The document discusses the preparation and staining of blood films, which is an important part of hematology. It describes how Romanowsky stains like Wright's, Leishman, Giemsa, and Jenner's stains are commonly used to differentially stain components of blood cells. Specifically, it provides details on the preparation and use of Leishman and Giemsa stains, which are frequently used stains that differentially color cells to distinguish types for examination under a microscope. Maintaining the proper pH and procedures are important for optimal staining and analysis of blood films.

2. THE PREPARATION AND STAINING OF
BLOOD
• FILMS Examining a properly prepared and stained
blood film constitutes the most important
investigation in Haematology. It is performed for:
Differential Leucocyte Counts (DLC) General
assessment and verification of various cell counts
Study of RBC morphology for classifying various
anaemias Study of WBC morphology for
diagnosing leukaemias and other WBC disorders
Study of platelet morphology for diagnosing
some platelet disorders

3. • Study of parasites found in plasma or RBCs
(haemoparasites) Study of other defects like
Rouleaux formations, agglutination,
fragmentation RBC inclusions,
• WBC inclusions,
• platelet clumps etc

4. THE STAINING OF BLOOD FILMS
The stains most commonly used for the staining of blood films are Romanowsky
Stains.
These stains are composed of Azure B and Eosin Y.
Azure B combines with anionic components of the cell, e.g. DNA and stain these blue,
whereas Eosin Y combines with the cationic components, various proteins and stains
them red.
Then there occurs a stain-stain interaction.
This composition and mode of action allows Romanowsky Stains to reveal the subtle
differences in shades of the staining and allows for a differential staining of granules.
The pH of the staining mixture is extremely important for the differential staining. An
alkaline pH accentuates the basic dye staining.
Therefore; an optimum pH is to be sought. A pH of 6.8 is recommended for the
optimal staining of all components.

5. Romanowsky Stains
The four most commonly used are Stain
 Wright's Stain
 Leishman Stain
 Giemsa Stain
Jenner's Stain

6. STAINING OF BLOOD FILM
• After preparation, the slide is stained to
distinguish the cells from each other.
Cytohaematology is the study of the cellular
components of blood cells. Parasites may also
be observed during microscopic examination
of the blood smear.

7. • Leishman Stain and May-Grunwald-Giemsa
Stain are the most-frequently used. The
preparation & method of using these stains is
described below:

8. LEISHMAN’S STAIN
• It is named after Sir William Boog Leishman from
Scotland.
• Stain Preparation
Dissolve 0.15 g of leishman’s stain powder in 100
ml of absolute methanol.
The stain is then filtered into stock bottle.
Place at 500C for 15 minutes in water bath.
Again filter into clean brown borosilicate glass
bottle and store in dark at room temperature.

9. LEISHMAN’S STAIN
• Staining Procedure
• Prepare the blood film and air-dry it.
• Keep it on a staining rack and completely cover it with the
stain.
• Leave it to stain for 2 minutes.
• Pour the buffered water onto the slide about twice the
amount of the stain.
• Mix by blowing gently through a pipette. Leave for 5-7
minutes. Pour off the stain mixture.
• Wash in the buffer, cleaning the underside of the slide
with a cotton swab or tissue paper.
• Place vertically to drain and dry.

10. good practice
• A good practice is to initially make 2-3 bottles
at one time. When one bottle is finished, it
should be replaced with freshly prepared stain
and left to mature. In the meantime, another
bottle of stain is used. The required volume of
stain for daily use should be filtered into a
smaller dropping bottle every morning

11. GIEMSA’S STAIN
• It is named after Dr. Gustav Giemsa from
Germany.
Stain Preparation
Transfer 0.3 g of Giemsa stain powder to a
motor and mix with 25 ml of glycerin
thoroughly. This makes the stock solution.

12. GIEMSA’S STAIN
• Staining Procedure
1) Prepare blood film, air dry and place in a
staining rack.
2) Fix with methanol for 3 minutes.
3) Dilute the stain 1 in 10 with distilled water or
buffer solution of pH 6.8.
4) Cover the blood film with diluted Giemsa
stain for 10 minutes.
5) Wash with distilled water, dry and examine
under microscope.