Leishman stain is a differential stain that is used to stain the various components of the cells especially the blood cells and it can be used to study the adherence of pathogenic bacteria to the human cells. Leishman stain is a mixture of Methylene blue (Basic), and Eosin (Acidic) dye.

It differentially stains the human and bacterial cells which appeared as purple and pink colored bodies respectively. The Leishman stain is one of the best stains for routine blood stain to stain the Peripheral blood smear for the examinations of blood film under the microscope and is satisfactory for malaria and other blood parasites. Giemsa stain gives better results in parasitic studies.

It can also be used for staining the thick blood smears for the identification of Blood parasites but Giemsa Stain gives much better results in Thick Smears. Leishman stain is most widely used for staining the Thin Blood Smear for the identification of Toxic Granules, Blood Cell morphology, Type of Anemia etc.

Nowadays, commercially prepared Leishman staining solutions are used in most of the laboratories. However, some laboratories still prepare it in their own setup as per their Standard Operating Procedure (SOP).

Here, I’m gonna explain the method of Preparing Leishman Stain or Leishman Stock solution in Laboratory. This protocol is correct, however; do not consider it as the only correct way of preparing Leishman stain in laboratories as the protocol varies from lab to lab.

Materials Required for Preparing Leishman Stain

  • Leishman stain powder – 0.15 gm
  • Absolute methanol (Acetone free) – 100 ml
  • Spatula or Measuring spoon
  • Weighing paper or butter paper
  • Graduated cylinder
  • Glass or plastic funnel
  • Screw-capped, dark or amber glass bottle – 250-ml capacity
  • Analytical balance / Weighing scale
  • Shaker

Check out the Leishman Staining Protocol


Procedure of Preparing Leishman stain in Laboratory

Weigh the 0.15 g of Leishman stain powder on an analytical balance or weighing scale, Grind it in a Glass Mortar and put it into a bottle through a funnel.

Gently pour in about 20 ml of acetone-free methanol through the same funnel, ensuring that all the dry stain is washed into the bottle.

Re-cap the bottle and shake it in a circular motion for 2–3 minutes to start dissolving the stain crystals in Methanol solution. Warm the content at 50 °C for 15 minutes with occasional shaking.

Add the remaining amount of methanol to the mixture through the funnel and make the volume 100 ml, ensuring that even the last drop of the methanol washes in the funnel into the stain mixture.

Tighten the screw-cap on the bottle and continue shaking for few minutes.

Do not open the bottle and do not filter the content for about a week. After a week it is ready to use. Filter the content before use.

Label the bottle clearly as Leishman Stock Solution with the batch number, the name of the person who prepared it, date of preparation and date of expiry, and document in the quality control log-book of your Laboratory.

Tighten the screw-cap on the bottle to prevent absorption of water vapors from the air, and store in a cool place away from direct sunlight.

Do not store the solution near bottles containing acid. If stored correctly Leishman Stain solution is stable approximately for 3 months.

Use this stock solution of Leishman stain in various dilutions (ideally in 1:2 i.e. 1 part stain and 2 parts buffer solution) as per the quality of the stain and the purpose of staining, e.g. for blood cell examination in thin smear or for the identification of parasites in thick smear etc.

Check out the Protocol of Leishman staining by HIMEDIA LABS


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