Giemsa Stain

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.

Principle:

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.

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)

Composition:

Ingredients	Gm/L
Giemsa powder	7.6
Glycerol	500 ml
Methanol	500 ml

Procedure:

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.

Results

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 cells	Pale blue
Cytoplasm of  white cells	Pale blue or grey-blue
Melanin granules	Black green
Bacteria	Pale or dark blue
Chlymadia trachomatis inclusion bodies	Blue-mauve to dark purple depending on the stage of development
Borrelia spirochetes	Mauve-purple
Yersinina pestis coccobacilli	Blue with dark stained ends (bipolar staining)
Malaria parasite	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 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.

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Possible References Used

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