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Leishmans Stain for Protozoa in Blood Film

Leishman’s stain for protozoa in blood film:
The schedule for staining is as under. Prepare a blood film as described in Chapter 4. Do not fix the film and stain with undiluted Leishman’s stain (S69) for 1-2 minutes. Add double the volume of buffer solution (S71) or distilled water (pH 7.0) to the slide and mix well by alternately sucking the fluid in pipette and expelling back. Allow the diluted stain to act for 10 minutes. Pour-off the stain and flood the slide with distilled water until the smear appears pink. Blot, dry and examine.

According to Schute (22), 15 seconds are sufficient for fixation with undiluted stain. He recommended following procedure. Take unfixed blood smear and add four drops of Leishman’s stain. Rock the slide for 12-15 seconds and then add 8-10 drops of distilled water (pH 7.2). Mix well and after 15 minutes, flood off the stain with water for 2-3 seconds only. Longer washing will not allow the demonstration of Schuffner’s dots in malarial parasites. The method is also useful in differential counting of blood cells

Straining with sections can be done by below listed schedule. Treat the sections with xylene to remove paraffin and then with alcohol and finally with distilled water. Remove excess of water and stain with Leishman’s stain (S69) diluted 1-3 with buffer solution (S70) for 5 minutes. Wash with buffer solution and keep the sections in 1:1,500 acetic acid till protoplasm of cell appears pink and nuclei blue in colour. Use low power objective for this observation.

Wash with buffer solution and remove water by blotting. Dehydrate the sections with absolute alcohol, clear in xylene and mount. It is important to mount the sections in a neutral medium such as DPX, as azures gradually fade under the acidic conditions.
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