Wright Giemsa Stain – Protocol, Principle, Result.

Wright Giemsa Stain

• Wright Giemsa Stain is a type of Romanowsky stain.
• This stain was discovered by German chemist Gustav Giemsa, that’s why its called Wright Giemsa Stain.
• It is mainly used for the demonstration of malarial parasites in blood smears. On staining with Giemsa stain, the malaria parasite appears with a red or pink nucleus and blue cytoplasm.
• P. vivax shows Schüffner dots are seen as an even carpet of pink dots in the cytoplasm of red blood cells. Meanwhile, P. falciparum shows unevenly distributed Maurer clefts and coarse bodies in the red cell cytoplasm.
• Wright Giemsa Stain is a mixture of Azure B, Methylene blue, and Eosin dye.
• Azure B and eosin are acidic dyes while methylene blue is the basic dye.
• The basic components (cytoplasm and cell granules) of the cell are stained with the acidic dyes and become pale colors while the acidic components (nucleus) of the cell are stained with the basic dyes of the Wright Giemsa Stain and become dark purple or blue.

Wright Giemsa Stain Principle

Wright Giemsa Stain is a modified Romanowsky Stain technique, which is used to stain the smears of blood and marrow. The Staining of cells involves physical adsorption and chemical affinity which allows the stain to penetrate and remain within cells. Because each cell and its components are different in chemical composition, their affinity to acid stain (Eosin) and alkaline stain (Methylene blue) of this kit varies significantly. After staining the smear with Wright’s Stain, different types of cells will appear in different colors. Consequently, one can identify cells based on their stain color, shape and other physical characteristics.

Requirements

• Wright-Giemsa Solution
• Phosphate Buffer Solution (pH 6.8)
• Methanol, Absolute
• Water, Deionized/Distilled
• Coplin jars
• Forceps
• Staining tray
• Synthetic resin

*Prepare Working Wright-Giemsa Solution by mixing 25ml of Wright-Giemsa Solution with 25ml of Phosphate Buffer Solution, pH 6.8

Wright Giemsa Stain Protocol

1. Smear a small drop of blood on a clean microscope slide and allow for the air dry.
2. Fix by placing in absolute Methanol for 5 minutes.
3. Place slide in staining tray & flood with Working Wright-Giemsa Sol for 5 min. Note: Agitate slides occasionally to insure proper staining.
4. Rinse slide in deionized/distilled water.
5. Flood slide with Phosphate Buffer Solution, pH 6.8 until no stain runs off.
6. Allow the slide to remain in the Phosphate Buffer Solution, pH 6.8 for an additional 1 minute.
7. Dip slide in distilled water and air dry at room temperature.
8. Dip slide several times in Xylene or Xylene Substitute.
9. Mount in synthetic resin.

Wright Giemsa Stain result

The following cells are tested with Wright Giemsa Stain, and they shows the mentioned colors;

Precaution of Wright Giemsa Stain

• The duration of the staining procedure depends on the types of specimen, thickness of smear, number of nucleoli, cell type and room temperature etc. For example, during the staining of blood smear, it typically requires 3-5mins after adding Solution B; for bone marrow it requires more than 10mins.
• During the smear preparation of bone marrow, fibrins can clot quickly, that’s why the process of smearing must be fast.
• Avoid using oxalate as anticoagulant, because it can lead to the nucleolus to distort, chromatin to become compact, cytoplasm vacuole to form and oxalate crystal to appear.
• To avoid the sample drying, the staining procedure should be conducted with a sufficient volume.
• During hematocyte stain, if the weather is cold or humid, the incubation should be done at  37℃ to prevent cells from shrinking.
• To avoid the vaporization of reagent, immediately cap the bottle.
• Avoid using expired reagents.  avoid exposure to extreme high or low temperature and sunlight.

Application of Wright Giemsa Stain

• In histology, Wright Giemsa Stain is used for the routine examination of blood smear.
• In cytogenetics, it is used for staining of chromosomes and identifying chromosomal aberrations through G-banding (Giemsa-Banding).