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Histological techniques sections 1 2 3
This document outlines histological techniques focused on tissue collection, grossing, and fixation. It details procedures for specimen handling, various methods of fixation, and the chemical reactions involved, emphasizing the purpose and impact of different fixatives on cell preservation. The importance of avoiding autolysis and putrefaction, as well as factors influencing fixation efficacy, are also discussed.
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Histological techniques sections 1 2 3
- 1. Histological Techniques Dr MathewJoseph MD Anatomy (3rd year) All India Institute of Medical Sciences, Rishikesh
- 3. Contents Tissue collection andGrossing: • Sources of sampling • Instruments • Procedure • Specimen dissection plan • Sampling of tissue outside the laboratory
- 4. Section 1 Tissue Collectionand Grossing
- 5. Sources • Surgical resectionmaterial and biopsy material. • Postmortem specimens (Preferably recent) • Well perfused experimental animals, Meat venders.
- 6. Workstation • Well illuminatedroom • Ventilation and exhaust fan • Appropriate sink • Waste disposal unit • Firm, even and clean surface
- 8. Instruments • Scalpel blades •Scissors • Probes • Stainless steel ruler • Forceps • Sponges • First-aid box with eye wash bottle
- 15. Procedure Overview • Collectionof tissue/organ/specimen • Identifying the specimen. • Labeling of specimen • Store in normal saline Avoid exposure to air • Nervous tissue – Fixative (4 degree C) Avoid water • Wash excess of blood and mucous
- 17. Specimen Dissection Plans SNoSpecimen Procedure 1 Small sample Rarely need further dissection 2 Large sample While embedding lay out tissue in longitudinal fashion so that the plane of section cuts along the majority of the tissue: If >4-5 mm divide into two halves along the long axis. 3 Skin Mounted on edge to provide an adequate view of the epidermis, dermis and subcuticular substrates 4 Bowel Sampled with multiple blocks in relation to the adjacent mucosa, wall and serosal aspect tissues. 5 Brain Fixed immediately : Cut into slices once the fixation is done 6 Bone Fixation : Decalcification in EDTA
- 18. Sampling of tissueoutside the laboratory • Advanced Breast Biopsy Instrumentation (ABBI) • Brush biopsy • Computed tomography (CT) • Cone biopsy • Core biopsy • Crosby capsule • Curettings
- 19. Sampling of tissueoutside the laboratory • Ductal lavage • Endoscopic biopsy • Endoscopic retrograde cholangiopancreatography (ERCP) • Evacuation • Excision biopsy • Fine needle aspiration • Fluroscopy
- 20. Sampling of tissueoutside the laboratory • Frozen section • Imprint • Incision biopsy • Liquid based cytology (LBC) • Loop electrosurgical excison procedure (LEEP) • Magnetic resonance imaging (MRI) • Mammography • Needle biopsy
- 21. Sampling of tissueoutside the laboratory • Positron emission tomography with fluorodeoxy- glucose (PET with FDG) • Punch biopsy • Sentinel node biopsy • Shave biopsy • Smears • Stereotactic biopsy • Transurethral resection
- 22. Sampling of tissueoutside the laboratory • Trephine (bone marrow) biopsy • Ultrasound • Vacuum-assisted biopsies • Wire localization biopsy
- 23. Advanced Breast BiopsyInstrumentation (ABBI)
- 35. Section 2 Fixation ofTissue
- 36. Contents Fixation of Tissue: •Introduction • Function of fixative • Methods of fixation • Reaction of the cell(its component) with fixatives • Simple aqueous fixatives or fixative ingredients • Factors affecting fixation • Effect of fixation • References
- 38. Introduction Fixation is thecomplex series of chemical events and differs for the different groups of chemical substances found in tissues. Definition “A process by which the constituents ofthe cells or tissues are fixed in a physical and chemical state so that they will withstand subsequent treatment with various reagents with a minimum loss, distortion or decomposition”
- 39. • Once thetissue is removed from the body: Self-destruction / Autolysis. • If tissue is left without any preservation: Bacterial attack / Putrefaction.
- 41. Aim & Objectives •Toprevent autolysis and putrefaction. • Rapid and even penetration. • Topreserve cells and tissues in a life like manner as possible. • Elements that are to be demonstrated must remain in maximum concentration and precise localization.
- 42. Aim & Objectives •Stabilize labile elements. • Must be rigid to allow sectioning. • Must allow staining. • Optical contrast must be induced for morphological examination. • Allow long storage of tissues
- 43. Method of Fixation 1.Physical: 2. Chemical:
- 45. Methods of Fixation 1.Physical: •Heat • Microwave • Freeze drying 2. Chemicals -Coagulant -Non-coagulant (Baker’s Classification) Coagulant •Alcohol •Zinc Salts •Mercuric Chloride •Chromium Trioxide •Picric Acid Non-coagulant •Formaldehyde •Gluteraldehyde •Osmium Tetroxide • Potassium Dichromate •Acetic Acid
- 47. Classification of chemicalfixatives 1. Aldehydes : Formaldehyde Glutaraldehyde 2. Oxidizing agents : Osmium tetroxide Potassium permanganate 3.Proteins denaturing agents: Coagulant Acetic acid Methyl alcohol Ethyl alcohol 4.Other cross linking agents : Carbodiimides 5. Miscellaneous: Mercuric sodium, Picric acid , Dye stuff. (by bancroff)
- 48. Acc. to no.of fixatives used: 1. Simple fixatives 2. Compound fixatives i) Micro anatomical fixatives ii)Cytological iii)Histochemical
- 49. Reaction of fixativeswith Protein Most important reactions which stabilizes proteins by forming cross links between soluble protein & structural protein. Ultimately providing some mechanical strength.
- 50. Aldehydes • Cross linksare formed between protein molecules and Aldehyde group of fixative. • Aldehydes react with the basic amino acid residues of proteins & there is an accompanying change in isoeletric point of proteins. • This may form the basis for the of the different staining of tissues after different fixations.
- 51. Process takes placesin 2 steps: • 1st step-small polymers are formed • 2nd step small polymerscross-link Formations of cross linkages between Aldehyde and protein is measured by changes in viscosity, mechanical strength and molecular size of protein.
- 52. Formaldehyde • Slow reaction •Reversible*(in first 24 hr with excess of water) • Not good morphological picture • Less effective at cross linking • Loss of enzyme and immunological activity is less Glutaraldehyde • Rapid • Irreversible • Good morphological picture • More effective at cross linking • Loss of enzyme and immunological activity more
- 53. Oxidizing agents • Reactwith protein • Forms cross-links with proteins • Reflected by rapid increase in viscosity • After that decrease in viscosity , that phenomenon is known as secondary liquefaction. • Osmium tetroxide is more reactive towards protein.
- 54. Mercuric chloride • Itreacts with histidine residues in proteins. • Also there is production of H+ ions making solution more acidic more efficient. • But after fixation ultra structural preservation is poor.
- 55. Other fixatives • Heatfixation /microwave fixation ------ reacts with polar side chains of proteins. This increases their thermal energy which cause denaturation of proteins. This brings about tissue stabilization.
- 56. Reaction of fixativewith nucleic acid • Fixation brings about change in physical or chemical state of DNA or RNA at roomtemperature. • Few fixative react with nucleic acid chemically- including mercury and chromium salts. • Heating at 45 and 65 degrees with Aldehyde fixatives, there is uncoiling of RNA and DNA respectively.
- 57. • Ethanol, methanoland Carnoy’s fixative are commonly used. DNA is largely collapsed in methanol and ethanol. • Presence of salts is known to be essential for the maximum precipitation of nucleic acid from alcohol.
- 58. Reaction of fixativewith lipids Most of lipids are labile. So lost during routine processing. Todemonstrate them frozen section or cryostat is used. Aldehyde fixation: Preservation of lipoproteins (fixation of protein counterpart. ) Eg: phospholipids which contain amino group such as phosphotidyl ethanolamine are fixed by aldehyde. 22
- 59. • HgCl₂ reactwith highly unsaturated compound which form complex. It also reacts with lipids known as plasmalogen acetal phosphatides. • Additives such as tannic acid may be used for demonstration of lipid with light microscopy.
- 60. • Ultrastructural demonstration---post fixationwith osmium tetroxide. • Cholesterol may be fixed with Digitonin for Ultrastructural demonstration. 24
- 61. Reaction with Carbohydrates •Single fixative not satisfactory. • Alcoholic or picric acid fixatives preservation of glycogen which appear coarse eg: Alcoholic formaldehyde, Rossman’s solution. • Ultra structural studies gluteraldehyde is satisfactory while potassium permanganate increase image contrast. 25
- 62. • Tanic acidand cetyl pyrimidium have been found useful. • Additives to vehicle like Alcian blue or ruthenium red enhance glutaraldehyde fixation of glycogen and mucins. 26
- 63. Reagents used infixation 1. Aldehydes : formaldehyde, glutaraldehyde 2. Metallic: mercuric chloride, lead fixative 3. Picric acid fixative 4. Alcoholic fixative 5. Chromate fixative 6. Osmium tetra oxide fixative 7. Acetate fixative
- 64. Formaldehyde • Powerful reducingagent. • Most common fixative for routine fixation of biopsy specimen. • Formalin: 40%formaldehyde gas in water. • Forms methelene bridges between protein molecules. • Method: 4mm block - 8hrs at room temperature 4mm block - 2hrs at 45°C 28
- 65. • Most commonlyis used fixative •10% formalin consist of Formalin (40% formaldehyde): 10 ml Water :90 ml
- 66. • Neutralization isnecessary due to formation of formic acid by addition of buffer to maintain pH of 7. • Buffer added: phosphate buffer magnesium carbonate • Protein groups involved in formation of cross links amino, imino, peptide, hydroxyl, carboxyl and sulphahydryl. • Formaldehyde is also obtainable in a stable solid form composed of high molecular weight polymers known as paraformaldehyde .
- 67. Advantages • Cheap, easyto prepare, relatively stable, staining without preliminary procedures. • Good preservation of cell morphology • Good penetration properties. • Do not cause excessive hardening. • Best fixative for nervous system 31
- 68. Disadvantages •Slow fixation reaction. •Morphological details less accurate than glutaraldehyde. •Dermatitis of hand. •Fumes irritating to nostrils. •In tissue containing blood , dark brown artifact pigment granules are formed. 32
- 69. Fixative Formula AdvantagesUses 10%formal saline Water- Nacl- 900ml 8.5gm Formalin-100ml •Less shrinkage •Even fixation •Easy sectioning •Good staining Disadvantages •Slow fixative •Hard tissues •Neurological tissues •Gross specimen fixation. 10% formalin 40%formaldehyd e-100ml Distilled water- 900ml •Prevents pigments •Good fixation •Good penetration •Preserves Enzymes and organelles •Longer time for fixation •Routine specimen •Used for IHC 10% buffered neutral formalin Formalin -100ml Water - 900ml NaH₂Po₄-3.5gm Na₂HPo₄-6.5gm •Most routine purpose •Stops formation of formalin pigment •Fixes tissue rapidly •Loss of basophillic staining of the cytoplasm and nucleus •Loss of reactivity of myelin to weigert iron haematoxylin method 33
- 70. Fixative Formula AdvantageDisadvantage Calcium acetate formalin (formal calcium) Distilled water- 90ml Calcium acetate monohydrate- 2gm Formalin - 10ml •Buffered at pH7 by acetate •Preserves phospholipids •Less hardening or damage •Sectioned easily •Artifacts due to calcium Alcoholic formaldehyde Formalin- 100ml 95%alcohol- 900ml Calcium acetate-0.5gm •Rapid Fixation •Glycogen is better preserved •RBC are lysed Formol calcium Formalin- 100ml Distilled water- 900ml 10%calcium chloride- 100ml •Preservation of lipids •Artifacts due to calcium Neutral buffered phenol formalin Neutral buffered formaldehyde-100ml Phenol- 20gm •Stops formation of formalin pigment •Fixes tissue rapidly 34
- 71. Glutaraldehyde • Introduced bySabatini, Bensch and Barrett • It is a dialdehyde. • Stable in acid solution: in pH 3 to 5 at 0 ° to 4° C • Used in electron microscopy with osmium tetraoxide. 35
- 72. • Fixation ofsmall tissue: 2.5% solution for 2-4 hrs at room temperature • Fixation of large tissue: 4% solution for minimum 6-8hrs fully fixed for 24hrs 36
- 73. Advantages • Better preservationof cellular and fluid proteins than formaldehyde • More stable cross linkages • More rapid fixing action than formalin. • Less shrinkages than formalin • Give better section of blood clot and brain • Does not corrode metal • More pleasant and less irritating 37
- 74. Disadvantages • More expensive •Less stable • Penetrates tissue more slowly than formalin • Inferior to formalin for PAS technique. 38
- 75. Metallic fixative mercury Mercuricions act chiefly by combining with the acidic group of proteins and strong combination with sulfur thiol radicles. Advantages • Better staining of nuclei and connective tissue. • Give best results with metachromatic staining • Routine fixative of choice for preservation of detail of photography. 39
- 76. Disadvantages Corrode allmetal except nickel alloy. Solution deteriorates rapidly. Reduce amount of demonstrable glycogen. Penetration is slow. Long time fixation results in unduly hard and brittle tissue. Diffuse black granules are seen in tissue fixed with HgCl₂. 40
- 77. Picric acid fixative •It reacts with histone and basic proteins and forms crystalline picrates with amino acid. • It preserves glycogen well. Disadvantage: • Considerable shrinkage of tissue. • It dyes the tissue - yellow colour. 44
- 78. Alcoholic fixative • Mechanismof action: alcohol denatures and precipitate protein, possibly by disrupting hydrogen and other bonds. 47
- 79. Fixative Type FormulaAdvantage Disvantage Use Ethanol and methanol Cytological Cytoplasmic Ethyl alcohol and Rapid penetration •Inflammable •Causes •Smears Glycogen fixative Methyl alcohol Shrinkage and hardens Carnoy’s fixative Cytological Nuclear fixative Abs.Alcohol- 60ml Chloroform- 30ml Glacial acetic acid-10ml •Excellent Nuclear fixation and •Rapid penetration •Destroys Cytoplasmic Elements & lipids • Glycogen Preservation 48
- 80. Fixative Formula TypeAdvantages Use Clarke’s Fixative •Abs.alcohol- 75ml •Glacial Acetic acid- 25ml Cytological Nuclear fixative •Good penetration and nuclear details •preserves cytoplasmic elements Smears and chromosome study. Alcohol formalin 95%ethanol Formalin-10ml Useful fixative for sputum 49
- 81. Chromate fixative • Chromiumsalts in water form Cr-O-Cr complexes which have affinity for acidic and hydroxyl group of proteins so that complexes between adjacent protein molecules are formed. • This leads to disruption of internal salt linkages of protein ,thereby increasing the reactive basic groups and enhancing acidophilia in staining. 50
- 82. Fixative Formula TypeAdvantage and use Disadvantage Orth’s fluid 2.5%potassium dichromate- 100ml Sodium sulphate-1gm Just before using,formalin- 10ml Cytoplasmic fixative Regauds fluid Potassium dichromate Just before use,formalin- 20ml Cytoplasmic fixative Demonstration of,RBC colloid containing tissue, preserve phospholipid •Solution darken on standing •Prolonged fixation tend to bleach all tissue pigment. •Contraindicated in carbohydrates •Decrease intensity of PAS reaction. 51
- 83. Osmium Tetraoxide • Itis highly reactive substance , being easily reduced. • It gels protein probably by a process of bridge formation between compounds. • With lipid it forms mono and diester linkages which are then rendered insoluble and non extractable by fat solvent such as alcohol and xylene. • Rapid fixing agent • Stains tissue structure in a additive way as a grey black deposit. 52
- 84. Fixative Formula TypeAdvantages Disadvantage Use Flemming’s 1%aquaous Nuclear •It is expensive. •In electron fixative: chromic acid-15ml fixative Penetration is microscopy 2%aquaous slow. •Difficult to osmiumtetraoxide -4ml counterstain. •Cause reversal of tissue Acetic acid -1ml acidophilia Champy’s fluid 3% Potassium dichromate- 7ml Cytoplas mic fixative Preserves mitochondria, fat, yolk, lipids •Needs to be freshly prepared •Preferred for mitochondria 1% Chromic acid - 7ml 2% Osmium tetraoxide - 4ml 54
- 85. EFFECT OF FIXATION RULE#1 IS THAT FIXATIVES DENATURE MACROMOLECULES; FIXATION CHANGES THE SHAPE OF LARGE MOLECULES. THIS RULE IS THE BASIS FOR THE VARIED FUNCTIONS OF FIXATION AND WHY FIXED SPECIMENS LOOK THE WAYTHEY DO UNDER THE MICROSCOPE.
- 86. • RULE #2IS THAT DIFFERENT FIXATIVES PRODUCE THEIR OWN MORPHOLOGICAL PATTERNS. THAT IS AN OBJECTIVE FACT THAT DOES NOT IMPLY GOOD OR BAD. WHETHER WE LIKE WHAT WE SEE IS A SUBJECTIVE MATTER PREDOMINANTLY BASED ON OUR INDIVIDUAL TRAINING. MANY CHEMICALS ACT AS FIXATIVES IN THAT THEY DENATURE MACROMOLECULES, BUT FEW PRODUCE
- 87. RULE #3 ISTHAT FIXATION IS A CHEMICAL REACTION THAT IS NOT INSTANTANEOUS. ITS RATE IS DEPENDENT UPON THE CHEMICAL NATURE OF THE FIXATIVE SOLUTION AND ITS TEMPERATURE. Freida L. Carson
- 88. Factors affecting fixation •Hydrogen ion concentration • Temperature • Penetration • Osmolality • Concentration duration Other factors: • Volume changes • Substances added to vehicle 58
- 89. Hydrogen ion concentration •Satisfactory fixation occurs between pH 6 to 8. • Stabilization of tertiary and quaternary structure of proteins • By addition of acids pH decreases destruction of proteins and cause precipitation. • Hence, fixatives must be neutralized by adding buffer. 59
- 90. Commonly used buffersystem are : Phosphate, s- collidine, veronal acetate, Tris and cacodylate. •pH chosen must be as near the biochemical optimum as possible. •For electron microscopy , tissue must be fixed with a gradually increasing pH 60
- 91. Temperature High temperature: Rapid fixationreactions favors fixation. Fixation should be carried out atgradually increasing temperatures Disadvantages : 1. Risk of tissue distortion 2. Deleterious effect on certain antigen. Use : 1.Rapid fixation of urgent biopsy specimen. 2.To fix tissue with tuberculosis formaldehyde at 100°C is used. 61
- 92. Low temperature more accurate•Lowtemp. Slows down Autolysis details. •Ultra structure and enzyme histochemistry and electron microscopy , temp. range of 0 – 4 degrees is required. 62
- 93. Penetration • Fixation dependson diffusion of fixative into the tissue. • Penetration of fixatives is a slow process. • Size of specimen is important to ensure complete penetration of fixatives. • Small or thin slices of blocks - satisfactory fixation • Large blocks of specimen - slow fixation 63
- 94. • Slow rateof diffusion and reaction give rise to various zones of tissue fixed to different degrees. • d=k √t (d-depth penetrated , t-time , k- coefficient of infusibility. • Fixed tissue acts as a barrier to subsequent inward diffusion of fixatives. 64
- 95. Osmolality • Hypertonic solutions- cell shrinkage. • Isotonic and hypotonic solutions - cell swelling • In general fixatives that act mainly on protein precipitants cause shrinkage irrespective of what the osmotic pressure is and for non protein precipitants, reverse is true. 65
- 96. • By varyingthe Osmolality, structure of membrane system within various cells can be altered. Thus, additives to fixatives can alter extracellular space in tissues. • Sucrose is commonly added to osmium tetroxide for ultra structural studies • Fixative solutions must be preferably isotonic, thus cell swelling is compensated by processing and wax impregnation. 66
- 97. Concentration • Low concentrationof fixative with neutral pH favors fixation. • Glutaraldehyde solution is used as 3% solution but it is effective even at concentration as low as 0.05% with correct pH of fixative. • Presence of buffer causes polymerization of Aldehyde with a consequent decrease in effective concentration. • Staining of tissue is altered with the concentration of fixative employed. 67
- 98. Duration 1. Long duration Aldehyde: • inhibit enzyme activity and immunological reactions • shrinkage and tissue hardening. Glutaraldehyde: • longer duration of fixation • effective polymer formation • advantageous. In oxidizing fixatives : • degrade the tissue by oxidative cleavage of proteins and loss of peptides. 68
- 99. 2. Changes involume • Ideally, changes in processing and fixation cancel each other giving no net change. • Formalin fixed tissues along with paraffin embedding causes 33% shrinkage . 69
- 100. 3. Substances addedto vehicle •Fixative fixative agent + Buffer + water. •Salts added have denaturing and stabilizing effect on proteins. Eg : NaCl2 + HgCl2 , Tannic acid, Alcian blue for ultra structure.
- 101.
- 102. Contents Tissue Processing: • Introduction •Factors influencing the rate of processing • Stages of Tissue Processing • Errors and remedies during the process. • References
- 104. Introduction Definition: Tissue processing isthe process of removal of all the extractable water or fluid from the fixed tissue by treating it through a series of reagent(alcohol) solutions and replacing the tissue fluid with a support medium(wax) that provides sufficient rigidity to enable sectioning of the tissue without parenchymal damage or distortion.
- 106. Factors influencing therate of processing • Rate of exchange between the fluid within the tissue and the processing reagents. • Exposed surface of the tissue that is in contact with the processing reagents.
- 107. Factors influencing therate of processing S no Factor Effect Application Complication 1 Agitation Increases the flow of fresh solutions around the tissue. Automated processors with vertical or rotatory oscillation. 2 Heat Increases the rate of penetration and fluid exchange. Must be used sparingly: Temperature limited to 45 degree Celsius. Shrinkage, Hardening. 3 Viscosity Property of resistance to the flow of a fluid depends on size of the molecules in the solution. Smaller the size (LV), faster the rate of fluid penetration. All processing solutions must have similar viscocity. 4 Vacuum Increase the rate of infiltration. Vacuum can aid in the removal of trapped air in porous tissue: useful in fatty tissue. High pressure leads to tissue deterioration.
- 109. Stages of TissueProcessing S no Stages Effect 1 Dehydration Removal of water and fixative from the tissue. 2 Clearing Removing of dehydrating solutions, making the tissue components receptive to the infiltrating medium. 3 Infiltration Permeating the tissue with a support medium. 4 Embedding Orienting the tissue in a support medium and allowing it to solidify.
- 111. ***Additional steps arerequired after special fixation techniques before processing is initiated: S no Fixative Post fixative treatment 1 Picric acid fixatives :Form water soluble picrates. (Bouin’s Solution): Tissues kept directly into 70% alcohol. 2 Alcoholic fixatives (Carnoy’s Fluid) Tissues kept directly into 100% alcohol.
- 112. Dehydration The process ofremoving the fixative and water from the tissue and replacing it with dehydrating agent. S No Dehydrating agent Advantages Disadvantages 1 Ethanol Good hydrophilic and attracts water from the tissue. • Shrinkage • Hardening of tissue. 2 Methanol Substitute for ethanol. • Highly Toxic • Volatile • Inflammable • Expensive • Poor lipid solvent 3 Isopropanol • Shrinks and hardens tissue less than ethanol . • Used to dehydrate hard and dense tissues • Slower in action • Less hydrophilic than ethanol
- 113. Steps of Dehydration •This process is carried out by immersing tissue in increasing concentrations of ethanol solutions (50% to 100%). • A gradated increasing concentration of the alcohol cause gradual replacement of water from the tissue thus prevents excessive distortion of the tissue.
- 114. Steps of Dehydration SNo Steps of dehydration Large tissues Rapid dehydration of thin tissue 1 50% Alcohol 12-24 hours 30 minutes 2 70% Alcohol 12-24 hours 30 minutes 3 90% Alcohol 12-24 hours 30 minutes 4 100% Absolute alcohol : 2 changes 12-24 hours 1 hour
- 115. Errors in Dehydration S No Errorsin dehydration Effect Remedy 1 Excessive concentration gradient of dehydrating solution. Increases diffusion currents across the cell membranes leading to cell distortion. Dehydrate slowly with graded series of dehydrating solution of incresing concentration. 2 Excessive dehydration. Hard, Brittle and Shrunken tissue. Monitor time. 3 Inadequate dehydration. • Impair penetration of clearing agent leading to soft tissue, non receptive infiltration. • Clearing agents turns milky when tissue placed in it. Repeat dehydration with absolute alcohol and clear again.
- 116. Clearing • Is theprocess of replacing the dehydrating agent with an intermediate fluid which is miscible with both dehydrating fluid and the embedding medium. • The reagent for clearing has the same refractive index as the tissue proteins and hence when tissue is immersed in it, it becomes transparent and clear.
- 117. S No Clearing agents AdvantagesDisadvantages 1 Xylene • Rapid in action. • Readily eliminated in the incubator. • Inflammable • Hardening of tissue on prolonged immersion. 2 Toluene • Rapid in action. • Less damaging on prolonged immersion. • Expensive • More flammable and volatile than xylene. 3 Chloroform • Less brittleness of tissue • Better for thicker tissue blocks and CNS tissues. • Non flammable • Slower in action • Toxic • Expensive • Heavy • Highly volatile • Slow penetration 4 Benzene • Best solvent • More gentle and rapid in action • Toxic (Carcinogenic)
- 118. Clearing Steps of clearing: •One to three changes of xylene immerssions are done. • Time required depends on the size of tissue. S No Size of specimen Time kept in Xylene 1 Small specimen : < 5 mm 30 minutes to 1 hour 2 Large specimen : 5 mm to 1 cm 3 hours to 6 hours 3 Very bulky: > 1 cm (whole embryo) 12 hours
- 119. Errors in Clearing S No Errorin clearing Effect Remedy 1 Insufficient clearing • Opaque tissue • Difficult section cutting • Repeat clearing process 2 Prolonged exposure to clearing agents • Hard and brittle tissue • Time monitoring
- 120. Infiltration • It isthe process of replacing the clearing agent with a supporting medium which finally infiltrates the tissue. • It is done in three different wax immersions to ensure complete replacement of clearing agent in the tissue.
- 121. Common Embedding Medium S No Embedding medium AdvantagesDisadvantages 1 Paraffin wax • Most commonly used embedding medium. • Inexpensive, easily handled. • Good cutting quality. • Durable and good for storage. • High melting point may cause heat trauma to tissue. 2 Ester wax • Lower melting point than paraffin wax. • Good for thin sections. • Minimal shrinkage. • Morphological preservation of tissue is less better than compared to other agents. 3 Water soluble wax • Dehydration and clearing not required. • Tissue can be directly from an aqueous medium to wax for infiltration. • Less shrinkage • Sectioning and manipulation of tissue is difficult. 4 Cellulose nitrate • No heating required. Low tolerance of water.
- 122. Common Embedding Medium SNo Embedding medium Advantages Disadvantages 5 Double embedding • Good features of more than one embedding media are used to maintain morphological appearance of specimen. • Eg: Paraffin wax and Cellodin (Lung tissue) • Expensive 6 Celloidin embedding • The minimum thickness is about (12 – 15 um) or thicker (20-100 um) sections used for Golgi staining of neuronal tissue. • For section cutting, the block is lubricated with a fluid, usually 60-70% ethanol, and is not allowed to dry out. • Slow process • Sections are stored in 60- 7-% ethanol instead of being adhered to glass slides. • They are usually stained free floating and put on slides at the same time as coverslip is applied.
- 123. Error in Infiltration SNo Error in infiltration Effect Remedy 1 Insufficient infiltration Moist paraffin block Tissue crumbling Difficult sectioning Repeat infiltration
- 124. Embedding It is thefinal step of tissue processing involving orienting the tissue in a support medium and allowing it to solidify. 1. The Leuckhart’s embedding mould is filled with some molten wax. 2. The processed tissue specimen is placed in the bottom of the mould. 3. Orientation of specimen is done with forceps to ensure right sectioning of the tissue. 4. Any trapped air is removed using needle or fine tip forceps.
- 128. Summary Table of stagesof Tissue Processing S No Process Solution Time 1 Dehydration 50% Alcohol 1 Hour 2 Dehydration 70% Alcohol 1 Hour 3 Dehydration 90% Alcohol 1 Hour 4 Dehydration Absolute alcohol 1 Hour 5 Dehydration Absolute alcohol 40 minutes 6 Clearing Xylene 1 1 Hour 7 Clearing Xylene 2 30 minutes 8 Infiltration Paraffin wax 1 30 minutes 9 Infiltration Paraffin wax 2 30 minutes 10 Infiltration Paraffin wax 3 30 minutes 11 Embedding Embedding mould
- 130. References 1. Carleton’s HistologicalTechnique, 4th Edition, R.A.B Drury: Oxford;1957 2. John D. Bancroft: Theory & Practice Of Histological Techniques. 3. Google images.
- 131.