimens
• Preservation allows fecal samples to be examined after a
delay in delivery or postage or testing.
• Many methods for the preservation of stool samples and
permanent staining procedures.
• The most common fixatives are:
 Polyvinyl Alcohol, PVA
 Merthiolate Iodine Formalin, MIF
 Sodium acetate Acetic acid Formalin, SAF
 Formalin.
 Bayer’s solution*
• The preservatives used have different effect on the various
stages of the parasites.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
• Formalin 4% has been used for many years as an all
purpose fixative that is appropriate for helminthes eggs
and larvae and for protozoan cyst.
• The fixative has a long shelf life.
• Concentration
methods,
like
formalinether
concentration can be performed from the preserved stool
samples without loss of concentration abilities.
• The major disadvantage of formalin is that permanent
staining procedures can't be performed from formalin
preserved stool samples.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
• This fixative is recommended for the preservation of the
trophozoite and cyst stages of the intestinal protozoa, and
also suitable for helminthes eggs and larvae.
• The preservation of the two stages of protozoa is
excellent.
• The PVA is a plastic resin that serves as adhesive for the
stool material.
• Has a long shelf life ( months to years ).
• Concentration methods can’t performed from the
specimen preserved by PVA.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
• The greatest advantage of this fixative is that a permanent
stain can be prepared from stool specimen preserved by
PVA, giving excellent result with trichrome staining.
• Specimen preserved by PVA can’t be used with
immunoassay kits.
• Toxic, because contain mercury compound.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
• Good routine fixative for protozoan cyst and trophozoites,
helminthes eggs, and larvae.
• Has long shelf life. ( months to years).
• The preserved stool samples permits concentration techniques,
most monoclonal detection kits, and permanent staining.
• Unlike the PVA, the SAF fixative has poor adhesive properties
when SAF preserved samples are used to prepare permanent
stained smears. ( Mayer’s albumin has been recommended as an
adhesive.
• The combination of SAF preserved material and CB, IHK, and
mod. Ziel Neelsen provides excellent staining of protozoan
where staining of SAF preserved material with Trichrome gives
poor results.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
 Specific advantages of the use of SAF are:
 SAF preserved material can be used for concentration
techniques and permentant stained smears (CB, IHK).
 SAF preserved material can be used for some
immunoassay methods.
 SAF is easy to prepare and has a long shelf life.
 Unlike the PVA, the SAF fixative contain no mercury
compounds. It is therefore much less toxic than PVA
Raed Z. Ahmed, Medical Parasitology Lab.,2012
• This
fixative
was
originally
developed
as
a screening procedure for intestinal parasites.
• MIF combines preservation and staining for most kinds and
stages found in faeces.
• It’s contains Merthiolate, Iodine, and Formalin.
• The preserved material permits concentration techniques.
• The major disadvantages are the short shelf life ( duo to
iodine) and permanent stained smears can’t be prepared
from MIF preserved material.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Fixative used for the preservation of stool samples: an overview of
the advantages and disadvantages:
Formalin 4%
Toxicity
+/-
Shelf life
Long
(months)
Preparation
Quality of fixation
Formalin ether
concentration
Permanent stained
smear
PVA
SAF
MIF
+++
( duo to Hg )
+/-
+/-
Long
(months/years)
Long
(months/years)
Limited
Easy
Difficult
Easy
Easy
Egg: ++
Egg: ++
Egg: ++
Egg: ++
Cyst: ++
Cyst: +++
Cyst: ++
Cyst: ++
Troph’s: +/-
Troph’s: +++
Troph’s: +++
Troph’s: ++
Possible
Not possible
Possible
Possible
Not possible
Only Trichrome
IHK, CB, mod.
Ziel Neelsen
Not possible
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Preservation of worms
1. Cestodes
 Wash in water to remove the mucus. Large tapeworms such as
Taenia can be washed for several hours to relax the musculature,
and can then be fixed in 10% formol saline b/w two glass slides to
give flatter specimens.
2. Trematodes
 These should be treated in a similar manner to cestodes, and
mounted with the ventral sucker uppermost
3. Nematodes
 Adult are washed in saline to remove mucus. Worms up to about
7 cm in length are fixed in hot(60-70˚C) 70% alcohol, which
straightens out living worms, except those which have natural
curvatures at the head or the tail. Alternatively, they can be fixed
in hot 5% formalin.
 Large worms such Ascaris lumbricoides can be fixed and
preserved in cold 5% formalin
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Stool Analysis
• A stool analysis is a series of tests done on a stool (feces)
sample to help diagnose certain conditions affecting the
digestive tract .
• These conditions can include infection (such as from
parasites, viruses, or bacteria), poor nutrient absorption, or
cancer.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Stool Analysis (cont.)
• Laboratory analysis includes macroscopic, microscopic
examination, chemical tests, and microbiologic tests.
• The stool will be checked for color, consistency, weight
(volume), shape, odor, and the presence of mucus and
parasites stages.
• The stool may be examined for hidden (occult) blood, fat,
meat fibers, bile, white blood cells, and sugars called
reducing substances.
• The pH of the stool also may be measured.
• A stool culture is done to find out if bacteria may be
causing an infection.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Fecal Sample Examination
1. Macroscopic Examination

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
Color
Consistency
abnormal features
adult worm or segment
2. Microscopic Examination

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WBC/ HPF
RBC/ HPF
Mucus
Yeast
Cyst, trophozoite, or both
Larvae, egg, or both
3. Chemical Examination



 Stool reducing substances
Fecal PH test
testing
Fecal fatty acid testing
Testing feces for Occult Blood
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Macroscopic Examination
• Brown is normal color, results from the intestinal oxidation
of stercobilinogen to urobilin.
• Bright red to dark red to black stools occur when iron or
bismuth is taken or when there is intestinal hemorrhage.
• Pale yellow stools indicate the biliary obstruction,
steatorrea, and also associated with diagnostic procedures
that use barium sulfate.
• White stools occur when there is obstructive jaundice.
• Green stool may observed in patient taking oral antibiotic,
because of oxidation of bilirubin to biliverdin.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Macroscopic Examination (cont.)
degree of moisture, will be a
guide as to whether the trophozoite stage or the
cyst stage of protozoa is likely to present.
 Formed, write “F”
 Soft , write
“S”
 Loose , write
“L”
 Watery , write “W”
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Macroscopic Examination (cont.)
• If mucus is present writ “M”, and “B” if blood is present.
• The presence of mucus coated stool is indicative for
intestinal inflammation or irritation.
• The feces may have adult helminthes or segments present
such as Ascaris lumbricoides, Enterobius vermicularis, or
Taenia spp. gravid segment, these can be seen by naked
eye.
• And frequently motile for several days and may migrate
to the top of the container.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
o If several specimens are received at the same time; those
containing blood and mucus should be examined first,
followed by liquid specimens. These specimens are the
most likely to contain amoebic trophozoites ( which die
soon after being passed), and must be examined within 1
hour after passage.
o Formed specimens may be examined at any time during
the first day, but should not be left overnight ( cyst may
disintegrate).
o Excessive bulky stools may indicate conditions such as
giardiasis.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Microscopic Examination of wet mount
• Wet mount is the simplest and easiest technique for
the examination of feces, and this method should
be performed in all laboratories at peripheral level.
• A wet mount can be prepared directly from fecal
material or from concentrated specimens.
• The basic types of wet mount that should be used
for each fecal examination are normal saline
(0.85% NaCl), iodine, and buffered methylene
blue.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Microscopic Examination of wet mount (cont.)
 The Saline Wet Mount
 Is used for the initial microscopic examination of
stool specimens.
 It is employed primarily to demonstrate worms
eggs, larvae. Protozoan trophozoites, and cysts.
 This type of mount can also reveal the presence of
red blood cells and white blood cells.
 If the presence of amoebic trophozoites is
suspected, warm saline (37˚C) should be used.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Microscopic Examination of wet mount (cont.)
 The Iodine Wet Mount
 Is used mainly to stain glycogen and the nuclei of
cysts, if present.
 Cysts can usually be specifically identified in this
mount.
 Trophozoite can not be revealed by this type of wet
mount, because iodine kill trophozoite.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Microscopic Examination of wet mount (cont.)
 The Buffered Methylene Blue Wet Mount
 Should be prepared each time amoebic trophozoites are
seen in a saline wet mount, or when their presence is
suspected.
 BMB stains amoebic trophozoites, but not stain
amoebic cysts, flagellate trophozoites or flagellate cysts.
 BMB stain is appropriate only for fresh unpreserved
specimens.
 BMB stain live organism only, it isn’t used on preserved
samples in which the organism have been killed
 Wait for five minutes to allow the stain to penetrate the
trophozoites. It will overstrain the trophozoites in 30
minutes.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Formed stool: take the portion of stool from an area
to include inside and outside parts of the specimen.
Stool with mucus: if mucus is present, label a second
slide with the patient’s name or number. Put a drop of
saline on the slide, pick up a small portion of mucus
and mix with the saline. Trophozoites, if present, are
sometimes more readily found in mucus than in the
solid parts of the stool.
Loose watery stool: if mucus is not present, pick up a
small portion of the stool (any part) and mix with the
saline.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Making Direct smear Microscopy
 Materials and reagents:
 Microscopic slides.
 Cover slips.
 Applicator sticks.
 Marker or pen for labeling.
 Reagents:
 Saline solution(isotonic)
 Lugols iodine(1% solution)
 BMB
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Wet mount procedures
Examine the slide on microscope:
o 10X
o 40X
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Result of Examination
• If no parasites are found:
 “No ova or parasites seen”, and specify
whether this result was obtained by direct
examination or by a concentration method
(name method used).
 Never state categorically: “No parasites”
• If any parasites are seen, write the scientific name
of the parasite with stages
• Example: Giardia lambilia cyst
Raed Z. Ahmed, Medical Parasitology Lab.,2012