PARASITOLOGY LABORATORY

FECAL OCCULT BLOOD TEST • Many consumers prefer the flushable reagent pads
because there is no stool handling and no laboratory
• Occult means “hidden”; Occult blood means hidden processing. However, health care providers usually
blood that usually cannot be seen by naked eyes. favor the guaiac tests because the large studies that
• The purpose of the test is to detect pathological have shown the benefits of colon cancer screening
lesions (ex. Carcinoma) before they produce were done with guaiac tests.
symptoms and while the condition is still amenable
to treatment. WHY IS THE TEST PERFORMED?
• A number of easy-to-use test kits for detection of
occult blood are available. Prior to such kits, the • This test is mainly performed for colorectal cancer
traditional method was to expose the sample to a screening. It may also be performed in the
sequence of solutions that included glacial acetic acid, evaluation of anemia.
gum guaiac solution and hydrogen peroxide. A blue
ADVANTAGE DISADVANTAGE
color indicated a positive test result. The test kits Noninvasive Detects blood in stool, but
use these same principles, with some using paper Low-cost not its cause. False-
impregnated with guaiac. For these reasons, analysis positive results are
of feces for occult blood is sometimes still referred to common with some testing
as “stool for guaiac”. methods. This may cause
• FOBT is widely used in the screening of colorectal unneeded anxiety about
cancer. cancer and lead to
unnecessary further tests.
BACKGROUND False-negative results are
also common and may
• Fecal occult blood tests have been validated for miss disease in its early
colorectal cancer screening to detect early-stage stages.
colorectal cancer or adenomas since 1967 POSITIVE RESULT MAY INDICATE:
• The use of the FOBT as a screening test can
promote early detection of colorectal cancer and • Bleeding esophageal varices
may reduce mortality in at risk populations • Colon polyp or colon cancer
• If patients are symptomatic with the suspicion of rectal • Esophagitis
blood loss, melena, and/or abdominal pain the • Gastritis
endoscopic evaluation is recommended. • Gastrointestinal trauma
• Hemorrhoids
FECAL OCCULT BLOOD • Inflammatory bowel disease
• Peptic ulcer
• term for blood present in the feces that is not visibly
apparent. • Complications of recent GI surgery
• Fecal Occult Blood Test (FOBT) is a non-invasive • Angiodysplasia of the colon
test (nothing enters the body). This test detects • Gastrointestinal tumor
hidden (occult) blood in the stool. Such blood may • Fissures (cracked around anus)
come from anywhere along the digestive tract. Hidden
Additional conditions under which the test may be
blood in stool is often the first, and in many cases the
only, warning sign that a person has colorectal performed include the following:
disease, including colon cancer.
• Colon cancer screening
HOW IS THE TEST PERFORMED? • Evaluation of anemia
• There are two types of FOBTs:
o 1) the traditional guaiac smear test
(Hemoccult, Seracult, Coloscreen), and
o 2) the newer, flushable reagent pads (EZ
DetecT, ColoCARE). They are both useful in
detecting hidden blood in the stool and are
mainly used for colorectal cancer screening.
• The tests differ in the way they are performed. The
flushable reagent pads are available without a
RISK
prescription at many drugstores. In contrast, the
traditional guaiac smear test is completed and • A negative test does not necessarily mean there are
interpreted by a medical professional, and these tests no colorectal diseases present. Not all polyps bleed,
are usually available from a laboratory or a doctor's and not all polyps bleed all the time. That is why a
office FOBT must be used with one of the other more
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invasive screening measures (sigmoidoscopy, • There are many ways to collect the samples. You can
colonoscopy, double barium contrast enema). catch the stool on plastic wrap that is loosely placed
o For back up lang, to screen lang ng status, over the toilet bowl and held in place by the toilet seat.
hindi siya confirmatory test. Then put the sample in a clean container. One test kit
supplies a special toilet tissue that you use to collect
CONSIDERATIONS the sample, then put the sample in a clean container.
Do not take stool samples from the toilet bowl water,
• Colonoscopy is generally recommended as the
because this can cause errors.
preferred follow-up test to a positive FOBT.
• infants and young children wearing diapers, you can
• Factors that can cause this test to be less accurate
line the diaper with plastic wrap. The plastic wrap is
include the following:
positioned so that it keeps the stool from any urine.
o Bleeding gums following a dental procedure
Mixing of urine and stool can spoil a good sample.
Eating red meat within 3 days of the test
o Eating turnips or horseradish • Laboratory procedures may vary. In one type of test, a
o Drugs that can cause GI bleeding include small sample of stool is placed on a paper card. A
anticoagulants, aspirin, colchicine, iron drop or two of testing solution is put on the opposite
supplements in large doses, NSAIDs (anti- side of the card. A color change (shade of blue)
inflammatory analgesics), and indicates the presence of blood in the stool.
corticosteroids.
o Drugs that can cause false positive
measurements including iron, colchicine,
oxidizing drugs (for example, iodine,
bromides, and boric acid), and reserpine.
Large amounts of vitamin C can cause false-
negative results on most FOBTs.

FACTORS THAT CAN AFFECT THE RESULT OF FOBT

CAUSES OF POSITIVE TEST ARE:

• 2-10% cancer (colorectal cancer, gastric cancer)

• 20-30% adenoma or polyps
• Bleeding peptic ulcer
• Angiodysplasia of the colon

STOOL COLOR INDICATION

BLACK STOOLS

• associated with upper G.I. bleeding when the

HOW IS THE TEST PERFORMED?
hemoglobin has come in contact with gastric acid and
has been converted to hematin. (5 days)

• If the test is performed in a laboratory, clinic or

hospital, stool may be collected by a HCW during an DARK RED/MAROON STOOLS
examination.
• If the test is performed at home, a stool sample from • liquid consistency - upper G.I. bleeding is massive
three consecutive bowel movements is collected, and the volume increases G.I. motility
smeared on a card, and submit to a laboratory for
processing. In order to ensure the accuracy of the
guaiac test, follow the manufacturer's instructions on
how to collect the stool.

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BRIGHT RED
• lower G.I. bleeding from hemorrhoids, ulcerative
colitis and carcinomas.
OCCULT BLOOD INDICATIONS AND PURPOSES
• known or suspected disorder associated with
gastrointestinal bleeding.
• therapy with drugs that may lead to gastrointestinal
bleeding (eg. Aspirin, anticoagulants).
• Hemorrhoids- usual age of occurrence- older adult;
severity: usually mild; blood is bright red, other
features maybe painless or symptomatic often
associated with constipation.
• Anorectal fissure- affects any age usually mild; blood
is bright red nearly always painful. It can be seen in
Crohn's disease & anal intercourse may predispose.
• In the event of a positive fecal occult blood test, the
next step in the workup is a form of visualization of the
gastrointestinal tract (ie: endoscopy, colonoscopy,
virtual colonoscopy).
THERE ARE THREE METHODS FOR MEASURING BLOOD
IN FECES
Hemoccult or Instaccult -This method can reduce death from
colorectal cancer
1. Fecal porphyrin quantification (Hemoquant)– high
false positive rate.
2. Immunochemical fecal occult blood tests
(HemeSelect), (QuickVue iFOB), (FOBGold or
Automated SENTiFOB) or (OC Auto 80 - Automated
iFOBT) - more specific.
3. Fecal DNA test (PreGen-Plus) is more sensitive than
fecal occult blood in one study (51.6% vs. 12.9%)
FOBT ONE METHOD
• test involves smearing some feces onto some
absorbent paper that has been treated with a
chemical. Hydrogen peroxide is dropped onto the
paper; if trace amounts of blood are present, the
paper will change color. This method works as
hemoglobin has a peroxidase-like effect, rapidly
breaking down hydrogen peroxide.
FECAL IMMUNOCHEMICAL TEST
• tests detect the globin in feces rather than heme. By
detecting globin the tests are both more sensitive and
specific for lower gastrointestinal bleeding.
INSURE
• This test is designed to address patient ease of use
by using a brush, not a wooden stick, to sample stools
while in the toilet bowl Using these tests there is no
direct fecal handling and there is no need for
changing diet or medication to perform the test.
CLEARVIEW IFOB TEST
• This test requires only one specimen, and because it
is specific to human hemoglobin, patients are not
required to adhere to strict dietary or medication
restrictions. For hospitals and large clinics, the OC
Automated 80 can perform fecal occult blood
detection by immunoassay. This method addresses
the dietary issues associated with the guaiac test and
has been shown to detect many more early-stage
cancers and polyps.
GUIAC-BASED FECAL OCCULT BLOOD TEST
• This test usually picks up a daily blood loss of about
10 ml (about two teaspoonfuls). The sensitivity of a
single FOBT has been quoted at 30%, but if 3 tests
are done (as is standard), the sensitivity rises to 92%.
• "Normally, there is only about 0.5-1.5 ml of blood a
day that escapes the blood vessels into the stool each
day. There are more sensitive tests than the guaiac
such as a heme-porphyrin test or an immunochemical
test, but the former test is not used much due to the
high false positive rate. The latter test is very sensitive
-- it picks up as little as 0.3 ml... It does not detect
blood from the stomach and upper small intestine, so
it is much more specific for bleeding from the colon or
lower gastrointestinal tract”.
PreGen-Plus
• The stool-based DNA test was capable of detecting
several stages of colorectal cancer, in otherwise
healthy adults, and most importantly in its early stage,
the easiest and most effective to treat, stage of
colorectal cancer.
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DIAGNOSTIC METHODS OF PARASITES
• Laboratory diagnosis of parasitic infection mainly
depends on,
o Detection and Identification of the parasite
o Morphological stage in the life cycle of the
parasite (Trophozoite, Cyst, Egg or Larva)
• In Clinical specimens (Feces, Blood, Urine, Bone
Marrow, Lymph Nodes).
• Other diagnostic methods include,
o Culture of Parasites
o Antibody detection in serum
o DNA probes
Additional Information:
• Protozoa (troph, cyts) metazoa (egg/ ova, larva, Adult
worm)
• Protozoa (intestinal, extraintestinal),
metazoan/helminths (nematode, trematode, cestode,
filariae), ectoparasite
• Sample types and associated parasites
o Feces: Giardia, Cryptosporidium,
Entamoeba, Ascaris, Enterobius, etc.
o Blood: Plasmodium, Leishmania,
Trypanosoma, microfilariae, babesiosis
o Skin: Onchocerca, sarcoptes, demodex
o Vaginal or urethral: Trichomonas
o Eye scrapings: Acanthamoeba
o Tissue: Naegleria (csf), Acanthamoeba, and
Leishmania (various tissue in the body)
o Urine: Schistosoma haematobium and
Trichomonas
o Sputum: Ascaris, Strongyloides, hookworm
larva (heart to lungs migration ASH)
paragonimus
• Culture, serological test, molecular test
1 EXAMINATION OF FECES
• Clean container – No contamination (Urine, water etc)
• Immediate examination – Motility of Trophozoite of
Protozoan parasites.
• Liquid Feces – Within 30 mins Formed Feces –
No time limit
• Should be maintained at 4ºC – No room temperature
o If immediate examination is not possible then
preservatives are used. The commonly
used preservatives are ;
o Formalin solution
o Polyvinylalcohol (PVA) Fixative
o Merthiolate iodine formalin (MIF) Solution
• Because urea and acidic pH inhibit some parasites
and distort their morphology, stool should be free of
urine.
WET MOUNT PREPARATION
• Liquid stools are best to detect trophozoites,
whereas formed stools are best to detect ova and
cysts.
• formalin “all purpose fixative”
• Polyvinyl alcohol (PVA) containing the fixative
mercuric chloride is considered the “gold standard” for
the fixation of ova and parasites in the preparation of
permanently stained smears of stool specimens.
• Merthiolate-Iodine-Formaldehyde (MIF)
solution enables fixing (with formaldehyde) of
protozoa and their staining with two coloring
agents (iodine and eosin). Stool preservation and
staining can be accomplished simultaneously with this
kit.
• Sodium-acetate-formalin (SAF) = Advantage: does
not contain HgCl2
MOTILITY IN WET MOUNTS
• Bihira kasi makikita lang siya sa freshly collected stool
MACROSCOPIC EXAMINATION
• Feces should be examined for its,
o Consistency
o Color
▪ Color and consistency (formed,
semi formed, mushy, mucoid, loose,
watery)
o Odor
o Presence of blood
o Mucus
• Adult roundworms – Ascaris lumbricoides or
segments of tapeworms may be seen in the feces.
• Presence of blood and mucus – Amebic dysentery
o Amebic dysentery is a severe form of
amebiasis associated with stomach pain,
bloody stools (poop), and fever.
MICROSCOPIC EXAMINATION
• This method includes;
o Wet Mount,
o Smear after concentration
o Permanent stained smears.
• Wet mounts are prepared as saline and iodine wet
mount.
• Various concentration can be used to
increase the sensitivity of microscopic examination.
• Trophozoite, Cyst of intestinal protozoa, Eggs, Larvae
of helminths – can be identified by microscopic
examination.
• Lugol's Iodine is a rapid, non-specific contrast dye
that is added to direct wet mounts of fecal material to
aid in differentiating parasitic cysts from host
white blood cells. Many protozoa and cysts take up
the dye and appear brown while other objects in the
sample remain clear.
• Saline as well as iodine preparation are
made on the same glass slide.
• A drop of normal saline (0.9 %) is put at one end
and iodine in another end.
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• A minute portion of feces is added to both and
mixed with the help of wooden stick to make a
uniform suspension.
• A cover slip is gently placed over suspension
(Avoid Bubbles)
• Examine the preparation under low power
objective lens of microscope. (Prepare another
slide if the specimen is dried up)
• Number of eggs under LPO
• Lugol’s iodine= differentiate cysts from WBC
PERMANENT STAINED SMEARS
• Permanent stained smears are employed for;
o Cytological details
o Accurate diagnosis
o Keeping permanent records
o Permanent stained smears are used to
enhance parasite morphology and to
allow for future study. Stained fecal
smears are important in the identification of
Entamoeba histolytica.
• Commonly used stain and methods are
o Iron – Hematoxylin stain - is used when
enhanced detail is needed; however, it is
difficult to obtain consistent staining results.
o Trichrome stain – Wheately or Gomori, most
commonly used stain for fecal parasite study.
• Modified acid-fast stains (Cryptosporidium, Isospora,
Cyclospora) = intestinal protozoa
CONCENTRATION METHODS
• When the parasite is scanty in feces, routine
microscopic examination may fail to detect eggs,
cysts, and trophozoite in the specimen.
• So, it is necessary to employ concentration methods
to selectively concentrate cysts, eggs and larvae.
• Trophozoites of protozoa are destroyed by these two
methods;
o Floating techniques
o Sedimentation techniques
• small or insufficient in quantity or amount.
• Concentration procedures for feces remove debris
that could obscure parasites.
FLOATING/FLOATATION TECHNIQUES
• In floating techniques, the feces is suspended in a
solution of a higher specific gravity so that the eggs
and cysts float to the top and get concentrated at
the surface. Following floating techniques can be
used;
1. SALT FLOTATION TECHNIQUE
o About 2ml of saturated salt solution (SG –
1.20) is taken in a flat bottomed vial and 1gm
of feces is emulsified in it. More salt
solution is added so that the container is
filled completely to the brim.
o A cover slip is carefully placed at the top of
the container so that it is in contact with the
surface of the solution.
o It is then allowed to stand for 20 – 30 mins,
after which the cover slip is removed, turned
over smoothly on a glass slide and examined
under the microscope.
o Brine flotation technique
▪ Mix saturated salt solution + feces=
homogenous mixture
▪ Add saturated salt solution
hanggang sa lid ng tube/ vial
▪ lagay ng cover slip (15-20) on top
then turn over sa glass slide
o It has been found that all the Helminthic eggs
float in the saturated salt solution except
unfertilized eggs of Ascaris lumbricoides,
eggs of Taenia solium and Taenia saginata
and all the intestinal flukes
o The larvae of Strongyloides stercoralis do
not float in saturated salt solution.
o for Protozoan cysts
o for Nematode eggs except T. trichiura and
C. philippinensis (sedimentation) because of
their plugs, unfertilized eggs ng ascaris (kasi
mabigat)
o NOT for trematodes such as Schistosomal
and operculated eggs (sedimentation)
2. ZINC SULFATE CENTRIFUGAL FLOTATION
TECHNIQUE
• About 1gm of feces is thoroughly mixed in 10 ml of
formalin solution. The coarse particles are
removed by straining the suspension through
gauze. The filtrate is collected in a small test tube and
centrifuged at 2500 Rpm for 1 min.
• Repeat the centrifugation until the supernatant is
clear.
• The clear supernatant is poured off and 3 – 4 ml of
zinc sulfate solution (SG – 1.18 – 1.12) is added to
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the sediment. Centrifuge again at 2500 Rpm for 1
min.
• With the help of wire loop, sample is transferred to a
glass slide and examined under microscope.
• Washing= mix formalin + feces> filter using gauze>
filtrate iccentrifuge> decant yung supernatant>
sediment > add zinc sulfate then mix> centri > 2
layers
• For protozoal cyst, a drop of iodine solution is added
before
• the cover slip is put on glass slide.
• This concentration technique is used for cysts of
protozoa, and eggs of;
o Nematodes
o Small tapeworms
• This method is not suitable for unfertilized eggs of
Ascaris lumbricoides and eggs of most trematodes
and large tapeworms.
• For cysts and egg ng nematode and cestodes
• Large tapeworm- diphyllobothrium latum
SEDIMENTATION TECHNIQUES
• In sedimentation technique the feces is suspended
in a solution of low specific gravity so that the eggs
and cysts gets sediment at the bottom.
• FORMALIN ETHER CONCENTRATION METHOD -
Commonly used sedimentation Technique
o 1 -2 gm of feces is thoroughly mixed in 10 ml
of formalin and strained to two layers of
gauze in a funnel. The filtrate solution is
centrifuged at 2000 rpm for 2 min. The
supernatant is discarded.
o The sediment is suspended in a 10 ml of
formalin and again centrifuged.
o The supernatant is discarded, and the
sediment is resuspended in 7ml of formalin.
It is allowed to stand for 10 min.
o 3 ml of ether is added to this solution.
The tube is shaken vigorously to mix and
then its centrifuged at 2000 rpm for 2 min.
four layers become visible.
▪ A top layer of ether
▪ A plug of Debris
▪ The formalin layer
▪ The sediment at the bottom.
o Washing= mix formalin + feces> filter using
gauze> centri yung filtrate> decant yung
supernatant> sediment + ether > mix> centri
o Wet mount is prepared. It is also mixed with
drop of iodine solution are examined.
o Ether dissolved fecal fat, CHO and formalin
fixes the eggs and cyst of the parasite and
removes fecal odor- helminthic eggs and
protozoan cysts.
o As ether is inflammable and explosive, it can
be replaced with ETHYL ACETATE.
o Papano mo makukuha yung sediment?
▪ rim yung fecal debris (2nd layer)
▪ Decant > ang matitira sediment
▪ Add ng konting formalin, then
aspirate lagay sa slide
o Best for eggs of: Schistosoma, Operculated
egg, Trematodes, Cestodes, T. trichiura, C.
philippinensis
EGG COUNTING
• Worm burden can be made by estimating the
number of eggs passed in faeces.
• However it is only rough indication of worm burden.
• Eggs count helps to classify helminthic infections as
heavy, moderate or light.
• For counting eggs there are two methods available;
o Egg count in wet mount preparation
o Stroll’s dilution technique
• Para ma-assess ang worm burden at ma-classify into
light moderate and heavy
• # of eggs per LPO
EGG COUNT IN WET MOUNT PREPARATION
• 2 mg of faeces is mixed in a small drop of saline on a
glass slide and cover slip is applied.
• It is examined under lower power of microscope and
the number of eggs is counted.
• Now the no of eggs per gram of faeces can be
calculated
STROLL’S DILUTION TECHNIQUE
• ]It is commonly used method for counting helminthic
eggs.
• 3 gm of feces is thoroughly mixed with 45 ml of N/10
NaOH in a tube.
• The mouth of the tube is closed with rubber cork and
shaken vigorously. Using a pipette, 0.15 ml of the
emulsion is removed and is placed on glass slide.
• A cover slip is put over it and the preparation is
examined under
• low power microscope.
• All the eggs in the preparation is counted. The
number of eggs per gram is calculated by multiplying
the count of eggs with 100.
• Quantitative test= Measured lahat and merong
calculation
• Other methods used for egg counting includes;
o Modified kato thick smear technique
▪ merong template na gamit. 43 g
o Mc Master’s egg counting chamber
▪ Calculation of results. The number
of eggs per gram can be calculated
as follows: Count the number of
eggs within the grid of each
chamber, ignoring those outside the
squares. Multiply the total by 50 –
this gives the eggs per gram of
feces (e.p.g.)
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ANAL SCRAPPINGS AND SWAB
• Enterobius vermicularis infection is usually diagnosed
by demonstrating the eggs on the perianal and
perineal skin. The following methods are generally
used.
SCOTCH CELLULSE ADHESIVE TAOE METHOD
• A 3 inches length of the tape is applied on the
perianal skin at several places.
• The adhesive tape is pressed against the perianal
skin.
• The adhesive side of the tape is placed between the
tape and slide. The toluene clears everything excepts
eggs and hair.
• Eggs of helminths other than Enterobius vermicularis
may also seen in the preparation.
NIH SWAB
• Eggs are deposited in large number of perianal
and perineal skin at night can be demonstrated by
scrapping this area by NIH swab in the morning
before taking bath.
• Spread over glass slide and examined
microscopically.
• This procedure should be repeated on the three
successive days.
2 EXAMINATION OF BLOOD
• Next to feces, the blood is the most common
specimen for recovery of various stages of parasite.
They are;
o Plasmodium sp.,
▪ Babesia sp.,
▪ Trypanasoma brucei gambiense
▪ Trypanasoma brucei rhodesiense
▪ Trypanasoma cruzi
▪ Wuchereria bancrofti
▪ Brugia malayi
o Two methods employed are :
• Wet preparation
• Stained blood smears
WET PREPARATION
• A drop of anticoagulated blood is placed on a clean
glass slide and cover slip is put over it.
• This preparation is examined microscopically for
parasite such as
o Trypomastigote= diagnostic stage
trypanosoma
o Microfilariae= ds ng lymphatic filiariasis
BLOOD-STAINED SMEARS
• Malaria detection
• Three types of blood smear is used:
o Thin blood smear
o Thick blood smear
o Thin and thick blood smear on the same
slide
THIN BLOOD SMEAR
• Identification of plasmodia and other parasites present
in the erythrocytes.
• The pulp of the finger or the lobe of the ear is wiped
out with spirit and allowed it to dry.
• It is pricked with a sterile cutting needle under all
aseptic conditions.
• A drop of blood is taken on the grease free glass slide
at one end and another end.
• Keep at an angle of 30 degree and pushed gently to
the end.
• The smear is formed with tails. It is allowed to dry.
THICK BLOOD SMEAR
• Take 4 drops of blood and join the corners with a
needle.
• It can also be prepared by spreading with a needle or
with the corner of another slide. It is 1 cm square. It is
allowed to dry.
• The thickness should be such it must allow to read the
newspaper.
• The smear is dehemoglobinized prior to staining.
It is done through placing the smear in distilled water
in a glass cylinder for 5 – 10 mins.
• With water based stains (Giemsa, JSB)
dehemoglobinization occurs when the stain is poured
on the smear, while for alcohol based stain
(Leishman) dehemoglobinization is with water.
• Giemsa, wrights, Leishman stains
THICK AND THIN BLOOD SMEARS ON THE SAME SLIDE
• Two drops of blood taken at two different places on
the same slide.
• One drop is made into thick preparation and another
into a thin smear.
• Thin smear on larger area and thick smear on smaller
area.
• Thick or thin blood smears are stained with
Romanowsky’s stain.
o Romanowsky stains are neutral stains
composed of a mixture of oxidized
methylene blue (azure) dyes and Eosin Y.
The azures are basic dyes that bind acid
nuclei and result in a blue to purple color.
The acid dye, eosin, is attracted to the
alkaline cytoplasm, producing red coloration.
• Example of these stain include.
o Leishman’s stain
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o Giemsa stain FIELD STAIN

o Field’s stain
o Jaswant singh and bhattacharjee stain • it is a quick method of staining thick blood smears
• These stains are combination of methylene blue and without fixation. It is useful when large number of
eosin. smears are to be examined
• They also contain oxidation products of methylene • Composition
blue named azures. o Solution A:
▪ Methylyne blue 0.8gm
LEISHMAN’S STAIN ▪ Azure I 0.5 gm
▪ Potassium hydrogen phosphate
• Prior fixation is not necessary as the stain contains an anhydrous 6.25
alcoholic solution which fixes as it stains. ▪ Disodium hydrogen 5.0
• Composition: ▪ Distilled water 500 ml
o Leishman dry powder – 0.015 gm o Solution B:
o Absolute methyl alcohol – 100ml ▪ Eosin 1.0gm
• Procedure: ▪ Potassium hydrogen 6.25 gm
o Smear is covered with Leishman stain for 2 ▪ Sodium hydrogen phosphate 5.0
minutes. gm
o Add buffer solution over the smear and leave ▪ Distilled water 500 ml
it for 15 – 20 • Procedure:
o minutes. o Thick smear is immersed in solution A for 1-2
o The slide is washed with buffered distilled seconds.
water. o It is removed and immediately washed in a
o It is air dried and examined under oil clean water for few seconds.
immersion. o It is then immersed in solution B for 1
• Merong fixative second.
• Flood ng stain – wash – airdry o It is then removed and rinsed gently in a
clean water for 2 – 3 seconds.
GIEMSA STAIN o It is air dried and examined under oil
immersion.
• Prior fixation of blood smear is required as the stain
used is in aqueous solution. The smear is fixed with JSB (JASHWANT SINGH BHATTACHARJEE) STAIN
absolute alcohol for 2-3 minutes.
• Composition • Rapid Romanowsky method of staining malarial
o Giemsa stain powder – 0.75 gm parasite. It is water soluble stain
o Glycerol – 25 ml • Composition:
o Methanol – 75 ml o Solution 1
• Procedure: ▪ Methylene blue 0.5gm
o Fixed smear is immersed in 1:10 dilution of ▪ Distilled water 500 ml
Giemsa in buffered water ▪ 1% H2SO4 3ml
o The stain is washed with buffered water ▪ Potassium dichromate 0.5 gm
o It is examined under IOI ▪ 1% potassium hydroxide 10ml
• Need fixation prior staining • Hiwalay din ang methylene blue at eosin
o Solution 2:
Added information: ▪ Eosin 1gm
▪ Distilled water 500ml
• For staining thick and thin smear it is
• Procedure:
dehemoglobinized and then
o Prior to staining, thin smear is fixed with
• Stained along with the thin smear.
methyl alcohol for 3-5
• A line with glass marking pencil is made or drawn
o minutes while fixation is not required in thick
between the two smears.
blood smear.
• Undiluted Leishman stain poured over thin smear.
o Smear is immersed in solution 1 for 30
• After dilution the stain is flooded over the thick smear seconds.
also. o Washed with acidulated water (pH 6.2-6.6)
• In case of Giemsa stain - Thin smear is fixed first for 4 seconds.
• After drying, the whole slide Is Flooded with diluted o Immersed again in solution I for 30 seconds.
Giemsa stain and is kept for 30 minutes to 2 hours. o Washed again with acidulated water for 10
seconds.

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• There are three jars containing solution I and II. Slide

is immersed in these jars. Smear is air dried and
examined under oil immersion.

3 EXAMINATION OF URINE

• Eggs of Schistosoma haematobium may be detected

• Another parasite Trichomonas Vaginalis may also be
recovered in urine both males and females
patients
• A drop of urine sediment is placed on a glass slide.
• A cover slip is placed over it and examined under
microscope.
• In case of Wuchereria bancrofti the microfilariae may
be discharged in urine (chyluria).’
• Egg lang ang makikita sa urine
• Same lang sa urinalysis

6 EXAMINATION OF ASPIRATES

• The aspirate from the liver is useful in diagnosis of

4 EXAMINATION OF SPUTUM
• Sputum is commonly examined for the detection of
eggs of Paragonimus Westermani
• Nag de-develop sa lung tissue
5 EXAMINATION OF CEREBRO SPINAL FLUID
• Direct microscopic examination of unstained and
stained smears of CSF is useful for the detection of
o Naegleria fowleri - trophozoite (nasal
mucosa) sa brain talaga
o Acanthamoeba species - cysts and
trophozoite (usually sa eye, nasal passages,
broken skin)
o Balamuthia mandrillaris - cysts and
trophozoite (usually sa eye, nasal passages,
broken skin)
o Tryponosoma brucei - blood, lymph, spinal
fluid
• Microfilariae
• Loa loa – urine, sputum, spinal fluid
amebic liver abscess and hydatid cyst
• Duodenal aspirates may reveal trophozoites of
Giardia Lamblia
• This material can be examined immediately in a
0.85% saline wet mount preparation (or part of this
material could be placed in formalin) or can be fixed in
PVA. Once fixed in PVA, the material can be stained
using trichrome stain and examined for trophozoites
of Entamoeba histolytica.
• Cystic echinocccosis (CE), also known as hydatid
disease, is caused by infection with the larval stage
of Echinococcus granulosus, long tapeworm found in
dogs (definitive host) and sheep, cattle, goats, and
pigs (intermediate hosts).
• Duodenal aspirates may be useful in
demonstrating Giardia duodenalis or Strongyloides
stercoralis larvae. Material collected following
intubation through the nose and stomach into the
upper small intestine may be submitted to the
laboratory. Centrifuge the specimen at 500 × g for 2 to
3 minutes and examine the wet mount. An unfixed
specimen can be examined immediately or if the
specimen cannot be examined within 1 to 2 hours
after collection, it should be preserved in 10%
formalin.
7 EXAMINATION OF BIOPSU

• Muscle biopsy may reveal

o Cysticerci of Taenia solium
o Larvae of Trichinella spiralis
o Sarcocysts of Sarcocystis lindemanni
• Liver biopsy may demonstrate

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o Entamoeba histolytica - small intestine

(intestinal disease), liver, lungs and brain
(extra intestinal diseases)
o Leishmania donovani - it affects various
tissue in the body
o Echinococcus granulosus - IH ang man,
hydatid cysts found in various organs
commonly liver and lungs
• Brain biopsy may reveal trophozoites of
o Entamoeba histolytica
o Naegleria fowleri
• Sisteserki= larval cyst (brain, muscle, other tissues)

8 CULTURE

• Culture methods are available for many protozoan

parasite such as
o Entamoeba histolytica - Balamuth’s medium
o Balantidium coli – LES medium
o Leishmania spp. – Shneider;s drosophilia
medium, trager’s medium, beren’s medium
o Trypanosoma spp. – NNN medium
• Culture is useful for accurate diagnosis or as a
supplement to other methods or to the diagnosis
those cases where routine
• Not a routine examination. It is employed for accurate
diagnosis of the parasite species
• There are three basic types of culture systems: xenic,
in which the parasite is grown in the presence of an
undefined flora; monoxenic, in which the parasite is
grown in the presence of a single additional species;
and axenic, in which the parasite is grown in the
absence of any other metabolizing cells.
• Novy-mcneal-nicolle= for trypanosoma and leismania

9 ANIMAL INOCULATION

• Animal inoculation is not a routine diagnostic

procedure is parasitic infection.
• It is useful In some parasites such as Toxoplasma
Gondii (Intraperitoneal inoculation of mice)

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• Leishmania donovani (Intraperitoneal inoculation of SCOTCH TAPE METHOD AND ANAL SWAB METHOD FOR
hamsters) ENTEROBIUS VERMICULARIS DETECTION
• Trypansoma species (Intraperitoneal inoculation or tail
vein inoculation in mice) ENTEROBIUS VERMICULARIS/ PINWORM/
THREADWORM/ SEATWORM
• Introduction of the parasite to the animal (esp. mice)
• Intaperitoneal= sa body cavity • Shape: round
10 SEROLOGY • Color: white
• Size:
• Various serological methods such as ELISA, IHA, o Females 8-13 mm long
FAT, and latex agglutination have been used for o Males: 2 to 5mm long
diagnosis of different parasitic infection • Posterior:
• Some examples are Amoebiasis, leishmaniasis, o Tapered in females
Toxoplasmosis. o Curved in males
• Enzyme-linked immunosorbent assay (ELISA) is a • Anterior part/ cervical ale:
labeled immunoassay that is considered the gold o Found in both males and females.
standard of immunoassays.
EGGS OF E. VERMICULARIS
• ELISA stands for enzyme-linked immunoassay. It is a
commonly used laboratory test to detect antibodies • Oviparous worms
in the blood • D shaped
• ELISA or Enzyme-Linked Immunosorbent Assay is an • 50-60 x 20-30 micrometer
immunoassay technique utilized to detect diseases.
• Colorless
The principle of ELISA is antigen-antibody
• Thin
interaction. Here, the specific antibodies associate or
• Single cell or larva
bind to its target antigen
• Indirect hemagglutination DEVELOPMENT OF EGGS
• Fluorescent antibody test

MOLECULAR METHODS

• DNA probes and PCR are available for the diagnosis

of parasitic infection.
• Malaria, Toxoplasmosis, Leishmaniasis and Filariasis
are some example.
• Sizes of various morphological forms of parasites
are very important in diagnosis of these infection.
• Quantitative test
• Polymerase chain reaction (PCR) is a technology
used for quick and easy amplifying DNA sequences,
which is based on the principle of enzymatic
replication of the nucleic acids. This method has in
the field of molecular biology an irreplaceable role and
HOW MAN BECOME INFECTED
constitutes one of the basic methods for DNA
analysis.
1. Enterobius vermicularis lay the eggs in the perianal
skin at night
2. Eggs are single celled when laid by the worms
3. After 4-6 hours they develop into larvated eggs
(infective stage)
4. The larvae eggs after ingestion by man, they develop
into worms
• Human becomes infected ingestion of infective eggs
containing larva
• The larva hatch in the small intestine
• Migrate to the large intestine
• Where they develop to adults (male and female)
• After 1 month the females become gravid
• The worm do not lay eggs in the intestinal lumen
• They lay eggs in perianal skin, the skin around the
anus

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E. vermicularis deposits thousands of eggs in the anal o Firmly press the sticky side of the cellophane
area. tape over the skin surround the anus several
times
o The eggs will stick to the tape
o The tape is then placed on glass slide, sticky
side down
o Put drop of glycerol, examine the slide under
the microscope
• Precaution:
o Put on gloves because eggs are directly
infective

ANAL SWAB/NIH SWAB TECHNIQUE

• National Institute for Health

• Is a modification of scotch tape, paddle coated with
adhesive material has advantage of scotch tape and
glass slide.

• Pinworm eggs can survive for up to two weeks on

clothing, bedding, food, and other surfaces. In which
ready to infect other people.

FACTORS HELPS SPREAD OF INFECTION

• The words deposit large numbers of eggs

• The worms lay the eggs of intestine anal area, where
it can spread to environment easily OTHER SPECIMENS
• The eggs become infective within short time (4-6
• Enterobius can be viewed in feces sometimes
hours)
• Urine of the infected females
• The eggs can remain infective for up to 2 weeks
• Vaginal secretion in case of vaginitis caused by the
• The eggs have a surface that adheres to
worms.
environmental objects
• Poor personal hygiene TREATMENT

BEST SAMPLE TO DIAGNOSE ENTROBIASIS: • Several medications used for the treatment of
pinworm are:
1. Swab from perianal area early morning before going
o Mebendazole
to toilet or taking a bath
o Pyrantelpamoate
SUITABLE DIAGNOSTIC TECHNIQUE o Albendazole
• All three of these drugs are to be given in single dose
1. Scotch tape technique and then repeated 2 weeks late
2. Anal swab or NIH swab technique
PREVENTION AND CONTROL
DIAGNOSTIC CSTAGES:
1. Hand hygiene
1. Eggs or adult worms 2. Trimming nails regularly and avoid biting the nails
3. Avoid scratching anal area
SCOTCH TAPE TECHNIQUE 4. Showering every morning and washing the anal area
to remove the eggs in the perianal region
• Eggs can be collected from the perianal skin ysing a
5. Do not co-bath with infective people and do not share
piece of transparent adhesive tape
personal belongings
• Procedure
6. Clean the bathroom and toilet
7. Washing of clothes and bed linens with hot water to
kill the eggs
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8. Ironing of clothes or expose then to sunlight for 24 BLOOD FILM TECHNIQUE FOR PARASITOLOGICAL
hours to kill the eggs. ANALYSIS
9. Treatment of infected individuals. In institutions,
daycare centers, and schools, control of pinworm can • The most commonly used technique for blood
be difficult but conducting mass drug administration examination is blood film.
during an outbreak can be successful. • A very thin layer of blood spread over a
10. Avoid ready made foods and drinks. microscope slide.
• Allows the various types of blood cells to be seen and
SUMMARY identified.
• Blood smear plays an important role in
• E vermicularis inhabits in the large intestine
diagnosing a wide range of illnesses.
• Pinworm can lay about 10,000-11,000 eggs per day
• Includes detection of blood-borne parasites, like
• 95-97% of eggs are deposited in the perianal skin
malaria.
• Only 3-5% can be found in stool
• The eggs usually deposited at night while the patient
is asleep
• Sometimes the worm can be found in stool
• Six or more daily consecutive specimens should be
collected or obtained before the patient is considered
infection free.

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LESIHMAN STAIN
METHYLENE BLUE EOSIN
Basic dye Acid due
Blue – purple color Pink-red color
Stains nuclei, ribosomes, Stain most cytoplasm
and rough ER (DNA and proteins which are mostly
RNA acidic) basic
Structures that stain with Structures that stain with
methylene blue are termed eosin are termed
basophilic eosinophilic

THIN BLOOD SMEAR THICK BLOOD SMEAR

– Intact RBCs lysed RBCs
– smaller volume – larger volume
– 0.005 μl blood/100 fields – 0.25 μl blood/100 fields
– good species – more difficult to diagnose
differentiation species
– low density – parasite density
infections can be missed

STAINING

• Staining is a biochemical technique of adding a

specific dye to a substrate (DNA, proteins, lipids,
carbohydrates) to qualify or quantify the presence of
a specific compound.
• Stains and dyes are frequently used in
• biology and medicine to highlight structures in
biological tissues for viewing, often with the aid of
different microscopes.
• Romanowsky stains are based on a
• combination of eosine and methylene blue
• • Wright's stain, Leishman stain and Giemsa stain.
• • All are used to examine blood or bone
marrow samples.
• • Nuclei stained dark blue/violet, erythrocytes pale
pink & cytoplasm pale blue
• • All are also suited to examination of blood to detect
blood-borne parasites like malaria.

STAINING PROCEDURE
Ang mag send sa iba, magkakaron ng enterobius. EME
• Thin smear are air dried AHAHHA
• Overflow the smear with Leishman stain (1
• ml) for 1-5 min. Goodluck mga bebiticakes qoe
• Add a double amount of distilled water and mix the
stain by blowing the fluid.
• Leave the mixture on the slide for 10-15 min.
• Wash off by slow-running water to remove the extra
liquid stain.
• Stand slide on end, and let dry in air.
• Examination

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