Vital staining involves using non-toxic dyes on living organisms and cellular structures. Common vital stains include neutral red, methylene blue, and Janus green B. Vital stains can be used to differentiate between living and dead cells, stain specific organelles like mitochondria, and study pathological cell changes without harming living tissues. Vital stains are classified based on their chemical properties and degree of dissociation into categories like basic, acidic, atmospheric and electro neutral. Techniques for vital staining include progressive, regressive, counterstaining, and double staining using combinations of contrasting dyes.

2. TOPIC:
VITAL STAINING AND DOUBLE STAINING

4. VITAL STAINING

6. VITAL STAINS
Non-toxic dyes/stains used for living organisms & their
cellular organelles.
eg: Neutral red for staining cytoplasmic inclusions,
living organisms, nuclei, plasma etc.
Methylene blue for nerve cells
Janus-Green-B :
A basic vital stain for living organisms and
cell organelles especially mitochondria.

7. LIST OF COMMON VITAL STAINS
Eosin dye exclusion
Propidium iodide, DNA stain that can differentiate
necrotic, apoptotic and normal cells.
Trypan blue, a living-cell exclusion dye.
Erythrosine, which is Red no.3 in food colouring,
can be used as an exclusion dye

8.  Vital stains is the term applied by cytologists to those
stains which are used on living cells regardless of whether
the stained parts of the cell are dead or alive.
For example only the vacuole, which is filled with dead
matter , is coloured and with a dye must penetrate a layer
of living protoplasm to reach the vacuole.
Vital staining of a cell wall when colouring of the wall
takes places during the life of the cytoplasm lining it.
Vital staining are completely non-poisonous dyes. It may
also be applied in studies of pathological alternation of cell
morphology

9. Vital staining as a physiological tool.
Vital stains can be used to measure the vacuolar PH by
determining the threshold PH for stain accumulation.
Vital stains can be applied as cytochemical test
substances for the detection of such cell constituents.
Vital stains interfere with metabolic, developmental and
stimulatory process by physical or chemical action.
The important distinction between living and dead cells
often is made possible by tests with Vital stains.

10. CLASSIFICATION OF VITAL STAINING
The classification of vital stains corresponding to their
chemical structure.
A classification that is physiologically meaningful uses a
physicochemical parameter, the ionic characteristics of
stains and the vital stains may thus be classified as
1) Basic
2) Acidic
3) Atmospheric
4) Electro neutral

11. The degree of dissociation is an important factor in vital
staining.
Vital stains are weak electrolytes
oAcidic Vital stains undergo electrolytic dissociation.
oBasic Vital stains dissociation is best described as
protonization.

12. METHODS OF STAINING
PROGRESSIVE STAINING:
The tissues are purposefully understained first.
Then more & more stain is added slowly in definite
intervals until the desired colour intensity is attained.
The staining interval is determined by trial and it may vary
with specimens.
This method is meant for only beginners.

13. REGRESSIVE/RETROGRESSIVE STAINING
The tissue is overstained first.
Then, it is slowly destained until the desired colour
intensity is attained.
The commonest destaining agent is
70% alcohol to which 1% acetic acid
0.5% Conc.HNO₃ or Conc. Hcl is added.
The tissues are immediately washed

14. COUNDER STAINING
Staining in which a particular part of the tissues/cells are
stained with a particular stain first & followed by the other
parts with a contrasting stain here.
Both stains will impart colour to all parts, but, due to
differential displacement, the two colours appear in
different parts discontinuously.

15. DOUBLE STAINING

16. DOUBLE STAINING
A mixture of two dyes, each of which stains different
portions of a tissue or cell

17. Single, Double, Triple & Quadruple Staining
Single Staining :only one stain is used here
Double Staining :uses two contrasting stains
simultaneously
Eg: Safranin+Fastgreen
Triple Staining: uses three contrasting stains simultaneously
Eg: Safranin, Gentian violet, Orange G
Quadruple Staining: uses four contrasting stains
simultaneously
Eg: Safranin, Methyl violet, Fastgreen & Orange G

18. SAFRANIN&FAST GREEN STAINING(free-hand sections)
1. Keep sections in 30% alcohol (5min)
2. Stain in a 1% solution of Safranin (2-24 hrs)
3. Wash off excess stain in water
4. Dehydrate in 95% alcohol (2min)
5. Counterstain in fast green (15 seconds)
6. Rinse excess stain with Colove Oil
7. Clear in a mixture containing 50% Colove Oil ,
25% absolute alcohol & 25% Xylol (a few seconds)
8. Remove clearing mixture & kept in Xylol (a few seconds)
9. Give two changes of pure Xylol (10min)
10.Mount in Canada balsam
Safranin colours chromosomes ,nuclei lignified and
cutinized cell walls.
Fastgreen gives green colour to cellulose cell walls
& cytoplasm.

19. Crystal violet – Orange G Staining of free hand sections
1.Killing and fixing
2.Dehydraction in alcohol series.
3. Clearing in Xylol / xylene.
4. Transfer to absolute alcohol and xylene
5.Transfer to absolute alcohol.
6.Transfer to water .
8. Wash in H₂O – 3 changes.
9. Dehydrate in alcohol ( 1 min) (50%,95%,100%).
10.Stain with Orange G( 1min)
11.Drain off excess stain and rinse in Clove oil.
12.Rinse in xylene.
13.Mount in Canada balsm/DPX.