STAINS

2. Staining means the use of dyes to render various
tissues constituents visible and distinct from one
another.
Different parts of cells or tissues react differently to
colours due to their chemical/physical differences.
Thus they become easily visible.
Widely accepted theory of staining is the view
advanced by Witt(1876).
Stains are crystalline aromatic salts which can dissolve
in water to form coloured cations or anions or both.

3. Organic stain has two active chemical components.
1) Chromophoric group
2) Auxochromic group
Chromophoric component impart colour to the
stain.
A material present in this are called chromogens.
Chromophoric group include nitro group, carboxyl
group, azo group, quinoid group etc.

4. Auxochromic component enable the stain to
combine with the cells.
They are either acid or alkali radicals.
This includes hydroxyl group.
When the auxochromic group of the stain
attaches to the cell components, the
chromophoric group also attaches
automatically and imparts colour to the cell
components.

5. COLOURING BY STAINS
1) The ionized stain gets precipitated upon materials
by surface adsorption.
2) In many cases, the dye may enter into the tissue and
impart colour.
3) The material gets saturated with the dye and the
subsequent precipitation of the dye result in
colouration.
4) In some cases, staining involves the action of a
special kind of substance, called mordant.
Differential absorption of the mordant and its
subsequent reaction with the dye results in the
production of an insoluble compound which imparts
colour.

6. MORDANT
chemical substances which can enhance the
colouring property of stains and increase the affinity
of tissues for stains.
Mordant and stain together form a chemical complex
called lake.
Property of the mordant to combine with cell
component and stain is called lake formation.
Mordants are usually double salts of
potassium/ammonium with aluminium/ferric
sulphate.
Ferric ammonium sulphate (ferric alum) is a common
mordant used with haematoxylin and carmine.

7. METACHROMASIA
Some basic stains impart the cells a colour, which is
different from the actual colours of the stains. This
property is of stain is called metachromasia and
such stains are called metachromatic stains.
Example: violet coloured methyl violet makes the
tissue red or purple.
Red coloured borax carmine stains nucleus and
cytoplasm pink.
Thiomine, toluidene, azure-A, etc are also
metachromatic stains.

8.  Compounds which catalyse the action of some
stains.
 Example: KOH for Loeffer’s methylene blue.
SPECIFIC STAINS
 Stains that impart colours only when specific
conditions are provided.
 Example: Sudan IV

9. GENERAL STAINS
Stains that impart colour everything
indiscriminately.
Example: safranin.

10. stains
Chemical
nature
basic
acidic
neutral
Affinity to
different parts
of the cell
nuclear
cytoplasmic
Micro technical
purposes
histological
cytological

11. Basic: when stains consists of a coloured organic
base combined with an uncoloured acetate,
chloride or sulphate radicals.
Acidic: when they consist of a metallic base like
sodium, potassium combined with a coloured
organic radicals.
Neutral: compounds of an acidic and basic dye.
Nuclear: they stain the nucleus.
Cytoplasmic: stains that impart colour to
cytoplasm.
Histological: when they serve to define tissues like
phloem, xylem, sclerenchyma and so on.
Cytological: when they serve to define cell
components.

12. classification
stains
Based on
source
Natural dyes
Synthetic/coal
tar dyes

13. NATURAL DYES
Obtained from plant or animal sources.
E.g : brazilin obtained from caesalpinia
crista & c. echinata.
Oracin from lichens like rocelle.
Hematoxylin derived from the timber of
hematoxylon campechianum.
Carmine from cochineal.
Indigo from indigofera.

14. SYNTHETIC DYES
Synthetic chemical compounds of coal tar origin.
E.g : Picric acid, orange G, safranin, fast green etc.

15. CLASSIFICATION
stains
Based on
colouring
duration
Temporary
stains
Permanent
stains

16. TEMPORARY STAINS
Whose colour fades or which gradually
damages the sections.
E.g : Methylene blue.
PERMANENT STAINS
Whose colour lasts for many years.
E.g : Hematoxylin.

17. Earlier workers used the natural dyes
obtained from plant or animal sources.
But now we use synthetic chemical
compounds.
It is made from the substances found in
coal tar.
Examples for natural dyes,
i. Brazilin - timber of caesalpinia crista and
c.echinata.
ii. Hematoxylin - timber of hematoxylon
campechianum.

18. iii. Carmine – made by adding alum to cochineal.
iv. Yellowish powder obtained by grinding the
dried bodies of the female dactyl opius cacti
insect and extracting the colouring matter.
Coal tar dyes are many.
Grouped in to six , depending upon their
chemical nature.
Example for synthetic dyes,
1) Picric acid
2) Orange G
3) Alizarin
4) Methylene blue
5) safranin
6) Light green etc.

19. I. ANILIN BLUE/COTTON BLUE/CHINA BLUE/SPIRIT BLUE
Both aqueous and alcoholic solutions are used.
a. Anilin blue 1 gm
b. Distilled water 100 ml
c. Alcohol 85 %
Excellent for filamentous algae and fungi.
Stains the cell wall and achromatic figure used in
combination with safranin.

20. Cotton blue in lacto phenol is prepared with
a. Phenol 100 ml
b. Glycerin 100 ml
c. Lactic acid 100 ml
d. Distilled water 100 ml
This is used for fungi in whole mounts.
II. CRYSTAL VIOLET/GENTIAN VIOLET
a. Crystal violet 1 gm
b. Clove oil 100ml
This solution works well , since aqueous or alcoholic
violets gets washed away during dehydration.
Good counter stain for safranin.

21. III. CARMINE/ACETO CARMINE
Boil 100 ml of 45 % acetic acid in a flask fitted with
a reflux condenser.
Add a small pinch of carmine powder from a
weighed out quantity of 2gm. Remove source of
heat and add rest of the dye , dissolve , cool and
filter.
Add a few drops of ferric acetate dissolved in
glacial acetic acid until the colour is wine red. Store
in a refrigerator.
This stain is customarily need for cytological
preparations.
IV. FAST GREEN/FCF
a. Fast green 1 gm
b. Clove oil 75 ml
c. Absolute alcohol 25 ml

22. Filter when sufficient time has been allowed for the
stain to dissolve completely.
Stain acts on non-lignified tissues and on spindle
fibres.
Fresh stain is not to be used , does not fade.
V. ORANGE G
a. Orange G 1 gm
b. Alcohol 100 % 100 ml
c. Clove oil 100 ml
Dissolve stain in alcohol and add clove oil. Allow
alcohol to evaporate.
Most useful cytoplasmic counter stain for sections
previously stained red, blue , or green.

23. VI. SAFRANIN O
a. Safranin o 2.25 gm
b. Alcohol 95 % 225 ml
Dissolve dye in alcohol and dilute to required
concentration with distilled water.
Stains lignified, cutinised, and suberised structures
as well as chromosomes ,nucleoli, and
centromeres.
A good combination with fast green FCF.

24. VII. HEMATOXYLINS
Great affinity for metals.
Most formulae include a mordant containing either
aluminium or iron or potassium alum.
Colour resulting from staining depends on the
mordant used.
Great use in histology and cytology.
There are several formulae named after many workers.
E.g. Hematoxylin – Harris , Hematoxylin Delafield's
(modified) , hematoxylin/iron-alum-hematoxylin
Heidenhain’s etc.

25. a. Hematoxylin 1 gm
b. Distilled water 200 ml
c. Sodium bicarbonate very small quantity
Measure out the water and add the sodium
bicarbonate.
Bring water to boiling point, remove source of heat
and add the dye.
Cool and store in a refrigerator.
Dilute stain before use.

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