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This document discusses staining techniques used in histopathology. It describes the purpose of staining as outlining tissues and cellular components to identify tissues and establish the presence or absence of disease. The most common staining technique is hematoxylin and eosin (H&E) staining. Hematoxylin stains nuclei blue and eosin stains cytoplasm pink, allowing clear visualization of tissue structures. The document provides details on the preparation and use of hematoxylin and eosin stains as well as other specialized staining techniques.
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Presentation by Dr. N. Manjula on various staining techniques in histopathology.
Staining applies dyes to tissue sections to outline structures, identify tissues, and detect diseases.
Stains categorized into routine, special, vital, supravital, metachromatic, basic, acid, and neutral.
Natural and synthetic dyes; direct and indirect staining methods using mordants.
Notable staining methods: Hematoxylin & Eosin, Gram's stain, Romanowsky stain, and their significance.
Hematoxylin's origins, extraction process, oxidation (ripening), and types including Alum and Iron hematoxylin.
Detailed discussion on Alum hematoxylin, stability issues, and progressive vs. regressive staining methods.
Types of Iron and other hematoxylins, along with usage details for specific staining applications.
Eosin as the primary counterstain, its types, solutions, and preparation necessary for staining.
Detailed preparation and staining procedure including steps from dewaxing to mounting the samples.
Visual results of various stains on different tissue components post staining process.
Brief mention of the methodology utilized for automatic staining processes.
Daily quality control measures for staining efficacy and guidelines for acceptable results.
Overview of the strengths and limitations of staining procedures in histopathology.
Key references and literature supporting the staining techniques discussed in the presentation.
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STAINING TECHNIQUES manju.pdf
- 1.
- 2. STAINING ▪ Staining isthe process of applying dyes on paraffin cut sections to study the architecturaland cellular pattern of the tissues.
- 3. PURPOSE OF STAINING ▪Outlines tissues and cellular components ▪ Identification of tissue ▪ Establishes the presence or absence of disease
- 4. TYPES ▪ ROUTINE: cellsand tissues combinewith active coloring agent. Eg:- H and E. ▪ SPECIAL: Eg:- impregnationof silver salts in reticulin stain. ▪ VITAL: staining selective living tissue components,demonstrating cytoplasmic structures by phagocytosis of dye. Eg:- Reticuloendothelial system withTRYPAN BLUE.
- 5. ▪ SUPRAVITAL: stainsliving cells after removal from body. Eg:-Retic stain. ▪ METACHROMATIC: certain dyes stain and tissues in a color quite different from fundamentalcolor of stain.This is metachromasia. Preferable to use frozen sections or fresh tissues.
- 6. BASED ON TISSUEAFFINITIES ▪ BASIC STAIN: active coloring substance combined with a colourless inorganic acid radical.Any substance stained by the basic dye – basophilic.In H and E basophilicstructures appearBLUE. ▪ ACID STAIN: substance that is stained by an acid dye is referred to as acidophilic. In H and E, acidophilic substance due to eosin appear in various shades of pink. ▪ NEUTRAL STAIN: it is formedby mixing aqueous solutions of certain acidic and basic stains. Eg:- Romanowsky dyes.
- 7. BASED ON SOURCE ▪NATURAL DYE: obtained from plants and animals. ▪ Eg:- hematoxylin. ▪ SYNTHETIC DYE: derived from coal tar and hydrocarbon benzene. ▪ Eg:- aniline, azo dyes.
- 8. METHODS OF STAINING ▪DIRECTSTAINING:process of giving color to the sections by using aqueous or alcoholic dye solutions. ▪ Eg:- methyleneblue and eosin. ▪ INDIRECTSTAINING:process by which action of dye is intensified by adding another agent (MORDANT). ▪ Mordantis a polyvalentmetal ion which forms coordinationcomplexes with certain dyes. ▪ Eg: Potassiumalum with Ehrlich hematoxylin,Iron inWeigert hematoxylin.
- 9. Common staining methods ▪Hematoxylinand Eosin staining in histopathology ▪ Gram's stain ▪ Romanowsky staining in Haematology ▪ Papanicoloaustaining inCytology
- 10. Introduction ▪ Hematoxylin andeosin is the most widely used stain because : ❖ It's simple ❖Ability to demonstrate clearly enormous number of different tissue structures . ▪ Haematoxylin stains nucleus blue and gives good intranuclear detail. ▪ Eosin stains cytoplasm pink.
- 11. Hematoxylin ▪ It isderived fromGreek word Haimato meaning blood and Xylonmeaning wood. ▪ A natural dyeextracted from the core of tree Haematoxylon campechianum. ▪ Hematoxylinis extracted from logwoodwith hot water and then precipitated out from the aqueous solution using urea. ▪ Major oxidization product of hematoxylin HAMATEINwhich is responsiblefor the color properties.
- 14. Ripening ▪ Process ofoxidation of hematoxylin. ▪ Carried out in two ways: ▪ Natural ▪ Artificial
- 15. ▪ Natural -exposure to light and air, slow process, 3 to 4 months, Eg:- Ehrlich's hematoxylin. ▪ Artificial – by addition of oxygen or removal of hydrogen. ▪ Adding chemicals mercuric oxide, sodium update, potassium permanganate, instantly prepared.
- 16. ▪ Haemateinis anionic,having poor affinity for tissue, it is inadequate to stain nucleus alone. ▪ Hence a mordant is required. ▪ Most useful are: salts of aluminium,iron.
- 17. TYPES OF HEMATOXYLIN ▪ALUM HEMATOXYLIN ▪ IRON HEMATOXYLIN ▪ TUNGSTENHEMATOXYLIN ▪ MOLYBDENUMHEMATOXYLIN ▪ LEAD HEMATOXYLIN ▪ HEMATOXYLINWITHOUTMORDANT
- 18. Alum hematoxylin ▪ Usedroutinely to producegood nuclear staining. ▪ Mordant used are either aluminium potash sulfate or aluminium ammonium sulfate is used in our dept. ▪ TYPES OF ALUM HEMATOXYLIN ▪ Ehrlich’s hematoxylin ▪ Mayer’s hematoxylin ▪ Harris hematoxylin is used in our dept. ▪ Delafield’s hematoxylin ▪ Cole’s hematoxylin ▪ Gill’s hematoxylin ▪ Carazzi’s hematoxylin
- 19. ▪ Major disadvantageis their instability to resist acid solution and relative ineffectiveness when used with fixatives particularly of acid type. ▪ HenceWeigert’s is used which is resistant to picric acid. ▪ Celestine blue B is successfully used as a mordant for alum hematoxylin when used with acid solutions.
- 20. ▪ PROGRESSIVESTAINING:sections are stainedfor predetermined time so that nuclei become well stained but background is relatively understained. ▪ REGRESSIVE STAINING:sectionsare first overstained then excess stain is removed in acid alcohol. ❑Advantage:degree of stainingis controlledand nuclei take good stainingwhere cytoplasmis clear.
- 21. ALUM HEMATOXYLIN SPECIALCHARACTERS HARRISHEMATOXYLIN EHRLICH HEMATOXYLIN MAYER’S HEMATOXYLIN GILL’S HEMATOXYLIN DELAFIELD’S HEMATOXYLIN COLE’S HEMATOXYLIN CARAZZI’S HEMATOXYLIN 2-4 ml OFGLACIALACETIC ACID IS ADDED TO 100 mlOF STAINTO INCREASE THE NUCLEAR STAIN PRECISION. ALWAYS FILTER BEFORE USE. IT IS A REGRESSIVE STAIN. EXCELLENT NUCLEAR STAIN USED AS REGRESSIVE STAIN. USED AS BOTH PROGRESSIVEAND REGRESSIVE STAIN. USED AS PROGRESSIVE NUCLEAR COUNTER STAIN FOR GLYCOGEN. THIS CAN BE READILY USED. NATURALLY RIPENED ALUM HEMATOXYLIN. ARTIFICIALLY RIPENED HEMATOXYLIN. USED IN FROZEN SECTION. PROGRESSIVE NUCLEAR STAINWITH SHORT STAINING TIME
- 22. Iron hematoxylin ▪ Weigert’shematoxylin ▪ Heidenhain’s hematoxylin ▪ Verhoeff’s hematoxylin ▪ Loyez hematoxylin
- 23. ▪ Iron saltsare used as both oxidizing agent and as mordants. ▪ Most commonlyused are ferric chloride and ferric ammoniumsulfate. ▪ Not only used as nuclear stain but also to demonstrate muscle striations, cellular organelles – mitochondria.
- 24. IRON HEMATOXYLIN SPECIALCHARACTERS WEIGERT’SHEMATOXYLIN HEIDENHAIN’S HEMATOXYLIN VERHOEFF HEMATOXYLIN USED AS NUCLEAR STAIN. CELESTINE BLUE ALUM HEMATOXYLIN HAS REPLACED IT. IT IS USEFUL WITH EOSIN FOR CNS TISSUES. IT IS A PROGRESSIVE STAIN. USED TO DIFFERENTIATE STRUCTURES BASED ON DEGREE OF DIFFERENTIATION OF STAIN. IT IS A REGRESSIVE STAIN. TISSUE ELEMENTS DEMONSTRATED ARE MITOCHONDRIA, STRIATIONS OF MUSCLE DEVELOPED PRIMARILYTO STAIN ELASTIC FIBRES. COUNTER STAINUSED ISVANGIESONSTAIN. IT GIVES HIGHCONTRAST WHICH CAN BE USED FOR ELECTRON MICROSCOPY. ELASTICFIBRES: BLUISH BLACK. CYTOPLASM AND MUSCLE:YELLOW. COLLAGEN: RED
- 25. TUNGSTEN HEMATOXYLIN ▪ Itsuse is applicable to bothCNS material and general tissue structure. ▪ Stainingis more precise after sections have been treated with acid dichromate solution. ▪ Good results are obtained with buffered formalinfixative. ▪ Ripened both naturally and artificially. ▪ Time consuming process.
- 26. OTHERS ▪ MOLYBDENUM HEMATOXYLIN– complexes of molybdenumand hematoxylin are principallyused to stain collagen and coarse chromatin. ▪ LEAD HEMATOXYLIN – used for demonstrationof granules in neuroendocrine system, keratohyalin granules and calcium deposits. ▪ COPPER HEMATOXYLIN – used to stain fatty acids, acidophils in pituitary, myelin sheaths, mitochondria.
- 27. ▪ CHROMIUMALUM HEMATOXYLIN– used to stain lipoproteins, myelin, phospholipidsand cytoplasmicgranules in beta cells of anterior pituitary and pancreatic islets. ▪ HEMATOXYLINWITHOUTMORDANT: free hematinstains collagen, elastin and erythrocytes from yellow to orange brown.
- 28. EOSIN ▪ Most commonlyused counterstain with hematoxylin. ▪ Other counter stains are : orange G, phloxine, Bordeaux red and Biebrich scarlet. ▪ But these give undesireable intense red colour and fail to achieve subtle differentiation as in eosin. ▪ They are fluorescent xanthene dyes.
- 29. ▪ Types: eosinY,ethyl eosin, eosin B. ▪ EosinY is widely used and is both water and alcohol soluble. ▪ Glacial acetic acid may be added which augments staining. ▪ Thymol added to prevent fungal growth.
- 30. STAIN PREPARATION ▪ REQUIREMENTS: ▪XYLENE ▪ ACETONE ▪ HAEMATOXYLINE SOLUTION ▪ ACIDALCOHOL ▪ EOSIN SOLUTION ▪ DPX – distyrene, a plasticizer and xylene.
- 31. ▪ PREPARATION: ▪ Haematoxylinepowder:2.5 g ▪ Absolute alcohol:50 ml ▪ Ammonium or potassium alum:50 g ▪ Distilled water:500 ml ▪ Mercuric oxide:1.5 g ▪ Glacial acetic acid: 10 ml
- 32. ▪ PREPARATION: ▪ Dissolvehaematoxylinein alcohol seperately with heat. ▪ Heat 500 ml of distilled water and add 50 g of ammonium or potassiumalum till it dissolves. ▪ Then add Haematoxyline solutionin to the alum water. ▪ After boiling add 1.5 gm mercuric oxideand mix it well. ▪ Cool the solutionand add 10 ml of glacial acetic acid and mix it well. ▪ Filter before use.
- 33. ▪ 0.1 %acid alcohol: ▪ 70% absolute alcohol 99 ml and conc.HCL 1.0 ml. ▪ Eosin: ▪ Eosin 0.5 g, distilled water 500ml, glacial acetic acid 1 drop.
- 34. PROCEDURE 1. DEWAXNATION: xylene I- 5 min xylene II – 5 min 2. DEHYDRATION: acetone I – 5 min acetone II – 5 min 3. WASHWITHWATER. 4. STAINWITH HAEMATOXYLINEFOR 5 min.
- 35. 5.Wash with water. 6.ONEDIP inACIDALCOHOL and immediatelywash in water. 7. BLUING – running tap water 10 min. 8. SATINWITH EOSIN FOR 30 sec. 9.Wash with tap water and allow for air dry. 10. MOUNTWITH DPX.
- 37. RESULTS ▪ Collagen: palepink. ▪ Muscle: deep pink. ▪ Acidophilic cytoplasm: red. ▪ Basophilic cytoplasm: purple. ▪ Nuclei: blue. ▪ Erythrocytes: cherry red.
- 38.
- 43. QUALITY CONTROL ▪ Dailyone QC is done in our dept. ▪ Efficacyof hematoxylin is tested by adding few drops of it to tap water and the colour change is noted. ▪ If it turns blue – purple immediately,it is in working condition. ▪ If it turns red to brown, it should be discarded.
- 44. ADVANTAGE DISADVANTAGE SIMPLE PROCEDURE COSTEFFECTIVE PLEASING COLOURS MOST HISTOLOGIC STRUCTURES ARE STUDIED DEPARAFFINIZATION IS IMPORTANT. AND SHOULD BE ADEQAUTE. AIR BUBBLES CAUSE NON STAINING. DEHYDRATION AFTER STAINING IS IMPORTANT.
- 45. REFERENCES ▪ MANUALOF HISTOLOGICTECHNIQUESBY SANTHOSHKUMARMONDAL. ▪ BANCROFT’STHEORYAND PRACTICEOF HISTOPATHOLOGICALTECHNIQUES. ▪ PRINCIPLESAND INTERPRETATIONOF LABORATORYPRACTICESINSURGICAL PATHOLOGY BY SHAMEEMSHARIFF. ▪ BASICANDADVANCEDLABORATORY TECHNIQUESIN HISTOPATHOLOGYAND CYTOLOGY BY PRANABDEY.