Procedure
for thick Blood Film Staining Technique
A thick film was made by placing a large
drop of blood (about 15mm in size) on the centre of grease-free microscopic
slide. Without delay, the blood was spread with a glass spreader held at a
steep angle to achieve a thick smear covering an area of 15 x 15mm. This was
allowed to air dry in a horizontal position. Then a Giemsa stain (3% solution
diluted with buffer for 30 minutes staining) was applied on the thick film and
allowed for 30 minutes. After this
time, the stain was washed off using
distilled water and again allowed to air-dry. The dried stained thick smear was
finally viewed under a light microscope using 100x oil immersion objective
lens. The chromatin of the parasites appeared as dark red dots while the
cytoplasm appeared as blue rings.
3.6
Procedure for thin Blood Film Staining Technique
A thin film was made by placing a small
drop of blood on the centre of a grease-free microscopic slide. The drop of
blood was then spread with a glass spreader held at an angle of 30o to
obtain a thin film with a smooth tail end. This was allowed to air
dry in a horizontal position and then fixed with absolute methanol for two
minutes. After this, a Giemsa stain diluted with buffer for 30 minutes staining
was applied on the thin film and allowed to air dry for 30 minutes. The stain
was then washed off using distilled water and also air dried. The stained thin
film was viewed under a light microscope using 100x oil immersion objective
lens. Identification of different Plasmodium species was based on referencing
to the distinctive characteristics of the various stained morphologic forms of
each species (Appendix IV-VII).