Leishman Stain

Red Blood Cell Resources
Associated Diseases

Principle

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 haemoglobin molecules results in its affinity for acidic dye like Eosin Y.

Materials

  • Leishman stain
  • Phosphate buffer 0.66 M pH 6.8

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

Method

  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 using depex and cover the smear with a cover slip.
  7. Examine blood film using a light microscope (using x10 and x40 lenses).

Normal peripheral blood smear x400 magnification