RELIABLE DIAGNOSTIC

FOR PARASITIC INFECTION

Outline
Stool Analysis
◦ Macroscopy analysis
◦ Microscopy analysis

Parasite Staining
o Non Permanent staining
o Permanent staining
 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.
 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.
 Fecal Sample Examination
1. Macroscopic Examination
 Color  abnormal features
 Consistency  adult worm or segment
2. Microscopic Examination
 WBC/ HPF  Yeast
 RBC/ HPF  Cyst, trophozoite, or both
 Mucus  Larvae, egg, or both
3. Chemical Examination
 Fecal PH test  Stool reducing substances
 Fecal fatty acid testing testing
 Testing feces for Occult Blood
 Macroscopic Examination
1. Color:
o Brown is normal color, results from the intestinal oxidation
of stercobilinogen to urobilin.
o Bright red to dark red to black stools occur when iron
or bismuth (precense in drug taken for gastric) is taken or
when there is intestinal hemorrhage.
o Pale yellow stools indicate the biliary obstruction and also
associated with diagnostic procedures that use barium sulfate.
o White stools occur when there is obstructive jaundice.
o Green stool may observed in patient taking oral antibiotic,
because of oxidation of bilirubin to biliverdin.
Macroscopic Examination (cont.)
2. Consistency: 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”
Macroscopic Examination (cont.)
3. Abnormal features:
o If mucus is present writ “M”, and “B” if blood is present.
o The presence of mucus coated stool is indicative for intestinal
inflammation or irritation.

4. Adult worm or segments:

o The feces may have adult helminthes or segments present
such as Ascaris lumbricoides, Entrobius vermicularis, or
Taenia spp. gravid segment, these can be seen by naked eye.
o And frequently motile for several days and may migrate to
the top of the container.
 Notice
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.

Microscopic Examination
 Intestinalprotozoa, and eggs and larvae of
helminths can be detected and identified by
microscopy examination of the stool.

 It includes saline wet mount, iodine wet

mount, smear after concentration and
permanent stain.
Microscopic Examination

Non permanent
Permanent stain
stain

 Trichrome stain
 Saline wet
 Iron hematoxylin
mount
 Modified acid-fast
 Iodine wet
mount stain
 Microscopic Examination of Saline
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.
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.
 HOW TO MAKE A WET MOUNT
 
Wet mounts are commonly used in the science of Biology. This is an important skill
to develop in order to be able to study specimens with a Microscope. This skill is only
developed with practice and by following precise instructions.

Preparing a Wet Mount:

1. Put one drop of water on the slide. (Using a water dropper)

2. Place an object on the slide. (using tweezers to pick up and place the
object)

3. Lower the cover glass slowly to avoid air pockets, pull the tweezers
out.

4. After placing the cover glass, the excess water should be absorbed
with paper.
 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.
Lugol’
s 1. Prior to use, dilute Lugol’s Iodine 1:5 with sterile de-ionized water. (This
working solution should be prepared fresh approximately every 3 weeks)

I 2. Prepare a direct smear of the specimen by mixing a small portion

O
D (2 mg) of feces with a drop of sterile physiological (0.85%) saline on a clean
I glass slide.
N
E 3. Place a coverslip over the sample and examine the wet mount preparation for
the presence of motile protozoa. The organisms are very pale and transparent and
T are more easily observed under low light intensity.
E
C
H 4. Once the wet mount has been thoroughly examined, a drop of Lugol’s Iodine
N (working solution) can be placed at the edge of the coverslip, or a new mount can
I be prepared using iodine alone. The prepared slide can be sealed if desired.
Q
U 5. Examine the slide for the presence of brown parasitic structures.
E
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.
Trophozoites
Permanent Stain
 Trichrome stain
 A Trichrome stain is a stain including three colored
components. It is a general name for a number of
techniques for the selective demonstration of muscle,
collagen fibers, fibrin and erythrocytes.
 Three dyes are employed, one of which may be a
nuclear stain.
 Types include:

Gömöri trichrome stain (√)

Lillie's trichrome
Masson's trichrome stain
Permanent Stain (con’t)
 Trichrome stain protocol

1. Erythrocytes – yellow or red

2. Cytoplasm, fibrin, muscle – red
3. Collagen – light green/blue
4. Black: nuclei
 Trichrome Stain Protocol
o Place the coverslip with section in a staining rack.

o Immerse sections in Harris Hematoxylin for 5 minutes.

o Wash with tap water until the water is clear.

o Immerse sections in Gomori trichrome stain for 10 minutes.

o Differentiate using 0.2% acetic acid. A few dips should be sufficient.

o Immerse rack with sections directly into 95 % alcohol

o Continue to dehydrate in ascending alcohol solutions (95% x 2, 100% x 2) in

staining dish(jar)s.

o Clear with xylene (3 - 4x) also in staining dish/jars.

o Mount coverslip onto a labeled glass slide with DPX medium.
https://www.youtube.com/watch?v=RJgLryj__fk
Trichuris trichiura

Entamoeba coli
Permanent Stain (con’t)

Iron- haematoxylin stain

o Iron Hematoxylin Stain is intended to be used for
the preparation of permanent stained slide for the
detection and quantification of intestinal amoebae
and protozoa.

o The use of Iron Hematoxylin Stain has been widely

documented and provided most of the original
morphological description of intestinal protozoa
found in humans.
 Iron- haematoxylin stain protocol
1. Fixed in Schaudinn’s solution for 15 minutes or longer.
2. The smear is then immersed in the 70% alcohol mixed with
iodine, and 50% alcohol 2-5 minutes in each.
3. It is washed in running water for 2-10 minutes and immersed
in 2% aqueous ferric ammonium sulphate solution for 5-15
minutes. Then followed by washing in running water for 3-5
minutes.
4. It is the stained in 0.5% aqueous haematoxylin for 5-15
minutes, and then washed in running water for 2-5 minutes.
5. Then it is differentiated in saturated aqueous solution of
picric acid for 10-15 minutes and dehydrated by immersion
for 2-5 minutes each in 50%, 70% and 80% and 95%
alcohol.
6. The smear is cleared with xylol and mounted with xylol-
balsam.
 Permanent Stain (con’t)
Modified acid-fast stain

 This technique is useful for the identification of oocysts

of the coccidian species (Cryptosporidium,
Cystoisospora, and Cyclospora), which may be difficult
to detect with routine stains such as trichrome.
 Unlike the Ziehl-Neelsen Modified Acid-Fast Stain, this
stain does not require the heating of reagents for
staining.

 https://www.youtube.com/watch?v=Qzc0ZC-FbMQ
Modified acid-fast stain (con’t)
 Prepare a smear with 1 to 2 drops of specimen on the slide and dry
on a slide warmer at 60°C until dry. Do not make the smears too
thick!
 Fix with absolute methanol for 30 seconds.
 Stain with Kinyoun's carbol fuchsin for one minute. Rinse briefly
with distilled water and drain.
 Destain with acid alcohol for 2 minutes. Rinse with distilled water
and drain.
 Counterstain with methylene blue for 2 minutes. Rinse briefly
with distilled water and drain.
 Dry on a slide warmer at 60°C for about 5 minutes. Mount with a
coverslip using desired mounting media.
 Examine 200 to 300 fields using 40× or higher objectives. To
confirm internal morphology, use 100× oil immersion objective.
Cryptosporidium
parvum
Thank you