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Chapter 5

Malaria Microscopy: Examination

and Species Identification

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 Content Outline

• Examining blood films for malaria parasites

• Diagnostic features of the plasmodium species

• Detection and identification of malaria parasites Stages.

• Identification of malaria parasite species and artifacts

• Malaria parasite counting methods

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 Learning Objectives
At the end of this module the participants should be able to:

• Examine blood films for malaria parasites

• Diagnostic features of the plasmodium species

• Detect and identify properly the stages and species of malaria

parasites.

• Recognize artifacts that may be mistaken for malaria parasites.

• Estimate parasite density for plasmodium species

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 Malaria Microscopy

Activity 5.1. Self Reflection

Instruction: Read the following question and give

your answer

Question: How do you examining thick and thin

blood films for malaria parasites?

Time:3 min

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 Malaria Microscopy…….cont’d

• Is the gold standard method for detecting, identifying and

quantifying of malaria parasites in blood sample.

• Requires trained lab personnel and equipment.

• Examination made using stained thick and thin blood films.

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 Malaria Microscopy……Cont’d
Examining blood film
• Malaria parasites are detected, identified and quantified by
microscopic examination of thick and thin stained blood films with
Giemsa stain .
• Both thick and thin films should initially be examined with 10x
and 40x to avoid missing large parasites such as microfilariae
and trypanosomes.

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 Malaria Microscopy……Cont’d
Required Materials and Equipment
• Lens paper
• Lens cleaning solution
• Immersion oil
• Binocular microscope
• Stained blood film
• Tally counter(s)
• Tissue paper
• Slide box(es)
• Pen
• Patient Register
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 Systematic approach to examine thick blood film

• Thick films are performed to

• To detect parasites
• To measure parasite density

• If malaria parasites is found a further 100 fields should be

examined to rule out the presence of a mixed infection.

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 Systematic approach …..Cont’d
Determination of "No Hemoparasite Found" (NHF):
• At least 100 fields should be examined before a thick film
is reported as negative.
– In nonimmune patients, symptomatic malaria can
occur at lower parasite densities, and screening more
fields (e.g., 200, 300, or even the whole film) is
recommended.

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 Systematic approach …..Cont’d
Procedure for examination of thick
blood film
1. Place the stained slide on the
mechanical stage.
2. Scan the entire film at a low
magnification.
3. Place a drop of immersion oil on the
middle of the thick film.
4. Examine the film using the 100X.

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 Systematic approach …..Cont’d

4. Select an area that is well-stained, free of stain precipitate,

and well-populated WBCs (10-20 WBCs/field).

5. Move the blood film following the pattern shown in the

diagram.

6. If you see parasites, make a tentative species determination

on the thick film and then examine the thin film to confirm the
species present.
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 Systematic approach to examine thin blood film

• Thin film is performed for

– Species identification
– Quantification e.g. Percentage of infected red cell
– Useful in high parasitaemia.
– Difficulty in examination of thick film.
• Since it takes more time (~10x) as compared to examination of
a thick film, routine examination of thin films is not
recommended.

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 Systematic approach …..Cont’d
 Procedure for examination of thin
blood film
1. Place a drop of immersion oil on
the edge of the middle of the film.
2. Carefully examine the film using
the 100X .
• If doubtful diagnosis examine
more fields

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 Characteristics of thick and thin blood films

Thick blood film Thin blood film

Lysed RBCs Fixed RBCs
Many layers Single layer
Large volume Smaller volume
Good screening test Good species differentiation
Low density infection can be
Low density infection can be missed
detected

More difficult to diagnose species Requires more time to read

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Diagnostic features of the plasmodium species

Activity 5.2. Think/Pair/share

Instruction: first think about the following
question and then discus in pair with a
participant sitting next to you and share your
idea.
Question: List a unique characteristic future
used to differentiate each plasmodium species.
Time: 5 minutes

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 Diagnostic features…………Cont’d

• Chromatin: round in shape and stains red.

• Cytoplasm : from a ring shape to a totally irregular shape and
stained blue .
• Malaria pigment: stain in various shades from yellow –gold
through brown to black
• Stippling: stains in shades of pink which vary among different
species

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 Species differentiation on thick films
Feature P.falciparum P. vivax P. ovale P. malariae
Uniform trophozoites +
Fragmented
++ +
trophozoites
Compact
+ +
trophozoites
Pigmented
+
trophozoites
Irregular cytoplasm + +
Stippling ("RBC
+ +
ghosts")
Schizonts visible Very rarely Often Often Often
Gametocytes visible occasionally usually usually usually

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 Species differentiation on thin films
Feature P. falciparum P. vivax P. ovale P. malariae
Enlarged
No Yes Yes No
infected RBC
Infected RBC
Round Round, distorted Oval, fimbriated Round
shape
Stippling infected
Mauer clefts Schuffner spots Schuffner spots none
RBC
Trophozoite Small ring, Large ring, Small ring,
Large ring, amoeboid
shape Delicate compact compact

Chromatin dot Often double Single Single Single

Rare, 12-30
Mature schizont 12-24 merozoites 4-12 merozoites 6-12 merzoites
merozoites

Gametocyte Crescent shape large, round large, round compact, round

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 Parasite morphology at specific stages Depends on:

• Number and size of chromatin dots

• Shape and size of cytoplasm

• Degree of pigmentation within cytoplasm

• Stages of parasite seen together

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 Parasite morphology……Cont’d
Feature of Plasmodium

Chromatin Dot

Vacuole

Cytoplasm

Stippling

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 The Trophozoite /Ring/ stage
• Most commonly seen
• As it is a growing stage,
the parasite within the
RBC may vary in size. Red blood cell

Cytoplasm (blue stain)

• Malaria pigment is a by-

product of the growth or Chromatin dot (red stain)

metabolism of the parasite.

• It does not stain, but has a Red blood cell
color of its own, which may
range from pale yellow to Chromatin dot

dark brown or black. (red stain) Cytoplasm (blue stain)

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 The Schizont stage
• Malaria parasite starts
to reproduce asexually
• There are several
obvious phases in this
stage, ranging from
parasites with two
chromatin pieces to
parasites with a number
of chromatin dots and
definite cytoplasm.

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 Shizonts stage……Cont’d

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 The Gametocyte stage
– Sexually dedifferentiated.
• Male (microgametocyte).
• Female(macrogametocyt ).

– Shaped as either round or

banana depending on the
species.

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 Gametocytes

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 Appearance of different species in a thick blood film

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 Appearance of different Species in Thin blood film

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 Plasmodium falciparum
Diagnostic points:-
• RBCs not enlarged.
• Maurer’ dot may be present
• No Schuffener’s dot
• Rings appear fine and delicate
• May be several in one cell.
• Some rings with 2 chromatin dots.
• Presence of marginal or appliqué
forms/acol formation/.
• Gametocytes crescent/banana shape
• Usually trophozoites + gametocytes seen
• Schizont rarely seen

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 Plasmodium vivax
Diagnostic points:-
• RBCs are usually enlarged.
• Schuffner's dots are
frequently present in the red
cells.
• The mature ring forms tend to
be large and coarse.
• Trophozoites schizonts &
gametocytes
• Developing forms are
frequently present

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 Plasmodium ovale
Diagnostic points:-
• Red cells enlarged.
• Comet forms common (top
right)
• Rings large and coarse.
• Schuffner's dots, when
crescent, may be
prominent.
• Mature schizonts similar to
those of P. malariae but
larger and more coarse .
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 Plasmodium malariae
Diagnostic points :-
• RBC not enlarged
• Ring forms may have a
Squarish appearance.
• Band forms are a
characteristic of this
species.
• Mature schizonts may have
a typical daisy head
appearance with up to ten
merozoites.

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 Key to identifying parasite stages in thin blood films

1. Are there one or more red-stained

chromatin dots and blue
cytoplasm?
Yes: go to 2
No: what you see is not a parasite
2. Are the size and shape right for a
malaria parasite?
Yes: what you see is probably a
malaria parasite; go to 3
No: what you see is not a parasite

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 Key to identifying parasite stages…
3. Is there malaria pigment in
the cell?
Yes: go to 7
No: go to 4
4. Does the parasite have one
chromatin dot attached to blue
cytoplasm in the form of a
regular ring with a vacuole?
Yes: this is a trophozoite stage
No: go to 5

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 Key to identifying parasite stages…
5. Does the parasite have one
chromatin dot attached to blue
cytoplasm in the form of a small
solid or regular ring or with a
vacuole?
Yes: this is a trophozoite stage
No: go to 6
6. Is the parasite with one
chromatin dot irregular or
fragmented?
Yes: this is a trophozoite stage
No: go to 8
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 Key to identifying parasite stages…
7. Does the parasite with malaria
pigment have one chromatin
dot?
Yes: go to 8
No: go to 9
8. Does the parasite have a
vacuole or is it still fragmented in
some way?
Yes: this is probably a late
trophozoite stage
No: go to 11

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 Key to identifying parasite stages…
9. Does the parasite have two
chromatin dots attached to a ring
and also have a vacuole?
Yes: this is a trophozoite stage
No: go to 10
10. Does the parasite have between
2 and 32 chromatin dots and
pigment?
Yes: this is a schizont stage

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 Key to identifying parasite stages…
11. Is the parasite rounded or banana-
shaped?
Rounded: go to 12
Banana-shaped: go to 14
12. Does the rounded parasite have
clearly stained chromatin and deep
blue cytoplasm?
Yes: this is a female gametocyte
No: go to 13

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 Key to identifying parasite stages…
13. Does the rounded parasite have a reddish
overall colour to the staining, so that the
chromatin cannot be clearly seen?
Yes: this is a male gametocyte
14. Does the banana-shaped parasite have
densely stained blue cytoplasm and bright red
chromatin?
Yes: this is a female gametocyte
No: go to 15
15. Does the banana-shaped parasite have a
reddish overall color to the staining, so that the
chromatin is indistinct?
Yes: This is a male gametocyte
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 Stage of parasite in peripheral blood
Species
Trophozoits Shizonts Gametocyte

• Size: small to medium • Immature pointed-end forms

uncommon.
• Shape: ring and comma • Size: small, compact • Mature forms: banana-
P. falciparum • Chromatin: often 2 dots
• Cytoplasm: regular
• Number: few,
• Mature forms: 12-30 or more
shaped or rounded.
• Chromatin: single, well
• Mature forms: sometimes merozoites defined
present in severe malaria, • Pigment: single dark mass • Pigment: scattered, coarse,
compact with pigment as few NB: Usually associated with rice-grain-like; pink extrusion
coarse grains or a mass many young ring forms. body sometimes present.

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 Stage of parasite in peripheral blood
Species
Trophozoits Shizonts Gametocyte

• Size: small to large • Immature forms difficult

• Number: few to moderate to distinguish from mature
• Size: large
• Shape: broken ring to trophozoites.
P. vivax irregular forms
• Number: few to
moderate
• Chromatin: single, • Mature forms: round,
• Mature forms: 12-
occasionally two large
24 merozoites,
• Cytoplasm: irregular or
usually 16.
fragmented • Chromatin: single, well
• Pigment: loose
• Mature forms: compact, defined
mass
dense
• Pigment: scattered, fine • Pigment: scattered, fine.

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 Species Stage of parasite in peripheral blood
Trophozoites Shizonts Gametocytes

Size: like P. malariae

Number: few
Size: may be smaller than P. Vivax Mature forms: 4-12 Mature forms: round,
Number: usually few merozoites, usually 8, in smaller than P. vivax
Shape: ring to rounded, compact loose cluster Chromatin: single, well
P. ovale forms Pigment: concentrated defined
Chromatin: single, prominent mass Pigment: scattered,
Cytoplasm: fairly regular, coarse.
Pigment: scattered, coarse

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 Species Stage of parasite
Trophozoites Shizonts Gametocytes

•Size: small; Immature and certain

•Number: usually few; Size: small, mature forms difficult to
• Shape: ring to rounded, compact; distinguish from mature
Number: Few trophozoites.
P. malarie compact forms;
Mature forms: 6-12 Mature forms: round:
• Chromatin: single, large;
merozoites, usually 8 compact;
•Cytoplasm: regular, Pigment: Chromatin: single, well
dense; concentrated. defined;
•Pigment: scattered, Pigment: scattered,
abundant, with yellow coarse, may be
tinge in older forms. peripherally distributed.

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Artifacts and contaminants confusing malaria parasites

Activity 5.3. Self-reflection

Instruction: Read the following question and answer it.

Question: List artifacts and contaminants which can

confusing with malaria parasites

Time:3 minutes

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 Artefacts.............Cont’d

• Vegetable spores, yeast, pollen, algae and bacteria in the

stain or on the slide

• Platelets

• Howell-jolly, Cabot ring bodies in anaemic patients

• Ghosts of immature red cells mimicking Schüffner’s dots.

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 Artefacts.............Cont’d

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 Estimating Parasite Density
Activity 5.4. Self-reflection

Instruction: Read the following question and answer it.

Question: How many recommended methods of malaria

parasite quantification methods do you know?

Time:3 minutes

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 Estimating Parasite Density…cont’d

• Important for clinical purposes

– To monitor the progress of the disease and
– The efficacy of therapy.
–To classify the severity of the disease status
• Methods
1. Number of parasites/µL of blood (thick film)
2. Number of parasites/µL of blood (thin film)
3. Proportion of parasitized erythrocytes (thin film)
4. Semi quantitative count (thick film):

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 Estimating Parasite Density…cont’d
• 1. Number of parasites/µL of blood (thick film):
•Requires observation of at least 100 fields while you count 200
WBCs.
• Number of asexual parasites and WBCs should be counted in
each field until the number of WBCs reaches 200.
• If number of WBCs is unknown, it can be assumed to be
8000/µL

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 Estimating Parasite Density…cont’d
Example:
• Patient WBC = 8000 ul
• Parasite count against 200 WBCs = 650
• Parasite count/ul = 650 x 8000/ul
200
= 26000 parasites/ul

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 Estimating Parasite Density…cont’d
• 2. Number of parasites/µL of blood (thin film)
• Requires the preliminary determination of RBCs number
present in the average microscopic field.
• The number of asexual parasites is counted in at least 25
microscopic fields.
• The number of RBCs in the average microscopic field is about
200, so total RBCs counted in 25 fields is about 200 x 25 =
5000.
• RBCs/µl is assumed to be 5 millions/µl for males and 4.5
millions/µl for females.

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 Estimating Parasite Density…cont’d
Example:
• Parasite counted against 5000 RBCs/25 fields/= 50
• # of RBCs in 25 fields= 5000
• RBC count = 5 million/Male patient/
50 x 5,000,000
5000
• 50,000/µl of blood.

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 Estimating Parasite Density…cont’d
3. Proportion of parasitized erythrocytes (thin film):

• Indicate the percentage of erythrocytes that are infected by

malaria parasites.
• The number of parasitized erythrocytes (asexual forms) present
in 25 microscopic fields is counted divided by the total number of
erythrocytes present in these fields (about 5000), and multiplied
by 100.

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 Estimating Parasite Density…cont’d
• Example
– Average # of RBCs/25 fields=5000
– # Parasitized RBCs/25 fields=100
– % of parasitized RBCS= 100 X 100

5000
– 2% of RBCs are infected with asexual for of malaria parasite.

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 Estimating Parasite Density…cont’d
4. Semi quantitative count (thick film):

• Very quick but less accurate.

• Used only when not possible to perform more accurate
methods.
• Reporting:
–+ 1-10 asexual parasites / 100 thick film fields
– ++ 11-100 asexual parasites / 100 thick film fields
– +++ 1-10 asexual parasites / single thick film field
– ++++ > 10 asexual parasites / single thick film field

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 Reporting of BF results

If positive :
• Check the presence of
• Different stages ( Throphozoits, schizonts and gametocytes).
• Mixed infection
• Report the species, stage and density of parasites and if present
malaria pigments .
• If negative after examination of a minimum of 100x thick film
fields.
– Report No parasite or hemoparasite found.
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 Diagnostic quality Control: Depends upon

• Compliance with standards

• Availability of supplies/ equipment/ infrastructure
• Condition of microscope
• Training of laboratory personnel
• Regular supervision
• quality of reagents & stains
• cleanliness
• Work load
• Technical ability & type of techniques used

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 Summary
• Microscopic examination of thick and thin stained blood films with
Giemsa stain requires trained lab personnel.

• Diagnostic features mainly Chromatin, Cytoplasm, Malaria pigment, and

Stippling’s should be considered during detection and identification of
malaria parasite species

• There are different artifacts that may be mistaken for malaria parasites

• There are three recommended methods of malaria parasite

quantification methods.

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 Activity 5.5. Lab Demonstration

Instruction: Form 3 groups each group containing

a maximum of 10 participants and assign one
participant on one microscope at time to complete
the following activity.
Activity
1. Examine blood film slides; detection, species
Identification and parasite estimation
Total Time: 2.45hrs

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 Thank You!!

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