Giemsa stain: Introduction, Principle, Reagent Preparation, Staining Procedure, Result Interpretation, Uses, and Keynotes

Introduction of Giemsa Stain

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.

Principle of Giemsa Stain

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.

Requirements

• Giemsa powder
• Glycerine
• Potassium dihydrogen
• phosphate
• Sodium hydroxide
• Methanol
• Weighing scale
• Measuring cylinder
• Conical flasks
• Glassware pipette
• Coplin jar
• Reagents bottles
• Gloves
• Masks
• Lab coat
• Cotton

Giemsa Stain Preparation

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

• Dissolve both powder in distilled water.
• 50ml of potassium dihydrogen phosphate is mixed with 23.6ml of sodium hydroxide. The pH of the solution is adjusted to 6.8.

Working Solutions

Giemsa Stain: –

Ingredients	Amount/Volume
Giemsa stock	10 ml
Working buffer	90 ml

•  The working Giemsa stain should be prepared fresh then use.

Buffer solution

Ingredients	Amount/Volume
Stock buffer	20 ml
Distilled water	480 ml

Staining Procedure

For Paraffin Section

• Deparaffinize and hydrated sections to tap water. Flood slide with Giemsa stain for 15-20 minute.
• Wash in tap water.
• Differentiate 0.2% acetic acid 1 dip.
• Wash in running tap water.
• Dehydrate, clear and mount with DPX.

For bone marrow imprints and smears

• Smears are fixed in methanol for 30 minutes.
• Smears are stained in working Giemsa solution for 20 minutes.
• Wash under running tap water for 5 minutes.
• Air dry smears, clear in xylene and mount in DPX.

Result-interpretation of Giemsa Staining

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

Leishmania donovani (LD bodies) amastigotes in Giemsa stained smear of bone marrow of a Visceral leishmaniasis (VL) or kala-azar patient

Cyst of Pneumocystis jiroveciin in Giemsa stained smear of BAL showing sporozoites

Helicobacter pylori in Giemsa stained gastric biopsy

Cryptococcus neoformans Capsules (clear zone covering body) in Giemsa stained smear of CSF

Uses of Giemas stain

• This stain is used for the demonstration of some microbes like Helicobacter pylori (bacteria) and parasites like Toxoplasma gondii, Leishmiania donovani , Giardia lamblia.
• 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.

Keynotes

1. Giemsa stain is the surname of German chemist and bacteriologist,Gustav Giemsa.
2. Leishman stain differs from Jenner’s having eosin B instead of eosin Y.
3. Occasionally bacterial capsules can also be noticed.

Bibliography

1. Bailey and Scott’s  Diagnostic Microbiology -13th Edn.
2. Bancroft’s Theory and Practice of Histological Techniques (6^th Edition)
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