Thick and Thin Blood

Smear for Malaria

Diagnosis
Written by Acharya Tankeshwar ● in
Parasitology ● Last Updated January 1, 2024

The direct microscopic visualization of the malarial

parasite on the thick and/or thin blood smears
has been the “gold standard” for malaria
diagnosis.

Preparation of Thick and Thin Blood

Smear

Table of Contents

1. Thick Blood smear

1.1. Making Thick Blood Smear
1.2. Quality Control
1.3. Limitation of Thick Smear
2. Thin Blood Smear
2.1. Making Thin Blood Smear
2.2. Limitation of a thin smear
3. Giemsa Staining of Thick and Thin Blood
Smear
4. Microscopic examination
4.1. Examining the thick film
4.2. Examining Thin Films

Thick Blood smear

Thick blood film samples a relatively large volume
of blood, thus allowing more efficient detection of
parasites (increased sensitivity).

Thick smears consist of a thick layer of

dehemoglobinized (lysed) red blood cells
(RBCs), which provides a better opportunity to
detect parasitic forms against a more transparent
background. However, they do not permit an
optimal review of parasite morphology.

Making Thick Blood Smear

1. Using the corner of a clean slide, spread the
drop of blood in a circle the size of a dime
(diameter 1-2 cm).
Do not make the smear too thick or it will fall
off the slide. (you should be able to read
newsprint through it.)

2. Allow the smear to dry thoroughly. Insufficiently

dried smears (and/or smears that are too thick)
can detach from the slides during staining. You
can accelerate the drying by using a fan or
hairdryer.
Do not fix thick smears with methanol or heat.

3. If there will be a delay in staining smears, dip

the thick smear briefly in water to hemolyse the
RBCs.

Quality Control
Visually, the smear should appear as a round to
oval smear of blood about 2 cm in diameter. It
should be of such thickness that newsprint can
barely be seen through the wet or dry smear.

Limitation of Thick Smear

Making a species identification of malarial
parasites may be impossible, even for
experienced technicians.

A thin film should always be examined if a

definitive identification based on morphology is
required.

Smears must be prepared from anticoagulated

blood within one hour after venipuncture. The
morphology of parasitic forms and the
erythrocytes become atypical after that time
from the direct action of the anticoagulant.

Blood smears should be stained as soon as

possible after they are prepared. Storage of
unstained slides for a few days in the hot and
humid atmosphere without staining will result
in auto-fixation, and the thick film will be
useless for microscopy.

Thin Blood Smear

Thin
smears
consist
of
Prepared smear
blood
spread
in a layer such that the thickness decreases
progressively toward a monolayer. It
allows optimal assessment of the morphology of
any parasitic forms that may be present. Thin
blood film is prepared similarly to the differential
white cell count.

Making Thin Blood Smear

1. Bring a clean spreader slide, held at a 45° angle,
toward the drop of blood on the specimen slide.

2. Wait until the blood spreads along the entire

width of the spreader slide.

3. While holding the spreader slide at the same

angle, push it forward rapidly and smoothly.

4. Wait until the thin films are completely dry

before staining.

5. Fix the thin film with methanol (100% or

absolute) for 15-30 seconds and let it dry
completely before staining.
Note: fixation time for ethanol is 20 minutes

Limitation of a thin smear

Parasitic forms may be missed in light
infections. In such instances, a thick film must
be examined.

Smears must be prepared from anticoagulated

blood within 1 hour after venipuncture. The
morphology of parasitic forms and the RBC
become atypical after that time from the direct
action of the anticoagulant.

Giemsa Staining of Thick

and Thin Blood Smear

P. falciparum trophozoite stage in thick

(right) and thin (left) smear.

Giemsa stain is the most reliable method for

staining thick and thin blood films. Giemsa
solution is composed of eosin and methylene blue
(azure). The cytoplasm appears blue (stained by
methylene blue), and the nucleus appears red
(stained by eosin).

Counts the number of slides to be stained. Each

slide requires approximately 3 mL of stain. If you
have 16 slides to stain, you can prepare 50 mL
Giemsa working solution. Two commonly used
working solutions are;

1. 10% Giemsa stain working solution : It is

used in hospital/diagnostic laboratories for a
quick diagnosis. It is slightly costly, as more
stain is consumed.

2. 3% working solution: This cost-effective slow

method is mostly used to stain slides for
teaching or epidemiological purposes.

Check the preparation of Giemsa working

solution in this blog post.

···

1. Fix the thin film by carefully dropping

methanol onto the thin film using a Pasteur
pipette.
Alternatively, you can dip the thin film for 2
seconds into a small container or beaker
containing methanol. Avoid contact between
the thick film and methanol. Methanol vapors
quickly fix the thick film, thus interfering in its
hemolysis.

2. Let the blood film dry in the air or on a drying

rack or tray.
Allow methanol-fixed thin smear to dry by
placing the slides on a flat surface. It will
approximately take 2 minutes to completely air
dry the specimen. Never dry slide in a vertical
position with the thin film down, as this may
lead to fixing of the thick film by methanol
vapor.

3. Place the slides individually on the staining

rack with blood films facing up.
Ensure that these slides are not touching each
other.

···

4. Gently pour the stain onto the top of the

slides until the blood films are covered.
Each slide will require approximately 3 mL of
stain. Avoid pouring the stain directly onto thick
films.

5. Set the timer for the staining. Leave the stain

on the slides for 45-60 min (if you are using
3% Giemsa working solution) or 8-10 min
(while using 10% Giemsa solution).
Optimum staining duration should be
determined previously by testing the batch of
stock staining solution used (i.e. your internal
quality control will dictate the exact duration
requirement for good staining).

6. Gently flush all the stains from the slides by

dropping buffered water (pH 7.2) over them.
Buffered water should be poured onto the
slides from the thin film end to avoid undue
disturbance and to wash off the thick films. The
surface becomes covered with a metallic green
scum during the staining process. You should
avoid getting it onto blood films during rinsing,
as it can impair examination.

7. When the stain has been washed away,

remove each slide individually and allow air
drying.
Ensure that thick films are not scraped against
the edge of the rack.

···

8. Discard any remaining Giemsa working

solution.

Microscopic examination

Examining the thick film

Examining the thick blood films

1. Place the Giemsa-stained blood film to be

examined on the microscope stage, with the
label to the left. Position the thick film in line
with the 10X objective lens.

2. Switch on the microscope, adjust the light

source optimally and find the focus by looking
through the ocular and the 10x objective.

3. Scan the blood film for parasites and blood

elements. Select part of the film that is well
stained and has evenly distributed white blood
cells.

4. Place a small drop of immersion oil on the

thick film. To avoid cross-contamination,
ensure that the immersion oil applicator never
touches the slide. Do not allow the 40x
objective to touch the oil.

5. Switch the 100x oil immersion objective over

the selected portion of the thick film. Use the
fine focus adjustment to see the image. Raise
the mechanical stage to avoid damaging the
slide.

6. Using the fine adjustment, focus on the cell

elements, and confirm that the film is
acceptable for routine examination; 15-20
white blood cells per thick film field will give a
satisfactory film thickness. Films with fewer
white blood cells per field will require more
extensive examination.

7. Examine the slide systematically. Start at the

top left of the film (marked with a vertical green
arrow) and begin at the periphery of the field,
then move horizontally to the right, field by field.

8. When the other end of the film is reached,

move the slide slightly downwards, then to the
left, field by field, and so forth. For efficient
examination, continuously focus and refocus
with the fine adjustment throughout the
examination of each field.

9. Examine the thick film under the oil

immersion objective, field by field,
horizontally or vertically. Use the fine
adjustment to focus.

10. A minimum of 100 high-power fields must be

examined before a thick film can be declared
as having “no malaria parasites seen.” If
possible, the whole thick film should be
scanned.

11. If parasites are found, scan additional 100 fields

to increase the chance of identifying mixed
infections.

12. Identify all species and stages observed, and

record them.

Examining Thin Films

The thin blood film should always be examined to
identify parasite species or mixed infections after
examining the thick film. Unlike the thick film, the
thin film allows visualization of parasite and red
cell morphology. Perform an examination at the
feathery end or edge of the thin film.

Examining the thin blood films

1. Place a drop of immersion oil on the feathered

edge of the thin film.

2. Move from the 10x lens to the 100x oil

immersion lens.

3. Examine the feathery end of the edge of the

thin film where red cells lay side by side, and
there is minimal overlap. Follow the pattern of
movement shown in Fig. 2. Move along the edge
of the film, then move the slide outwards by one
field, inwards, returning in a lateral movement,
and so on.

4. Continue examining the thin film until the

presence and species of malaria parasites have
been confirmed. Identify and record all species
and stages observed in the malaria microscopy
blood register.

Further Resources:

Staining for Malarial Parasites; a guideline by

DPDx

World Health Organization; Global Malaria

Programme

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Acharya Tankeshwar
Hello, thank you for visiting my blog. I am
Tankeshwar Acharya. Blogging is my passion. As
an asst. professor, I am teaching microbiology and
immunology to medical and nursing students at
PAHS, Nepal. I have been working as a
microbiologist at Patan hospital for more than 10
years.

···

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