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CHAPTER 2 FRESH TISSUES EXAMINATION -vary according to structural and chemical components of the cells to be studied -may be done

on fresh/preserved tissue METHODS 1. Teasing/Dissociation - selected tissue is immersed in watch glass containing NSS then carefully dissected/separated and examined under the microscope. 2. Squash Preparation/Crushing - small pieces not more than 1 mm are placed in a slide and forcibly compressed with another slide or with a cover glass. 3. Smear Preparation - examining sections of sediments, whereby cellular materials are spread lightly over a slide by means of wire loop or applicator. Streaking zigzag line Spreading teasing mucous strands Pull-Apart two slides are used Touch Preparation cells are transferred to the slide 4. Frozen Section Tissue is frozen with liquid nitrogen and a section is examined under the microscope. PROCESSING OF TISSUES Fixation Dehydration Clearing Infiltration (Impregnation) Embedding Trimming Section-Cutting Staining Mounting Labelling

CHAPTER 3 FIXATION AND FIXATIVES Fixation fixing or preserving fresh tissue for examination - Quality of section on the slide is only as good as the quality of the fixed specimen Primary aim: preserve the morphological and chemical integrity of the cell in as life-like manner. - Shape, structure, intercellular relationship and chemical constituents of tissues are preserved. - Prevents degeneration, decomposition, putrefaction, and distortion of tissues after removal from the body. Secondary goal: harden and protect the tissue from the trauma of further handling Stabilizing proteins: most important reactions for maintaining morphology in the fixation of tissues for routine histopathology - They are fixed to structural proteins and thus rendered insoluble 1. To preserve the tissue 2. To prevent breakdown of cellular elements 3. To coagulate or precipitate protoplasmic substances Additive fixation fixative becomes part of the tissue Negative fixation fixative does not incorporate into the tissue but alters the tissue composition and stabilizes the tissue by removing the bound water attached.

MAIN FACTORS INVOLVED IN FIXATION: 1. Hydrogen Ion Concentration pH 6 and 8 2. Temperature Formalin heated at 60C 3. Thickness of section 2cm2 for light microscopy 4. Osmolality slightly hypertonic 5. Concentration low conc. of glutaraldehyde 6. Duration of fixation 2-6 h in buffered formalin PRACTICAL CONSIDERATIONS OF FIXATION: 1. Speed 2. Penetration 3. Volume 4. Duration of Fixation

EFFECT OF FIXATIVES - harden soft and friable tissues - make the cells resistant to damage and distortion - inhibit bacterial decomposition - increase optical differentiation of cells and tissues - act as mordants or accentuators - reduce the risk of infection CHARACTERISTICS OF A GOOD FIXATIVE 1. Cheap 2. Stable 3. Safe to handle 4. Kills the cell quickly producing minimum distortion of cell constituents. 5. Inhibit bacterial decomposition 6. Produce minimum shrinkage of tissues 7. Harden tissues making cutting sections easier 8. Isotonic, causing minimal physical and chemical alteration of the cells and their constituents. 9. Make cellular components insoluble to hypotonic solutions

2. According to Action a. Microanatomical Fixatives permits the general microscopic study of tissue structures 1. 10% Formol Saline 2. 10% Neutral Bufered Formalin 3. Heidenhains Susa 4. Formol sublimate 5. Zenkers solution 6. Zenker formol 7. Ouins solution 8. Brasils solution b. Cytological preserve the specific parts and particular microscopic elements of the cell itself 1. Nuclear Fixative preserve nuclear structures Flemmings fluid Carnoys fluid Bouins fluid Newcomers fluid Heidenhains Susa 2. Cytological Fixatives preserves cytoplasmic structures Flemmings fluid without acetic acid Kellys fluid Formalin with post-chroming Regauds fluid (Mullers fluid) Orths fluid 3. Histochemical Fixatives preserve chemical contents of cells and tissues Formol Saline 10% Absolute Ethyl Alcohol Acetone Newcomers Fluid LIPID FIXATION Use mercuric chloride and potassium dichromate - Bakers formol calcium for phospholipids CARBOHYDRATE FIXATION Alcoholic formaldehyde PROTEIN FIXATION Neutral buffered formol saline or formaldehyde GLYCOGEN FIXATION Rossmans fluid or absolute alcohol

TYPES OF FIXATIVES 1. According to composition a. Simple Fixative made up of only one component substance. i. Aldehydes 1. Formaldehyde 2. Glutaraldehyde ii. Metallic Fixatives 1. Mercuric Chloride 2. Chromate Fixatives a. Potassium dichromate b. Chromic acid 3. Lead Fixatives a. Picric Acid b. Acetic Acid c. Acetone d. Alcohol e. Osmium tetraoxide 4. Heat b. Compound Fixative made up of two or more fixatives

ALDEHYDE FIXATIVES Formaldehyde widely used (10% formalin) - DA: fumes are irritating to the nose and eyes - prolonged storage may induce precipitation of white paraformaldehyde - Removal of precipitate is addition of 10% methanol 10% Formol-Saline - 40% Formaldehyde + NaCl + Distilled water fixation of CNS Tissues and General post-mortem tissues - preserves enzymes and proteins 10% Neutral Buffered Formalin/Phosphate-Buffered Formalin - Sodium dihydrogen phosphate + Disodium hydrogen phosphate + 40%Formaldehyde + Distilled water - preservation of surgical, post-mortem and research specimens - best fixative for iron-containing tissues Formol-Corrosive (Formol Sublimate) - Sat. Aq. Mercuric Chloride + 40% Formaldehyde - routine post-mortem tissues - excellent in silver reticulum methods - fixes lipids, especially neutral fats and phospholipids Alcoholic Formalin (Gendres Fixative) - 95% Ethyl Alcohol saturated with picric acid + Strong formaldehyde solution + glacial acetic acid - immunoperoxidase studies on tissues - used for rapid diagnosis - good for preservation of glycogen and for microincineration -used to fix sputum, since it coagulate mucus Glutaraldehyde -two formaldehyde residues linked by 3C chains -used for enzyme histochemistry and electron microscopy -preserves plasma proteins

METALLIC FIXATIVES 1. MERCURIC CHLORIDE - Mercuric Chloride + Potassium Dichromate + Sodium Sulfate + Distilled Water - most common metallic fixative - Tissues fixed with mixtures containing mercuric chloride (except Susa) contain black precipitates of mercury. -Routine fixative of choice for preservation of cell detail in tissue photography. - Renal tissues, Fibrin, Connective tissues and muscles - Black deposits may be removed by adding saturated iodine solution in 96% alcohol, the iodine being decolorized with absolute alcohol in the subsequent stages of dehydration. Zenkers Fluid - Mercuric Chloride + Glacial Acetic Acid - fixing small pieces of liver, spleen, connective tissue and nuclei - may act as mordant - Mercuric deposits may removed by immersing tissues in alcoholic iodine solution. de-zenkerization Zenker-formol (Hellys solution) - Mercuric chloride + Potassium dichromate + Sodium sulphate + Distilled water + Strong formaldehyde (40%) - fixative for pituitary gland, bone marrow and blood containing organs such as spleen and liver. - preserves cytoplasmic granules - Brown pigments are produced if tissues are allowed to stay for more than 24 hours. - Pigments can be removed by immersing the tissue in saturated alcoholic pricric acid or sodium hydroxide Heidenhains Susa Solution - Mercuric chloride + Sodium chloride + Trichloroacetic acid + Glacial Acetic Acid + Formaldehyde (40%) + Distilled water - tumor biopsies especially of the skin - excellent cytologic fixative -Mercuric chloride deposits may be removed by immersion on alcoholic iodine solution - the tissue should be transferred directly to a high-grade alcohol, to avoid undue swelling of tissues caused by treatment with low-grade alcohol or water. B-5 Fixative - Distilled water + Mercuric Chloride + Sodium acetate - commonly used for bone marrow biopsies

CHROMATE FIXATIVES Chromic Acid - used in 1-2% aqueous solution - precipitates all proteins and adequately preserves carbohydrates. - Strong oxidizing agent, strong reducing agent must be added. Potassium Dichromate - used in 3% aqueous solution - preserves lipids - preserves mitochondria Regards (Mullers) Fluid - Potassium dichromate + Strong formaldehyde 40% - Demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid-containing tissues - Prolonged fixation gives out black deposits and can be removed by running tap water. Orths Fluid - study of early degenerative proceses and tissue necrosis - demonstrates rickettsiae and other bacteria - preserves myelin LEAD FIXATIVES - used in 4% aqueous solution - recommended for acid mucopolysaccharides - fixes connective tissue mucin - forms insoluble lead carbonate due to prolonged standing which can be removed by filtration or adding acetic acid drop by drop PICRIC ACID FIXATIVES - Yellow color may be removed by treatment with another acid dye or lithium carbonate - excellent fixative for glycogen demonstration - suitable for Aniline stains Bouins Solution - Sat. Solution of picric acid + Strong formaldehyde 40% + Glacial acetic acid - Fixation of embryos and pituitary biopsies - Preserving soft and delicate structures (endometrial curettings) -yellow stain is useful for handling fragmentary biopsies. - collagen, elastic connective tissues Brasils Alcoholic Picroformal Fixative - Formaldehyde + Picric Acid + Ethanol/Isopropyl Alcohol + Tricbloroacetic acid - Fixative for glycogen - Less messy than Bouins solution

GLACIAL ACETIC ACID - Precipitates chromosomes and chromatin materials - Essential constituent of most common compound nuclear fixatives ALCOHOLIC FIXATIVES - Ideal for small tissue fragments - Used as a fixative and dehydrating agent Methyl Alcohol - fixing dry and wet smears, blood smears and bone marrow tissues Isopropyl Alcohol 95% - fixing touch preparations Ethyl Alcohol blood, tissue films and smears Carnoys Fluid - Absolute alcohol + Chloroform + Glacial acetic acid - fixing chromosomes, lymph node glands and urgent biopsies -fix brain tissue for diagnosis of rabies Newcomers Fluid - Isopropyl alcohol + Propionic acid + Petroleum ether + Acetone + Dioxane - fixing mucopolysaccharides and other proteins OSMIUM TETRAOXIDE (OSMIC ACID) - Pale yellow powder which dissolves in water to form strong oxidizing solution. -fixes conjugated-fats and lipids permanently by making them insoluble during subsequent treatment with alcohol and xylene -helps preserve cytoplasmic structure -fixes myelin and peripheral nerves well -fixes materials for ultrathin sectioning in electron microscopy, since it rapidly fixes small pieces of tissues and aids in their staining -black osmic oxide crystals may be dissolved in cold water -Osmic acid-fixed tissues must be washed in running water for at least 24 hours to prevent formation of artefacts Flemmings Solution - common chrome-osmium acetic acid fixative used - Recommended for nuclear preparation of such sections - Aqueous chromic acid 1% + Aqueous osmium tetraoxide 2% + Glacial acetic acid -an excellent fixative for nuclear structures -permanently fixes fat Flemmings solution without acetic acid - Made up only of chromic and osmic acid - Removal of acetic acid from the formula serves to improve the cytoplasmic detail of the cell

TRICHLOROACETIC ACID - incorporated into compound fixatives -precipitates proteins -marked swelling effect on tissues serves to counteract shrinkage produced by other fixatives -may be used as a weak decalcifying agent ACETONE - Used at a cold temperature ranging from 5*C to 4*C - Recommended for the study of water diffusible enzymes especially phosphatases and lipases - Used in fixing brain tissues for diagnosis of rabies HEAT FIXATION - Involves thermal coagulation of tissue protein for rapid diagnosis - Employed for frozen tissue sections and bacteriologic smears - Destroys RBC - Dissolves starch and glycogen SECONDARY FIXATION - Process of replacing an already fixed tissue in a second fixative order - Usually with 10% formalin or 10% formol saline as primary fixative POST CHROMATIZATION -form of secondary fixation whereby a primarily fixed tissue is placed in aqueous solution of 2.5- 3 % potassium dichromate for 24 hours to act as mordant for better staining effects WASHING-OUT -process of removing excess fixative from the tissue after fixation in order to improve staining and remove artefacts from the tissues 1. Tap water- removes:-excess chromates from tissues fixed in Kellys, Zenkers, and Flemmings solutions -excess formalin -excess osmic acid 2. 50-70% alcohol used to wash out excess amount of picric acid (Bouins solution) 3. Alcoholic iodine- used to remove excessive mercuric fixatives

Factors that affect Fixation of the Tissues A.RETARDED BY: 1. Size and thickness of the tissue specimen-largest tissues require more fixatives and longer fixation time 2. Presence of Mucus-prevents complete penetration of fixative; hence, tissues that contain mucus are fixed slowly and poorly.Excess mucus may be washed away with normal saline solution. 3. Presence of fat- fatty tissues should be cut in thin sections and fixed longer. 4. Presence of blood- tissues containing large amount of blood should be flushed out with saline before fixing 5. Cold temperature- inactivates enzymes B.ENHANCED BY: 1. Size and thickness of tissues- smaller and thinner tissues requires less fixative and shorter fixation time 2. Agitation- fixation is accelerated when automatic or mechanical tissue processing is used. CHAPTER 5 DEHYDRATION - Process of removing intercellular and extracellular water from the tissue following fixation and prior to wax impregnation Dehydration Agents - Solutions utilized General Rule: Whatever the dehydrating agent is used, the amount in each stage should not be less than 10 times the volume of the tissue in order to ensure complete penetration of the tissue by the dehydrating solution. Characteristics of Ideal Dehydrating Agent 1. Should dehydrate rapidly 2. Should not evaporate very fast 3. It should be able to dehydrate even fatty tissues 4. It should not harden tissues excessively 5. It should not remove stains 6. It should not be toxic to the body 7. It should not be a fire hazard Commonly used: - Alcohol (most common) - Acetone - Dioxane 4 cellosolve - Triethyl phosphate - Tetrahydrofuran

ALCOHOL - Routine dehydration of tissues - Mixes with water and other organic solvents Methyl Alcohol blood and tissue films; smear preparation Butyl Alcohol plant and animal micro-techniques 37C temp hastens dehydration Anhydrous copper sulphate accelerates dehydration - blue discoloration will indicate full saturation ACETONE - Urgent biopsies - More miscible with epoxy and other resins & highly flammable - Small pieces of tissues due to extreme volatility and inflammability DIOXANE - Less tissue shrinkage as compared to alcohol dehydration - Its vapour produces a cumulative and highly toxic action in man I. Gaupners Method 1st Pure dioxane solution 1 hr 2nd Pure dioxane solution 1 hr 3rd Pure dioxane solution 2 hrs 1st Paraffin wax 15 minutes 2nd Paraffin wax 45 minutes 3rd Paraffin wax 2 hrs II. Weisebergers Method - the tissue is wrapped in a gauze bag and suspended in a bottle containing dioxane and a little anhydrous calcium oxide. CELLOSOLVE - can be stored in for months without distortion. TRIETHYL PHOSPHATE - removes water very readily and produces very light distortion and hardening of tissues TETRAHYDROFURAN - dehydrates and clears tissue - THF is toxic if ingested or inhaled. Vapors cause nausea, dizziness, headache and anesthesia. - May cause conjunctival irritation Phenol 4% + 95% Ethanol acts as softener for hard tissues such as tendons , nails or dense fibrous tissue.

CHAPTER 6 CLEARING de-alcoholization - Alcohol or dehydrating agent is removed from the tissue and replaced with a substance that will dissolve the wax with which the tissue is to be impregnated. CHARACTERISTICS OF A GOOD CLEARING AGENT 1. Should be miscible with alcohol. 2. Should be miscible with, and easily removed by melted paraffin wax 3. Should not produce excessive shrinkage 4. Should not dissolve out aniline dyes 5. It should not evaporate quickly in a water bath. 6. Should make tissues transparent. 1. 2. 3. 4. 5. 6. 7. 8. Xylene ( most common) Toluene Benzene Chloroform Cedarwood Oil Aniline Oil Clove Oil Carbon tetrachloride

XYLENE (XYLOL) - Routine histologic processing - Urgent biopsies which it clears within 15-30 minutes - Becomes milky when an incompletely dehydrated tissue is immersed in it. TOLUENE - Substitute for xylene or benzene - Miscible with both absolute alcohol and paraffin - Much more expensive than xylene BENZENE - Urgent biopsies and routing purposes - Excessive exposure to benzene may be extremely toxic to man and may become carcinogenic or it may damage the bone marrow resulting in aplastic anemia. CHLOROFORM - Slower action than xylene - Recommended for tough tissues, for nervous tissues, lymph nodes and embryos because it causes minimum shrinkage and hardening of tissues CEDARWOOD OIL - Used to clear both paraffin and celloidin sections - Recommended for CNS tissues and cytological studies, particularly smooth muscles and skin. - Becomes milky upon prolonged storage and should not be filtered before use.

ANILINE OIL clearing embryos, insects and very delicate specimens due to its ability to clear 70% alcohol without excessive tissue shrinkage CLOVE OIL Tissues become brittle, aniline dyes re not removed and celloidin is dissolved. CARBON TETRACHLORIDE Similar to chloroform except its a lot cheaper. METHYL BENZOATE AND METHYL SALICYLATE - used in double embedding techniques

BY AUTOMATIC PROCESSING - Use of automatic tissue processing machine which fixes, dehydrates, clears and infiltrates tissues. - Only 2-3 changes of wax are required to remove the clearing agent Agitation & Heat hastens automatic process Ex: Elliott Bench-Type Processor BY VACUUM EMBEDDING - Principle is the negative atmospheric pressure hastens the tissue processing - Removal of air bubbles and clearing agent from the tissue block thereby promoting a more rapid wax penetration of tissue. - gives the fastest result SUBSTITUTES FOR PARAFFIN WAX PARAPLAST - Mixture of highly purified paraffin and synthetic plastic polymers - Melting point of 56-57c - More elastic and resilient than paraffin wax Embeddol similar to paraplast; melting point 56-58C Bioloid recommended for embedding eyes Tissue Mat Product of paraffin, containing rubber, with the same property as paraplast. ESTER WAX - Melting point 46-48C - Harder than paraffin wax - 3-4 changes of wax are required to ensure complete tissue impregnation WATER SOLUBLE WAXES - Melting points 38-42C or 45-56C Carbowax

CHAPTER 7 IMPREGNATION AND EMBEDDING IMPREGNATION - process whereby the clearing agent is completely removed from the tissue EMBEDDING process by which impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify 1. 2. 3. 4. Paraffin wax Celloidin Gelatin Plastic

55-60C above the melting point of wax 56C temp of wax normally used for routine work 1. By manual processing 2. By automatic processing 3. By vacuum embedding BY MANUAL PROCESSING - 4 changes of wax are required at 15 minutes interval in order to insure complete removal of the clearing agent. Example of time schedule for manual processing: FIXATION: 10% Buffered Formalin 24 hours DEHYDRATION: 70% Alcohol 6H 95% Alcohol 12H 100% Alcohol 2H 100% Alcohol 1H 100% Alcohol 1H CLEARING: Xylene or Toluene (2x) 1H IMPREGNATION: Paraffin Wax (4x) 15minutes EMBEDDING: Paraffin Wax 3H

CELLOIDIN IMPREGNATION - purified form of nitrocellulose soluble in many solvents, with large hollow cavities which tend to collapse, for hard and dense tissues such as bones and teeth for large tissue sections of the whole embryo - supplied in thin 2%, medium 4%, or thick 8% 2 Types: 1. Parloidoin hard, amber, platelet-chips 2. Low Viscosity Nitrocellulose nitrated variety - it can be used in higher concentrations and still penetrate tissues rapidly. - usually marketed while wet with alcohol.

2 Methods: 1. Wet Celloidin Method - Recommended for bones, teeth, alrge brain sections and whole organs Process: Fixation placed in equal parts of ether and alcohol (1224hours) thin, medium celloidin (5-7 days) thick celloidin (3-5 days) stored in 70% alcohol 2. Dry Celloidin Method - Recommended for processing the whole eye sections - Same Principle except that of 70% Alcohol, it is GILSONS MIXTURE (equal parts of chloroform and cedarwood oil) - Best stored in air-tight jar GELATIN IMPREGNATION - Used as an embedding medium for delicate specimens and frozen tissue sections because it prevents fragmentation of tough and friable tissues - Fixation 10% gelatin with 1% phenol (24H) transferred to 20% gelatin with 1 % phenol (12H) 20% gelatin with 1 % phenol cooled to refrigerator transferred to 10% formalin (12-24H) -Volume of impregnating medium should be at least 25 times the volume of the tissue EMBEDDING - After impregnation, the tissue is placed into a mold containing the embedding medium and this medium is allowed to solidify. Types of Blocking-Out Models 1. Leuckharts Embedding Model L-shaped strips of heavy brass arranged in a flat metal plate and which can be moved to adjust the size of mold to the size of the specimen. 2. Compound Embedding Unit interlocking plates resting on a flat metal base 3. Plastic Embedding Rings and Base Mold - special stainless steel base mold fitted with plasticembedding ring 4. Disposable Embedding Models -

PLASTIC (RESIN) EMBEDDING - superior results for light microscopic studies - high resolution light microscopy of tissue - Epoxy, Polyester, Acrylic EPOXY - Benzoyl peroxide + epoxy (catalysts) - epoxy plastics, catalysts and accelerators - Cyclohexene dioxide-based plastics (Spurtt) can be obtained pure, have very low viscosity and infiltrate fastest. Disadvantages: 1. Hydrophobic 2. Reduce antigenicity of embedded tissue and may compromise the result of immunohistochemical staining 3. Cause sensitization if absorbed bt skin or inhalation 4. VCD is known to be carcinogenic POLYESTER PLASTICS - Introduced for electron microscopy ACRYLIC PLASTICS - made up of acrylic acid or methacrylic acid - used for light microscopy Ex. 1. Polyglycol methacrylate (GMA) 2. Methyl methacrylate (MMA) undecalcified bone

LABORATORY 1 FRESH TISSUE EXAMINATION Fresh Tissue Examination has advantage of examining the tissue in living state allowing protoplasmic activities to be observed. Methods - Teasing (Dissociation) - Squash Preparation (Crushing) - Smear Preparation -Streaking -Spreading -Pull-apart -Touch Preparation -Frozen Section 2- FIXATION Fixation first and most critical step in histotechnology. - 10% Neutral buffered formalin is mostly widely used fixative because it is compatible with most stans. - Physical characteristics to note: color, consistency, texture and size of tissue to be processed - Size: 1 x 1 x 0.5 3- DEHYDRATION - Dehydration is the removal of intercellular and extracellular water from the tissue following fixation. - Alcohol is the most commonly used dehydrating agent - Acetone provides a rapid method stat method - Dioxane is a rapid dehydrating agent but its fumes are highly toxic -Cellosolve dehydrated rapidly and is not harmful to the tissues - Tetrahydrofuran possesses same properties as dioxane -70%Ethyl alcohol 6H 90%Ethyl Alcohol 12H Absolute Ethyl Alcohol 2H Absolute Ethyl Alcohol 1H Absolute Ethyl Alcohol 4- CLEARING (DE-ALCOHOLIZATION) - Clearing is the removal of dehydrating agent from the tissue -Xylene is the most rapid clearing agent, suitable for urgent biopsies - Xylene 1H Xylene 1 H

5- INFILTRATION/IMPREGNATION - Impregnation is the complete removal of the clearing agent by substitution as the medium (paraffin) penetrates the tissue with the use of no less than two, and preferably three baths of paraffin. - Infiltrating Mediums are paraffin wax, celloidin, gelatin and plastic -Tissue into a beaker with melted paraffin wax 15minutes Second beaker of paraffin wax 15 min Third beaker of paraffin wax 15 min

6 EMBEDDING - Embedding is the orientation of the tissue in melted paraffin, which when solidified, provides firm medium for keeping intact all the parts of the tissues when sections are cut. - Leuckhearts Embedding Model - Compound Embedding Unit - Plastic Embedding Unit - Disposable Embedding Molds -Paper boats, Peel-away, Plastic ice trays -Arrange tissue in embedding mold Tissue is oriented at the center of the mold Pour melted paraffin wax in a mold and allow to solidify for 3 H MANUAL TISSUE PROCESSING FIXATION: 10% Buffered Formalin DEHYDRATION: 70% Alcohol 95% Alcohol 100% Alcohol 100% Alcohol 100% Alcohol CLEARING: Xylene or Toluene (2x) IMPREGNATION: Paraffin Wax (4x) EMBEDDING: Paraffin Wax

24 hours 6H 12H 2H 1H 1H 1H 15minutes 3H