Giemsa staining is essential staining technique widely used in microbiology, hematology, parasitology, and cytogenetics. It is particularly renowned for its role in the diagnosis of malaria and other blood parasites, as well as for detailed examination of blood cells and bone marrow biopsy. Despite major advancements in molecular diagnostics, Giemsa stain remains a standard in routine laboratory practice, especially in developing countries.
HISTORY OF GIEMSA STAINING
A German bacteriologist Gustav Giemsa developed Giemsa staining in 1904 by modifying Romanowsky staining technique, which was known for inconsistent staining results.
Giemsa improved the stain by stabilizing the dye mixture using glycerol and methanol, resulting in enhanced reproducibility.
PRINCIPLE OF GIEMSA STAINING
The principle of Giemsa staining is based on the Romanowsky effect, which occurs due to the interaction between acidic and basic dyes with cellular components.
Dyes Used in Giemsa Stain
- Azure B (basic dye)
- Methylene blue (basic dye)
- Eosin Y (acidic dye)
Staining mechanism
- Acidic components like Nucleic acids (DNA & RNA) stain blue to purple
- Basic components like Cytoplasmic components & hemoglobin stain pink to red
- Parasites show distinct chromatin dots and cytoplasm
- The stain works optimally at pH 6.8, which is critical for proper color differentiation.
Samples for Giemsa staining
Giemsa stain can be used for:
- Peripheral blood smears
- Thick and thin blood films
- Bone marrow aspirates
- Tissue imprints
- Lymph node aspirates
- Cell cultures
- Chromosomal preparations (G-banding)
GIEMSA STAINING PROCEDURE
REAGENTS AND MATERIALS
- Giemsa stain (stock solution): It contains
- Azure B
- Methylene blue
- Eosin Y
- Glycerol (stabilizer)
- Methanol (solvent and fixative)
- Methanol not only dissolves the dyes but also fixes the cells, preserving morphology.
- Buffered distilled water (pH 6.8)
- Absolute methanol
- Clean glass slides
- Staining rack or Coplin jar
- Microscope with oil immersion lens
STEPS OF GIEMSA STAINING METHOD
1. Preparation of Smear
Prepare a thin or thick smear and allow it to air-dry completely
2. Fixation
Thin smear: Fix with absolute methanol for 1–3 minutes
Thick smear: Do not fix (RBCs must lyse)
3. Preparation of Working Stain
Dilute Giemsa stain with buffer
Common dilutions: 1:20 or 1:50
4. Staining
Flood the slide with diluted stain and leave it for 20–30 minutes
5. Washing
Gently rinse with buffered water
6. Drying
Air-dry in vertical position
7. Examination
Examine under oil immersion (100×)
INTERPRETATIONS OF RESULTS
1: BLOOD CELLS
- RBCs appear Pale pink
- WBC nuclei appear Purple
- Lymphocytes cytoplasm appear blue.
- Neutrophils appear as Lilac granules
- Eosinophils appear as Bright red granules
- Basophils appear as Dark blue granules
- Platelets appear Pale blue
2: PARASITES
- Plasmodium spp.: Blue cytoplasm, red chromatin dots
- Leishmania donovani : Blue cytoplasm with red nucleus and kinetoplast
- Trypanosoma: Blue body with red nucleus
CLINICAL APPLICATIONS OF GIEMSA STAINING
1. Malaria Diagnosis
Giemsa stain is the gold standard for malaria diagnosis, it allows :
- Species identification
- Parasite staging
- Parasite density estimation
2. Detection of Blood Parasites
- Leishmaniasis
- Trypanosomiasis
- Babesiosis
3. Hematological Disorders
- Anemia evaluation
- Leukemia diagnosis
- Myelodysplastic syndromes
4. Bone Marrow Examination
- Detection of abnormal cells
- Parasite identification
5. Cytogenetics
- G-banding of chromosomes
- Detection of chromosomal abnormalities
ADVANTAGES OF GIEMSA STAINING
- It gives excellent nuclear detail
- It is highly sensitivity for parasites
- Cost-effective
- Long-lasting slides
- Widely accepted diagnostic method
- Suitable for routine laboratory use
LIMITATIONS OF GIEMSA STAINING
- Precise pH control
- Time taking
- Interpretation needs expertise
- Not ideal for routine bacterial identification
- Overstaining
- precipitate formation
QUALITY CONTROL
- Use fresh buffer
- Store stain in dark bottles
- Filter stain regularly
- Use known positive slides
- Strict pH monitoring
FAQs
1: Why is Giemsa stain preferred for malaria?
Because it provides excellent visualization of Plasmodium species and developmental stages.
2: What is the optimal pH for Giemsa staining?
pH 6.8 is ideal for proper staining.
3: Is Giemsa stain used in cytogenetics?
Yes, it is used for G-banding of chromosomes.
4: Can Giemsa stain bacteria?
It is mainly used for parasites and blood cells, not routine bacteria.