1) The document provides instructions for preparing three types of biological stains - Wright stain, Leishman stain, and Giemsa stain. It details the materials, reagents, and procedures needed to make each stain. 2) For Wright stain and Leishman stain, the dye powder is dissolved in acetone-free methanol and stored in tight bottles. For Giemsa stain, the dye powder is dissolved in a mixture of glycerin and methanol to make a stock solution, which is then diluted before use. 3) The stains are used to stain blood smears and bone marrow samples to study blood cells, parasites, and other anatomical details microscopically.

1. Tansen School of Health Science
Practical write up
Title: preparation of stain
Aim: to prepare the stain
Introduction:
All the biological stains are prepared from dyes which have been manufactured for special
purpose that means to study the anatomical details of blood cells and tissue
microscopically.
Romanowsky stain: - when alkali is added to methylene blue, it produceoxidation of
methylene blue and forms polychrome methylene blue which further combine with
eosin to form methylene blue azure. These dyes are not soluble in water but dissolve in
methanol (acetone free methanol). Acetone acts as strong dehydrating agent and
discolouring agent.
-use to stain the blood smear for differential leucocyte count and also for lupus
erythematous (LE cell)
- to stain the bonemarrow
- to study the blood parasite e.g.: malaria parasite.(we do Giemsa stain)
Materials required:
1. Physical balance
2. Conical flask
3. Measuring cylinder
4. Spatula
5. Storing bottle
6. Filter paper

2. Reagents:
Forwright stain
Dye : - 0.25 gm
(Azure A Eosinate: 24%
Azure B: 35%
Azure C: 6%
Methylene blue: 35%)
Methanol : -100 ml
ForLeishman stain
Dye/powder : - 0.15 gm
(Preparation of powder
Methylene blue: - 1 gm
Sodium carbonate (0.5%):- 100 ml
Eosin Y (0.1%): - 100 ml)
Methanol : - 100ml
ForGiemsa stain
Powder : - 0.3 gm
Glycerine : - 25 ml
Methanol : - 25 ml
Principle:
Principle of Romanowsky stain: - acidic component of cells are basophilic (acidic in nature) and
stained by basic dye. Basic component of the cells are acidophilic (basic in nature) and stained by
acidic dye.

3. Procedure:
For Leishman stain:-
- Weight 150 mg of powder in minimum volume(30-40 ml)of acetone free methanol(in mortar)
- Take the supernatant in separate container (flask), add small volume of methanol and
dissolved remaining powder.
- Make volume up to 100 ml .store in tight stoppered bottle.
- Label (date of manufacturing, name of stain or reagent)
- Keep the stain at room temperature for 24 hours before use( Ripeing time)
- If stain is needed urgently, it can be warmed at 50 c for 15-20 minutes with occasional
shaking.
For wright stain:-
- Wright’s stain powder: - 0.25gm
- Acetone free methanol: - 100 ml
Dissolve powder in methanol. Keep for few days (for ripening) before using.
For Giemsa stain:-
 Preparation of Giemsa stain:-
Giemsa powder: - 0.3gm
Glycerin: - 25 ml
Acetone free methanol: - 25 ml
This makes stock solution and before use, it has to be diluted by adding 1 ml stock to 9 ml distilled
water.
Internal Quality Control:
1) Shouldn’t kept in refrigerator.
2) Shouldn’t use acetone contain methanol.
References:
Cheesbrough, M., 2006. District laboratory practice in tropical countries. Cambridge university press.
Godkar, P.B. and Godkar, D.P., 2006. Textbook of medical laboratory technology. Bhalani publishing house.
Dacie and Lewis, Practical Haematology.