1. COLLEGE OF MEDICAL LABORATORY TEHNOLOGY
LEVEL 2/SEMESTER 2
DIAGNOSTIC PARASITOLOGY(DMLT 2822)
ASSIGNMENT: DIAGNOSIS OF INTESTINAL PROTOZOA
NAME: NITHYA LATCHUMY A/P GEEVARETNAM CHETTIAR
MATRIX NUMBER: MLT 11011
LECTURER’S NAME: PROFESSOR MADYA DR. INIT A/P ITHOI
DATE OF SUBMISSION: 5TH APRIL 2013

2. INTRODUCTION
Intestinal protozoa can be divided into pathogenic and non-pathogenic
protozoans. Laboratory diagnosis is important to differentiate between
the harmless and the medically important parasites. This is most often
based upon the morphology of respective organisms. There are some
steps that need to be followed in order to successfully diagnose
intestinal protozoan. The steps include:
 Collecting specimens
 Preparation of specimens
 Evaluation under microscope
Preparation of stool specimen includes direct smear (wet mount),
concentration method, fixative and preservation, staining methods and
cultivation.
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3. SPECIMENS AND COLLECTION OF SPECIMENS
The specimen is stool as the intestine is the habitat of the protozoa. Duodenal aspirate and
material from ulcer or abscess can also be examined.
The stool is collected in a dry, clean and leakproof screw-capped container.
Loose and watery stool are examined for trophozoites while formed stool is examined for cysts.
(Diagram 1.1)
Diagram 1.1
Stool is best examined fresh as motile trophozoites may be seen and increases the chances to
discover protozoa.
The stool specimen must be free of urine and contaminants.
If specimens need to be stored, they must be preserved immediately. They are then sealed well
and stored properly.
If preservative is unavailable, the specimen is kept in a refrigerator. These specimens are not
suitable to examine motile form of protozoa.
Screw-capped container to
collect and store stool
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4. PREPARATION OF STOOL SPECIMENS
a. DIRECT SMEAR (WET MOUNT)
Direct fecal smears can be used as a quick screening test to check for any
intestinal parasite, but the small size of the sample limits its usefulness. Wet
mounts are useful for detecting motile organisms. The disadvantages are dirty
background and less detection of parasites. Protozoa are often detected via a
direct fecal smear, and are best acquired from the surface of fresh feces. The
procedure of direct smear is as follow:
First of all, a drop of saline or iodine is placed on a clean glass slide using a
dropper.
A pea-sized amount of fresh stool is taken using an applicator stick and
mixed together with the saline/iodine drop.
The smear is mounted with a coverslip and examined under microscope at
40x objective.
Trophozoites, cysts, ova can be seen.
Feces is mixed with saline using an The fecal material is mixed until it is well
applicator stick dispersed to produce a smear
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5. b.CONCENTRATION METHODS
A concentration procedure is performed mainly to separate the parasites from fecal
debris. The concentration procedure not only increases the numbers of parasites in
the sediment but it also unmasks them, making them more visible by removing
debris.
Formalin ether sedimentation technique
Formalin acts both a fixative and preservative of protozoan eggs, larvae and cysts. The specific
gravity of protozoan cysts and helminthes eggs is greater than that of water. Fecal debris is
extracted into the ether phase so that the parasitic forms can be separated and then
regimented by centrifugation. The procedure is as follow:
 A small amount of stool is mixed with 5 ml of 10% formal saline in a centrifuge tube
using an applicator stick.
 The mixture is then strained into a paper cup containing cotton gauze as a filter.
 The filtered suspension is poured back into centrifuge tube.
 10% formal saline is added until it reaches 8 ml.
 2 ml of ether is added.
 The centrifuge tube is plugged with a rubber stopper and shaked vigorously for few
minutes.
 It is then centrifuged at 2500 rpm for 2 minutes.
 The debris plug is loosened with an applicator stick and the supernatant is discarded in a
single hand motion.
 The sediment is allowed to mix with the remaining fluid and placed on a clean glass
slide.
 The sediment is mounted with a coverslip and viewed under microscope.
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6. BRINE FLOATATION TECHNIQUE
This technique is best used in diagnosis of helminthes but can also be
used to recover protozoa which are light weighted. The protozoa which
are lighter than the solution will float and can be lifted with glass slide
to be examined. The procedure is as follow:
 Saturated solution of sodium chloride is prepared (brine solution).
 A small amount of feces is mixed with 2ml of brine solution in a
bijou bottle.
 More brine solution is added till the brim of bijou bottle while
stirring.
 Drops of brine solution are added up to the surface of the bottle
without overspilling.
 A clean glass slide is placed over the solution surface and left for
30 minutes exactly.
 The slide is lifted in a single hand motion and examined under
microscope.
Bijou bottle used in brine
floatation technique.
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7. FIXATIVE AND PRESERVATION
Preservation of specimens is necessary when stool specimens cannot be examined within the prescribed
time interval. Various preservatives are available with the two most commonly used being Merthiolate
iodine formaldehyde (MIF) and Polyvinyl alcohol (PVA).
FIXATIVE ADVANTAGE DISADVANTAGE
Polyvinyl alcohol (PVA)  Permanent smears can  Staining not consistent
be made and stained  Organism morphology
with trichrome may be poor
 Zinc is preferred over  Copper-morphology of
copper cysts and trophozoites is
 No mercuric chloride poor
Merthiolate iodine  Components both fix  Not suitable for some
formaldehyde (MIF) and stain organisms permanent smears
 Easy to prepare stained with trichrome
 Long shelf life  Inadequate preservation
 Useful for field surveys of morphology of
 Suitable for protozoan trophozoites
concentration  Iodine interferes with
procedures other stains and
fluorescence
 Iodine may cause
distortion of protozoa
10% Formalin  All purpose fixative  Not suitable for some
 Easy to prepare permanent smears
 Long shelf life stained with trichrome
 Good preservation of  Inadequate preservation
morphology of helminth of morphology of
eggs, larvae, protozoan protozoan trophozoites
cysts, and coccidia  Can interfere with PCR,
 Suitable for especially after extended
concentration fixation time
procedures and UV 
fluorescence microscopy
 Suitable for acid-fast,
safranin, and
chromotrope stains
 Compatible with
immunoassay kits and
UV fluorescence
microscopy

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8.  Polyvinyl alcohol (PVA)
This is a mixture of fixative and water-soluble resin that is specifically used to fix and preserve
trophozoites of intestinal amoebic organisms. These trophozoites are very fragile and will
become distorted or disintegrate completely within a few hours after stool passage. This
fixative will preserve trophozoites for long periods of time and will make it easier to identify
them. PVA is primarily used for preserving fresh specimens to be shipped to central
laboratories. Permanently stained films can be prepared from the preserved material. The
solution serves as an adhesive as well as a preservative and prevents the loss of organisms
during staining. This is advantageous when preparing smears from liquid specimens.
Preparation of reagents:Polyvinyl Alcohol Fixative (PVA
Ethyl alcohol (95 %) 50 ml
Saturated mercuric chloride 100 ml
Glacial acetic acid10 g
Glycerol3 ml
Polyvinyl alcohol powder (PVA) 9 g
Procedure:
STEP 1: Mix a portion of feces with three times the amount of formalin.
STEP 2: Allow settling for at least one hour and store in screw-cap bottles.
STEP 3: If shipment or prolonged storing is required, dip the top portion of the tightly fastened
container, to include the cap, two or three times into a hot paraffin bath. This will prevent
spillage and reduce the rate of evaporation
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9. PERMANENT STAINING
It is a routine diagnosis. Two methods are used in permanent staining of intestinal protozoa, trichrome
and iron-hematoxylin.
TRICHROME STAINING METHOD
The Trichrome stain is a rapid staining procedure which provides excellent differentiation of
internal structures of intestinal parasites as well as facilitating the separation of these organs
from background material and artifacts.
The Trichrome stain (Wheatley Modification) is to be used for staining of intestinal protozoan
cysts and trophozoites in PVA fixed specimens. Sediments from the Formalin-ethyl acetate
concentration technique cannot be stained by this method. All liquid stool specimens should
receive a trichrome stain as trophozoites will occur in liquid specimens and not formed
specimens. Soft stool specimens may be stained if requested or the concentrates are
suspicious.
Preparation of staining solution
1) Tincture of iodine:
 Iodine crystals 7g
 Potassium iodine 5g
 70 % alcohol 100ml
Procedure:
1. The iodine and potassium iodide is grinded in a mortar
2. The mixture is then dissolved and rinsed in alcohol
3. Tincture is stored in a brown bottle
2) Modified trichrome stain
 Chromotrope 2R 0.6g
 Light green SF 0.3g
 Phosphotungstic acid 0.7g
 Acetic acid 1.0ml
 Distilled water 100 ml
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10. Trichrome staining procedure
1. A thin smear is made of fresh stool on a slide
2. After the smear is dried, it is placed in PVA fixative
3. It is then let to stand overnight
4. The smear is then stained as follow :
Solution Duration
Tincture of iodine 1 minute
70 % alcohol 1 minute
Trichrome stain 8 minute
Acid alcohol 10 seconds
Absolute alcohol 1 minute
xylene 1 minute
5. It is then mounted withdeepex or Canada balsam
The outcome of trichrome staining(100x oil immersion)
Protozoa-Entamoeba histolytica cyst
Cytoplasm stain blue-green
Nucleus stain red
Glycogen vacuoles are clear( dissolved by fixatives
Background is usually various shades of green
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11. IRON HEMATOXYLIN STAIN
This staining method gives maximum detail and more reliable outcome but is more time consuming than
the trichrome staining. It is often used with fresh stool
Procedure:
1. Direct smear of fresh stool
2. Slides are immersed in schaudinn’s solution for overnight
3. Dip slides in iodine tincture for 5 min
4. Place slides in 70% ethanol for 5 min.
5. Place slides in 50% ethanol for 2 min.
6. Wash well with distilled water.
7. Place slides in hematoxylin working solution for 10 min.
8. Place slides under running tap water (best if tepid) for 10 min.
9. Differentiate slides (one by one) in destaining solution or for 30 s.
10. Place slides under running tap water for 10 min.
11. Place slides in 95% ethanol for 5 min.
12. Place slides in 100% ethanol for 5 min.
13. Place slides in 100% ethanol for 3 min.
14. Place slides in xylene for 5-10 min.
15. Mount slides with DPX
Entamoeba histolytica
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12. RESULTS (MICROSCOPIC EVALUATION)
Entamoeba histolytica cyst- wet mount (high power)
Entamoeba histolytica trophozoite- iron hematoxylin oil
immersion
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13. Entamoeba coli cyst-iodine stain oil immersion
Entamoeba coli cyst and trophozoite- trichrome
stain oil immersion
Iodamoeba buetschlii cyst- iodine stain high power
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14. Iodamoeba buetschlii trophozoite and cyst- trichrome stain
oil immersion
Giardia lamblia trophozoite- trichrome stain oil immersion
Giardia lamblia cyst- iron hematoxylin stain oil immersion
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15. Balantidium coli trophozoite- trichrome stain oil immersion
Balantidium coli cyst in formalin preserved stool- unstained
high power
Cryptosporidium parvum oocyst- acid fast stain oil immersion
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