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HEMATOXYLIN AND EOSIN STAINING a brief discription
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- 3. A dyeis a colured substance which has an affinity to the substrate to which it is being applied May be natural or synthetic (majority) Hematoxylin – natural – heartwood of tree Hematoxylon campechianum Eosin – synthetic – xanthene DYES
- 4. Chemically, dyesare classified as acidic or basic Which combine with basic or acidic components respectively All ordinary stains used are salts and are composed of an acid and a base, the action depends on pH of solution in which they are employed
- 5. ACIDIC–anionic acid component iscoloured, base colourless eg : eosin BASIC- cationic base has colouring substance eg :hematoxylin, basic fuschin NEUTRAL- mixtures of acidic and basic dyes -eg: Romanowsky stains AMPHOTERIC – both anionic and cationic groups, but on same ion
- 6. Most widelyused nuclear stain, natural Itself has no staining properties Oxidization product hematein gives colour Oxidization – natural / synthetic HEMATOXYLIN
- 7. NATURAL (RIPENING)-exposure of prepared solutions to sunlight and air eg: Erhlich’s hematoxylin Adv – Long shelf life Disadv – slow process – 6 to 8 weeks (sometimes 3-4 months)
- 8. CHEMICAL- oxidisingagents sodium iodate – Mayer’s hematoxylin KMnO4 or mercuric oxide – Harri’s hematoxylin ADV- Rapid process, can be used immediately after preparation DISADV – Shorter useful life because of the continuing natural process of oxidation GLYCEROL – stabilizer, prevents over oxidation and evaporation -- improves and keep qualities of staining solution
- 9. MORDANTS Hematein haspoor affinity to tissues- improved by using mordants ( non dyeing compound which acts as a link between dye and tissue) Metal with two valencies MC used for hematoxylin – aluminium, iron, tungsten
- 10. CLASSIFICATION BASED ONMORDANT (1) Alum H (2) Iron H (3) Tungsten H (4) Molybdenum H (5) Lead H (6) H without mordant Most mordants are incorporated into the H But in few, they are used before staining eg- Heidenhain’s iron H
- 11. Used commonlyin routine H and E stain Alums are double sulphates with active trivalent metal ions such as Fe, Al, Cr together with K or ammonium as second cation NATURAL Erhlich’s ,Delafield’s CHEMICAL Mayer’s, Harri’s, Cole’s, Gill’s,Delafeld’s,Carazzi’s ALUM HEMATOXYLINS
- 12. Mordant used– aluminium as alum. Pot. Sulphate or alum. Ammon. Sulphate Produces good nuclear staining It stains nucleus red – converted to blue black when washed in a weak alkali solution –blueing Regressive or progressive stain Time required depends type and age of alum H and type of tissue
- 13. 1.ERHLICH’S HEMATOXYLIN Erhlichin 1886 Naturally ripened, 2months(emergency – add sodium iodate), last for years ADV AND USES Stains nuclei intensely and crisply Staining bone and cartilage For tissues exposed to acid (fixed long in formalin) Stains mucin in salivary gland & goblet cells Fades much more slowly Used for regressive staining
- 14. DISADV –Not ideal for frozen sections - staining time 20 to 30min Composition hematoxylin – 6g absolute alcohol – 300 ml glyecrin/glycerol- 300ml distilled water – 300 ml glacial acetic acid- 30 ml pot alum – 10 to 14 g
- 15. 2.DELAFIELD’S HEMATOXYLIN Naturallyripened alum H USE – For decalcified (acid treated) tissue, long stored and Bouin’s fixed tissues COMPOSITION hematoxylin-4g 95% alcohol-125 ml saturated aqueous ammon. Alum ( 15g/100 ml ) – 400 ml glycerin - 100ml
- 16. 3. MAYER’S HEMATOXYLIN Chemically ripened using sodium iodate USES 1.both as regressive and progressive stain. As progressive stain, they are suitable for automated procedures 2.as a nuclear counterstain in the demonstration of glycogen and in many enzyme histochemical procedures 3.as a counterstain in immunohistochemistry
- 17. COMPOSITION Hematoxylin – 1g distilled water – 1000 ml pot or ammonium alum – 50 g Na. iodate – 0.2 g citric acid – 1g chloral hydrat SLR – 50 g OR chloral hydrate AR- 30 g Staining time – progressive : 10 to 20 min regressive : 5 to 10 min
- 18. PREPARATION Hematoxylin K alum Na.iodate + Distilled water Chloral hydrate Citric acid Stain is ready Filter before use Warm/gentle heat &stir/ overnight room temp Boil 5 min Cool, filter
- 19. 4. HARRI’SHEMATOXYLIN Originally chemically ripened with mercuric oxide, bu replaced by Na/K iodate Composition hematoxylin – 2.5 g absolute alcohol – 25 ml K alum - 50 g distilled water – 500ml sodium iodate - 0.5 g glacial acetic acid- 20 ml
- 20. Hema toxyli n+abs alcoh ol K alum+ Dist. H2O Mix and heatmixture rapidly to boiling Sod iodate/ merc oxide Cool rapidly Add glacial acetic acid Ready for use
- 21. USES 1. Good hematoxylinstain for routine use. Gives clear nuclear staining. routinely used as regressive stain when used progressively , produces a light nuclear stain 2. Used as progressive nuclear stain in diagnostic exfoliative cytology
- 22. Staining time- 10 min Disadvantage quality of nuclear stain and speed of staining deteriorates after few months – formation of precipitate in stored solution should be filtered and also increase the staining time hence, preparation of a fresh batch of stain every month is advised
- 23. 5.COLE’S HEMATOXYLIN alum H , artificially ripened with alcoholic iodine solution Staining time – 20-45 min Composition hematoxylin – 1.5 g aquous K/Ammon alum- 700 ml 1% iodine in 95% alcohol- 50 ml distilled water – 250 ml
- 24. 6 CARAZZI’S HEMATOXYLIN Chemicallyripened by pot iodate Staining time 1-2min USES : progressive nuclear counterstain- pale and precise nuclear staining and doesn’t stain any cytoplasmic components COMPOSITION hematoxylin – 5g k. alum – 25g glycerol- 100 ml distilled H2O- 400 ml potassium iodate – 0.1 g
- 25. 7. GILL’SHEMATOXYLIN Chemically ripened by sodium iodate ADV :- 1.Fast in action 2.stable for 12 months , more stable than Harri’s 3.little or no surface precipitation Staining time regressive – 5 to 15 min
- 26. Disadv stains gelatinadhesive and even the glass may produce dark staining of mucus Formula dist water - 750 ml ethylene glycol – 250 ml hematoxylin – 2g sodium iodate- 0.2g alum sulphate – 17.6 g glacial acetic acid – 20 ml .
- 27. Staining times dependson (1)type of hematoxylin (2)age of stain (3)intensity of use of stain (4)progressive / regressive (5)pre treatment of tissue or sections (6)post teatment of tissue section eg subsequent van Geison (7)personal preference
- 28. DISADV sensitivity to subsequentacidic solution : van Gieson and other trichrome stains >in van Gieson :- application of picric acid- acid fuschine mixture after staining with H removes most of H > hence, Weigerts iron H / celestin blue solution with an alum H is used
- 29. Iron saltsare used as both oxidising agents and mordant MC used- Fe chloride & Fe.ammon.sulphate Commonly used Weigert’s Heidenhain’s Loyez H for myelin Verhoeff’s H for elastin fibres IRON HEMATOXYLIN
- 30. PRECAUTION : Over oxidationof H – hence H and Fe solutions are prepared separately and either mix immediately before use/ use consecutively
- 31. 1.WEIGERT’S HEMATOXYLIN Fe.Cl3 – mordent and oxidising agent Fe and H prepared separately and mixed immediately before use Staining time 15 to 30 min USE – as nuclear stain where acidic solutions are to be applied to the sections subsequently (eg- van Gieson stain)
- 32. PREPARATION SOL A :Hematoxylin 1 g absolute alcohol – 100 mL Use gentle heat to dissolve. And allowed to ripen naturally for 4 weeks before use SOL B : 30% aquous FeCl3 – 4mL conc. HCl – 1 mL distilled H2O – 100 mL MIX EQUAL VOL. JUST BEFORE USE HAVE A VIOLET BLACK COLOUR BROWN - DISCARD
- 33. 2.CELESTIN BLUEALUM HEMATOXYLIN Oxazine dye with little colouring property on its own USE – when acid counterstain is to be used Largely replaced Weigert’s H in van Gieson Composition celestin blue B – 2.5g fe.ammon.sulphate – 25g glycerin/glycerol- 70ml distilled H2O- 500 ml
- 34. 3.HEIDENHAIN’S IRONHEMATOXYLIN FE. AMMON. SULPHATE mordant and oxidising agent Used as regressive stain and same solution used as differentiating fluid Good cellular details and good photomicrography SOL A – hematoxylin – 0.5 g abs alcohol – 10 ml distilled water- 90 ml SOL B – fe. ammon.sulphate- 5 g dist water- 100ml HERE, IRON ALUM APPLIED FIRST, F/B HEMATOXYLIN
- 35. mitochondria, chromosomes,muscle striation, myelin, nuclear chromatin – GREY BLACK Time reqd in mordant – varies acc to fixative formalin, formalin sublimtes, Bouin’, carnoy- 1hr Helly’s or Zenker’s- 3 hrs osmium tetroxide, flemings fluid – upto 24hrs
- 36. Differentiation –difficult to judge. So, dip slide in and out of mordant until slide is clear & check under microscope Cytoplasmic counterstains usually not needed Fading – resistant to fading only if washed well after differentiation Staining time :- reduced at 60 deg C
- 37. 4.LOYEZ HEMATOXYLIN Fe.ammon.SO4 Use – to demonstrate myelin , and may be applied to paraffin, frozen or nitrocellulose section 5. VERHOEFF’S HEMATOXYLIN for demonstarting elastin fibres Stains coarse elastic fibres black FeCl3 – differentiator Provides excellent contrast But requires careful differentiation
- 38. Termed asMallory PTAH which may be used with (1) hematin (2)chemical oxdn, (3)Natural oxdn FIXATIVE – Zenker’s – preferred 10% BNF TUNGSTEN HEMATOXYLIN
- 39. 1.USING HEMATEIN hemateininstead of hematoxylin doesnt require oxidation process staining solution can be immediately used MIX BOTH, BOIL, COOL,FILTER AND USE Hematein 0.8g+dist H2O 1ml PTA 0.9g+dist H2O 9ml
- 40. 2.CHEMICALLY OXIDISED PTAH Oxidation using KMnO4 Solution should be used within 24 hrs Composition hematoxylin – 0.5g PTA- 10g Distilled H20 – 500ml 0.25% aqu. KMnO4 – 25ml
- 41. 3 NATURALLYOXIDISED PTAH Most satisfactory, but time consuming Takes months, can be used for years Use- CNS material, general tissue structures and tissues fixed in any of the std. fixative SOL A – 10% HCl in abs alc. 12 ml+3% aqu. Pot dichromate 36ml SOL B- 0.5% aqu.KMnO4 50ml+3%H2SO4 2.5ml SOL C- H 0.5 G, PTA -5G, dist H20(mordant)-500ml SEPARATELY PREPARED AND MIXED
- 42. RESULTS TISSUE COLOR Musclestriations,neuroglial fibres,fibrin , ameoba DARK BLUE Nuclei, cilia, RBC BLUE Myelin Lighter blue Collagen, osteoid,cartilage, elastic fibres Deep brownish red cytoplasm Pale pinkish brown
- 44. MOLYBDENUM H: are not used now LEAD H : Demonstration of granules in endocrine cells of alimentary tract & other regions : now replaced by IHC H WITHOUT MORDANT :to demonstrate various minerals in tissue section
- 45. Stains cellcytoplasm and most conn.tissue fibres In varying shades of pink, orange and red Most suitable stain that combine with alum H Also demonstartes general histologic architecture of tissue EOSIN
- 46. Xanthene dyes– Eosin Y, Ethyl Eosin , Eosin B Eosin y- water and alcohol solube & contain eosin Y, dist H2O, glacial acetic acid and 95% alc
- 47. USES Counterstainin routine H & E – pink – orange colour to cytoplasm RBC – intensley red To distinguish b/w cytoplasm of diff types of cells and conn tissue PHLOXINE & BIEBRICH SCARLET – SUBSTITUTES FOR EOSIN
- 48. Glasswares shouldbe clean &dry Weigh accurately & use correct solvent Keep silver & osmic acid stains in dark bottles Dilute ammonia should be freshly prepared Alcoholic solutions to be kept in glass stoppered bottle-to prevent evaporation of alcohol PREPARATION OF STAINS
- 49. SOLVENTS 1. Distilled water–MC 2. Ethanol 3. Methanol 4. Acetone 5. Phenol
- 50. Should allow easyflow of work from one procedure to another without any crossover Staining bench should face window &well illuminated Sinks –atleast 2 in number with atleast 2 cold water taps -1 for routine and 1 for special stain Staining rack Adequate supply of distilled water Microscope
- 51. STOCK STAINS ANDREAGENTS 1. STORING – in glass/ polypropylene stoppered bottles in cupboard 2. LABELLING – should be proper, pour from opposite side of labelling 3. CONTAINERS- frequently used stains & reagents kept in polythene wash bottle/coplin jar
- 52. MANUAL/ AUTOMATIC MANUAL Basic staining equipments : *glass troughs and racks of 12,25 or 50 slides *xylene-at start and end of staining STAINING PROCEDURE
- 54. ROUTINE H &E 1. REMOVAL OF PARAFFIN WAX WITH XYLENE- sections to be placed in xylene( Xylene I for 3 min & xylene II for 3 min) to dissolve wax
- 55. 2. REHYDRATION WITHDESCENDING CONCN OF ALCOHOL absolute alcohol – 1/2min > 95% alc. Half min > 90% alcohol -2 dips > 80 % alc 2 dips > 70 % alc. 2 dips> 50 % alc 2 dips > drained and taken to running tap water for 5 to 10 min Removal of deposits due to fixatives ; mercuric pigment by iodine – sodium thiosulphate & formalin by methods like
- 56. 3 STAININGBY H& E DIFFERENTIATION FOR REGRESSIVE STAIN 1% acid alcohol x 5 – 10 sec HEMATOXYLIN ACC. TO TYPE – regressive/progressi ve BUEINGx 5-15 min COUNTERSTAIN WITH 1% EOSIN Y X 10 MIN WASH with running tap water x 2-3 min to differentiate eosin After this, nuclei, keratin, RBC, conn tissue easily seen
- 57. 4.DEHYDRATION THROUGH GRADEDALCOHOL ascending grades of alcohol 2-4 dips in 95% & 2-4 dips in absolute alcohol 5 CLEARING WITH XYLENE/XYLOL Sections are transferred to 2 xylene bath Xylene 1 – 1 to 2 min, xylene II – 2 min Clearing tested by holding against watch Incomplete clearing => return to last alcohol and agitate for 30 sec , then to xylene 6. MOUNTING
- 58. STRUCTURE COLOR NUCLEI BLUE/BLACK CYTOPLASM VARYING SHADES OF PINK MUSCLE FIBRES DEEP PINK/ RED RBC ORANGE/ RED FIBRIN DEEP PINK RESULT OF H & E
- 60. MOUNTANTS - stainedelements of tissue sections can be seen only if sections are impregnated by transparent mounting medium Functions : 1.To make stained sections visible 2.Protection from physical injury 3.Fix slide to coverslip 4.Remove trapped air bubbles 5.Fill tissue spaces & cavities
- 61. QUALITIES RI closeto glass(1.518) Freely miscible with clearing agents Non reactive Colourless, transparent
- 62. AQUEOUS RESINOUS glycerine jellyNatural –canada balsam Apathy’s medium Dammar balsam Farrant’s medium colophonium resin Fructose syrup terpene resin Polyvinyl alcohol synthetic – euparal Highman’s mod. Of polystyrene
- 63. MOUNTING OF PARAFFINSECTION manually or automatic cover slips Manual method: - equipments required 1. Cover slips or cover glasses • square/rectangular • fraction of a mm thick • round – microbiology • routine – 0.125 mm thick • Size – various, 10x10mm,13x13mm, 15x15mm,18x18mm,22x22mm,22x30mm,22x40, 22x50 Supply- in 10 g packet, each costs about Rs.65
- 64. 2.DISSECTING NEEDLES to adjustcoverslip during & after mounting 3.MOUNTANTS depends on the type of section MOUNTING PROCEDURE 1. clean the coverslips 2.lay coverslips in a row 3.bring sections to xylene 4.removal of excess xylene
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- 68. FOR URGENTFROZEN SECTIONS – RAPID H & E Procedure 1.Freeze the required tissue block in freezing microtome/ cryostat. cut sections 3-6 micometre 2.Fix sections in AAF x 30 -60 sec alternative – 10% BNF x 20 sec 3.Rinse rapidly in water 4.Hematoxylin – harri’s x 2min/ carazzi’s x 1min
- 69. 5.rinse inLiCO3/ scott’s/alkaline tap water 6.counterstain in 1% aq.eosin 10-25sec 7.rinse rapidly in tap water 8.dehydrate with alcohol 9.clear with xylene 10.mount in DPX
- 70. TROUBLESHOOTING IN H&E, REMEDIES DEFECT CAUSE REMEDY PATCHY STAIN Less deparaffinization Destain slide & repeat procedure SECTIONS HAZY Less dehydration Repeat dehydration with fresh alcohol Very dark staining of H & E with no contrast Less differentiation Fresh differentiation soln Faint nuclear staining Over differentiation Restain with H
- 71. Pale or nonuclear staining Defect in H Repeat with fresh H Bubble artefacts-water/ air Water bubble- d/t improper dehydration because xylene wont mix with water Check % of alcohol and change it Air bubble due to improper mounting Remount the slide Sections floats after differentiation Not well embedded Re embedd
- 72. Unexpected colour– yellowish/brownish due to contamination by oxyhematin Weak staining during rapid staining RECTIFIED WITH FRESH BATCH OF STAIN
- 73. ARTEFACTS INHISTOLOGIC SECTIONS 1.formalin pigment- appears as fine black ppt on slides often with no relation with tissue Polarising microscopy to confirm Prevented by using enough BNF 2.mercurial pigment – large irregular clumps of black ppt 3.malarial pigment 4.tearing artefacts due to insufficient dehydration 5.brittle sections d/t alcohol fixatives 6.bubbles due to thin mounting media
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