Romanowsky stains are commonly used to stain blood films. They contain basic dyes like methylene blue that stain nucleic acids blue-grey and acidic dyes like eosin that stain proteins and granules orange-red. Leishman's stain contains methylene blue and eosin in methanol and is useful for identifying cells in blood smears and genetics. The staining process involves making a blood smear, fixing it with alcohol, staining it using the Romanowsky stain followed by a rinse to differentiate cells by their staining. Care must be taken to maintain the proper pH and timing during staining.

1. BT 1103 HEMATOLOGY 1
TOPIC 10: ROMANOWSKY
STAINING

2. LEARNING OUTCOME
Explain the principle of Romanowsky stain
Understand the usage of Romanowsky stain
Explain the principle of Leishman staining
Perform the Leishman staining
Differentiate the cells after staining

3. ROMANOWSKY STAINING
Romanowsky stains are universally employed for staining blood films
and are generally very satisfactory.
There are a number of different combinations of these dyes, which vary,
in their staining characteristics.
1. May-Grunwald-Giemsa is a good method for routine work.
2. Giemsa stain is thought to produce more delicate staining
characteristics.
3. Wright's stain is a simpler method.
4. Leishman's is also a simple method, which is especially suitable when
a stained blood film is required urgently or the routine stain is not
available (e.g. at night).
5. Field's stain is a rapid stain used primarily on thin films for malarial

4. MAIN COMPONENTS OF
ROMANOWSKY STAIN
The main components of a Romanowsky stain are:
 A cationic or basic dye (methylene blue or its oxidation products
such as azure B), which binds to anionic sites and gives a blue-grey
color to nucleic acids (DNA or RNA), nucleoproteins, granules of
basophils and weakly to granules of neutrophils
Structure stained with methylene blue are termed basophilic
 An anionic or acidic dye such as eosin Y or eosin B, which binds to
cationic sites on proteins and gives an orange-red color to
hemoglobin and eosinophil granules.
Stucture stained with eosinophils are termed eosinophilic.

7. A) LEISHMAN STAIN
Leishman's stain : a polychromatic stain
Consist of:
1. Methanol : fixes cells to slide
2. Methylene Blue stains RNA,DNA: blue-grey color
3. Eosin stains hemoglobin, eosin granules: orange-red color
 A stain containing alkaline methylene blue and eosin in methanol
 USES:
 in genetics to identify chromosomal bands
 in blood smear analysis to identify

8. B) WRIGHT STAIN
Wright’s stain is a polychromatic stain
consist of:
eosin
methylene Blue.
When applied to blood cells, the dyes produce multiple colors based
on the ionic charge of the stain and the various components of the
cell.
The eosin ions are negatively charged and stain basic cell
components an orange to pink color.
The methylene blue ions are positively charged and stain the acid
cell components in varying shades of blue.
The neutral components of the cell are stained by both components
of the dye producing variable colors.

9. STEPS
Preparation of blood smear
Fixation using alcohol
Staining using special stain

10. 1. PREPARATION OF BLOOD SMEAR

11. 2. FIXATION USING ABSOLUTE
ALCOHOL

12. 3. PHOSPHATE BUFFER

13. PHOSPHATE BUFFER
USES: as a buffer in various Romanowsky type staining
procedures to controls the PH of the stain. Phosphate buffer
pH 7.0 at 25 °C.
PRINCIPLE: If the PH is too acid, those cells or cell parts taking
up an acid dye stain will stain pinker and the acid components
that stain with the basic dye show very pale staining.
If the stain – buffer mixture is alkaline, the red blood cells will
appear grayish blue and the white cell nuclei will stain very
deeply purple.
Therefore, to stain all cells and cell parts well, the pH of the
phosphate buffer is critical.

14. Too acidic Adequate Basic

15. 4. STAIN

16. PROCEDURE
1. Thin smear are air dried.
2. Flood the smear with stain.
3. Stain for 1-5 min.
4. Add an equal amount of buffer solution and mix the stain by
blowing an eddy in the fluid.
5. Leave the mixture on the slide for 10-15 min.
6. Wash off by running water directly to the centre of the slide to
prevent a residue of precipitated stain.
7. Stand slide on end, and let dry in air.

17. 5. RINSE

18. PRECAUTIONS
1. The staining rack must be exactly level to guard against uneven
staining of the smear.
2. Insufficient washing of the smears when removing the stain and
buffer mixture may cause stain precipitate on the dried smear.
3. Excessive rinsing of the stained smear will cause the stain to fade.
4. Whichever method is used, it is important to select dyes that are not
contaminated with other dyes or metallic salts.
5. Staining time must be specific for each lot of stains and so we must
follow the kit procedure.
6. Bone marrow time staining must be increased.

19. STAINING FAULTS
Too faint:
Staining time too short.
Excessive washing after staining.
Stain deposit:
Stain solution left in uncovered jar.
Stain solution not filtered.
Dirty slides.