2. 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.
3. The dye used to denoted powder and the stain to denote
the solution of one or more dyes. The dyes are classified
into two groups:-
1) Natural dyes
2) Synthetic dyes
4. 1) Natural dyes
they are prepared or extracted from natural sources
such as hematoxylin(It is a compound extracted from
the heartwood of the logwood tree (Haematoxylum
campechianum), litmus(Litmus is a water-
soluble mixture of different dyes extracted from lichens),
orcein(Orcein, also archil, orchil, lacmus and C.I. Natural
Natural Red 28, are names for dyes extracted from
several species of lichen, commonly known as "orchella
weeds", found in various parts of the world), carmine,
safranin.
5. A tree covered with leafy foliose
lichens and shrubby fruticose
lichens
7. 2) Synthetic dyes
These are manufactured or prepared or sometimes referred as
coal tar dyes.
stain can further be divided into 3 parts:-
1) Acidic dyes/stains:- acidic dyes stains basic component of
the cell such as cytoplasm.
the coloring substance present in the acidic component and
basic part is colorless. E.g. eosin, safranine, phylozine.
8. 2) Basic dyes/stains:-
coloring substance is present in the basic part of the
component and acidic part is colorless.e.g. methylene blue,
crystal violet, gentian violet, methyl violet, brilliant crysal blue.
So basic dyes stain the acidic component of the cell(nucleus).
9. 3) Neutral dyes/stains:-
in these days, both acidic and basic components are colored.
These dyes are also called amphoteric dyes e.g. Romanowsky
dyes.
10. AMPHOTERIC DYES
These dyes stain both acidic and basic component of the cell
at suitable PH. In acidic PH,it behaves like acidic dye and in
basic PH,it behaves like basic dye.
Acidic and basic dyes are usually prepared in aqueous
solution. But, neutral dyes are prepared in organic
solvent(methanol) and differentiated by aqueous solution.
Acidic/basic dyes are differentiated by organic media.
11. ROMANOWSKY’S STAIN
In 1819,Romanowsky discovered that when alkali is added to
methylene blue, it produce oxidation 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
discoloring agent. So, not used in above stain.
13. Uses of Romanowsky stain
use to stain the blood smear for differential leucocyte count
and also for lupus erythematous (LE cell)
to stain the bone marrow
to study the blood parasite e.g.: malaria parasite.(we do
Giemsa stain)
14. 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.
Neutrophilic structure is stained by the combination of both
acidic and basic dye. Hence,nucleic acid of nucleus is
basophilic and stain blue. The basophilic granules are highly
acidic(basophilic contain heparin) and stain blue.
15. Hemoglobin is basic,which stain red i.e. color of eosin.
Eosinophilic granules are basic due to the presence of
histamine and stain orange red.
17. Note:- most commonly ‘eosin’ is used as the acidic stain
though azure I and azure II also can be used.
Most commonly ‘methylene blue’ is used as basic stain
though toluidine blue can also be used.
18. Leishman’s stain
Preparation of Leishman’s powder
Methylene blue: - 1 gm
Sodium carbonate (0.5%):- 100 ml
Eosin Y (0.1%): - 100 ml
19. Procedure:-
- Dissolve methylene blue in sodium carbonate solution by
heating mixture at 56-65 degree Celsius for 12 hour. cool and
keep at room temperature for 10 days.
- Add equal volume of eosin.
- Mix well and keep at RT for 6-12 hr.
- Filter the mixture through filter paper and collect precipitate
on filter paper.
20. - Wash precipitate with several changes of distilled water
until no more colour is extracted.
- Dry the precipitate at 37 c in incubator and grind to a
powder in glass mortar.
22. procedure
- 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.
23. - 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.
25. Preparation of wright’s stain
- Wright’s stain powder: - 0.25gm
- Acetone free methanol: - 100 ml
Dissolve powder in methanol. Keep for few days(for
ripening) before using.
26. 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.
27. How to make buffer water
Composition of buffer water which used in Romanowsky
stain.
- Anhydrous disodium hydrogen
phosphate(Na2HPO4)=0.47 gm
- Anhydrous potassium dihydrogen
phosphate(KH2PO4)=0.46 gm
- Distilled water = up to 1 liter
- Dissolve the phosphate in distilled water and check the PH.
The PH should be 6.8.
28. Staining method for wright’s
stain
1) Prepare smear and air dry it.
2) Cover the smear with wright’s stain for 1-2 minutes(
fixation time), taking care that it should not dry on the
slide. Main aim is fixation.
3) Dilute with an equal volume of buffer water,PH 6.8.
4) Keep the diluted stain for 3-5 minute(staining time)
29. 5) Wash the smear with buffer water or tape water.
6) Clean the back side of slide by using tissue paper.
7) See under microscope for proper staining i.e. to check the
smear under or over stained.
8) Let it dry at RT by keeping upright position in slide rack for
drainage and examine under microscope.
31. Comparative chart of common
Romanowsky stain
Wright stain Leishman stain Giemsa stain
-Wright’s stain powder:-
0.25gm
-Acetone free methanol: -
100 ml
Fixation:- 1-2 mints
Staining time:- 5-7 min
Dilution:- by equal
of buffer water
Leishman’s powder:-0.15
gm
Acetone free methanol : -
100 ml
Fixation:- 2 mints.
Staining time:- 7-10 min
Dilution :- by double
volume of buffer water.
Giemsa powder:-0.3gm
Glycerin:- 25 ml
Acetone free methanol:-
25 ml
Fixation: separate
prefixation in methanol
3-5 mints due to less
volume of methanol
contained in composition.
Staining time:- 15-20
Dilution- 1:10 with buffer
water before staining.
32. Staining method with
Leishman’s stain.
1) Prepare smear and air dry it.
2) Cover the smear with stain and wait for 2 mints for
fixation.
3) Dilute with double volume of buffer water and wait for 7-
10 minutes for staining.
4) Wash the smear with tap water.
5) Clean the back side of slide and then drain and air dry the
smear
6) Examine under microscope.
33. Giemsa staining technique
1) Prepare the smear and air dry at RT.
2) Fix in methanol for 3-5 mints.
3) Take out and dry it.
4) Cover the smear with diluted stain(1 in 10 dilutions), 1 part
Giemsa stain + 9 part distilled water or buffer water.
5) Keep for 15-20 mints or longer, depending up on the
potency of stain.
6) Wash with tap water and dry at RT.
34. Field’s stain
Field stain is a histological method for staining of blood
smears. It is used for staining thick blood films in order to
discover malarial parasites. Field's stain is a version of
a Romanowsky stain, used for rapid processing of the
specimens.
35. MGG stain
Most commonly used for bone marrow. First, fix in
methanol and then stain with may Grunwald stain(0.3 gm
powder+100ml methanol).after that ,stain with Giemsa
stain.
37. JSB stain
It is a method of rapid staining of malaria parasites by a
water soluble dye/stain.
Editor's Notes
Denoted ;mark
Coal tar is a thick dark liquid which is a by-product of the production of coke and coal gas from coal( a substance that can be burned to provide heat or power)black in color.
