WRIGHT'S STAIN: RATIONALE, MATERIALS, TECHNIQUE, AND USES - BIOLOGY - 2025

The Wright stain is a staining technique created by the American pathologist James Homer Wright in 1902, based on the Romanowsky stain. Since the Romanowsky stain was unstable, Wright incorporated methanol as a solvent and fixative.

This coloring is polychromatic, which means that it generates several colors depending on the structure that absorbs the dye. This staining technique has been widely used to perform differential white blood cell counts and to study the morphology of red blood cells, platelets, and leukocytes in peripheral blood and bone marrow.

Various peripheral blood smears stained with Wright's stain. A. Acute leukemia B. Plasmodium vivax within the erythrocyte. C. Plasmodium falciparum, macrogametocyte. D. Lymphocyte

Its application is very important, since abnormalities can be seen in the different cell lines of the blood, facilitating the diagnosis of diseases such as leukemia or bacterial or parasitic infections.

Perhaps these are the most common applications in which this technique is used, however they are not the only ones. It is also useful in samples other than blood and bone marrow, such as nasal discharge, fecal mucus, sputum, skin samples, among others.

Rationale for Wright's stain

Wright's stain was born from Romanowsky's stain, which consists of a methyl alcohol solution of an acidic dye (eosin Y) and a basic dye (methylene blue) and their oxidation products.

The mixture of dyes used in Wright's stain causes the effect known as Romanowsky, that is, it gives a beautiful purple coloration to the nuclei of leukocytes and neutrophilic granules, while the red blood cells stain pink.

The components responsible for giving the typical color gamut of Wright's stain are blue B and eosin Y. The effect observed will depend on the binding of the dyes to the chemical structures and the interactions of blue B and eosin Y.

Acidic structures such as nucleic acids, nuclear proteins, and the reactive immature cytoplasm of some cell types, fix blue B (basic stain).

While basic structures such as hemoglobin, the granules of segmented eosinophils, among other cellular structures, bind eosin Y (acid dye).

The staining result can be influenced by different factors, such as the pH of the Wright dye, the buffer and the wash solution; as well as the staining and fixation time.

Therefore, each step in the preparation of the reagents is crucial and must be carried out with attention to every detail.

materials

Wright's stain. For 100 mL it is required:

Weigh out 0.3 g of Wright's stain, measure 97 ml of methanol and 3 ml of glycerol.

Preparation

Place the heavy amount of Wright's stain in a mortar and gradually add the glycerol until the powder is completely dissolved.

Subsequently, the methanol is added, mixed and poured into an amber bottle.

Before use, the solution should be shaken with gentle movements and filtered.

Buffered buffer solution

In one liter of distilled water, 3.76 g of disodium hydrophosphate (Na ₂ HPO ₄ 2H ₂ 0) plus 2.1 g of dihydrogen potassium phosphate (KH ₂ PO ₄) are added.

Mix very well until all incorporated reagents are dissolved. Adjust the pH to 7.2. Pour into a glass jar and keep at room temperature.

Additional materials needed to perform the coloring

Additionally, other materials are required to be able to carry out the coloring technique, these are: object slides or covers objects, coloring bridge, t-shirts with water or buffer for washing, a timer to keep the coloring times and some drying material (absorbent paper, gauze or cotton).

Components of Wright's stain

Methanol

Alcohol (methanol) serves as a fixative of the blood smear to the slide.

It is basically a coagulant reducing, dehydrating and fixing reagent. Therefore, its function is to coagulate proteins and make them insoluble, but without actually denaturing them.

Methanol is the most widely used smear fixation reagent in all laboratories, as it provides better results than ethanol. The ideal concentration is 99%.

Damper

The buffer (buffered solution) has the function of adjusting or maintaining the pH of the dye, since a pH adjusted to 7.2 is essential for the cell structures to be able to absorb the dyes properly.

On the other hand, the methanol fixation step dehydrates the cells and the buffer helps to rehydrate them.

Eosin (Y)

Eosin has an affinity for building blocks because it is an acid dye. Two types of eosin are known very similar to each other, so much so that either of the two can be used, obtaining the same result.

One is called eosin Y, yellow eosin, or tetrabromofluorescein, and the other is called eosin B, bluish erythrosin B, or dibromodinitrofluorescein. However, eosin Y is the most commonly used.

The most important property of this dye is its negative polarity, this makes it attracted to positively charged cell structures.

Methylene blue

It is the basic coloring. Its main property is metachromasia, that is, not all structures will be stained the same color, it depends on the chemical composition of the structures that are being colored.

Some will turn light or dark blue, and some will turn dark purple or pale lilac.

Technique

1-Perform the spreading of the sample so that a thin film remains, either on a slide or coverslip.

2-Let it dry in the air for approximately 2 hours.

3-Place the dry smear on the staining bridge or staining tray with the spread of the sample facing up.

4-Cover the sheet with the Wright stain drop by drop until the entire surface is covered. Leave on for 5 - 8 minutes.

5-The stain should completely cover the slide, without spilling over the edges. If during the coloring time it begins to evaporate, add a few additional drops.

6-Subsequently add an equal amount of the shock absorber, blow a little until the characteristic metallic shine appears. Time 10 to 15 minutes.

7-Wash with tap water, placing the gentle stream until the sheet looks pink.

8-With a gauze soaked in alcohol, remove the dye adhered to the back of the slide.

9-Let the smear dry very well before placing the immersion oil to view it under the microscope.

Utility

Hematology

It is ideal for the staining of peripheral blood smears, for the examination of thick blood smears and for the study of cells from bone marrow samples.

Due to the chemical properties of this combination of dyes, cell structures can be easily recognized, and the different types of cells present can be distinguished.

Runny nose

This technique is very useful to identify the cells of the nasal discharge (epithelial cells, segmented eosinophils, polymorphonuclear cells) in the diagnosis of allergic rhinitis.

parasitology

In this sense, it has been useful for the study of Leishmania sp within the histiocytes of the subcutaneous cellular tissue in skin ulcers. Likewise, it is used to identify leukocytes in stool samples (fecal leukogram).

In this case, it is of interest to the physician to know whether the leukocytosis present in the stool is polymorphonuclear or mononuclear. This finding in the fecal leukogram will guide whether it is a bacterial or viral infection respectively.

On the other hand, parasites circulating in the blood can be found within the erythrocyte or free in plasma. The parasites sought are Plasmodium spp, Trypanosoma cruzii and filariae, and in veterinary medicine it is useful in the search for Theileria equi and Babesia caballi, causal agents of bebesiosis, especially in horses.

The Wright stain and also the Giemsa stain make it possible to differentiate haemoparasites from normal cellular components. Two types of spreads can be used for this:

Spread thin

Blood is spread as a thin film on a slide. It is stained with Wright's stain, revealing the characteristics of the nucleus and cytoplasm.

Thick drop

This methodology is used to investigate the presence of parasites in a larger quantity of blood.

To do this, a large drop of blood is placed on a slide. Once there, it must be defibrillated, making larger and larger circles from the center outward, using the edge of another slide.

Finally, to be able to observe the parasites in the thick smear, the erythrocytes must be lysed with water.

Respiratory infections

At the respiratory level, this technique is also useful, because the cells present in the samples of sputum, bronchial lavage or bronchoalveolar, are important to establish the diagnosis.

Similarly, polymorphonuclear cells and mononuclear cells can be distinguished here.

Bacteriology

The use of this technique in bacteriology is limited, because it is not good for staining bacteria, which is why other specialized staining techniques are used for their staining.

However, it has been used to search for epithelial cells with Chlamydia trachomatis inclusion bodies in urethral or endocervical mucosa smears, although it must be recognized that it is not the best method for this.

It is also possible to observe spiral-like bacteria such as Borrelia burgdorferi among red blood cells in infected patients, as well as morulae or inclusion bodies of Ehrlichia sp in the cytoplasm of lymphocytes, monocytes, or neutrophils in a blood smear.

Mycology

Histoplasma capsulatum is a pathogenic fungus frequently diagnosed by microscopic observation of various tissue samples, stained with Wright's stain.

How are the structures of the blood sample observed with the Wright's stain?

Source: Retamales E, Mazo V. Institute of Public Health, Government of Chile. Recommendations for the staining of blood smears for reading the hemogram.

Recommendations for good staining

Blood sample slides should air dry spontaneously. Smears should be as thin as possible to obtain better fixation of the dye and avoid over-staining.

For high quality staining, it is advisable to stain within two hours of smear preparation. On the other hand, the ideal sample is capillary blood, without anticoagulant.

However, if venous blood is used, it should be used as an anticoagulant EDTA and not heparin, since the latter can deform cell structures.

To avoid deterioration of the prepared colorant, it should be stored in dry places.

During the washing process the use of water adjusted to a neutral pH is recommended.

Finally, it is advisable to test the staining methods used in the laboratory from time to time.

This is done by staining samples or extended patterns, as a quality control. This step is important, as it ensures that the staining is properly prepared and the staining times are well standardized.

If the pattern sheet is poorly colored, then there are problems that must be solved.

Common mistakes in Wright staining

Very pale staining

Very pale smears are usually due to a very short staining time or excessive washing. It is corrected by lengthening the contact time with the dye or decreasing the washing time.

Colorant precipitates

The presence of precipitates of dye in the smear can have several causes, however, the most frequent causes are: use of unfiltered dye, staining on uneven staining bridges, using sheets dirty with dust or grease, not washing well complete staining.

Extremely red or blue smear

The buffer is responsible for the pH of the dye. Dyes with a pH below that indicated (acidic) will result in very reddish smears.

If the pH of the dye is above (alkaline) an extremely bluish smear will be obtained.

Storage mode

The reagent should be stored at room temperature.

References

1. Gutiérrez V. Comparative study between the Wright staining method and the Elisa test for the diagnosis of canine Ehrlichiosis in the city of San Pedro Sula, Honduras. 2008. Degree Thesis to qualify for the Veterinary Medical Degree. University of San Carlos of Guatemala.
2. López-Jácome L, Hernández-Durán M, Colín-Castro C, Ortega-Peña S, Cerón-González G, Franco-Cendejas F. Basic stains in the microbiology laboratory. Research in Disability. 2014; 3 (1): 10-18.
3. "Wright's stain." Wikipedia, The Free Encyclopedia. 18 May 2018, 12:05 UTC. 8 Dec 2018, 20:37
4. Calderón A, Cardona J, Vergara Ó. Frequency of Babesia spp. in hunting horses, Córdoba (Colombia). Rev. udcaactual divulg cient. 2013; 16 (2): 451-458.
5. Forbes B, Sahm D, Weissfeld A (2009). Bailey & Scott Microbiological Diagnosis. 12 ed. Argentina. Editorial Panamericana SA
6. Retamales E, Mazo V. Institute of Public Health Government of Chile. Recommendations for the staining of blood smears for reading the hemogram.