The document discusses methods for quantifying and identifying parasites from fecal and blood samples. It describes the MacMaster technique for determining the number of nematode eggs per gram of feces. This technique involves dissolving feces in salt solution, filling counting chambers, and examining under a microscope. The document also outlines how to make thin and thick blood smears for microscopy to detect parasites like Plasmodium and microfilaria. Various staining techniques are discussed for blood films, as well as rapid tests and other diagnostic methods.

1. College of Health Sciences
Dep. of Medical Laboratories
Parasitology Practice
3rd stage
Lecture 6
Dr.: Shameeran S. Ismael
BVM & S, M.Sc Medical Microbiology(Parasitology),
PhD Molecular Parasitology
Shameeran S. Ismael Parasitolog
Practice.Medical Laboratories, 2020

2. • It is used to counting the number of eggs or
larvae per gram of feces is performed (EPG,
LPG).
• The number of eggs or larvae per gram of feces
may be counted for further examination as well
as for determination of severity and size of
infection. The method used for quantitative is:
1. MacMaster Method
2. Stoll’s Method
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
II.Quantitative Fecal Methods

3. • The McMaster technique provides an estimate
of worm burden by determining the number of
nematode eggs/larvae per gram of feces. The
advantage of this technique is that it is quick
since the eggs are floated free of debris before
counting.
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
MacMaster Method

4. Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
MacMaster Slide:
MacMaser counting slide consisting of two
glass or plastic slides joined together; between
the marked areas of the upper slide and button
slide, two or three chambers of 0.15 ml volume
each are formed (10*10*1.5mm).

5. 1- Suspend 2g of feces in 60ml of saturated
sodium chloride solution.
2- The suspension strained through a fine sieve.
3- By pasteur pipette fill one compartment of the
counting cell at once.
4- Let all bubbles escape.
5- Repeat the same operation to fill the second
counting chamber
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
Procedure:

6. 6- After a few minutes, the eggs float up to the surface
and stick to the cover slide.
7- Count the number of eggs or larvae under low power.
Calculation:
2g of feces is dissolved in 60ml
Thus: 1g in 60/2 or 30ml
1grepresnts in other word the content of 0.15ml
Number of eggs in one gram of feces = X
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019

7. X*60/2*1/0.15= EPG
EPG= X*200
X= Number of eggs in one counting cell
When 2 or 4 counting cells are filled and
counted:
X= Total number of eggs/Total No. of counting
cells
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019

8. • It is impossible to calculate from the EPG the
precise size of the worm population in the host
because of many factors influencing egg
production. These factors are:
1. Male is not involved in the calculation
2. Worms produce various numbers of eggs
according to the species
3. Immune responses
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
Interpretation of the egg count:

9. 4. Different amounts of eggs production according to
seasons, temperature, host hormonal status
5. Host nutritional conditions and consistency of feces
6. Using of anthelmintics
7. EPG of cestodes infections has only a diagnostic
value.
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019

10. Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
Blood examination

11. Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
Blood sample will be used for:
Microscopic Examination
Thin Smear
Thick Smear
Wet Mount
Indirect
Immunological
Methods
Detect of Ab
Detect
Parasitic Ag

12. 1. Blood Wet Mount
• Direct wet mounts of fresh whole blood (or
centrifuged blood)
• Usually used for detection of microfilariae and
trypanosomes, this only gives evidence of
infection.
• One drop of blood with one drop of normal saline
• Collected blood with anticoagulants
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
I.Microscopic Examination of Blood

13. Peripheral blood film is a thin layer
of blood smeared on a microscope slide and
then stained to detect Plasmodium, Filaria,
Trypanosoma …etc,.
Three basic steps to make blood film:
1. Preparation of blood smear.
2. Fixation of blood smear.
3. Staining of blood smear
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
2.Blood Film:

14. • Thin • Thick
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
Prepare Blood Film:
Blood drop
spread
Air dry
methyl alcohol
Geimsa
Air dry
Geimsa
Circular motion

15. 1. Place a drop of blood at one
end of a clean glass slide.
2. Place another slide (spreader)
with smooth edge at an angle
of 30-45⁰ near the drop of
blood.
3. Move the spreader backward
so that it makes contact with
drop of blood.
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
A.Thin Blood Smear Procedure:

16. 4.Then move the spreader forward rapidly over
the slide.
5. A thin peripheral blood film is thus prepared
6. Dry it and stain it.
Shameeran S. Ismael Parasitolog
Practice.Medical Laboratories, 2020

17. •A peripheral blood film consists of 3 parts :
1. Head: the portion of blood film near the drop of blood.
2. Body: the main part of the blood film.
3. Tail: the tapering end of the blood film.
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
Parts of a Thin Blood Film:

18. 1. Place a large drop of blood in the centre of a clean
glass slide.
2. Spread it in a circular area of 1.5 cm with the help
of a stick or end of another glass slide.
3. Dry it
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
B.Thick Blood Smear Procedure

19. Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
Observe Thin and Thick Smear

20. • The thick film is more sensitive in detecting parasite
and also saves time in examination.
• The thin film technique cause very little distortion of
the parasite, and permits species identification when it
may not be possible in thick films, but many fields
must be examined to detect parasite when they are few
in number.
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019

21. • To preserve the morphology of the cells, films must be fixed as
soon as possible after they have dried.
• It is important to prevent contact with water before fixation is
complete.
1. Methyl alcohol (absolute methanol)
2. Ethyl alcohol (absolute alcohol)
Methylated spirit (95% ethanol) must not be used
• To fix the films, place them in a covered staining jar or tray
containing the alcohol for 2-3 minutes.
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
Fixation of blood smear

22.  Romanowsky stains are universally employed for
staining of blood films.
 All Romanowsky combinations have two essential
ingredients
1. methylene blue is the basic dye and has
affinity for acidic component of the cell (i.e.
nucleus)
2. eosin or azure is the acidic dye and has
affinity for basic component of cell (i.e.
cytoplasm).
• Most Romanowsky stains are prepared in methyl
alcohol so that they combine fixation and staining.
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
Stains for Blood Film

23. 1. Leishman stain
2. Giemsa stain
3. Wright stain
4. Field stain
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
Stains included under Romanowsky

24. 1. Immunochromatographic assay ICT
• Rapid diagnostic tests for malaria employing ICT
(immunochromatographic assay methods based on the
detection of malarial antigens present in peripheral
blood.
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019
Special Tests:

25. ICT (immunochromatographic assay)
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019

26. 2.Casoni test: is an immediate hypersensitivity skin test
used in the diagnosis of hydatid disease.
•The test involves the intradermal injection of 0.5 ml of
sterilized fluid from hydatid cysts/human cyst on forearm
and equal volum of saline injected on the other forearm and
check the reaction after 30 minute.
Shameeran S. Ismael Parasitology theory.
Vet. Med, 2019

27. Thanks for attention,,,
Shameeran S. Ismael Parasitolog
Practice.Medical Laboratories, 2020