Seminar 3 Special Stains

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Special Stains for use in Oral Pathology

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Special stains:

Special stains

Why are the special stains special ?:

Why are the special stains special ?

Objectives of Special Staining :

Objectives of Special Staining To provide a definitive diagnosis . In a differential diagnosis of oral lesions in or around the oral cavity. e.g. To distinguish a tumor of salivary gland origin from an odontogenic tumor or a tumor arising from non-glandular epithelium

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Stains for collagen Stains for muscle Stains for elastic tissue Stains for reticulin fibres Stains for carbohydrates Stains for amyloid Stains for lipid Stains for pigments & minerals Stains for nerve tissue Stains for microorganisms Stains for decalcified bone Broadly classifying the special stains under following category…..

Demonstration of Collagen:

Demonstration of Collagen Massons trichrome stain Van Geisons stain

MASSON'S TRICHROME STAIN :

MASSON'S TRICHROME STAIN PRINCIPLE: The term ‘ trichrome stain’ is a general name for a number of techniques for selectively demonstration of 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.

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As a general rule, a three – dimensional, insoluble protein ‘network’ is formed (i.e.; protein component of a tissue + fixative in use) different proteins will form networks with different physical feature. For example, Erythrocyte protein will produce a very dense network , with only small pores between the protein elements. Muscle cells will form a more open structure with large pores. Collagen will show the least dense network and is quite porous .

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Another hypothesis is 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 color , while the red color is pulled out of the collagen. At the same time the collagen stained with the aniline blue.

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The three dyes : Aniline Blue (stains collagen and mucus to blue or blue green) Beibrich Scarlet (stains cytoplasm, muscle and keratin to red) Weigert's Iron Hematoxylin (stains nuclei blue to black). PURPOSE: - 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

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. Factors affecting trichrome staining Tissue permeability and dye-molecular size Heat (Increases the rate at which staining occurs) Acidic pH solution is used (pH- 1.5 to pH 3.0) FIXATION:- If the fixation in formaldehyde has been prolonged the result will be even less satisfactory ,due to the saturation of tissue group with formaldehyde To improve the intensity of the staining reaction, Zenker’s solution, formol-mercury,Bouin’s fixative or picro -mercury alcohol are the most satisfactory fixative for this stain.

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Solution: Bouin’s solution Picric acid saturated aqueous solution 75cc Formaldehyde, 37- 40% 25cc Glacial acetic acid 5.0cc Weigert’s Iron Hematoxylin Solution A , Solution B and Working solution Biebrich Scarlet-Acid Fuchsin Solution Biebrich Scarlet, aqueous 1% 90 cc Acid fuchsin, aqueous, 1% 10 cc Glacial acetic acid 1.0 cc

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Phosphomolybdic – Phosphotungstic Acid Solution Phosphomolybdic acid – 5.0gm Phosphotungstic acid - 5.0gm Distilled water - 200 cc 1% Acetic Water Solution Glacial acetic acid 1.0cc Distilled water 100cc Aniline Blue Solution Aniline blue 2.5 gm Acetic acid 2.0 cc Distilled water 100 cc Light Green Solution Light green 5.0 cc Distilled water 250 cc Glacial acetic acid 2.0cc Heat water, dissolve light green, cool, filter and add acid

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Staining procedure Xylene Absolute alcohol 95% alcohol Rinse in distilled water Mordant in Bouin’s fixative for 1 hour at 56°c Cool and wash in running water until yellow colour appears Rinse in distilled water Weigert’s iron haematoxylin solution for 10 min. wash in running water 10 min. 9. Rinse in distilled water Biebrich Scarlet- acid fuchsin solution for 10 min. Rinse in distilled water

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12. Phosphomolybdic acid- Phosphotungstic acid solution for 10 – 15 min before aniline blue solution. Aqueous phosphotungstic acid 5 % for 15 min before light green counterstain. 13. Aniline blue solution for 5 – 10 min or light green solution for 1 min. 14. Rinse in distilled water. 15. Acetic water 1 % for 3 to 5 min. 16. Alcohol, 95 % 17. Absolute alcohol - 3 changes 18. Xylene - 2 changes 19. Mount in DPX

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Human skin Results Nuclei – Blue- Black Cytoplasm, keratin, – Red muscle fibers, intercellular fibers RBCs, Fibrin Collagen, mucous, – Blue Cartilage, Amyloid, adult or mature bone

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Van Gieson’s Stain (1889) Van Gieson’s mixture of picric acid and acid fuchsin The simplest method for the differential staining of collagen. Principle Picric acid is employed. It provides acidic pH and acts as a counterstain for muscle and cell cytoplasm. It forms with dyes a complex which has affinity for collagen.

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Disadvantage: It is unable to stain young fibrils ,the deep red that is imparted to mature collagen The tendency for the red colour to fade ; whatever mounting medium is used To avoid this fading, Curtis (1905) suggested the use of ponceau S as a substitute for acid fuchsin but this dye, unfortunately, stains young collagen fibres less well than does acid fuchsin.

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Solution: Weigert’s Iron Hematoxylin Solution A Hematoxylin 1.0 gm Absolute alcohol 100.0 cc Solution B 29% ferric chloride 4.0 cc Distilled water 95.0 cc Conc. Hydrochloric acid 10.0 cc Working solution Equal parts of Solution A and Solution B Van Gieson’s Solution Acid Fuchsin, 1% aqueous solution 25 cc Picric acid, saturated aqueous solution 97.5 cc Fixative :- Any well-fixed tissue may be used.

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Staining Procedure Xylene Absolute alcohol 95% alcohol Rinse in distilled water Stain in Weigert’s hematoxylin solution for 10 min. Wash in distilled water Counterstain in van Gieson’s solution for 1-3 min. 95% alcohol Absolute alcohol – 2 changes Xylene – 2 changes Mount in DPX

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Results: Collagen – deep red Muscle, – yellow cornified epithelium Nuclei – blue to black

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Note: - Running water will remove van Gieson’s solution - Nuclei stained with alum hematoxylin readily decolorized with picric acid in stain

Demonstration of fibrin ::

Demonstration of fibrin : Gram Weigert Phosphotungstic acid hematoxylin Early fibrin deposits may be stained by these dyes, although the phosphotungstic acid blocks the staining of muscle, collagen .and most of the connective tissue fibres .

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Demonstration of muscle striations - Haematoxylin and eosin and trichrome methods can demonstrate muscle striations. - They may also be stained by using Heidenhain iron haematoxylin and Mallory’s phosphotungstic acid haematoxylin. Both these methods will give better definition of muscle striations than the trichromes.

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Mallory's PTAH stain Purple skeletal muscle striations in tumor with rhabdomyoblasts .

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Demonstration of elastic tissue fibres - Numerous techniques have been evolved for the demonstration of elastic tissue fibres, although few are in current use. - Of these, the most popular are . Verhoeff , . Orcein, . Weigert’s resorcin-Fuchsin, . Aldehyde fuchsin.

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Verhoff’s Elastic Stain Verhoeff's Elastic stain is used to demonstrate pathologic conditions such as atrophy, breaks, thinning, loss etc. in elastic fibers.

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Principle The tissue is overstained with a soluble lake of hematoxylin-ferric chloride-iodine. Both ferric chloride and iodine serve as mordants, but they also have an oxidizing function that assists in converting hematoxylin to hematein. The mechanism of dye binding is probably by formation of hydrogen bonds, but the exact chemical groups reacting with the hematoxylin have not been identified. This method requires that the sections be overstained and then differentiated, so it is regressive.

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Differentiation is accomplished by using excess mordant, or ferric chloride, to break the tissue-mordant-dye complex. The dye will be attracted to the larger amount of mordant in the differentiating solution and will be removed from the tissue. The elastic tissue has the strongest affinity for the iron-hematoxylin complex and will retain the dye longer than the other tissue elements. This allows other elements to be decolorized and the elastic fibers to remain stained. Sodium thiosulfate is used to remove excess iodine. Van Gieson's solution is the most commonly used counterstain, but others may be used.

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Solution : Elastic tissue stain Hematoxylin - 1 gm Absolute alcohol - 22 cc 10 % Aqueous solution of FeCl 3 - 8 cc Iodine solution - 8 cc Ferric Chloride solution Ferric Chloride - 2 gm Distilled Water- 100 cc Van Gieson’s Stain Acid Fuschin Aqueous solution 1%- 5cc Saturated Aqueous Picric acid solution – 100 cc Sodium Thiosulphate solution Sodium Thiosulphate – 5 gm Distilled water - 100 cc

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Staining Procedure Deparafinization and wash Verhoff’s Elastic tissue stain for 15 min Wash in Distilled water Differentiating 2% FeCl 3 - only a few minutes Place in 5% sodium thiosulphate for 1 min Wash in tap water for 5 min Counterstain in Von Gieson’s stain for 1 min Absolute alcohol - two changes Xylene – two changes Mount in DPX

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Results : Elastic Fibres – Bluish Black to Black Nuclei - Blue to Black Collagen - Red Other tissue elements – Yellow

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This section shows elastic cartilage stained with hematoxylin and potassium iodine to reveal elastic fibers (arrow) , which are dark-stained linear structures embedded in the cartilage matrix.

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Demonstration of reticulin fibres : Reticulin fibres are demonstrated either by using dyes as means of coloring agent or by metal impregnation methods. Dye techniques :1.Gordon and sweet’s method, 2. Gomori’s method, Reticulin fibre demonstration cannot be considered completely reliable by dye method Silver impregnation is the best method because it provides good contrast enabling even the finest fibers to be resolved.

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Wilder’s Reticulin Stain Used to demonstrate reticular fibers in tissue sections Differential diagnosis of carcinomas, sarcomas, and lymphosarcomas. Certain liver diseases where a change from normal reticular fiber pattern is seen.

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Solutions Phosphomolybdic Acid solution Phosphomolybdic Acid – 10 mg Distilled water - 100 cc Uranium Nitrate solution Uranium nitrate – 1 gm Distilled water - 100 cc Ammoniacal silver solution 10.2 % aqueous solution of silver nitrate- 5 cc 28 % ammonia water – drop by drop 3.1 % sodium hydroxide – 5 cc Make the solution to 50cc with distilled water.

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Reducing solutions Distilled water - 50 cc Neutral formaldehyde, 40% - 0.5 cc Uranium nitrate 1% aqueous solution – 1.5 cc Gold Chloride solution 1% Gold Chloride solution - Distilled water – 40 cc Sodium Thiosulphate solution Sodium Thiosulphate – 5 gm Distilled water - 100 cc Nuclear fast red stain Nuclear fast red - 0.1 gm 5 % solution of aluminium sulphate – 100 cc

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Staining Procedure 1. Xylene, Absolute alcohol, 95% alcohol, Distilled water 2. Phosphomolybdic acid for 1 min (oxidizer) 3. Rinse well in running water 4. Dip in 1% aqeuous uranium nitrate for 5 seconds or less 5. Wash in Distilled water for 10 to 20 min 6. Place in ammoniacal silver solution for 1 min 7. Dip quickly in 95% alcohol and then immediately into reducing solution for 1min

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8. Rinse well in distilled water 9. Tone in Gold chloride solution for 1 min 10. Rinse in distilled water 11. Place in 5% Sodium Thiosulphate for 1 to 5 min 12. Wash well in tap water 13.Counter stain with Alum Hematoxylin and Eosin or Nuclei fast red. Rinse with distilled water 14.95% alcohol 15.Absolute alcohol - 2 changes 16.Xylene - 2 changes 17.Mount in DPX

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Result Reticulin fiber – Black Collagen – Rose color Other tissue elements – Depending on counter stain used

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Buccal mucosa. Reticulin stain delineates the cell membrane of nonkeratinized squamous epithelial cells and the delicate basement membrane. Papillae of the lamina propria contain blood vessels .

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Mechanism of PAS technique ? Differentiation of the mucin by different stains ? Stains for carbohydrates ? Clinical application of PAS stain?

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STAINS FOR CARBOHYDRATE Periodic Acid Schiff Stain Alcian Blue Mucicarmine Stain – Southgate’s

 PERIODIC ACID SCHIFF (PAS )FOR THE DEMONSTRATION OF GLYCOGEN :

PERIODIC ACID SCHIFF (PAS )FOR THE DEMONSTRATION OF GLYCOGEN McManus, 1946) Glycogen is a simple intracytoplasmic polysaccharide found in greatest amount in liver, cardiac and skeletal muscle , with significant quantities also present in hair follicles, endometrial glands, vaginal and cervical epithelium, umbilical cord, neutrophil leucocytes and megakaryocytes.

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PRINCIPLE Tissue containing vicinal glycol groups or their amino or alkylamino derivatives are oxidized by periodic acid to form dialdehydes, which combine with Schiff's reagent to form an insoluble magenta compound. The chromphoric groups of basic fuchsin in Schiff's reagent are broken by sulphuration to form a colorless solution. In the presence of free aldehyde groups an insoluble colored compound similar to the original dye is formed.

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PAS Technique: - Periodic Acid Solution Periodic acid - 1 gm Distilled water - 200 ml - Schiff Reagent Dissolve 1gm of basic fuchsin in 200 ml of boiling distilled water. Allow the solution to cool 50 °c, and add 2-gm potassium metabisulphite with mixing. Allow to cool to room temperature then add 2 ml conc. Hydrochloric acid, mix, add 2 gm activated charcoal and leave overnight in the dark at room temperature. Filter, the solution should be either clear or pale yellow color. Store in a dark container at 4°c.

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SPECIMEN Standard paraffin section fixed in 10% neutral buffered formalin. PROCEDURE 1. Deparaffinize and hydrate to distilled water. 2. Oxidise with periodic acid for 5 minutes. 3. Rinse in distilled water. 4. Treat with Schiff's reagent for 5 minutes. 5. Wash in running water for 10 minutes; this intensifies the colour reaction. 6. Stain the nuclei with Mayer's haematoxylin for 1 minute (no need to blue). 7. Wash in water. 8. Dehydrate, clear and mount.

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Results: PAS positive substances - magenta Nuclei - blue

PERIODIC ACID SCHIFF (PAS) WITH DIASTASE FOR THE ENZYME EXTRACTION OF GLYCOGEN:

PERIODIC ACID SCHIFF (PAS) WITH DIASTASE FOR THE ENZYME EXTRACTION OF GLYCOGEN PRINCIPLE Glycogen is digested with certain forms of amylase. Commercially available diastase, which á amylase or salivary amylase from saliva can be used to digest glycogen in tissue sections. Purpose : - Glycogen is present in mucosa, skin, liver, parathyroid gland and skeletal & cardiac muscle. - The PAS stain is used for demonstration of basement membrane, mucosubstances secreted by the epithelia of various organs, fungi, etc.

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REAGENTS (A) AMYLASE SOLUTION - Amylase Type VI-B from porcine pancreas (Sigma A 3176)- 0.25g (stored in fridge) - Distilled water 10mL If larger quantity required, use 1g Amylase in 40mL distilled water. Prepare fresh just before use. Warm water for 5 sec in microwave oven then add amylase. (B) PAS TECHNIQUE for PAS reagents NOTE : Place waste amylase into container and place into yellow biohazard bag for disposal by incineration..

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PROCEDURE 1. Deparaffinise two positive controls and two test sections and hydrate to distilled water. 2. Treat one control and one test section with Amylase for 25 minutes to digest Glycogen. 3. Wash in running water for 20 minutes. 4. Proceed with P.A.S. technique as follows. 5. Oxidise with periodic acid for 5 minutes. 6. Rinse in distilled water. 7. Treat with Schiff's reagent for 5 minutes. 8. Wash in running water for 10 minutes, this intensifies the color reaction. 9. Stain the nuclei with Mayer's haematoxylin 1 minute (no need to blue). 10. Wash in water. 11. Dehydrate, clear and mount.

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RESULTS Presence of glycogen will be evidenced by loss of staining after enzyme treatment when compared to the untreated sections. PAS Stain PAS with Diastase

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Glycogen in Ewing Sarcoma

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Note The most important PAS positive carbohydrates in tissues are polysaccharides (glycogen),Neutral mucopolysaccharides, mucoproteins, glyco proteins, and glycolipids. Acid mucopolyssacharides are only weakly positive or negative. The PAS reaction can be used to demonstrate many other normal and pathological tissue constituents, the most important of which are amyloid, basement membrane, cartilage, cerebrosides, epithelial mucins, fungi, hyaline membrane of neonatal lung, lipochrome pigments, mucoid cells, mucoid granules, zymogen granules, starch, thyroid colloid etc.

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The types of mucopolysaccharides are as follows: Neutral - These can be found in glands of the GI tract and in prostate. They stain with PAS but not with Alcian blue, colloidal iron, mucicarmine, or metachromatic dyes. Acid (simple, or non-sulfated) - Are the typical mucins of epithelial cells containing sialic acid. They stain with PAS, Alcin blue at pH 2.5, colloidal iron, and metachromatic dyes. They resist hyaluronidase digestion. Acid (simple, mesenchymal) - These contain hyaluronic acid and are found in tissue stroma. They do not stain with PAS, but do stain with Alcian blue at pH 2.5, colloidal iron, and metachromatic dyes. They digest with hyaluronic acid. They can be found in sarcomas.

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Acid (complex, or sulfated, epithelial) - These are found in adenocarcinomas. PAS is usually positive. Alcian blue is positive at pH 1, and colloidal iron, mucicarmine, and metachromatic stains are also positive. They resist digestion with hyaluronidase. Acid (complex, connective tissue) - Found in tissue stroma, cartilage, and bone and include substances such as chondroitin sulfate or keratan sulfate. They are PAS negative but do stain selectively with Alcian blue at pH 0.5.

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PAS stain for fungi The fungal cell wall contains polysaccharides that are oxidized by the periodic acid to aldehydes. The aldehydes react with Schiff's reagent to stain the fungi rose pink.

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Candida Hyphae in chronic hyperplastic Candidosis PAS-positive materials can simply be recognized by their shape (morphology) , eg.Fungal hyphae

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Clinical Application of PAS Stain Salivary Gland Tumors Odontogenic Tumors Adenomatoid Odontogenic Tumor Calcifying Epithelial Odontogenic Cyst Soft tissue tumors Alveolar Soft-Part Sarcoma (Diastase resistant crystals) Fungal Infection Candidiasis Actinomycosis Histoplasmosis Blastomycosis

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ALCIAN BLUE Ph2.5 – ACID MUCOPOLYSACCHARIDES 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. Acid mucin - strongly sulphated connective tissue mucins react at low pH values with suitable cationic dyes and are usually PAS-negative. .

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PRINCIPLE Alcian blue is a copper phthalcyanin dye and contains positively charged groups capable of salt linkage with certain polyanions. These polyanions consist of the sulphate and carbocyl radicals of the acid mucins - the phosphate radicals of the nucleic acids do not react. Consequently, only the acid mucins are stained. By varying the pH of the solution of Alcian blue more information can be gained concerning the types of acid mucin present.

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PURPOSE: Alcian blue stains acid mucosubstances and acidic mucins. Excessive amounts of non-sulfated acidic mucosubstances are seen in mesotheliomas, certain amounts occur normally in blood vessel walls but increase in early lesions of atherosclerosis.

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REAGENTS: 3% Glacial Acetic Acid Acetic acid -3.0 ml Distilled water -100.0 ml Alcian Blue Solution: 3% glacial acetic acid-100.0ml Alcian blue 8GX -1.0 gm Mix, adjust pH to 2.5, using acetic acid. Filter, add a crystal of thymol, label with initial and date. Nuclear Fast Red (Kernechtrot): Aluminum sulfate 25.0 gm Distilled water 500.0 ml Nuclear fast red 0.5 gm Dissolve the aluminum sulfate in the water. Add the nuclear fast red, dissolve with aid of heat. Filter, add a crystal of thymol SPECIMEN Standard paraffin section fixed in neutral buffered formalin

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PROCEDURE: 1. Hydrate slides to distilled water. 2. 3% acetic acid, 3 minutes. 3. Alcian blue solution, 30 seconds. 4. Wash in running water for 2 minutes, rinse in distilled. 5. Nuclear-fast red, 5 minutes, wash in tap water. 6. Dehydrate, clear, and coverslip.

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Submandibular salivary gland mucous cell showing sulfomucin Brown black, sialomucin blue, -Iron alcian blue technique RESULTS: Acid mucins /mucosubstances - blue Nuclei (using Nuclear fast red) -reddish pink Background - Very pale red or colourless

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Colonic mucosa showing sialomucin that have acid and neutral mucopolysaccharide stain purple

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Mixed minor salivary gland of the uvula. Acid mucopolysaccharides in mucous cells are stained turquoise ( Alcian blue at pH 2.6).

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The goblet cells of the gastrointestinal tract are filled with abundant acid mucin and stain pale blue with this Alcian blue stain.

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MUCICARMINE STAIN – SOUTHGATE’S – MUCIN PRINCIPLE :- Mucicarmine has large molecular size which allows the dye to penetrate and bind to acidic substrates of low density, i.e. mucins. Neutral mucins and some strongly acidic sulphated mucins do not show appreciable staining.

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PURPOSE: To stain mucin which is a secretion produced by a variety of epithelial cells and connective tissue cells. Excess mucin is secreted by epithelial cells in certain inflammations and in certain intestinal carcinomas . The mucicarmine technique is also useful in determining the site of a primary tumor. Finding mucin positive tumor cells in an area that does not contain mucin producing cells would indicate the tumor did not arise from that area. It is also useful in staining encapsulated fungi; cryptococcus .

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SPECIMEN Standard paraffin section fixed in 10% neutral buffered formalin. REAGENTS: Southgate's MucicarmineSolution: Carmine, alum lake -1.0 gm Aluminum hydroxide -1.0 gm 50% alcohol -100.0 ml Aluminum chloride, anhydrous- 0.5 gm Mayer's Hematoxylin: Metanil Yellow Solution: Metanil yellow -0.25 gm Distilled water -100.0 ml Glacial acetic acid -0.25 ml

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PROCEDURE: 1. Deparaffinize and hydrate to distilled water. 2. Mayer's hematoxylin for 10 minutes. 3. Wash in running tap water, 5 minutes. 4. Mucicarmine solution, 45 seconds. 5. Rinse quickly in distilled water. 6. Metanil yellow, 30 seconds to 1 minute. 7. Dehydrate quickly in three changes of absolute alcohol, clear and coverslip

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Colonic Mucosa showing sialomsucin stain -- - magenta RESULTS: Mucin - deep rose Nuclei - black Other tissue elements- yellow

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Mucoepidermoid carcinoma, mucin positive

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Demonstration of amyloid Congo red Thioflavin T Crystal Violet

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Congo-Red - Putchler’s Modification – Amyloid Principle : Amyloid is homogeneous and eosinophilic , the deposits are extracellular and may become sufficiently large enough to cause damage to surrounding tissues . When stained with the congo red stain the amyloid, with aid of polarizing lenses, will birefringe an apple green color , under the microscope. Purpose : To demonstrate amyloid deposits in tissue sections

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REAGENTS Mayer's Hematoxylin : Hematoxylin - 1 gm Distilled water- 1000 ml Sodium iodate -0.2 gm Ammonium or Potassium aluminum sulfate - 50 gm Citric acid – 1 gm Chloral hydrate – 50 gm 1% Sodium Hydroxide Sodium hydroxide 1.0 gm Distilled water 100.0 ml Sodium Chloride, Stock Solution, Saturated: Sodium chloride 30.0 gm Distilled water 200.0 ml Mix well, then add: 100% alcohol 800.0 ml

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Working Solution: Stock sodium chloride 50.0 ml 1% sodium hydroxide 0.5 ml Congo Red Stock Solution,Saturated: Congo red stain 1.0 gm Stock sodium chloride 500.0 ml Working Solution: Stock Congo red solution 50.0 ml 1% sodium hydroxide 0.5 ml

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PROCEDURE: 1. Deparaffinize and hydrate to water. 2. Mayer's Hematoxylin for 10 minutes. 3. Wash in tap water until blue 4. Working sodium chloride solution, allow slides to sit in solution for 2-5minutes. 5. Place directly into Working Congo red solution, Allow slides to sit in solution for 2-5 minutes. 6. Dehydrate rapidly in absolute alcohol, 10 dips, 3 changes. 7. Clear in xylene & mount.

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RESULTS: Amyloid -red to pink Nuclei -blue

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Puchtler and Sweat Congo Red Method Polarised Light showing “apple green”birefringence of amyloid deposits

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Positive red staining is present around the large central artery and a smaller vessel to its upper right. The right panel shows the green birefringence that is diagnostic of amyloid when the Congo red stain is viewed with polarized light. All amyloids have a fibrillar ultrastructure that gives this reaction.

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Clinical Application Calcifying Epithelial Odontogenic Tumors : Extracellular amyloid of epithelial origin present in tumor. Keratin protein forms an important component of the amyloid in this tumor.

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Sections must be examined with a fluorescent microscope revealing a yellow to yellow green appearance

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In epidermis a cornoid lamella without underlying granular layer and hydropic degeneration of basal cell layer. In upper dermis there are a mononuclear inflammatory infiltrate, and a deposit of eosinophilic amorphous material (H&E, × 100). Dermal amyloid deposits under polarizing microscopy ( Thioflavin -T stain, × 200).

Crystal violet:

Crystal violet Metachromatically results in a red – purple colour of amyloid APPLICATION: Amyloidosis (pink staining in a blue background)

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Stains for demonstration of lipid and principle?

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Stains for Lipid - Oil Red O - Sudan Black B

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PRINCIPLE : Staining with oil-soluble dyes is based on the greater solubility of the dye in the lipid substances than in the usual hydroalcoholic dye solvents .

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PURPOSE : To demonstrate fat or lipids in fresh tissue sections. Fat occurring in an abnormal place, such as fatty emboli that may develop after either a bone fracture or an injury that crushes a fatty body area. Tumors arising from fat cells (liposarcomas) can be differentiated from other types of tumors.

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REAGENTS : 85% Propylene Glycol: Propylene glycol -85.0 ml Distilled water -15.0 ml Hematoxylin: Glycerin Jelly Oil Red O Solution: Oil red O -0.7 gm Propylene glycol -100.0 ml Dissolve oil red O in propylene glycol , slowly, while stirring. Heat to 100°C, but not over 110°C, for a few minutes, stirring constantly. Filter through Whatman #2 filter paper . Cool, and filter again through a frittered glass filter of medium porosity with suction.

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PROCEDURE : 1. Fix slides in 10% formalin if fresh. 2. Wash well in tap, rinse in distilled, drain off excess water. 3. Propylene glycol, two changes, 5 minutes each. 4. Oil red O, 7 minutes, agitate. 5. 85% Propylene glycol, 3 minutes. 6. Rinse in distilled water. 7. Hematoxylin, 1 minute. 8. Wash in water. 9. Bluing solution, 20 dips, or running tap water. 10. Wash in tap water, rinse in distilled. 11. Mount with aqueous mounting media, Glycerin Jelly.

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sebaceous gland stained with oil red O stain Cytoplasm – red Nuclei - blue

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Oil red O stain of fat emboli in lung.

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Lipid in skin

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FAT - SUDAN BLACK B - PROPYLENE GLYCOL PRINCIPLE : Sudan Black is slightly basic dye and will combine with acidic groups in compound lipids , thus staining phospholipids also. PURPOSE : For the demonstration of fat.

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REAGENTS : 85% Propylene Glycol: Propylene glycol -85.0 ml Distilled water -15.0 ml Hematoxylin: Sudan Black B/Propylene: Sudan Black B -0.7 gm Propylene glycol -100.0 ml

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PROCEDURE : 1. Fix slides in 10% formalin if fresh. 2. Wash well in tap water, rinse in distilled, drain off excess water. 3. Propylene glycol, two changes, 5 minutes each. 4. Sudan Black, 7 minutes, agitate. 5. 85% Propylene glycol, 3 minutes. 6 Rinse in distilled water. 7. Nuclear Fast Red, 3 minutes. 8. Wash in water. 9. Wash in tap water, rinse in distilled. 10. Mount with aqueous mounting media, Glycerin Jelly.

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RESULTS : Fat blue -black Nuclei -red

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STAINS FOR PIGMENTS AND MINERALS Masson Fontana Method DOPA method Perl’s Prussian Blue Method Von Kossa stain for Calcium

MASSON FONTANA METHOD:

MASSON FONTANA METHOD PRINCIPLE: Melanin is insoluble in organic solvents but soluble in 1M sodium hydrooxide . It is slowly bleached by strong oxidising agents. The solutions of ammoniacal silver nitrate are reduced by melanin to black metallic silver this is the basis of Massons Fontana method for demonstrating melanin.

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PURPOSE: - To identify argentaffin granules and melanin . Melanin is a nonlipid, non hematogenous pigment.It is a brown-black pigment present normally in the hair, skin, retina, iris and certain parts of the CNS. Argentaffin granules are found in carcinoid tumors.

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REAGENTS: Ammoniacal Silver Stock Solution: 10% Silver nitrate 25.0 ml Add ammonium hydroxide drop by drop, until solution precipitates and clears again. 10% silver nitrate 1.0 ml 0.1% Gold Chloride: 1% Gold Chloride, Stock 5.0 ml Nuclear-Fast Red: Ammoniacal Silver Working Solution: Ammoniacal silver stock solution 12.5 ml Distilled water 37.5 ml 10% Silver Nitrate: Silver nitrate 20.0 gm Distilled water 200.0 ml

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PROCEDURE: 1. Deparaffinize and hydrate to distilled water. 2. Working Silver Solution, 2 minutes. 3. Rinse in distilled water. 4. 0.1% Gold chloride, 10 minutes. 5. Rinse in distilled water. 6. Wash in tap water, rinse in distilled water. 7. Nuclear-fast red, 5 minutes. 8. Wash in tap water. 9. Dehydrate, clear and coverslip.

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Melanin pigment of skin showing black color RESULTS: Melanin, argentaffin cells -black Nuclei -red

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Melanin pigment in cells of malignant melanoma, Fontana-Masson stain.

DOPA METHOD:

DOPA METHOD Principle : The enzyme tyrosinase is located within some cells producing melanin will oxidize DOPA to form an insoluble brown – black pigment . Purpose : Cells that are capable of producing melanin can be demonstrated by DOPA (dihydroxy- phenylalanine) method.

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Reagents pH buffer 7.4 Sodium Cacodylate – 42.8 gm Hydrochloric acid – 9.6 mmol Distilled water – 1000 ml Primary fixative 10% formalin in pH 7.4 buffer Sucrose - 0.44 M Incubating medium 0.1% DOPA in the pH 7.4 buffer

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Procedure : Place section in distilled water for few seconds Place section in DOPA solution for 30 min at 37° C Replace with fresh solution and then examine microscopically every 30 min ( Color of solution changes from reddish to light brown) Wash section well in several changes of distilled water Stain in Mayer’s Hematoxylin for 2 min Wash well to “blue” the section, dehydrate, clear and mount in synthetic resin

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Result DOPA oxidase – Brown Nuclei - blue

VON KOSSA METHOD FOR CALCIUM:

VON KOSSA METHOD FOR CALCIUM PRINCIPLE: Tissue sections are treated with silver nitrate solution, the calcium is reduced by the strong light and replaced with silver deposits , visualized as metallic silver. PURPOSE: Abnormal deposits of calcium may be found in any area of the body. With the H&E stain, calcium appear deep blue-purple.

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REAGENTS: 5% Silver Nitrate Solution: Silver nitrate 25.0 gm Distilled water 500.0 ml Nuclear Fast Red:

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PROCEDURE: 1. Deparaffinize and hydrate to distilled water. 2. 5% Silver solution , place in bright sunlight , or in front of a 60-wattlamp, place foil (or mirror ) behind the jar to reflect the light. Leave for 1 hour or until calcium turns black . 3. Rinse in distilled water, 3 changes. 4. Wash in tap water, rinse in distilled water. 5. Nuclear-fast Red, 5 minutes. 6. Wash in water, 7. Dehydrate, clear, and coverslip.

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Coronary artery showing calcified atheromatous plaque RESULTS : Calcium salts -black Nuclei -red Cytoplasm -pink

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PERL’S- PRUSSIAN BLUE REACTION PRINCIPLE : The reaction occurs with the treatment of sections in acid solutions of ferrocyanides . Any ferric ion in the tissue combines with the ferrocyanide and results in the formation of a bright blue pigment called 'Prussian blue" or ferric ferrocyanide .

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PURPOSE: To demonstrate ferric iron in tissue sections. Small amounts of iron are found normally in spleen and bone marrow. Excessive amount are present in hemochromatosis , with deposits found in the liver and pancreas, hemosiderosis , with deposits in the liver, spleen, and lymph nodes.

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REAGENTS: 5% Potassium Ferrocyanide: Potassium ferrocyanide - 25.0 gm Distilled water - 500.0 ml Nuclear-fast Red: 5% Hydrochloric Acid: Conc. Hydrochloric acid, - 25.0 ml Distilled water - 475.0 ml Working Solution: 5% potassium ferrocyanide -25.0 ml 5% hydrochloric acid -25.0 ml.

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PROCEDURE: 1. Deparaffinize and hydrate to distilled water. 2. Working solution. Allow slides to stand in solution for 5 minutes. 3. Rinse in distilled water. 4. Nuclear-fast red, 5 minutes. 5. Wash in tap water. 6. Dehydrate, clear, and coverslip.

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Hemosiderin , liver, iron stain. RESULTS: Iron (hemosiderin) -blue Nuclei -red Background - pink

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Demonstration of Nerve tissue Bielschowsky’s Silver Stain Golgi cox potassium dichromate- mercuric chloride method Holmes method for axons Weils haematoxylin method for myelin Luxol fast Blue method for myelin

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Bielschowsky’s Silver Stain for Neurofibrils , Dendrites and Axons Principle: The nerve fibers are sensitized with a silver solution . The sections are treated with ammoniacal silver, and then reduced to a visible metallic silver. Solution Silver A 20% silver nitrate Reducer A ( make fresh) Pyrogallol – 100 mg Formalin - 10 ml 80% alcohol – 1200 ml

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Reducer B (make fresh) Formaldehyde – 5 ml Distilled water - 95 ml Silver B (make fresh) 20% Silver nitrate - 5ml 40% NaOH - 200 µl Distilled water - 20 ml 0.88 Ammonia - 500 µl 5% Sodium thiosulphate 0.2% Gold chloride Section: Paraffin sections at 10 um thick .

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Method : Take section to distilled water Cover section with Silver A for 1 -2 hrs in 37° C in a moist chamber Rinse twice in distilled water and wash in a pot of reducer A for 3 - 5 min. Agitate regularly. The section should turn yellow Wash in 3 changes of distilled water Place in Silver B for 30 sec(Avoid prolonged treatment as it cause precipitation) Wash section in a pot of reducer for 2 – 5 min

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7. Wash in distilled water – 3 changes Wash section in a pot of reducer B until the neurons are dark brown Wash in distilled water and tone in 0.2% Gold chloride for 2 – 3 min Wash in distilled water Fix in 5% Sodium thiosulphate for 5 min Wash in tap water Dehydrate, clear and mount in DPX

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Result: Neurofibrils, dendrites and axon – black

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Staining for Microorganism Gram staining Ziehl- Neelsen (ZN) stain Warthin-Starry for spirochetes Gomori Methenamine (hexamine) silver for fungi Giemsa stain for parasite

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Gram method for Bacteria Gram staining differentiate bacteria into 2 classes depending on their cell wall structure and composition Gram positive Gram negative, Crystal violet is first applied to the section as a primary stain, followed by iodine mordant forming a dye lake . The section are then decolorized using alcohol / acetone mixture Gram positive cell do not decolorize at this step and gram negative bacteria will not retain crystal violet

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PRINCIPLE Crystal Violet stains the nucleic acids of the bacteria (and background tissue) and after treatment with iodine, the sections are differentiated in acetone and counterstained with basic fuchsin . The tissue background and Gram-negative bacteria lose their blue staining and are subsequently stained with basic fuchsin. Gram-positive bacteria retain the crystal violet/iodine blue staining.ple:-

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A .Gram method for bacteria in smear Procedure : Fix dry film by passing it 3 times through a flame Stain for 15 sec in 1% crystal violet or methyl violet Flood for 30 sec with Lugol’s iodine. Pour off excess Flood with acetone for 2 – 5 sec, wash with water Counter stain for 20 sec with dilute carbol fuchsin, or freshly filtered neutral red 1-2 min. Wash with water and carefully blot section until it is dry

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gram stain of the abscess shows thin, gram positive rods in chains. Result Gram positive organism - blue-black Gram negative organism - red

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B .Gram method for paraffin sections Reagents: Crystal violet solution 0.5% crystal violet in 25% alcohol Gram’s & Lugol’s iodine Iodine - 1 gm Potassium iodide - 2gm Distilled water - 10 ml 1% aqueous neutral red.

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Procedure: Deparaffinize & rehydrate through graded alcohols to distilled water. Stain with crystal violet solution, 2 min. Rinse in tap water. Iodine solution, 2 min. Rinse in tap water, & flood with acetone, 1-2 sec. Wash in tap water. Counter stain in neutral red, 3 min. Blot, dehydrate rapidly, clear & mount. Result: Gram-positive organisms, fibrin, some fungi, keratohyalin, and keratin - blue Gram – negative organisms -red.

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Gram Positive Gram Negative

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Ziehl-Neelsen (ZN) Stain for Mycobacterium Bacillus Mycobacteria are difficult to demonstrate by the Gram technique because they possess a capsule containing a long chain fatty acids that make them hydrophobic . Phenolic acid or heat may be used to reduce the surface tension and increase the porosity.

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PRINCIPLE Mycobacteria tuberculosis (tubercle bacilli) have a lipid-rich cell wall which is capable of taking up strong phenol dye solutions (eg. carbol fuchsin solution) such that the dye is retained upon subsequent differentiation in acid or alcohol ie. they are said to be acid and alcohol fast (AAFB = acid and alcohol fast bacilli).

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Solutions Carbol – fuchsin 1 g basic fuchsin dissolved in 10 ml of absolute alcohol; add 100 ml of 5% aqueous phenol. Mix well and filter before use. Acidified methylene blue 0.25% methylene blue in 1 % acetic alcohol.

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Method Deparaffinize and rehydrate through graded alcohols to distilled water. 2. Flood sections with freshly filtered carbol-fuchsin and heat to steaming with intermittent flaming, 15 minutes, or stain in Coplin jar at 56°-60°C, 30 min. 3. Wash well in tap water. 4. Differentiate in 1 % acid alcohol, 10 min. 5. Wash well in tap water. 6. Counterstain in methylene blue solution, 30 seconds. 7. Blot and differentiate by alternate dehydration and rehydration until the background is a delicate pale blue. 8. Dehydrate, clear and mount.

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Results Mycobacteria, hair shafts, Russell bodies, Splendore -Hoeppli immunoglobulins around actinomycetes and some fungal organisms -Red Background -pale Blue

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Mycobacterium tuberculosis in lung, Ziehl-Neelsen acid fast stain.

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Note The blue counterstain may be patchy if extensive caseation is present. Care should be taken to avoid over-counterstaining, as scant organisms can easily be obscured. Decalcification using strong acids can destroy acid-fastness; formic acid is recommended. Victoria blue can be substituted for carbol fuchsin and picric acid for the counterstain.

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Warthin-Starry method for spirochetes (Warthin & Starry 1920) PRINCIPLE Organisms are demonstrated by silver impregnation technique. Solutions Acetate buffer. pH 3.6 Sodium acetate 4.1 g Acetic acid 6.25 ml Distilled water 500 ml 1 % silver nitrate in pH 3.6 acetate buffer.

Warthin-Starry Method for Spirochaetes:

Warthin-Starry Method for Spirochaetes Developer : Dissolve 3 g of hydroquinone in 10 ml pH 3.6 buffer, and mix 1 ml of this solution and 15 ml of warmed 5% Scotch glue or gelatin; keep at 40°C. Take 3 ml of 2% silver nitrate in pH 3.6 buffer solution and keep at 55°C. Mix these two solutions immediately before use.

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Procedure: Deparaffinize & rehydrate through graded alcohol to distilled water. Impregnate in preheated 55-60°c silver solution, 90-105 min. Prepare and preheat developer in water bath. Treat with developer for 3 min at 55°c (section should be golden brown at this point) Remove from developer and rinse in tap water for few min at 55-60°c, then buffer at room temperature. Tone in 0.2% gold chloride. Dehydrate, clear and mount.

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Result: Spirochetes - black Background - golden yellow Cat scratch associated bacilli

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Long, spiraling spirochetes are seen with a Warthin -Starry silver stain.

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Spirochetes are black in a gold background.

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Gomori methenamine silver for fungi GMS staining is a silver staining technique for demonstrating fungi in tissue sections. It is primarily based on staining the polysaccharides in fungal cell walls . PRINCIPLE This method depends upon the reduction of the silver by the aldehyde groups produced after oxidation of fungal wall components with chromic acid.

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Reagents: 5% sodium tetraborate in distilled water (borax) Methenamine silver Incubating solution borax - 5ml distilled water - 25ml methenamine silver - 25ml Arzac’s counterstain orange G - 0.25 gm light green - 1g 50% alcohol - 100ml glacial acetic acid - 1ml

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Procedure: Deparaffinize & rehydrate through graded alcohol to distilled water. Oxidize in 5% aqueous chromic acid, 1 hour. Wash in tap water. Rinse in 1% sodium metabisulfate. Wash in tap water, 5 min. Rinse in tap water, then place in preheated silver incubating solution in a dark place, upto 1 hour Rinse well in distilled water. Tone in 0.1% gold chloride for 4 min. Place in 3% sodium thiosulfate, 5 min. Counterstain in Arzac’s stain, 15-30 sec. Blot, dehydrate, clear and mount.

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Pneumocystis carinii Result: Fungi, pneumocytstis, melanin - black Mucin and glycogen - grey-black RBCs - yellow Background - pale green

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GMS stain for Cryptococcus neoformans

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Giemsa stain A stain for hemopoietic tissue and hemoprotozoa consisting of a stock glycerol methanol solution of eosinates of Azure B and methylene blue with some excess of the basic dyes. A mixture of glycerin, methanol, methylene azure, and eosin used to stain chromosomes, blood cell producing tissues, and certain species of spirochetes and protozoans

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PRINCIPLE This method is a modified version of the original Giemsa technique used for haematological smears and gives good results for sections. Giemsa is a Romanowsky stain which contains azure B and eosin Y and is capable of making subtle distinction in shades of staining. The acidic groupings of the nucleic acids and proteins of the cell nuclei determine their uptake of the basic dye azure B and the presence of basic groupings result in an affinity for acidic dyes and their staining by eosin.

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Giemsa stain is used for the histopathological diagnosis of Malaria and other parasites . It is named after Gustav Giemsa , an early malariologist. Giemsa stain is also a differential stain. It can be used to study the adherence of pathogenic bacteria to human cells. It differentially stains human and bacterial cells pink and purple respectively. It can be used for histopathological diagnosis of malaria and some other spirochete and protozoan blood parasites.

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Giemsa stain is a classical blood film stain for peripheral blood smears and bone marrow specimens. Erythrocytes stain pink, platelets show a light pale pink, lymphocyte cytoplasm stains sky blue, monocyte cytoplasm stains pale blue, and leukocyte nuclear chromatin stains magenta.

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Solution: Giemsa stock Giemsa stain powder - 4gm Glycerol - 250 ml Methanol - 250 ml Working Giemsa for parasites Giemsa stock - 4 ml Acetate buffered distilled water, pH 6.8 - 96 ml

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Method : Deparaffinize and rehydrate through graded alcohols to water. Rinse in pH 6.8 buffered distilled water. Stain in working Giemsa, overnight. Rinse in distilled water. Rinse in 0.5% aqueous acetic acid until section is pink. Wash in tap water. Dehydrate rapidly through alcohols, clear and mount.

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Designed to differentiate blood cells and to stain intracellular parasites in red blood cells and plasma, e.g. Plasmodium falciparum (malaria parasite).

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Toxoplasma gondii

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Demonstration of decalcified bone Masson’s trichrome stain PAS Silver impregnation Schmorl’s picro-thionin Weigert – van gaeson

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Masson’s trichrome stain :- adult or mature bone often shows areas of blue or green. PAS :- the +ve red PAS reaction demonstrate mucopolysaccharides in new bone.The PAS reaction is not affected by decalcification ,but prolonged treatment with strong acid should be avoided. Silver impregnation of reticulin fibers helps in diagnosis of bone tumors,tumor metastasis to bone and myelofibrosis.stain is not affected by decalcification.

Schmorl’s picro-thionin :

Schmorl’s picro-thionin The major problem in demonstrating canaliculi is the attempt to show spaces too fine for identification by routine staining of surrounding bone so it is necessary to fill the spaces with a substance that appears dark against a lighter or unstained background. The picro-thionin method (Schmori 1934) depends on deposition of a thionin precipitate within the lacunae and canaliculi .

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Fixation Any fixative, but avoid mercuric chloride Decalcification Any decalcification solution Sections Frozen or celloidin freshly cut. Solutions Stock solution 0.25% aqueous thionin. Working solution (0.25% thionin):filter 50ml stock solution and dilute with 50ml distilled water .Add 1 or 2 drops concentration ammonia immediately before use . Saturated aqueous picric acid.

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Method Wash sections in distilled water,10 min. Stain in thionin solution ,5-20 min or longer. Wash in distilled water Immerse section in picric acid solution,30-60 seconds. Wash in distilled water Differentiate in 70% alcohol until the bluish-green clouds of stain cease to form, 5-10 min or longer. Dehydrate rapidly, clear in xylene and mount in permanent mounting media.

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Note Agitate section gently during steps 3-6 .this is particularly important in stage 6 and change 70% alcohol frequently. If bone matrix is decolorized during differentiation (step 6) restore yellow color by returning the section to the picric acid solution for a few seconds before proceeding with dehydration.

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Result Lacunae and canaliculi – dark brown-black Bone matrix – yellow or brownish – yellow Cells - red

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Connective tissue and lipids Collagen Gomori's One-Step Trichrome Stain manual, Trichrome automated Elastic fibers Verhoeff - Van Gieson stain Reticulin Wilder's method, both manual and automated Fat Oil red O stain for frozen section Carbohydrates Glycogen/neutral mucin Lillie PAS stain manual, PAS automated Glycogen digestion Amylase digestion procedure, both manual and automated . Mucopolysaccharid-acid Modified Mowry's Colloidal iron stain Mucopolysaccharid-acid Alcian Blue pH 2.5 or pH 1.0 Acid and neutral mucopolysaccharides Alcian blue 2.5 and PAS Sialomucin Mayer's mucicarmine stain

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Amyloid Amyloid Alkaline Congo Red stain Amyloid Lieb's crystal violet method Minerals, pigments and miscellaneous Calcium Kossa's method Iron Gomori's method Melanin Fontana Masson Silver stain Melanin removal Permanganate method Charcott Leyden crystals Luna stain Muscle striations Mallory's PTAH stain

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ELEMENTS STAINED NAME OF STAIN Microorganisms Acid-Fast Organism Fite stain Bacteria, blood elements May - Grunwald Giemsa Stain Bacteria Gram stain Bacteria/Protozoa Lillie's Azure A Eosin B Stain Helicobacter pylori Toluidine Method Helicobacter pylori Alcian yellow, automated Fungi Gomori's methanamine silver method, both manual and automated Fungi PAS green, both manual and automated Fungi/Cyptococcus Mucicarmine Spirochetes Steiner, automated . Spirochetes Warthin - Starry microwave method

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Connective tissue and lipids Collagen Gomori's One-Step Trichrome Stain manual, Trichrome automated Elastic fibers Verhoeff - Van Gieson stain Reticulin Wilder's method, both manual and automated Fat Oil red O stain for frozen section Carbohydrates Glycogen/neutral mucin Lillie PAS stain manual, PAS automated Glycogen digestion Amylase digestion procedure, both manual and automated . Mucopolysaccharid-acid Modified Mowry's Colloidal iron stain Mucopolysaccharid-acid Alcian Blue pH 2.5 or pH 1.0 Acid and neutral mucopolysaccharides Alcian blue 2.5 and PAS Sialomucin Mayer's mucicarmine stain

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Amyloid Amyloid Alkaline Congo Red stain Amyloid Lieb's crystal violet method Minerals, pigments and miscellaneous Calcium Kossa's method Iron Gomori's method Melanin Fontana Masson Silver stain Melanin removal Permanganate method Charcott Leyden crystals Luna stain Muscle striations Mallory's PTAH stain

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ELEMENTS STAINED NAME OF STAIN Microorganisms Acid-Fast Organism Fite stain Bacteria, blood elements May - Grunwald Giemsa Stain Bacteria Gram stain Bacteria/Protozoa Lillie's Azure A Eosin B Stain Helicobacter pylori Toluidine Method Helicobacter pylori Alcian yellow, automated Fungi Gomori's methanamine silver method, both manual and automated Fungi PAS green, both manual and automated Fungi/Cyptococcus Mucicarmine Spirochetes Steiner, automated . Spirochetes Warthin - Starry microwave method

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ELEMENTS STAINED NAME OF STAIN Microorganisms Acid-Fast Organism Fite stain Bacteria, blood elements May - Grunwald Giemsa Stain Bacteria Gram stain Bacteria/Protozoa Lillie's Azure A Eosin B Stain Helicobacter pylori Toluidine Method Helicobacter pylori Alcian yellow, automated Fungi Gomori's methanamine silver method, both manual and automated Fungi PAS green, both manual and automated Fungi/Cyptococcus Mucicarmine Spirochetes Steiner, automated . Spirochetes Warthin - Starry microwave method Connective tissue and lipids Collagen Gomori's One-Step Trichrome Stain manual, Trichrome automated Elastic fibers Verhoeff - Van Gieson stain Reticulin Wilder's method, both manual and automated Fat Oil red O stain for frozen section Carbohydrates Glycogen/neutral mucin Lillie PAS stain manual, PAS automated Glycogen digestion Amylase digestion procedure, both manual and automated . Mucopolysaccharid-acid Modified Mowry's Colloidal iron stain Mucopolysaccharid-acid Alcian Blue pH 2.5 or pH 1.0 Acid and neutral mucopolysaccharides Alcian blue 2.5 and PAS Sialomucin Mayer's mucicarmine stain Amyloid Amyloid Alkaline Congo Red stain Amyloid Lieb's crystal violet method Minerals, pigments and miscellaneous Calcium Kossa's method Iron Gomori's method Melanin Fontana Masson Silver stain Melanin removal Permanganate method Charcott Leyden crystals Luna stain Muscle striations Mallory's PTAH stain

Wright stain:

Wright stain Erthrocytes yellowish-red Polymorphonuclears: Nucleus dark purple Granules reddish-lilac Cytoplasm pale-pink Eosinophiles: Nuclei blue Granules red to orange-red Cytoplasm blue Basophiles: Nucleus purple to dark blue Granules very dark purple Lymphocytes: Nuclei dark purple Cytoplasm sky blue Platelets violet to purple granules RESULTS: Erthrocytes : pink-tan Eosinophiles : Granules red Lymphocytes: Granules red-purple Cytoplasm blue Neutrophils : Granules purple-pink

Stains for smears :

Stains for smears Blood smears Mucosal scrapes : buccal ,tongue Mucinous sample : sputum Fine needle aspiration

Hematologic stains :

Hematologic stains Romanowsky stains Giemsa stain Leishman stains Wright’s stains Leukocyte alkaline phosphatase Tartrate-resistant acid phosphatase Myeloperoxidase

Romanowsky stains :

Romanowsky stains It will stain both nucleus and cytoplasm. These histology stains are used for blood smear and bone marrow. Examples of Romanowsky histology stains include Wright's stain, Giemsa stain and Jenner's stain. These histology stains are based on a combination of eosin and methylene blue. .

Giemsa Stain:

Giemsa Stain Giemsa stain is a classical blood film stain for peripheral blood smears and bone marrow specimens.. It is also used to visualize parasites and malaria. This is a Romanowski type stain. Methylene blue and eosin are used. Note that the acidic components of the cellular constituents such as the cytoplasm and chromatin, pick up the basic methylene blue azure compliments of the Giemsa stain, which reveals the characteristic blue coloration of this stain.

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Result :- Erythrocytes - pink/red. Platelets and - blue leukocytes.

Wright's Stain:

Wright's Stain It is named for James Homer Wright , who devised the stain, a modification of the Romanowsky stain , in 1902. Because it distinguishes easily between blood cells, it became widely used for performing differential white blood cell counts, which are routinely ordered when infections are expected. This histology stain is used for blood smears and bone marrow smears. This histology stain uses a blend of basic dyes, such as methylene blue derivatives and acid dyes, such as eosin.

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FIXATION Streak thin (approx. one cell thick) smears across a sterile slide by means of a second slide or cover glass. Air-dry quickly. STAINING: 1. Place 1.0 ml of the Wright-Giemsa Stain upon the smear, in sufficient quantity to cover the entire surface, for 3-4 minutes. 2. Add 2.0 ml distilled water or Phosphate Buffer pH 6.5 (B-149-2) and let stand twice as long as in step 1. 3. Rinse stained smear with water or the Phosphate Buffer pH 6.5 (B-149-2) until the edges show faintly pinkish red. 4. The film is allowed to dry in the air. The preparation may be blotted gently to hasten drying. 5. Permanent mounts can be made with mounting medium.

lymphocyte‎ basophils:

lymphocyte ‎ basophils eosinophil ‎ neutrophil‎ Erthrocytes - yellowish-red Polymorphonuclears : Nucleus - dark purple Granules - reddish-lilac Cytoplasm - pale-pink Eosinophiles : Nuclei - blue Granules- red to orange-red Cytoplasm- blue Basophiles: Nucleus - purple to dark blue Granules - very dark purple Lymphocytes: Nuclei - dark purple Cytoplasm- sky blue Platelets - violet to purple granules

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Red blood cells - reddish/pink. Eosinophilic granules -bright orange/red. white blood cells- purple.

Leishmans stain:

Leishmans stain Where to look for cells in a blood smear The density of cells varies across the smear. Cells will be "heaped and piled" close to the point were the drop of blood was placed on the slide. White blood cells appear shrunken, and some types are difficult to distinguish from each other.

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There are fewer cells close to the tip of the smear. In this region, white blood cells are sometimes damaged and erythrocytes may be deformed. The best area to look at is between these two regions. Where it is located exactly and how wide it is will depend on the smear, but the middle of the smear is a good starting point

Myeloperoxidase stain :

Myeloperoxidase stain The myeloperoxidase (MPO) stain is helpful to identify cytoplasmic granules characteristic of myeloid cells. This is useful when there are large, immature white blood cells in the peripheral blood, and it is not clear whether they are of myeloid or of lymphoid origin . Staining with MPO in this setting suggests a myeloid leukemia.

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This peripheral blood smear is stained with the myeloperoxidase stain, and there is red reaction product in these leukemic cells, indicative that this is a myeloid leukemia.

Leukocyte alkaline phosphatase stain :

Leukocyte alkaline phosphatase stain The leukocyte alkaline phosphatase (LAP) stain is helpful in determining whether a high peripheral blood leukocytosis is a reactive process or a leukemia (chronic myelogenous leukemia, or CML). The more differentiated cells in the reactive process will stain more readily with LAP, while leukemic cells will not. The cells on a smear can be assessed and an "LAP score" can be generated. A high score generally indicates a "leukemoid reaction" or reactive condition (with an infection or other inflammatory process) while a low score suggests CML.

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This peripheral blood smear is stained for leukocyte alkaline phosphatase . The neutrophils have bright red reaction product in their cytoplasm

Tartrate-resistant acid phosphatase stain :

Tartrate-resistant acid phosphatase stain The tartrate-resistant acid phosphatase (TRAP) stain has one major usefulness--to help diagnose a rare leukemia known as hairy cell leukemia. This neoplastic B lymphocyte proliferation affects mainly bone marrow and spleen. There is typically pancytopenia, so the peripheral WBC count is not high. The circulating hairy cells get their name from the cytoplasmic projections. However, positive staining with TRAP helps make the diagnosis.

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This collage of cells shows the characteristic lymphocytes of hairy cell leukemia with their indistinct cytoplasmic borders and small projections. The cell at the lower right is stained for tartrate -resistant acid phosphatase (TRAP) and is positive, with red reaction product in the cytoplasm, characteristic for hairy cell leukemia.

Alkaline Phosphatase:

Alkaline Phosphatase This histology stain will stain endothelial cells. Sites of alkaline phophatase activity will appear red. Nuclei will stain blue.

PAPANICOLAOU STAIN:

PAPANICOLAOU STAIN Papnicolaou formula Harris’s hematoxylin Hematoxylin - 5g Ethanol - 50ml Potassium alum - 100g Distilled water (50̊ C) – 1000ml Mercuric oxide – 2-5g Glacial acetic acid - 40ml Orange G 6 Orange G (10% aqueous) – 50ml Alcohol – 950ml Phosphotungstic acid – 0-15g EA 50 0.04 M light green SF -10ml 0.3 M eosin Y – 20ml Phosphotungstic acid - 2g Alcohol -750ml Methanol -250ml Glacial aceticmacid -20ml Filter all stains before use.

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Specimen Collection: In the collection and preparation of smears for cytological examination, the major objectives are: 1. Specimens should have a sufficient number of cells from the area in question. 2. Smears should contain well preserved cells uniformly distributed so that each cell can be individually examined. 3. The staining procedure should clearly define the details of all structures. Cytological preparations are obtained from patient by approved methods and techniques. Scraping, obtained from the vagina, uterus, cervix, mouth or ulcerated skin area is spread directly on a clean microscope slide.

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4.Sediment (obtained by centrifugation or filtration) from bodily secretions is spread on a clean microscope slide. 5.The smear is immediately fixed with a cytological spray fixative or in an alcohol-ether dip. 6.Fixation or preservation is one of the most important steps in the procedure. Drying of the cells prior to fixation will usually result in artifacts such as nuclear distortion and vacuolization. 7.After fixation there are no special handling requirements for cytological smears. However,smears which are to be mailed to a laboratory, should remain in the fixative for about one hour. A second clean glass slide may be placed on each fixed slide for protection.

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Procedure: Notes: Filter the Harris Hematoxylin immediately before use. 1. Dip slide(s) gently 5-10 times in 95% ethanol. 2. Dip slide(s) gently 5-10 times in 70% ethanol. 3. Dip slide(s) gently 5-10 times in distilled water. 4. Stain 5 minutes in Harris Hematoxylin. 5. Place smears in distilled water. Rinse in successive changes of distilled water until the water remains colourless. 6. Dip slide(s) gently 5-10 times in 70% ethanol. 7. Dip slide(s) in a 1% solution of HCl in 70% ethanol until the smear shows a salmon colour. 8. Rinse slide(s) well in 2 changes of 70% ethanol.

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9. Dip slide(s) gently in a 3% solution of ammonium hydroxide in 70% ethanol until the smear takes on a blue colour. 10. Rinse the slide(s) in two changes of 70% ethanol. 11. Dip slide(s) 5-10 times in 95% ethanol. 12. Stain slide(s) in OG-6 for 2 minutes. 13. Rinse slide(s) in two changes of 95% ethanol. 14. Stain slide(s) in EA-50 or EA-65 for 3-6 minutes. 15. Rinse slide(s) well in two changes of 100% methanol. 16. Rinse slide(s) in one part absolute methanol one part xylene. 17. Clean smear in xylene.

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Mounting Procedure: 1. After the smear has been completely cleaned in xylene it is mounted with a microscope slide cover glass preferably 22x40mm, #1 thinness. 2. A permanent clean mounting medium should be used. 3. The excess xylene should be drained, in order to avoid th appearance of air spaces when xylene evaporates. 4. Place the required amount of mounting medium along an edge of one of the longer borders of the coverslip. 5. Place the slide at right angles to the edge of the coverslip so that the side containing the cells is facing the mounting medium. 6. Slowly lower the slide and permit the mounting medium to spread between the slide and coverslip Limitations: 1. Proper specimen collection and fixation of cells is essential for interpretation .

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Results: Nuclei are stained blue while cytoplasm displays varying shades of pink, orange, yellow and green

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Cytologic image of a scraping of the buccal mucosa. Intermediate squamous cell with sex chromatin body (Barr body) (arrow) lying against the inner nuclear membrane (Papanicolaou stain). - Cytology smear showing clusters of keratinizing squamous carcinoma indicating metastasis in the lymph node.

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: Cytology smear showing metastasis of adenocarcinoma. Note the neutrophils sticking around the mucin of the tumor cells. Cytology smear showing the streaking effect commonly seen with metastasis of nasopharyngeal carcinoma, small cell carcinoma and lymphoma.

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: FNA smear showing metastasis of malignant melanoma. Note the presence of giant cells (inset) and the presence of melanin pigment. (MGG X 400)

Stains for biogenic amines :

Stains for biogenic amines Types of stains for argentaffin include: Diazo (diazonium salts) Fontana-Masson Schmorl's Autofluorescence Types of stains for chromaffin include: Modified Giemsa Schmorl's Wiesel's Types of stains for argyrophil include: Grimelius (Bouin's fixative preferred) Pascual's

Chromatin And Argentaffin:

Chromatin And Argentaffin The distinction between chromaffin and argentaffin is artificial, since this depends upon the fixative used. "Chromaffin" cells have cytoplasmic granules that appear brown when fixed with a dichromate solution. "Argentaffin" cells reduce a silver solution to metallic silver after formalin fixation. Either reaction can be produced depending upon which fixative was used. Traditionally, chromaffin reaction is associated with adrenal medulla or extraadrenal paraganglion tissues (pheochromocytomas) whereas argentaffin reaction is associated with carcinoid tumors of the gut. Using a pre-reduction step may get more cells to stain, but they are called "argyrophil" then.

Fontana-Masson:

Fontana-Masson This histology stain uses silver. Fontana-Masson stains argentaffin granules and melanin black. The nucleus will stain pink/red. The cytoplasm will stain light pink.

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Giemsa stain is also used to visualize chromosomes . Giemsa stain also stains the fungus histoplasma . It can also be used to identify Mast cells . [3]

Romansky :

Romansky Paul Ehrlich had used mixtures of acidic and basic dyes for this purpose in 1879: eg Fuchsine (acid) and methylene blue (base). [1] In 1891 Ernst Malachowski [2] and Dmitri Leonidovich Romanowsky [3] independently developed techniques using a mixture of Eosin Y and modified Methylene blue that produced a surprising hue unattributable to either staining component: a beautiful, distinctive shade of purple . [4] [5] Requirement for the occurrence of the Malachowski-Romanowsky-Giemsa effect are: I A cationic dye: The best dye is azure B and, though azure A gives the nuclear purple colour, the cytoplasmic blue is inferior. No other cationic dye such as methylene blue is suitable. 2 An anionic dye: Most commonly eosin Y is used. [6] Because the aqueous dye solutions were unstable, methanol was introduced as a solvent, and William Boog Leishman [7] and James Homer Wright [8] advocated use of methanol as a fixative prior to staining. Gustav Giemsa improved this technique by standardizing the dye solutions and adding glycerol to increase stability. [9]

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Giemsa-stained thin blood film showing Plasmodium falciparum infections [10] The demethylation of Methylene Blue in aqueous solution using heat and alkali produces a mixture of Azure A , Azure B, Methylene Violet and Methylene Blue. Eosin Y is then added to produce a "neutral" dye. The precipitate is then dissolved in a mixture of methanol and glycerol to form a stock solution; this is diluted with water or an aqueous buffer to form a 'working' solution that is used in the staining of pathology specimens. The 'working' solution is stable for 3 hours. [11] Immunochromatographic capture procedures ( Rapid Diagnostic Tests ) are nonmicroscopic diagnostic options for the laboratory that may not have appropriate microscopy expertise available. [12]

Mallory's Trichrome stain :

Mallory's Trichrome stain Solution A Acid fuchsin-1g Distilled water-100mL Solution B Phosphomolybdic acid-1g Distilled water-100mL Solution C Orange G-2g Methyl blue-0.5g Oxalic acid-2g Distilled water100mL

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Method Bring sections to water via xylene and ethanol. Place into solution A for 2 minute. Rinse with distilled water. Place into solution B for 2 minutes. Rinse quickly with distilled water. Place into solution C for 15 minutes. Wash well with distilled water. Dehydrate and differentiate with ethanol. Clear with xylene and mount with a resinous medium.

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Results Nuclei  –  red Erythrocytes  –  orange Muscle  –  red Collagen  –  blue Notes Phosphotungstic acid may be substituted for phosphomolybdic acid in solution B. This is the method that introduced the concept, if not the name, of trichrome staining.

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Gingiva (gum). The masticatory mucosa has tall rete ridges. A dense network of collagen fibers (blue) tightly anchors the epithelium to the underlying bone (not shown); the keratin layer (orange band) on the surface of the epithelium imparts further strength to it (Mallory's trichrome ).

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Buccal mucosa showing maturation of squamous epithelium: there is a row of small basal cells, larger cells of stratum spinosum, and parallel arranged flat surface cells. No keratinization is seen. Lamina propria shows delicate strands of connective tissue, blood vessels, and a few lymphocytes. (Mallory's trichrome.)

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Endochondral ossification in Mallory stain. Cartilage is light blue and bone is dark blue. A thin layer of bone has already been laid down on the surface of the cartilage spicules along the leading edge of cartilage. Blood cells in the marrow cavity are red. The very dark blue at the lower left and right is spongy bone of the periosteal bone collar of the shaft. This will later be remodeled into Haversian systems of compact bone

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Periodic Acid Schiff Stain (Mc Manus 1946) PAS reaction is a useful indicator of the presence of tissue carbohydrate, particularly for glycogen when the technique incorporate a diastase digestion stage.

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PAS with Diastase staining is used for the demonstration of glycogen in tissue sections. Diastase and alpha-Amylase are enzymes that digest glycogen polymer to maltose and glucose that are easily washed from the sections, thus giving a negative reaction with PAS.

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PAS Stain PAS with Diastase - Diastase (amylase) specifically digests glycogen. - Predigesting one section with amylase and comparing with an undigested section. (both of which have been stained by PAS) allows identification of the glycogen.

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Principle: The PAS stain is a histochemical reaction in that the periodic acid oxidizes the carbon to carbon bond forming aldehyde which react to the fuchsin-sulfurous acid which form the magenta color. Glucose + Periodic acid - aldehyde (Carbohydrate) Aldehyde + Schiff’s reagent - magenta colored compound

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PAS Technique: - Periodic Acid Solution Periodic acid - 1 gm Distilled water - 200 ml - Schiff Reagent Dissolve 1gm of basic fuchsin in 200 ml of boiling distilled water. Allow the solution to cool 50 °c, and add 2-gm potassium metabisulphite with mixing. Allow to cool to room temperature then add 2 ml conc. Hydrochloric acid, mix, add 2 gm activated charcoal and leave overnight in the dark at room temperature. Filter, the solution should be either clear or pale yellow color. Store in a dark container at 4°c.

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Procedure: Dewax sections and bring to distilled water. Treat with periodic acid, 5 min. Wash well with several changes of distilled water. Cover with schiff’s reagent, 10-20 min. Wash in running tap water, 5-10 min. Stain nuclei with Harris’s haematoxylin or an iron haematoxylin. (Do not use Ehrlich’s haematoxylin, which will also stain some PAS positive compounds.) Differentiate in acid-alcohol . Wash in water, dehydrate in alcohol, clear in xylene and mount as desired.

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MUCICARMINE STAIN – SOUTHGATE’S – MUCIN 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

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Stain for Mast Cells Mast cell containing heparin and histamine in the form of granules. They are found in connective tissue around the blood vessels in many inflammatory conditions Mast cell granules stains metachromatically with toludine blue, azure A, Bismarck brown and thionin. Mast cells are usually visualized with alcian blue methods.

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Alcian blue – safranin method for mast cells Solution: Alcian blue - 900 mg Safranin - 45 mg Ferric ammonium sulfate - 1.2 gm Acetate buffer (pH 1.4) - 250 ml

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Method: Take sections to water. Stain in alcian blue solution, 15 min. Rinse in tap water. Dehydrate in alcohol. Clear and mount.

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Mast cells are apparent in the upper dermis in this section of skin because their cytoplasmic Granules stain metachromatically , giving the intense purple color seen here. Result: Mast cell granules containing biogenic amines – blue Mast cell containing heparin - red

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Toluidine Blue (Lariviere, 1999). Its specific role is as a metachromatic stain ( meta=change chromatic=colour ). Toluidine blue is a polychromatic stain which stains many tissue components. Toluidine Blue causes mast cells to stain purple in an otherwise blue background. TOLUIDINE BLUE

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SPECIMEN :- Standard paraffin sections or 6æ frozen section R EAGENTS Toluidine blue (CI 52040) 0.25 g Sodium acetate 2 g Sodium barbiturate 3 g Distilled water to 100 mL

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PROCEDURE 1. Paraffin or frozen sections may be used. 2. Bring sections to water. 3. Stain in toluidine blue solution, for 20 seconds . 4. Blot with filter paper; allow to dry and clear in histolene. 5. Mount in Safety Mount RESULTS Metochromatic substances - Red, pink or purple Nuclei and other components - Blue Mast cell granules - Purple

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Staining reactions of mast cells Method Result Toludine blue Azure A Thionin Csaba’s alcian blue safranin Aldehyde fuchsin PAS Purple Red Blue or red Purple to red Yellow to brown Variable

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References: 1. Papanicolaou GN: Atlas of Exfoliative Cytology, Harvard University Press, Cambridge, 1954. 2. United States, Naval Medical School, National Naval Medical Center, Bethesda, Maryland, Manual of gynecological exfoliative cytology. US Government Printing Office, Washington, D.C., 1965. 3. A Manual for Cytotechnology, C. Keebler, J. Reagen, Editors, American Society of Clinical Pathologists Press, Chicago (IL), 1983.

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Lillie, R. D. :Factors influencing the staining of blood films and the role of methylene violet. J. Lab. Clin. Med. 29:1181, 1944. Lillie, R. D., H.J. Conn’s Biological Stains, 8th edition, William’s & Wilkins, Baltimore, p 438,1972.

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