DOUBLE STAINING

DOUBLE STAINING

1. WELCOME

2. TOPIC: VITAL STAINING AND DOUBLE STAINING

3. VITAL STAINING

4. VITAL STAINING A vital staining in a causal usage may mean a stain that can be applied on living cells without killing them. Supra vital staining living cells that have been removed from an organism. Intra vital staining is done by injecting or otherwise introducing the stain into the body.

5. 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.

6. 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

7. 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

8. 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.

9. 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 Basic Acidic Atmospheric Electro neutral

10. The degree of dissociation is an important factor in vital staining. Vital stains are weak electrolytes Acidic Vital stains undergo electrolytic dissociation. Basic Vital stains dissociation is best described as protonization.

11. 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.

12. 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

13. 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.

14. DOUBLE STAINING

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

16. 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

17. SAFRANIN&FAST GREEN STAINING(free-hand sections) Keep sections in 30% alcohol (5min) Stain in a 1% solution of Safranin (2-24 hrs) Wash off excess stain in water Dehydrate in 95% alcohol (2min) Counterstain in fast green (15 seconds) Rinse excess stain with Colove Oil 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.

18. 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.

19. THANK YOU...