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Leishman Stain

Image depicting a normal peripheral blood smear, highlighting the consistent size and shape of red blood cells with central pallor occupying approximately one-third of their diameter

Principle of Leishman Stain

Romanowsky stains which include Leishman and Wright are commonly used to stain the peripheral blood smear.  Chemical components of the Leishman dye are Azure B (blue in colour) and Eosin Y (orange) to stain different groups of molecules in the cells. The DNA and acidic groupings of the proteins of cell nuclei and primitive cytoplasm determine the uptake of the basic dye Azure B. Conversely, the presence of the basic groupings on the hemoglobin molecules results in its affinity for acidic dye like Eosin Y.

Materials

  • Leishman stain
  • Phosphate buffer 0.66 M pH 6.8
  • Pasteur pipette
  • Kim wipes
  • Hair dryer
  • Unstained peripheral blood smear slide

Method

Preparation of phosphate buffer for 100 mL volume

  • Solution A: KH2PO4 9.1 g/L
  • Solution B: Na2HPO4 9.5 g/L

Add 50.8 mL of solution A to 49.2 mL of solution B to obtain pH 6.8

  1. Cover blood film completely with the Leishman stain using a Pasteur pipette and wait for 2-3 minutes. Approximately 3 mL of stain is required to stain a single slide.
  2. Add an equal volume of phosphate buffer onto the slide (stain:buffer ratio = 1:1). Use a Pasteur pipette to blow gently over the slide to completely mix the stain and buffer. DO NOT touch the stain using the Pasteur pipette.
  3. Leave the slide to stain for 15-20 minutes.
  4. Remove the stain with slow running tap water. Wipe the back portion of the slide and the edges dry using Kim wipes without touching the blood smear.
  5. Dry the slide with a hair dryer on low speed.
  6. Mount the slide with Depex and cover the zone of morphology with a cover slip.
  7. This slide is now ready for viewing.
Image depicting a normal peripheral blood smear using Leishman stain, highlighting the consistent size and shape of red blood cells with central pallor occupying approximately one-third of their diameter
A normal peripheral blood smear using Leishman stain x400 magnification. Red cell central pallor approximately 1/3 in size of the red cells. The cells are almost all the same shape and size. The red cells are slightly smaller than the small lymphocyte in size.

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