Leishmania donovani (LD bodies) amastigotes in Giemsa stained smear of bone marrow of a Visceral leishmaniasis (VL) or kala-azar patient
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Giemsa stain is a type Romanowsky stain that stains nuclei and cells. It was initially designed for the detection of malarial parasites in blood smears, but it is also used in histology for routine examination of blood smears. This technique uses for the demonstration of other than malarial parasites, microbes like Helicobacter pylori, Chlamydia trachomatis, Borrelia species, Histoplasma capsulatum, Pneumocystis jiroveci, Penicillium marneffei and occasionally bacterial capsules and parasites like Toxoplasma gondii, Leishmiania donovani , Giardia lamblia, etc. It is also used to differentiate nuclear and cytoplasmic morphology of the various blood cells like RBCs, WBCs, and platelets. In cytogenetics, it stains the chromosomes and identifies chromosomal aberrations.
Giemsa contains Methylene blue(AzureII)/Eosin. Methylene blue on oxidation produces colored compounds termed ‘Azure’ that have ability to combine with Eosin. Methylene blue azure is blue violet and stain acidic cell components while eosin is red and stains basic cell components.
a. Preparation of Giemsa stain
| Ingredients | Volume/Amount |
| Giemsa stock powder | 0.5 gm |
| Glycerin | 27 ml |
| Methanol | 42 ml |
Giemsa powder is mixed in 27 ml of glycerin and pre-heated up to 600C. Then add methanol, shake the mixture and allow to stand for 7 days. Filter before use.
b) Buffer solution (stock)
| Ingredients | Amount/volume |
| Potassium dihydrogen phosphate | 1.36 gm |
| Distilled water | 50 ml |
| Sodium hydroxide | 0.4 gm |
| Distilled water | 50 ml |
Giemsa Stain: –
| Ingredients | Amount/Volume |
| Giemsa stock | 10 ml |
| Working buffer | 90 ml |
Buffer solution
| Ingredients | Amount/Volume |
| Stock buffer | 20 ml |
| Distilled water | 480 ml |
For Paraffin Section
For bone marrow imprints and smears
| Nucleus | Blue |
| Cytoplasm | Pink |
| H. pylori | Blue |
| LD bodies | Blue |
| Mast cell | Magenta pink |
| Tissue elements | Shades of blue to pink |
| Collagen, Muscles and Bone | Pale pink |
| Erythrocytes | Salmon pink |
| Malaria parasite | Malaria parasites have a red or pink nucleus and blue cytoplasm |
| Borrelia spirochetes | Mauve-purple |
| Chlymadia trachomatis inclusion bodies | Blue-mauve to dark purple depending on the stage of development |
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