Introduction of Wright’s stain
Table of Contents
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
- Prepare a smear from given blood or bone marrow on a microscopic slide and allow to air dry.
- Put the air-dried smear on the slide staining rack, smear side facing upwards.
- Cover the smear with undiluted staining solution since the undiluted stain fixes and partially stains the smear.
- Leave for 2-3 minutes.
- 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.
- Wait for 5 minutes.
- 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.
- Allow the smear to dry at room temperature.
- 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
I got good info from your blog
I’m not that much of a internet reader to be honest but your sites really nice, keep it up! I’ll go ahead and bookmark your website to come back down the road. All the best
It is really a great and useful piece of information. I?¦m happy that you just shared this useful info with us. Please keep us up to date like this. Thanks for sharing.
Hmm it appears like your site ate my first comment (it was extremely long) so I guess I’ll just sum it up what I had written and say, I’m thoroughly enjoying your blog. I as well am an aspiring blog writer but I’m still new to everything. Do you have any recommendations for rookie blog writers? I’d definitely appreciate it.
I do believe all of the ideas you have presented for your post. They’re really convincing and will certainly work. Nonetheless, the posts are very short for starters. May you please lengthen them a bit from next time? Thank you for the post.
Excellent blog here! Also your site loads up very fast! What host are you using? Can I get your affiliate link to your host? I wish my web site loaded up as fast as yours lol
very informative articles or reviews at this time.
Wow, great article post.Really looking forward to read more. Awesome.
After examine a couple of of the weblog posts in your web site now, and I truly like your manner of blogging. I bookmarked it to my bookmark website list and can be checking back soon. Pls take a look at my site as properly and let me know what you think.
wow, awesome post.Much thanks again. Really Cool.
Really enjoyed this article.Really looking forward to read more. Keep writing.