Wright Stain: Introduction, Principle, Preparation, Procedure, Result-Interpretation, Keynotes, Wright’s Stained Footages

Introduction of Wright’s stain

Wright’s stain is also called a hematologic stain and it is one of the Romanowsky stains, which is commonly applied in clinical hematology laboratory for the routine staining of the peripheral blood smear (PBS) as well as staining bone marrow aspirates, urine specimens and to demonstrate malarial parasites in blood smears. Wright’s stain is named from the surname of American pathologist, James Homer Wright, who devised the stain in 1902 based on a modification of the Romanowsky stain. The stain differentiates simply between the blood cells and thus it became widely used for performing differential white blood cell (WBC) counts and evaluating the morphology of blood cells and microbes if present.

Composition of Wright Stain Powder

Constituents	%
Azure A Eosinate	24
Azure B	35
Azure C	6
Methylene blue	35

Principle of Wright’s Stain

Wright’s stain is a polychromatic stain containing a mixture of eosin and methylene blue. When used in blood cells, the dyes produce numerous colors based on the ionic charge of the stain and the various components of the cell. The ions of eosin are negatively charged and stain basic cell components an orange to pink color. The methylene blue ions are positively charged and stain the acid components of a cell in varying shades of blue. The neutral components of the cell are stained by both components of the dye producing variable colors.

Preparation of wright’s stain

Constituents	Amount
Wright stain powder	0.25 gm
Acetone free methanol	100 ml

• Weigh 0.25gm Wright stain powder using balace and transfer to it to a dry brown bottle.
• Using a dry neasuring cylinder, measure the methanol 100 ml and add this to the stain.
• Mix well it properly.
• Allow it 3-5 days prior using frshly made stain to allow time for the stain ripening.
• Filter small volume (50-100 ml) of the stain into a stain dispenising bottle that can be closed when not in use.

Requirement for Staining

• Wright stain
• Sample: Blood (EDTA) or bone marrow
• Phospahte buffer or Buffered water
• Timer
• Distilled Water (D/W)/ tap water (if unavailable D/W)
• Tissue paper
• Staining rack/rods

Procedure

1. Prepare a smear from given blood or bone marrow on a microscopic slide and allow to air dry.
2. Put the air-dried smear on the slide staining rack, smear side facing upwards.
3. Cover the smear with undiluted staining solution since the undiluted stain fixes and partially stains the smear.
4. Leave for 2-3 minutes.
5. Add approximately equal volume of buffered water/ phosphate buffer (pH 6.5). The diluted stain shouldn’t overflow. and mix by gentle blowing. Note-A metallic sheen i.e. green ‘scum’ should appear on the slide if mixing is proper.
6. Wait for 5 minutes.
7. Without disturbing the slide, flood the distilled water (D/W) and wash until the thinner parts of the film are pinkish red as shown above image.
8. Allow the smear to dry at room temperature.
9. Observe the smear first under the low power (10X) objective, and then under the oil immersion (100X) objective.

Result -Interpretation of Wright Stain

Cells	Color
Red blood cells/RBCs/Erythrocytes	Red to buff pink
Platelets	Light blue to colorless with red-violet granules
Neutrophil/polymorphonuclear (PMN) leukocyte	Nucleus-dark purple Cytoplasm-light pink with lilac granules
Eosinophil	Nucleus-Blue Cytoplasm-pink to clear with much large red to orange granules
Basophil	Nucleus: Purple to dark blue Cytoplasm-clear with few blue-black granules
Lymphocyte	Nucleus-dark purple Cytoplasm -varies from light to dark blue
Monocyte	Nucleus-light blue to purple Cytoplasm-gray-blue

Keynotes on Wright Staining

• Types of Romanowsky stain are Leishman’s stain, Wright’s stain, Giemsa’s stain, Jenner stain, May Grunwald Giemsa stain, Field’s stain and Jaswanta Singh Bhattacharya (JSB) stain.
• The methanol should be water free.
• The stain container should be labelled with flammable and toxic due to being methanol in it.
• Warming the solution in water bath at 37 °C will assist the dye to dissolve.
• Wright stain is methanol based and thus it doesn’t require a fixation step prior to staining.
• Fixation helps to reduce water artefact that can occur on humid days or with old stain.
• Composition of Phosphate buffe (0.15M, pH 6.5 to 6.8)

Constituents	Amount
Potassium dihydrogen phosphate, anhydrous	0.663 gm
Disodium hydrogen phosphate, anhydrous	0.256 gm
Distilled Water (D/W)	100 ml

Wright’s Stained Footages

Red blood cells, platelets and white blood cells in PBS demonstration

Lymphocyte on PBS demonstration

Wright’s stained Eosinophil on Peripheral Blood Smear (PBS) Demonstration

Basophil on PBS Demonstration

Kidney shaped monocyte on PBS Demonstration

Horseshoe Shaped Nuclei of Monocyte on PBS Demonstration

Band cell or Stab cell on Wright Stained PBS Demonstration

Blasts on Wright stained smear of PBS demonstration

Nucleated RBC on PBS demonstration

Teardrop cells or Dacrocytes on Wright’s stained PBS Demonstration

Acanthocyte on PBS Demonstration

Malarial parasite Gametocyte (Plasmodium falciparum) on Peripheral Blood Smear (PBS) Demonstration

Fungus Elements on Peripheral Blood Smear Demonstration

Further Readings

• https://www.frontiersin.org/articles/10.3389/fphys.2018.00113/full
• https://en.wikipedia.org/wiki/Romanowsky_stain
• https://en.wikipedia.org/wiki/James_Homer_Wright
• https://www.slideshare.net/nawakharneupane/dyes-and-stain
• https://resources.psmile.org/resources/process-control/section-specific-information/hematology/manual-differential/Pro6.4-B-04%20Wrights%20Stain%20-Diff%20Quick-
• https://himedialabs.com/TD/S030.pdf
• ttps://jcp.bmj.com/content/jclinpath/28/11/920.full.pdf