malaria

1. National Malaria Control Programme-Ghana
National Malaria Control Programme-Ghana
Preparation and Staining
of Malaria Blood films
On-site Malaria Diagnostic
Refresher Training, 2021.

2. Learning objectives
2
The learning objectives seek to provide understanding of
the following:
• Preparation of thick blood films
• Preparation of thin blood film
• fixing thin films
• Staining of thick and thin blood films
• Prevention of artefacts

3. Reagents And Equipment For Preparation Of
Thick And Thin Blood Films
• Glass slides: new, clean, grease-free, with frosted end
• Slide template
• Spreader slide: beveled corners, ground edges
• Sharp containers
• Absorbent cotton wool
• Slide folders and slide box with cover
• Lint-free clean cotton cloth
• Lead pencil
• Capillary/venous blood
• Gloves and laboratory coats
• Adjustable micropipette (0-10µl) and tips
3

4. Set up for Blood film Preparation
• Well-arranged working area with all required apparatus shown
4

5. Always use pre-cleaned frosted end new slides
Label slides per participant/client on frosted
end of slide.
Use a slide template
• Collect enough volume of blood directly
from the finger stick by use of a micro
pipette onto a labeled glass slide.
Frosted Slides

6. • Labeled slides
• 6µl blood (thick) 12mm
• 2µl blood (thin)
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7. Smear Preparation, Fixing and Drying of
Blood Films
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8. Smear Preparation - Thin film
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(Angle controls the spread; 30-45 degree angle preferred)

9. •Note: Never use one spreader/slide for two smear
preparation (cross contamination)
Thick film preparation
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10. Drying films
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• Flat surface
• Protection from dust & flies (Slide folders)
• Rapid drying: (slide warmers/hot air blowers/regulated
hot air oven at 37oC
• Overnight stay (folders)

11. Thick smear of proper density Can see through
the text
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12. Test for Thick Film correctness (1)
16µl 12µl 8µl 6µl 4µl 2µl
12
Ability to read print at various volumes of blood
after spread when wet shows 6 - 8 microliters is
the best for 10-12mm thick film
But for standardisation, use 6ul for 12 mm thick film

13. Test for Thick Film correctness (2)
13
Ability to read print at various volumes of blood
after spread when dried shows 6 - 8 microliters is
the best for 10-12mm thick film
But for standardisation, use 6ul for 12 mm thick film
16µl 12µl 8µl 6µl 4µl 2µl

14. Common issues
14
Badly prepared films can
cause presence of streaks - as
a result of chipped spreader
Holes in the film indicate
faulty preparation and dirty or
greasy slides, respectively.

15. The two films on the left, not dried flat;
On the far right, too small and too thick
Identify the problems:
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16. • TWO METHODS - dipping
- spraying
Time – two seconds to avoid
fumes fixing thick film
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Fixing of Thin Blood Films
• Thin film should be fixed with
absolute methanol.
• Thick film should never ever be
fixed.
Dipping
Spraying

17. Air dry films OR dry the
films with low heat from a
hair-dryer for 5s, at a
distance of 30 cm.
17
Drying of Blood Films
- Avoid air drying of slides on a
slide rack with the fixed thin
film facing down.
- It should lie on a flat surface
or horizontally

18. Staining of Blood Films
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19. Giemsa stain
• 10% (rapid staining)
• 3% (research slides)
depending on purpose
• Slides should be stained within
72 hours.
Working Giemsa Solution
19

20. • Arrange the slides on a
staining rack with the sample
side facing up
• pipette flood the films
separately with the prepared
stain (about 3mls per slide)
and time appropriately for
staining period.
20
Staining of Blood Films

21. Flood slides with buffered water to float off iridescent scum
and rinse slides one after the other
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Rinsing of Stained Blood Films
Gently rinse the
slides in order to
keep the thick
films in place;
individually wash
to prevent cross
contamination

22. Stained films with different volumes of blood
16µl 12µl 8µl 6µl 4µl 2µl
GIEMSA
STAINED
10%
GIEMSA
STAINED
10%
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Test for Film staining correctness

23. One on the left and right, not dried flat;
One in the center, too small and too thick
Identify the problems (1)
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24. Thick film has been
fixed!
Identify the problems (2)
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Top: Lifted up slide
during spreading
Bottom: too much
blood

25. Varying pH buffer solution with visual
inspection
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26. Drying of stained Slides
• Air-dry in a horizontal position(film side down).
• A dryer (warmer, regulated hot air oven) may be
used if rapid drying is required.
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You can dry in
a slide box
and
Arrange
slides in a
slide box

27. Drying of stained Slides
27
Dry slides
laterally with
side of both
films down
AND NOT AS
SHOWN
BELOW

28. Staining Problems
• Lack of concentration - lead to less stain added
• Stain pours off from slides before staining time elapses
• washing single stained slides takes time
• Improper washing- artefacts/poor background
NB: always remain organized and behave professionally to
avoid any of the above problems
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29. Quality Standards For Grading Blood Film
Preparation
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30. Criteria For Assessing And Scoring The Quality Of
Blood Smears
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31. Practical Session
1. Make 9 films each (thick & thin)
2. Use fixed volumes (6µl,2µl) & use slide template
3. Stain 5 of your best smears using 10% Giemsa working
solution for 10min
4. Pick all slides for assessment
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32. • Good film preparation and quality staining
is key for quality malaria microscopy
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33. REFERENCES
• IQLS Norvatis – CKAE609A2202 - Malaria
Laboratory Manual Version 5.4. September
2017.
• Malaria Microscopy Standard Operation
Procedure - MM - SOP - 07A
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34. National Malaria Control Programme-Ghana
THANK YOU
On-site Malaria Diagnostic
Refresher Training, 2021.