Giemsa stain: Introduction, preparation, principle, procedure and result Interpretation

Giemsa stain

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 smear. This technique uses for the demonstration of other than malarial parasites,  microorganisms like Helicobacter pylori, 

Chlamydia trachomatis, Borrelia species, Histoplasma capsulatum, Pneumocystis jiroveciPenicillium marneffei and occasionally bacterial capsules and parasites like Toxoplasma gondii, Leishmiania 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 stain the chromosomes and identify chromosomal aberrations.

a) Preparation of Giemsa 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 powder 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 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.

Procedure of Giemsa stain

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 D.P.X.

For Paraffin Section
De-paraffinize 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 D.P.X

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

Chlymadia trachomatis inclusion bodies : Blue-mauve to dark purple depending on the stage of development

References

  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 Text Book 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
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