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Introduction of Giemsa Stain

Giemsa stain comes under a type of Romanowsky stain. The name of this stain has come from the surname of a German chemist Gustav Giemsa, who created a dye solution. 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,  microorganisms like Helicobacter pylori, 

Chlamydia trachomatis, Borrelia species, Histoplasma capsulatum, Pneumocystis jiroveci,  Penicillium marneffei and occasionally bacterial capsules and parasites like Toxoplasma gondii, Leishmania donovani , Giardia lamblia, etc. It is also applied 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.

a) Preparation of Stain

• Giemsa stock powder: 1 gm
• Glycerin: 54 ml
• Methanol: 84 ml

Giemsa powder is mixed in 54 ml of glycerin and pre-heated up to 60°C.

Then add methanol, shake the mixture and allow to stand for 7 days.

Filter before use.

b) Buffer Solution (Stock)

• Potassium dihydrogen phosphate: 2.72 gm
• Distilled water:  100 ml
• Sodium hydroxide: 0.8 gm
• Distilled water:  100ml

Dissolve both powders in distilled water.

50 ml of potassium dihydrogen phosphate is mixed with 23.6 ml of sodium hydroxide.

The pH of the solution is adjusted to 6.8.

Working Giemsa Stain Solutions

• Giemsa stock: 10 ml
• Working buffer: 90 ml

Should be prepared fresh then use.

Buffer solution

• Stock buffer:  20 ml
• Distilled water: 480 ml

Principle of Giemsa Stain

Giemsa contains Methylene blue(AzureII)/Eosin. Methylene blue on oxidation produces colored compounds termed ‘Azure’ that have the ability to combine with Eosin. Methylene blue azure is blue-violet and stains acidic cell components while eosin is red and stains basic cell components.

Requirements for Giemsa Stain 

a) Compound light microscope

b) Reagents and glasswares

• Bunsen flame
• Wire loop
• Clean grease-free slides
• Marker pen
• Working Giemsa stain

Procedure for CSF Giemsa Stain

Smear preparation

1. Centrifuge the CSF and make smear-taking sediment using the inoculating loop on a clean dry microscopic glass slide.
2. Leave it to air dry.
   Dip the smear (2-3 dips) into pure methanol for fixation of the smear.
3. Leave to air dry for 30seconds

Staining

• Flood the smear with Giemsa stain solution for 20-30 minutes.
• Flush with tap water.
• Leave to dry.
• Focus the stained smear at 10X objective and finally observe at 100X objective using cedarwood oil i.e. oil immersion fields.

Result Interpretation of Giemsa Stain 

• Nuclei: Blue
• Cytoplasm: Pink
• H. pylori and  L.D bodies:  Blue
• Mast cell: Magenta pink
• Tissue elements: Shades of blue to pink
• Collagen, Muscle, and  Bone: Pale pink
• Erythrocytes: Salmon pink
• Malaria parasite: Malaria parasites have a red or pink nucleus and blue cytoplasm
• Borrelia spirochetes: Mauve-purple
• Chlamydia trachomatis inclusion bodies: Blue-mauve to dark purple depending on the stage of development
• Note: Capsules of Cryptococcus neoformans in Giemsa stained CSF is showing a clear zone around the yeast cells of this organism as shown above picture.

Bibliography

1. Bancroft’s Theory and Practice of Histological Techniques (6th Edition)
2. Bailey and Scott’s  Diagnostic Microbiology -13th Edn.
3.  Mackie & Mc Cartney  Practical Medical Microbiology- 14th  Edn.
4. Diagnostic Microbiology -Connie R. Mahon & George Manuselis
5. Koneman Color Atlas and Textbook of Diagnostic Microbiology-6th  Edn.
6. Medical Microbiology-The Practice of Medical Microbiology Vol-2-12th Edn. –Robert Cruickshank
7. District Laboratory Practice in  Tropical Countries  –  Part-2-   Monica Cheesebrough-   2nd Edn Update