Wright's Stain - Principle, Reagents, Procedure, Result, Application

Introduction to Wright’s stain

Commonly used in hematology, Wright’s stain is a type of Romanowsky stain used for the differentiation of blood cell types. It is mostly used to stain peripheral blood smears, bone marrow aspirates, and urine samples, and demonstrate malarial parasites in blood smears.

Wright’s stain is also used in cytogenetics to stain chromosomes for diagnosis of syndromes and diseases.

Stains similar to Wright’s stain are also known as the buffered Wright stain, the Wright-Giemsa stain, and the buffered Wright-Giemsa stain. These may include eosin Y, azure B, methylene blue,  May–Grünwald stain.

History of Wright’s Stain

Wright’s stain is named after James Homer Wright who developed the technique in 1902. This technique is based on a modified Romanowsky stain.

Since Wright’s stain easily distinguishes between blood cells, this procedure became popular for differential WBC counts and the study of the morphology of blood cells. This is routinely done in suspected cases of infection or leukemia.

Principle of Wright’s Stain

The polychromatic stain classically consists of a mixture of two dyes - eosin (red) and methylene blue. Eosin Y, an acidic anionic dye, and methylene blue, a basic cationic dye, ionize when diluted in buffered water.

Since Wright’s Stain is methanol-based, the fixation step is not required. Methanol, present in Wright’s Stain, fixes the cells to the slide while preserving the red cell morphology. Thus, the relationship between the parasite and host blood cells can be easily seen. However, fixation reduces water artifacts which may be present on humid days or with aged stains.

Eosin stains the basic components including hemoglobin and eosinophilic granules from orange to pink color while Methylene blue stains acidic cellular such as nucleic acid and basophilic granules to varying shades of blue. If the neutral components of the cells are present, they are stained by both components of the dye resulting in variable colors.

Reagents / Contents of Wright’s Stain

1. Staining Solution

   Wright’s stain powder = 1.0 gm

   Absolute methanol = 400 ml

2. Phosphate buffer (0.15M, ph 6.5/6.8)

   Potassium dihydrogen phosphate, anhydrous = 0.663 gm

   Disodium hydrogen phosphate, anhydrous = 0.256 gm

   Distilled water = 100 ml

Procedure for Wright’s staining

The procedure for Wright’s staining is as follows:

1. Prepare a thin film of the specimen on a microscopic slide and allow it to air dry

2. Place the air-dried smear on the staining rack and cover the film with undiluted staining solution (undiluted stain fixes as well as partially stains the smear)

3. Let it stand for 2-3 minutes

4. Add an equal amount of buffered water (pH 6.5) and mix by gently blowing

5. After metallic sheen (green scum) appear on the slide, leave it alone for 5 minutes

6. Gently flood the slide with distilled water until thinner parts of the film turn pinkish-red

7. Leave the slide to dry at room temperature and examine it under a light microscope

Result of Wright’s Stain

After Wright’s staining, Blood cells are differentiated based on color.

If malaria parasites are present in the blood smear sample, the RBC cytoplasm stains pale blue the nuclear material stains red, and the malarial parasite clear read chromatin. Wright’s Stain does not stain or poorly stain RBC inclusions as well as Schüffner’s dots.

Application of Wright’s Stain

1. Study and distinguish between blood cells

2. Performing differential white blood cell counts in suspected cases of infection or leukemia

3. Diagnosis of interstitial nephritis or urinary tract infection (UTI) when urine sample is stained

4. Demonstrate malarial parasites, trypanosomes, or intracellular leishmania in blood smears

5. Detection of intracellular fungi Histoplasma capsulatum and Cryptococcus neoformans