Diagnostic methods of Parasites

DIAGNOSTIC METHODS OF PARASITES
Presented By,
S. Shruthi Vasan
I MSc. Medical Laboratory Technology
KMCH Institute of Allied Health Science
Shruthivasan4s@gmail.com

CONTEXTURE
1 . Examination of Faeces
A. Macroscopic examination
B. Microscopic examination
2 . Examination of blood
A. Wet preparation
B. Stained blood smear
3 . Examination of Urine
4 . Examination of Sputum
5 . Examination of Cerebrospinal Fluid
6 . Examination of Aspirates
7 . Examination of Biopsy Material
8 . Culture
9 . Animal Inoculation
10 . Serology
11 . Molecular Methods

INTRODUCTION
Laboratory diagnosis of parasitic infection mainly depends on,
 Detection and Identification of the parasite
 Morphological stage in the life cycle of the parasite
(Trophozoite, Cyst, Egg or Larva)
In Clinical specimens (Faeces, Blood, Urine, Bone Marrow, Lymph
Nodes).
Other diagnostic methods include,
 Culture of Parasites
 Antibody detection In serum
 DNA probes

1. EXAMINATION OF FAECES
STOOL CONTAINER

1. EXAMINATION OF FAECES
 Clean container – No contamination (Urine, water etc)
 Immediate examination – Motility of Trophozoite of Protozoan
parasites.
 Liquid Faeces – Within 30 mints
Formed Faeces – No time limit
 Should be maintained at 4ºC – No room temperature
 If immediate examination is not possible then preservatives are
used. The commonly used preservatives are ;
 Formalin solution
 Polyvinylalcohol (PVA) Fixative
 Merthiolate iodine formalin (MIF) Solution

A. MACROSCOPIC EXAMINATION
 Faeces should be examined for its,
Consistency
Colour
Odour
Presence of blood
Mucus
 Adult roundworms – Ascaris lumbricoides or segments of
tapeworms may be seen in the faeces.
 Presence of blood and mucus – Amoebic dysentery

B. MICROSCOPIC EXAMINATION
 This method includes ;
1. Wet Mount,
2. Smear after concentration
3. 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.

1 . WET MOUNT PREPARATION
 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.
 A minute portion of faeces 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)

IODINE WET MOUNT SALINE WET MOUNT

2 . PERMANENT STAINED SMEARS
 Permanent stained smears are employed for;
 Cytological details
 Accurate diagnosis
 Keeping permanent records
 Commonly used stain and methods are
 Iron – Haematoxylin stain
 Trichrome stain

3 . CONCENTRATION METHODS
When the parasite is scanty in faeces, routine microscopic
examination may fail to detect eggs, cysts, 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;
3.1. Floating techniques
3.2. Sedimentation techniques

3.1. FLOATING TECHNIQUES
In floating techniques, the faeces 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;
3.1.1. Salt floatation technique
3.1.2. Zinc sulphate centrifugal floatation technique

3.1.1. SALT FLOATATION TECHNIQUE
 About 2ml of saturated salt solution (SG – 1.20) is taken in a
flat bottomed vial and 1gm of faeces is emulsified in it. More
salt solution is added so that the container is filled completely
to the brim.
 A glass slide is carefully placed at the top of the container so
that it is in contact with the surface of the solution.
 It is then allowed to stand for 20 – 30 mins, after which the
glass slide is removed, turned over smoothly and examined
under the microscope. A cover slip is not placed over the fluid
present on the slide.

3.1.1. SALT FLOATATION TECHNIQUE
It has been found that all the Helminthic eggs float in the
saturated salt solution expect unfertilized eggs of Ascaris
lumbricoides, eggs of Taenia solium and Taenia saginata
and all the intestinal flukes
 The larvae of Strongyloides stercoralis do not float in
saturated salt solution.

3.1.2. ZINC SULPHATE CENTRIFUGAL
FLOATATION TECHNIQUE
 About 1gm of faeces is thoroughly mixed in 10 ml of distilled
water. 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
sulphate solution (SG – 1.18 – 1.12) is added to 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.

3.1.2. ZINC SULPHATE CENTRIFUGAL
FLOATATION TECHNIQUE
 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
eggs of;
 Nematodes
 Small tapeworms
 This method is not suitable for unfertilized eggs of Ascaris
lumbricoides and eggs of most trematodes and large
tapeworms.

3.2 SEDIMENTATION TECHNIQUES
 In sedimentation technique the faeces is suspended in a
solution of low specific gravity so that the eggs and cysts gets
sediment at the bottom.
 FORMAL ETHER CONCENTRATION METHOD –
Commonly used sedimentation Technique.

FORMAL ETHER CONCENTRATION
TECHNIQUE
 1 -2 gm of faeces is thoroughly mixed in 10 ml of water 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.
 The sediment is suspended in a 10 ml of saline and again
centrifuged.
 The supernatant is discarded and the sediment is resuspended
in 7ml of formal saline. It is allowed to stand for 10 min.
 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.

FORMAL ETHER CONCENTRATION
TECHNIQUE A top layer of ether
 A plug of Debris
 The formal - Saline layer
 The sediment at the bottom.
 Wet mount is prepared. It is also mixed with drop of iodine
solution are examined.
 Ether dissolved faecal fat and formalin fixes the eggs and cyst of
the parasite and removes faecal odour - helminthic eggs and
protozoan cysts.
 As ether is inflammable and explosive, it can be replaced with
ETHYL ACETATE.

4. 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;
4.1. Egg count in wet mount preparation
4.2. Stroll’s dilution technique

4.1. EGG COUNT IN WET MOUNT
PREPARATION
 1- 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

4.2. STROLL’S DILUTION TECHNIQUE
 It is commonly used method for counting helminthic eggs.
 4 gm of faeces is thoroughly mixed with 56 ml of N/10 NaOH in a
flask.
 The mouth of the flask is closed with rubber cork and shaken
vigorously. Using a pipette, 0.075 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 200.

4.2. STROLL’S DILUTION TECHNIQUE Considering the consistency of faeces it is multiplied by
correction factor to convert the estimate to formed faeces.
The correction factor is 1 for hard formed faeces,
2 for mushy formed faeces,
3 for loose faeces,
4 for liquid faeces.
 The total egg production per day can be calculated by
multiplying the no. of eggs per gram with a 24 hour faecal
specimen.
 Other methods used for egg counting includes;
Modified kato thick smear technique
Mc Master’s egg counting chamber

5. 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;
5.1. Scotch cellulose adhesive tape method
5.2. NIH Swab

5.1 SCOTCH CELLULOSE ADHESIVE TAPE
METHOD
 A 3 inch 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.

5.2 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 faeces, the blood is the most common specimen for
recovery of various stages of parasite. They are;
 Plasmodium sp.,
 Babesia sp.,
 Trypanasoma brucei gambiense
 Trypanasoma brucei rhodesiense
 Trypanasoma cruzi
 Wuchereria bancrofti
 Brugia malayi
 Two methods employed are :
A. Wet preparation
B. Stained blood smears

A. 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
Trypanosomes and Microfilariae
Trypanosomes Microfilariae

B. STAINED BLOOD SMEARS
Three types of blood smear is used:
1. Thin blood smear
2. Thick blood smear
3. Thin and thick blood smear on the same
slide

1. 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.

2. 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 dehaemoglobinized 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) dehaemoglobinization with water is

3. 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.

STAINING OF BLOOD SMEARS
 Thick or thin blood smears are stained with romanousky’s stain.
Example of these stain include.
A. Leishman’s stain
B. Giemsa stain
C. Field’s stain
D. Jaswant singh and bhattacharjee stain
 These stains are combination of methylene blue and eosin.
 They also contain oxidation products of methylene blue named
azures.
They provide contrast in PS.
Cytoplasm of protozoal parasites - Blue
Nuclei - Red

A. LEISHMAN’S STAIN
 Prior fixation is not necessary as the stain contains an alcoholic
solution which fixes as it stains.
 Composition ;
1. Leishman dry powder - 0.015 gm
2. Absolute methyl alcohol - 100 ml
 Procedure :
1. Smear is covered with Leishman stain for 2 minutes.
2. Add buffer solution over the smear and leave it for 15 – 20
minutes.
3. The slide is washed with buffered distilled water.
4. It is air dried and examined under oil immersion.

B. GIEMSA STAIN
 Prior fixation of blood smear is required as the stain used is in
aqueous solution. The smear is fixed with absolute alcohol for
2-3 minutes.
 Composition ;
1. Giemsa stain powder - 0.75 gm
2. Glycerol - 25 ml
3. Methanol - 75 ml
 Procedure :
1. Fixed smear is immersed in 1:10 dilution of Giemsa stain
in buffered water.
2. The stain is washed with buffered water.
3. It is examined under oil immersion lens.

Note :
 For staining thick and thin smear it is dehemologinilized and then
Stained along with the thin smear.
 A line with glass marking pencil is made or drawn between the
two smears.
 Undiluted Leishman stain poured over thin smear.
 After dilution the stain is flooded over the thick smear also.
 In case of Giemsa stain - Thin smear is fixed first
 After drying, the whole slide Is Flooded with diluted Giemsa stain
and is kept for 30 minutes to 2 hours.

C. FIELD STAIN
 It is quick method of staining thick blood smears without fixation.
It is useful when large number of smears are to be examined.
 Composition :
I. Solution A :
 Methylene blue - 0.8 gm
 Azure I - 0.5 gm
 Potassium hydrogen - 6.25 gm
phosphate (Anhydrous)
 Disodium hydrogen - 5.0 gm
 Distilled water - 500 ml

II. Solution B:
 Eosin - 1.0 gm
 Potassium hydrogen - 6.25 gm
 Sodium hydrogen phosphate - 5.0 gm
Procedure :
1. Thick smear is immersed in solution A for 1-2 seconds.
2. It is removed and immediately washed in a clean water for few
seconds.
3. It is then immersed in solution B for 1 second.
4. It is then removed and rinsed gently in a clean water for 2 – 3
seconds.
5. It is air dried and examined under oil immersion.

D. JSB (JASHWANT SINGH BHATTACHARJEE)
STAIN
 It is rapid Romanousky method of staining malarial parasite. It is
water soluble stain.
 Composition :
I. Solution 1:
 Methylene blue - 0.5 gm
 1 % H2SO4 - 3 ml
 Potassium dichromate - 0.5 gm
 1% potassium hydroxide - 10 ml

Solution II:
 Eosin - 1 gm
Procedure :
1. Prior to staining, thin smear is fixed with methyl alcohol for 3-5
minutes while fixation is not required in thick blood smear.
2. Smear is immersed in solution 1 for 30 seconds.
3. Washed with acidulated water (pH 6.2-6.6) for 4 seconds.
4. Immersed again in solution I for 30 seconds.
5. Washed again with acidulated water for 10 seconds.
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 Trichomonis 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 Wucherria boncraofti the microfilariae may be
discharged in urine (chyluria).

Schistosoma haematobium
Trichomonis Vaginalis

4. EXAMINATION OF SPUTUM
Sputum is commonly examined for the detection of eggs of
Paragonimus Westermani

5. EXAMINATION OF CEREBRO SPINAL FLUID
(CSF)
 Direct microscopic examination of unstained and stained smears
of CSF is useful for the detection of
Naegleria fowleri
 Acanthomoeba species
Balamuthia mandrillaris
Trypanasoma brucei

Naegleria fowleri
Acanthomoeba species

Balamuthia mandrillaris
Trypanasoma brucei

6. EXAMINATION OF ASPIRATES
 The aspirate from the liver is useful in diagnosis of amoebic liver
abscess and hydatid cyst.
 Duodenal aspirates may reveal trophozoites of Giardia Lamblia

7. EXAMINATION OF BIOPSY
 Muscle biopsy may reveal
 Cysticerci of Taenia solium
 Larvae of Trichinella spiralis
 Sarcocysts of Sarcocystis lindemanni
 Liver biopsy may demonstrate
 Entamoeba histolytica
Leishmania donovani
Echinococcus granulosus
 Brain biopsy may reveal trophozoites of
Entamoeba histolytica
 Naegleria fowleri

8. CULTURE
 Culture methods are available for many protozoan parasite
such as
 Entamoeba histolytica - Balamuth’s medium
 Balantidium coli - LES medium
 Naegleria fowleri - Non nutrient agar & PYG medium
 Leishmania species - Schneider’s drosophilia medium
Trager’s medium
Steiger’s medium
Beren’s medium
 Trypanosoma species - NNN medium
 Culture is useful for accurate diagnosis or as a supplement to
other methods or to the diagnosis those cases where routine

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)
 Leishmania donovani (Intraperitoneal inoculation of
hamsters)
 Trypansoma species (Intraperitoneal inoculation or tail
vein inoculation in mice)

11. SEROLOGY
 Various serological methods such as ELISA, IHA, FAT and
latex agglutination have been used for diagnosis of different
parasitic infection.
 Some examples are Amoebiasis, leishmaniasis,
Toxoplasmosis.

12. 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.

Diagnostic methods of Parasites

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