Giemsa Stain: Principle, Procedure and Observation

Giemsa stain is a type of Romanowsky stain, named after Gustav Giemsa, a German chemist
who created a dye solution. 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 smear.

Uses of Giemsa Stain

Apart from staining malarial parasites, Giemsa stain has a variety of applications in
Microbiology and Pathology:

 Giemsa stain is used to obtain differential white blood cell counts.

 It is also used to differentiate nuclear and cytoplasmic morphology of the various blood
cells like platelets, RBCs, WBCs.
 In Microbiology, Giemsa stain is used for staining inclusion bodies in Chlamydia
trachomatis, Borrelia species, and if Wayson’s stain is not available, to stain Yersinia
pestis. Giemsa stain also is used to stain Histoplasma capsulatum, Pneumocystis jiroveci,
Klebsiella granulomatis, Penicillium marneffei and occasionally bacterial capsules.
 This stain is also used in cytogenetics to stain the chromosomes and identify
chromosomal aberrations. It is commonly used for G-banding (Giemsa-Banding)

Principle of Giemsa Stain

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.

Azure and eosin are acidic dye which variably stains the basic components of the cells like the
cytoplasm, granules etc.

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 the cellular stain. The fixative does not allow any further
change in the cells and makes them adhere to the glass slide.

Composition of Giemsa Stain

Giemsa stain can be prepared in house using Giemsa stain powder or can be commercially
obtained. The basic ingredients of both are the same; however, dilutions can be made depending
on use.
Ingredients Gm/L

Giemsa powder 7.6

Glycerol 500 ml

Methanol 500 ml

PROCEDURE

A. For In-house preparation of stain:

1. Weigh the required amount of powder stain, and transfer to a clean, dry 1litre capacity
bottle. Add methanol and mix well.
2. Measure and add glycerol and mix well.
3. Place the bottle of stain in water bath at 50-60°C or at 37°C for up to 2hours with
frequent mixing.
4. Label the bottle and store in a cool, dark place with a firm stopper.

Note: If water gets in contact during any steps of preparation of stain, the stain gets spoilt,
therefore use, dry glassware and store in conditions where there would be no water contact.

 Filter the stain using Whatman filter paper no.1 and dilute with water buffered to pH 7.2
to make working solutions

B. For staining slides

The method for staining, concentration and timing of stain used varies according to the purpose,
for example, thin blood smears use 1:20 dilution of stock whereas for thick blood smear 1:50
dilution is used.

For Thin blood smear

1. Fix air-dried film in absolute methanol by dipping the film briefly (two dips) in a Coplin
jar containing absolute methanol.
2. Remove and let air dry.
3. Stain with diluted Giemsa stain (1:20, vol/vol) for 20 min (For a 1:20 dilution, add 2 ml
of stock Giemsa to 40 ml of buffered water in a Coplin jar).
4. Wash by briefly dipping the slide in and out of a Coplin jar of buffered water (one or two
dips).
Note: Excessive washing will decolorize the film.
5. Let air dry in a vertical position. Observe under the microscope first at 40X and then
using oil immersion lens
For Thick blood smears

1. 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.
Note: If a rapid diagnosis of malaria is needed, thick films can be made slightly thinner
than usual, allowed to dry for 1 hour, and then stained.
2. DO NOT FIX.
3. Stain with diluted Giemsa stain (1:50, vol/vol) for 50 min (For a 1:50 dilution, add 1 ml
of stock Giemsa to 50 ml of buffered water in a Coplin jar)
4. Wash by placing the film in buffered water for 3 to 5 min.
5. Let air dry in a vertical position and observe under the microscope first at 40X and then
using oil immersion lens

For Chlamydia trachomatis

Follow the aforementioned steps but with the dilute stain of 1:40 dilution (add 0.5 ml stock
Giemsa solution to 19.5 ml buffered water) and leave the stain for 90-120 minutes.

OBSERVATION:

On microscopic observation, cell organelles, bacteria and, parasites are distinguished based on
their morphology and colour;

Cell Componenets Color observed after staining

Red blood c ells Mauve-pink
Neutrophils Reddish purple nuclei with pink cytoplasm
Eosinophils Purple nuclei, faintly pink cytoplasm and red to orange 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
Pale blue
cells
Cytoplasm
Pale blue or grey-blue
of white cells
Melanin granules Black green
Bacteria Pale or dark blue
Chlymadia
Blue-mauve to dark purple depending on the stage of development
trachomatis
inclusion bodies
Borrelia spirochetes Mauve-purple
Yersinina pestis
Blue with dark stained ends (bipolar staining)
coccobacilli
Malaria parasites have a red or pink nucleus and blue cytoplasm. If P. vivax
is seen, the Schüffner dots are seen as an even carpet of pink dots in the
Malaria parasite cytoplasm of red blood cells. If P. falciparum is observed, Maurer clefts
will be seen as unevenly distributed, coarse bodies in the red cell
cytoplasm.

A
B

Giemsa Stain of blood smear showing A-Erythrocyte, B-Platelet, C-Large lymphocyte and
D-Three lobed neutrophil
Plasmodium vivax in Giemsa-stained thin blood smear with all developmental
stages present in peripheral blood. (A) Growing amoeboid trophozoite in
enlarged red blood cell (RBC) with eosinophilic stippling. (B) Immature
schizonts with clumps of brown pigment almost fill the enlarged RBCs. (C)
Mature schizont with merozoites (about 14) and clumped pigment. (D)
Macrogametocyte with diffuse brown pigment and eccentric compact chromatin.