Carmine –dried bodies of female insect dactylopiuscacti,haematoxyline-haematoxylonecampechianum(logwood).organic compound from benzene.color bearing chemical configurations chromophore.quinonoid com produce more complex-brilliant darker colors.auxochrome-ionzing radicle.NH3-COMMNEST AUXOCHROME
The amount of stain taken by fat is determined by partition coefficient that applies to stain in presence of two solvent
Firm bond, broken by -acid
After formalin fixationmordanting the tissue sections with a picric acid solution such asBouin’s will enhance the trichrome staining intensity and radiance.The recommended fixatives for trichrome staining are: Bouin’s,Zenker’s, Formal-mercury, Zinc formalin and Picro-mercuric alcohol.
the high lipid content of nervous tissue makes it less suited to H&E than most others. Consequently, numerous stains have been developed which take advantage of the chemical properties of the lipids in neural tissue to highlight structural features. One of the most common stains used for nervous system tissues is the Cresyl Violet method. Cresyl Violet binds strongly to the RNA in the neuron's rough endoplasmic reticulum, since it's a basic stain. Luxol Fast Blue gives particularly good delineation of nerve tracts in the CNS, and the two are commonly used together
Term 1st used by LEEUWENHOEK in1719.used saffron for muscle fibre GOPPERT& COHN in 1849 used carmine GERLACH in1858 –selective nuclear staining for nerve cells,regressive stain-weak acetic acid WALDEYER in 1863-used hematoxylin
Stains are colored substances which dye tissue Dyes-staining agent whose chemical formula is known,mixture of very closely related compounds with alike properties Stain-dyes which are metallic salts of animal and vegetable origin
Acidic dyes-color acid is combined with non coloring metallic base(sodium & potassium). Eosin-Y,light green.Acid fuchsin is sodium salt of acidic sulphonated derivative of rosaniline Basic dyes-color base is combined with non coloring metallic acid (acetate,chloride,sulphate radicle) Basic Fuchsin(colored rosaniline base and colorless acidic CI radicle &,haematoxylin Neutral dyes- compounds of color base with color acid. neutral red.
Stored for long time ;-ve charge possesed by haematein loses its affinity,mordanting is an essential A mordant ammonium alum, potash alum ,iron alum(aluminium compound) forms lake with strong basic dye. Water soluble lakes from aluminium compounds are blue.progressive staining Lakes from ferric compounds-regressive staining
SUPRAVITAL-when stain is applied to a tissue which has already been removed before it is stained.(dissociation)e.g.-R.E. cells by trypan blue,lithium carmine stains histiocytes,alizarine stains bones red INTRAVITAL-by injecting dye into the living org e.g. Janus Green stain mitochondria. NON-VITAL-for fixed cells
Substances that dissolve in tissue – lysochrome Fat droplets electively stained in alcoholic solution if stain is more soluble in fat than in alcohol
Metallic compounds can be reduced by tissues to stable metallic state Ammoniacal silver –deposited silver is stable after reduction. Argentaffin cell-tyrosin derivative melanin, phenolic compound- kultschitzky cells Argyrophil cells
Certain basic dyes react with tissue components such that their normal color changes from blue to red or purple Reason-b‟cos of presence of polyanions within the tissue Metachromatic Dyes –thiazines-toludine blue Eg metachr staining in cartilage,epithelial mucins,mast cell granules
Assist interaction of tissues & dyes Mercuric chloride,formaldehyde,ethyl alcohol split chromatin-DNA & protein Trichloroacetic acid,picric acid,chromium compounds facilitate-acidic dyes After fixation ethyle alcohol or acetic acid – both acidic & basic dyes Blockage of carboxyl group with preserved amino group-basic dyes
• Tissue sections placed inProgressive ascending soln of dye • Selective affinity of dye for staining different tissue • Less sharper • Tissue over stainedRegressive • Differentiated • Routinely used staining
Methyline blue, eosin Indirect-dye+mordant=colored lake;combine with tissue-mordant-dye complex Insoluble in ordinary acqeous or alcoholic solvent,allowing counterstaining & dehydration
De-staining basic dyes with- weakly acidic medium mordant oxidizing agent dyes Aqueous haematoxylin differentiated in acidified alcohol(1% HCl in 95% alcohol) Eosin differentiated in alcohol 0.5%conc ammonium hydroxide
Ripening of stain Well at room temperature some require refrigeration –schiff‟s reagent,aldehyde fuschin,methyl- green,azocarmine,silver nitrate.
EQIPMENT AND MATERIALS 15cm deep sink,two taps,white background A slide washing tray Staining rack-two stout glass rods,4cm apart Bunsen burner
Bright daylight Microscope Glass lidded jars-for stains,grooved to hold 6 slides-coplin jars Stainless steel racks-10-20 slides COPLIN JAR
UniMailer Slide Storage System Slide Folder Rack
Removal of paraffin wax-two changes Removal of xylene with absolute alcohol Treatment with descending grades of alcohol Water Staining Dehydration Clearing mounting
• Xylene, decreasing concentration of I. Hydration alcohol-water II. Staining with • over stained 2-20 minutes haematoxylinIII. Differentiation • By acidified alcohol IV. Blueing • Water or lithium carbonate V. Dehydration • Increasing con. Of alcohol up to 95%VI. Staining with eosin • 0.5-1 % eosin in 90% alcohol 3 sec. to 1 min. VII. Clearing • Xylene VIII. Mounting • With Canada balsam
MOUNTING-used between section and coverslip- 1.resinous media(xylene preparation) 2.aqueous media(water preparations)- KAISER‟S GLYSERINE-JELLY APATHY‟S MOUNTANT 1.RESINOUS MEDIA-xylene balsam colophonium –terpentine euparal xam D.P.X.(distrene-polystyrene plasticizer-tricresyl phosphate,xylene B.P.S.(butyl,phthalate,styrene
RINGING MEDIA-mount which fail to set completely hard sealed at margin Solid media-paraffin wax,kronig‟s cement Commercially available-cellulose adhesive Durofix
Labelling of slides- Fading of stained section-
1- PERIODIC ACID SCHIFFS (PAS )- Principle: periodic acid oxidizes the carbon to carbon bond forming aldehydes which react to the fuchsin-sulfurous acid which form the magenta color. (Periodic Acid cleaves sugars into aldehyde groups. Aldehydes react with Schiff Reagent- RED) Amyloid ,BM,cartilage,cellulose,cerebrosides,epithelial mucins,fungi,glycogen,hyaline membrane fetal lung,lipochrome pigment,mucoid cells of ant lobe of pituitary,pancreatic zymogen granules,starch,thyroid colloid Results: Glycogen: magenta (red)
Feulgen Reaction: Active aldehyde group by breaking purine- deoxyribose bond - DNA (not RNA) is cleaved by HCl, reacts w/Schiff. Acidic phosphate radicle is reason for basophilia-methyl green pyronin technique
Methyl Green Pyronin StainDNA: blue-green to greenRNA: pink to red
PURPOSE: Alcian blue stains acid mucus substances and acidic mucins. PRINCIPLE: Alcian blue is a group of polyvalent basic dyes that are water soluble. The blue color is due to the presence of copper in the molecule. - Alcian blue stains both sulfated and carboxylated acid mucopolysaccharides and sulfated and carboxylated glycoproteins. - It is believed to form salt linkages with the acid groups of acid mucopolysaccharides. RESULTS: Acid mucins/mucus substances: blue cell nuclei:red background:yellow
Purpose: To differentiate between neutral and acidic mucus substances. Routine stain for G.I. biopsies. Results: Acid mucus substances: blue Neutral polysaccharides: magenta
PURPOSE: acid mucopolysaccharides (mucin), which is a secretion produced by a variety of epithelial cells and connective tissue cells. The mucicarmine technique is also useful in determining the site of a primary tumor in that finding mucin positive tumor cells. Principle: aluminum is believed to form a chelation complex with the carmine, changing the molecule to a positive charge allowing it to bind with the acid substrates of low density such as mucins. Results: Mucin: deep rose Nuclei: black Other tissue elements: yellow
Mucin: deep roseNuclei: blackOther tissue elements: yellow
The Azan-Mallory stain is one of several commonly used techniques in which three or more dyes are combined. These multiple-dye stains have the advantage of showing a large number of tissue structures. The Azan-Mallorys stain combines aniline blue, orange G (stains proteins) and acid fuchsin (stains DNA and RNA). Collagen-containing connective tissue is shown as blue, erythrocytes as orange, and chromatin, nucleoli, basophilic cytoplasm, and muscle cell cytoplasm as red. With azocarmine and aniline blue (Azan) stain, a combination of the basophilic dye (azocarmine) with aniline blue stains nuclei and basic structures are stained red and collagen, mucus, and cartilage matrix are stained blue
Figure 1. Weigert‟s Iron Hematoxylindemonstrating nuclear detail prior tomuscle and collagen staining. 20X
Used to differentiate between collagen and smooth muscle in tumors, and the increase of collagen in diseases such as cirrhosis. Routine stain for liver and kidney biopsies. the name implies, three dyes are employed selectively staining muscle, collagen fibers, fibrin, and erythrocytes. The general rule in trichrome staining is that the less porous tissues are colored by the smallest dye molecule; whenever a dye of large molecular size is able to penetrate, it will always do so at the expense of the smaller molecule. Others suggest that the tissue is stained first with the acid dye, Biebrich Scarlet, which binds with the acidophilic tissue components. Then when treated with the phospho acids, the less permeable components retain the red, while the red is pulled out of the collagen. At the same time causing a link with the collagen to bind with the aniline blue.
The trichrome stain is utilized as the stain of choice of distinguishing histologic changes in tumors, connective tissue diseases, muscle and fibroblast tumors, renal diseases and dermatology cases. Even the disciplines of forensics, archaeology and hematopathology incorporate the trichrome stain for specific tissue entities and structures. With the utilization of immunohistochemistry expressions, the trichrome techniques still offer a great deal of diagnostic results
There are hundreds of other staining routines, most of which involve the use of gold or silver salts. Among the most elegant of these stains are the ones developed by Camillo Golgi (1843-1926) or Santiago Ramon y Cajal (1852-1934), who shared a Nobel prize for their work in 1906. These methods are especially useful for visualizing glial elements. Both these men are great figures of the history of the life sciences and the study of the nervous system in particular. Golgi developed several stains that are still used today, was the discoverer of several important nervous system structures, and won the Nobel Prize for his work. Golgis stains comprise a set of methods for nerve cells and fibers; theyre characterized by fixation in an aldehyde- osmium-dichromate solution, followed by impregnation with silver salts. As you can see here, the process renders the subject as several shades of golds, browns and blacks. Neuron somata are golden and their processes black. This stain permits the definition of much detailed information about the structure of the nervous system.
Basic stain-base contains coloring substance combined with acidic radicle-Basic fuchsin Acidic stain- Romanowsky-combination of polychrome methylene blue and eosin Colorless leucobases-dyes can be reduced easily
Massons Trichrome Stain Muscles (red) Massons Trichrome Stain Collagen (green or blue) Massons Trichrome Stain Mucus (green or blue) Massons Trichrome Stain Cytoplasm of most cells (pink) Massons Trichrome Stain Glycogen (deep red or magenta) Periodic Acid Schiff (PAS) Reaction Contents of goblet cells (red or magenta) Periodic Acid Schiff (PAS) Reaction Basement membrane (positive or pink) Periodic Acid Schiff (PAS) Reaction Brush borders in kidney tubules (positive or pink) Periodic Acid Schiff (PAS) Reaction Elastic fibers (jet black) Verhoeffs Stain for Elastic Tissue Nuclei (gray) Verhoeffs Stain for Elastic Tissue Remaining structures (pink) Verhoeffs Stain for Elastic Tissue Fibrous c.t. (deep blue) Mallory-Azan Stain Mucus (deep blue) Mallory-Azan Stain Erythrocytes (red-orange) Mallory-Azan Stain Cytoplasm of liver (pink) Mallory-Azan Stain Cytoplasm of kidney (pink) Mallory-Azan Stain Nuclei (red) Mallory-Azan Stain Erythrocyte cytoplasm (pink) Mallory-Azan Stain Lymphocyte nuclei (dark purple-blue) Mallory-Azan Stain Lymphocyte cytoplasm (pale blue) Mallory-Azan Stain Monocyte nuclei (medium blue) Mallory-Azan Stain Monocyte cytoplasm (pale blue) Mallory-Azan Stain Neutrophil nuclei (dark blue) Mallory-Azan Stain Eosinophil nuclei (dark blue) Mallory-Azan Stain Eosinophil granules (bright pink) Mallory-Azan Stain Basophil granules (deep purple) Mallory-Azan Stain Platelets (light blue) Mallory-Azan Stain Myelinated fibers (blue-black) Cajals and Del Rio Hortegas Methods (silver and gold) Unmyelinated fibers (blue-black) Cajals and Del Rio Hortegas Methods (silver and gold) Neurofibrils (blue-black) Cajals and Del Rio Hortegas Methods (silver and gold) General background (nearly colorless) Cajals and Del Rio Hortegas Methods (silver and gold) Astrocytes (black) Cajals and Del Rio Hortegas Methods (silver and gold) End product of stain (can be black, brown or gold) Cajals and Del Rio Hortegas Methods (silver and gold) Lipids in general (black) Osmic Acid (Osmium Tetroxide) Stain Lipids in the myelin sheath of nerves (black) Osmic Acid (Osmium Tetroxide) Stain Elastic fibers (brown-reddish) Orcein Stain
Widely utilized techniques are the Masson, Gomori One Step, Martius Scarlet Blue and Mallory. ionized acid dyes react with the ionized basic tissues. fibrils of collagen stained blue, fibroglia, neuroglia and muscle fibers stained red and fibrils of elastin stained pink or yellow. The trichrome stain is also used to distinguish tumors that have arisen from muscle cells and fibroblasts. Gomori‟s trichrome is the trichrome stain of choice for distinguishing histological changes that occur in neuromuscular diseases.