1. Preparation and staining technique of Leishman stain.

Principle: Leishman stain is a mixture of methylene blue, and eosin dye, prepared in alcohol
medium and diluted with buffer or distilled water during staining procedure. Leishman stain
is a differential stain that is used to variably stain the various components of the cells and it
can be used to study the adherence of pathogenic bacteria to the human cells. It differentially
stains the human and bacterial cells and appeared as purple and pink colored bodies,
respectively. The Leishman stain is one of the best stains for routine blood stain to stain the
peripheral blood smear for the examinations of blood film under the microscope and is
satisfactory for malaria and other blood parasites.

The thin blood smear is prepared for studying the morphology of the blood cells and for the
identification of microbial agents. The thick blood smears are prepared for detecting the
blood parasites such as Plasmodium spp.

The thin peripheral blood smear is made by placing a well-mixed drop of blood 1 to 2 mm in
diameter & 1/4 inch from the edge of the clean microscopic glass slide. The drop should be in
the center line of the glass slide. These margins are left onto the glass slide to get a region
where the cells are spaced far enough apart to be counted and differentiated. Using a second
slide as the spreader, the blood is streaked into a thin film in the tongue-like shape and
allowed to dry. A thick blood film smear requires a large volume of blood as compared to thin
blood films which enable the more efficient detection of parasites in the blood specimen. A
thick blood smear is made by spreading a large blood drop in a small area of about 1 cm.

Material required: Leishman stain, microscopic glass slide, phosphate buffer (pH 6.8),
measuring cylinder, distilled water, Pasteur pipette, blood specimen

Preparation of Leishman stain:

 Weigh the 0.15 g of Leishman stain powder on an analytical balance or weighing

scale, grind it in a Glass Mortar and put it into a bottle through a funnel.
 Gently pour in about 20 ml of acetone-free methanol through the same funnel,
ensuring that all the dry stain is washed into the bottle.
 Re-cap the bottle and shake it in a circular motion for 2–3 minutes to start dissolving
the stain crystals in Methanol solution. Warm the content at 50 °C for 15 minutes with
occasional shaking.
  Add the remaining amount of methanol to the mixture through the funnel and make
the volume 100 ml, ensuring that even the last drop of the methanol washes in the
funnel into the stain mixture.
 Tighten the screw-cap on the bottle and continue shaking for few minutes.
 Do not open the bottle and do not filter the content for about a week. After a week it is
ready to use. Filter the content before use.

Procedure:

 Prepare a thin blood smear on a clean and dry microscopic glass slide and air dry it.
 Now, cover the well dried, thin blood smear with undiluted Leishman stain solution.
 Let it stand for 2 minutes, the methanol present in the stain fixes the smear onto the
glass slide.
 After 2 minutes, add twice the amount of distilled water or phosphate buffer solution
and mix the content by swirling or by blowing gently. Incubate the slides for at least
10 min at 37 °C. This will stain the blood cells.
 Rinse the slides thoroughly with phosphate buffer solution up to 2 minutes or until it
acquires a purple-pinkish tinge.
 Air dry the slides in a tilted position so that the water easily remove out of the slides.
 Let it dry in air for few hours and then observe the slides under oil immersion
objective lens of the microscope.

Result: The various blood cells will be observed under the microscope.

2. Preparation and staining technique of Geimsa stain.

Giemsa’s stain is a type of Romanowsky stain named after Gustav Giemsa. It was primarily
designed for the demonstration of malarial parasites in blood smears, but it is also employed
in histology for routine examination of blood smears. Giemsa stain is a differential stain and
contains a mixture of azure, methylene blue, and eosin dye. It is specific for the phosphate
groups of DNA and attaches itself to where there are high amounts of adenine-thymine
bonding. Methylene blue acts as the basic dye, which stains the acidic components, especially
the nucleus of the cell. Methanol act as a fixative as well as a cellular stain. The fixative does
not allow a further change in the cells and makes them adhere to the glass slide.

Composition of Giemsa stain: Giemsa powder - 7.6 g/L, Glycerol - 500 ml and Methanol -
500 ml

Depending upon the method of staining used to stain malaria blood films, the Giemsa
working solution is either 10% (for the rapid method) or 3% (for the slow method).

For thin blood smear:

 Fix air-dried film in absolute methanol by dipping the film briefly in a Coplin jar
containing absolute methanol.
 Remove and let air dry.
 Stain with a working solution of Giemsa stain
 Wash by briefly dipping the slide in and out of a Coplin jar of buffered water.
 Let air dry in a vertical position. Observe under the microscope first at 40X and then
using an oil immersion lens.

For thick blood smear:

 Allow the film to air dry thoroughly for several hours or overnight. Do not dry films
in an incubator or by heat, because this will fix the blood and interfere with the lysing
of the RBCs.
 DO NOT FIX.
 Stain with diluted Giemsa stain
 Wash by placing the film in buffered water for 3 to 5 min.
 Let air dry in a vertical position, observe under the microscope at 40X, and then use
an oil immersion lens.

On microscopic observation, cell organelles, bacteria, and parasites are distinguished based
on their morphology and color:

Cell Components The color observed after staining

 Red blood cells Mauve-pink

Neutrophils Reddish purple nuclei with pink cytoplasm

Purple nuclei, faintly pink cytoplasm, and red to orange

Eosinophils
granules.

Basophils Purple nuclei, blue coarse granules.

Lymphocytes Dark blue nucleus with light blue cytoplasm.

Monocytes Pink cytoplasm with a purple color nucleus.

Platelets Violet to purple color granules.

Nuclei of host cells Dark purple

Nuclei of WBCs Dark purple

Cytoplasm of host cells Pale blue

Cytoplasm of white cells Pale blue or grey-blue

Malaria parasites have a red or pink nucleus and blue

Malaria parasite
cytoplasm.