GHH (prelim handout)


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General Histology and Histotecnique 2012-2013; 1st sem; Prelim Handout

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GHH (prelim handout)

  1. 1. General Histology and Histotechnique (1st Semester; SY 2012-2013)Preparation for whole mount B. According to the reaction of the tissue stain.Materials needed: 1. ) Basophilic1. Glass slides - stains the acid component- usually 25 x 75 mm 2. ) Acidophilic- usually 1- 1.2 mm thick - Stains the basic components Types: - Stains the cytoplasm of the cell a. flat slides b. concave slides Some common stains: 1. ) Hematoxylin and Eosin (H & E)2. Cover slips a. Hematoxylin – stain nuclei blue (basophilic)Two types: b. Eosin – stain cytoplasm pink (acidophilic) Number 1 = 0.13-0.17 mm thick Number 2 = 0.17-0.25 mm thick 2. ) Connective Tissue stains Functions: a. Masson’s trichome a. hold samples in place b. Mallorys triple CT stain b. flattens out specimen - both employ a nuclear, cytoplasmic and a third stain c. Protects the objective from immersion into the specific for matrix water drop. 3. ) Silver impregnation3. Stains - Used to trace nerves, stain Golgi, reticular fibers.- Different stains have different affinities for the different organisms thus maybe used to 4. )Wright stain – blood smear differentiate different types of organisms or to view specific parts of organisms. 4. other chemicals- Substance that give color to the specimen to  Fixatives improve visibility.  Embedding materials Example:  Mountants Counter stain – not all organisms can absorb.  ClearersType of stainsA. According to the number of dyes used1. ) Positive staining - Stains the specimen itself. Type of Slide Preparation a. Simple Stain- Employs single dye (on color)  Temporary Slide- Primary dye - water Ex. Crystal violet, Methylene blue - fresh specimen b. Differential Stain  Permanent slide- Two or more stains - reagents are used- Primary dye – counter stain - dead tissues- Gram-staining Ex.  Fixatives – preserve specimen Primary dye = Crystal violet; - Function: to avoid post mortem conditions Counter stain = Safranin Ex: FAA, Bouin’s sol’n, Formalin (10%)Ex. Acid fast stain (Mycobacterium), Ziel Neelsen2. ) Negative staining  Dehydrants- the background of the organism/specimen is the - reagents that remove undesired fluid that may stain interfere with later processing- Function: To emphasized the appendages Ex: Ethyl alcohol (ethanol/alcohol)
  2. 2. General Histology and Histotechnique (1st Semester; SY 2012-2013) Clearing agents - utilize when rapid diagnosis of tissues- to clear - specially recommended for lipids and nervous tissueEx: Xylene and Cloroform elements USES: Embedding medium  Rapid pathological diagnosis during surgery- supports the specimen  Diagnostic and research enzyme histochemistry- paraffin (wax time)  Diagnostic and research demonstration of soluble substances such as lipids and carbohydrates Microtome  Immunofluorescent and immunohistochemical staining Stains  Some specialized silver stains, particularly in- Gives color to the specimen to differentiate the neuropathology different parts of specimen.Ex: Methylene blue, Hematoxylin, Wright stain, Crystalviolet, Eosin, Safranin, Fast green. Processing of Tissues  Adhesive/Affixative  Fixation- to stick or adhere the specimen to the slide Purposes:Ex: Mayer’s albumin a. Confers chemical stability on the tissue b. Hardens the tissue  Mountant c. Stops enzyme autolysis- render permanency d. many enhance later staining technique – act asEx: Balsam mordantFresh Tissue Examination Five (5) major groups of Fixatives: 1. Aldehydes1. Teasing/Dissociation - include formaldehyde or formalin and- use of needle to separate tissues in bulk glutaraldehyde2. Squashing - non precipitating- cut a tiny piece on slide and with the use of another  10 % formalin slide to compress - buffered to lessen acidity that causes precipitation3. Smear preparation and autolysis- to spread specimen thinly onto the slide  Glutaraldehyde a. Streaking - 2-4 % buffered- Applicator stick or the wine loop usage. - fixes very fast- Spread specimen to the slide directly on a zigzag - good for electron microscopy manner. - it penetrates poorly- Uniform the distribution - It gives best results on cytoplasmic and nuclear b. Spreading details.- to spread thinly into the slide- teasing is also needed 2. Mercurials- for fresh sputum mucoid secretion - contain mercuric chloride and Zenker’s c. Full-apart - fixed the tissue by an unknown mechanism- for blood smear, enzymes of the GIT - penetrate very poorly- it used pusher - causes tissue to harden d. Touch Preparation/impression smear - fixed fast- used of freshly cut fruit - give excellent nuclear detail- touched on to the surface of the slide - best in nematopoietic reticuloendothelial- letting the cells stick on to the slide4. Frozen section 3. Alcohols
  3. 3. General Histology and Histotechnique (1st Semester; SY 2012-2013)- include methanol and ethanol- protein denaturants 1. Buffering – pH 6-8- cause too much brittleness and hardness - phosphate, bicarbonates- good in cytological smears - Commercial formalin is buffered with phosphate at- give good nuclear detail a pH of 7.- precipitating 2. Penetration4. Oxidizing agents - depends of diffusability of fixatives- include Potassium permanganate, Potassium - formalin and alcohol: best dichromate and Osmium tetroxide - glutaraldehyde: worst- none precipitating - diffusability – constant- used in frequently - in between – mercurials- causes an extensive denaturation - in order to the organisms to be penetrated it should be cut in 2-3 mL5. Picrates- include fixatives and picric acid (e.g. Bouin’s sol’n) 3. Volume- precipitating fixative - ratio 10:1- enhanced nuclear detail - 10 – fixative- don’t cause hardens - 1 – specimen / tissue- Dry form – explosive- in Solutions– stains everything it catches including 4. Temperature the skin; yellow color - High temperature – Increases speed of fixation Ex. Hot formalin will fix the tissue faster.Fixative for General Usage 5. Concentration of Fixationa. 4 % / 10 % formalin - adjusted down to the lowest level possibleAdvantages: - 10% formalin: best1. easy to make - glutaraldehyde (3%)2. Has good penetration power3. Does not shrink the specimen 6. Time interval4. Does not affect the material it kept for long time - FAA – up to 12 hrs.5. Can be kept without deterioration for considerable - Carnoy’s – 1-3hrs time - Zenker’s – 14-24hrs6. Easy to handle7. Less hazard Organs for processing: Kidneyb. FAA – Formalin-Acetic-Acid-Alcohol Liver- Best for plant material (tissues) Stomach- Composition: 50ml ethanol, 5ml glacial acetic acid, Small Intestine 10ml 37-40% formaldehyde, 35ml water Large intestine Lungsc. Zenker’s and Carnoys’s fluid Spleen- best for animal tissues Gonads Zenkers’s: 950ml distilled water, 25g potassium Muscles dichromate, 50g mercuric chloride, 50g glacial acetic Specimen Class Carnoy’s: 60 ml ethanol, 30ml chloroform, 10ml Class A – delicate tissues: lungs; membranes glacial acetic acid Class B – moderately thick; spleen, live, kidney Class C – bulky and thick; skinFactors affecting fixation
  4. 4. General Histology and Histotechnique (1st Semester; SY 2012-2013)Fixation process: 100% ethanol – 30 min- submerge the specimen fully in fixative 100% ethanol– 30 min- cover the container tightly ***To ensure complete removal of water during dehydration, use two changes of 100% ethanol of atProper timing for each specimen if FAA is used: least ½ hour each.Class A – 6-9 hrs. (10hrs)Class B – 12-18 hrs. (16-18 hrs) Timing of dehydration:Class C – 24-36 hrs. Class A – 30 min in each alcohol concentration Class B – 1 hrDehydration Class C – 3-4 hrs.- removing undesired water inside the specimen ClearingCharacteristics of an ideal dehydrating solution: It should dehydrate rapidly without producing 1. Alcohol –to provide rapid miscible in the tissue considerable shrinkage or distortion of tissues. 2. To facilitate impregnation or infiltration It should not evaporate very fast. It should be able to dehydrate even fatty tissues Xylene It should not harden tissues exclusively. - Commonly used; tends to harden tissues if left too It should not remove stain. long. It should not be toxic to the body It should not be a fire hazard. Advantages: - makes tissue transparentCommonly used dehydrating agents: - miscible with absolute alcohol and paraffin- alcohol - evaporate quickly in paraffin oven and can- acetone - cheap- triethylphosphate- tetrahydrofuran Disadvantages:- dioxane - highly in flammable - If used longer than 3 hrs. it makes tissues Alcohol excessively hard and brittle- a standard series of alcohols are used most - Causes considerable hardening and shrinkage of- Alcohol series: 70% - 80% - 95% tissues- Higher grades of alcohol: shrinkage and hardening - Becomes milky when an incompletely dehydrated of tissues tissue is immersed in it.- concentrated alcohol tend to harden only the surface of tissue Toluene Ethyl alcohol - time recommended is 1-2 hours- best dehydrating agent  fast acting Advantages:  mixes with water - it is miscible with both absolute alcohol and paraffin  penetrates tissue easily - It acts fairly and rapidly and is recommended for  not poisonous routine purposes  not very expensive - tissue do not become excessively hard and brittle even if left in Toluene for 24 hrsDehydrating procedures: - it is not carcinogenicWater50% ethanol Disadvantages:70% ethanol - it is relatively slower than Xylene and benzene80% ethanol - it tends to acidify in a partially filled vessel90% ethanol
  5. 5. General Histology and Histotechnique (1st Semester; SY 2012-2013)- highly concentrated solutions will emit fumes that - complete clearing is difficult to evaluate are toxic upon prolonged exposure - tissues tend to float in chloroform- it is more expensive - it evaporates quickly from a water bathBenzene Impregnation, Embedding, Trimming- clearing agent- clear and penetrate tissues rapidly Impregnation- carcinogenic - Diffusion or accumulation in a cell or tissue substances that are not normal.Advantages: - To fill-out the tissues in each spaces or in-between- It is rapidly acting, so recommended for urgent spaces biopsies (15 to 60 min) and routine purposes.- It volatilizes rapidly in paraffin oven and is therefore Three (3) types of Tissue Impregnation: easily eliminated from the tissue. 1. Paraffin wax impregnation- It is miscible with absolute alcohol Precautions:- it does not make tissues hard and brittle a. Prolong treatment in melted paraffin causes- it causes minimum shrinkage shrinkage and hardening of tissues making cutting- it makes tissues transparent difficult. b. Infiltration in overheated paraffin (above 60°C) willDisadvantages: produce shrinkage and hardening of tissues and- it is highly in flammable destroy lymphoid tissues completely- if a section is left in benzene for a long time, c. Paraffin wax must be pure considerable tissue shrinkage may be observed d. Paraffin wax may be used only twice.- Excessive exposure to benzene may be extremely 2. Celloidin impregnation toxic to man and may become carcinogenic or it 3. Gelatin impregnation may damage the bone marrow resulting in a plastic anemia. Embedding - The process by which the impregnated tissue isChloroform placed into a precisely arranged position in a mold- Used for routine clearing of tissues during the containing a medium which is then allowed to embedding process. solidify. - Cooling allow hardening of tissue, giving them aAdvantages: firmer consistency and better support, thereby- It is recommended for routine work (6-24 hrs.) facilitating the cutting of sections.- It is miscible with absolute alcohol  Orientation- It is recommended for tough tissues (ex. skin, - A process by which a tissue is arranged in precise fibroid and decalcified tissues) for nervous tissues, positions in the mold during embedding on the lymph nodes and embryos microtome before cutting and on the slide before- It is suitable for large tissue specimens staining.- It is not inflammable. Embedding Proper:Disadvantages: 1. Prepare embedding boxes- It is relatively toxic to the liver after prolonged 2. Pour melted paraffin into embedding box inhalation 3. Using a needle or forceps, position properly the- wax impregnation after chloroform clearing is tissue inside the paraffin box according to the relatively slow desired cut later. For cross section, position tissue- it does not make tissues transparent vertically. Do not remove the needle or forceps ‘till- it is not very volatile in paraffin oven paraffin has surrounded the tissue.- it may even produce considerable deterioration of 4. Allow paraffin to solidify. the wax
  6. 6. General Histology and Histotechnique (1st Semester; SY 2012-2013) 5. Label the box bearing the name and position of tissue contained. Trimming- Embedded tissue is trimmed and cut into thin uniformly slices.- The slides, top and bottom of the tissue block are trimmed until perfectly level and all slides are parallel, almost to the edge of the tissue.- The block is placed in the microtome for final trimming and cutting.Microtomy- A process of cutting specimen to small or thin sizes.Microtome- An instrument used to cut the specimen.Steel Blades- Animal or plant tissue sections- Light microscopeGlass Knives- thin sections- Light microscope and Electron MicroscopeIndustrial Grade- Electron microscopeDiamond knives- Hard materials3 Essential parts of Microtome: 1. Block holder – holds block in precision 2. Knife carrier & knife – actual cutting of the tissue sections 3. Pawl, Ratchet feed wheel and adjustment screws – line up the tissue flock in proper positioning with the knife adjusting the proper thickness of the tissue for successive section.Kinds of Microtome’s:1. Rocking (by Paldwell Trefall, 1881)- Simplest- Cut the paraffin embedded tissues2. Rotary (by Minot, 1885-’86)- Most common- It involves staged rotary action3. Sliding (by Adams, 1789)- Cutting celloidin embedded tissue a. Base-sledge – a very hard tissue b. Standard sliding – with movable exposed knife. For cutting celloidin embedded tissue. This is the most dangerous style of microtome.
  7. 7. General Histology and Histotechnique (1st Semester; SY 2012-2013)4. Freezing (by Queckett, 1848)- Cutting embedded frozen sections Cryostat or cold microtome- Refrigerated apparatus used in fresh microtomy- For freezing tissue in the block holder to correct degree of hardness to facilitate easier and faster sectioning.- Provides a means of preparing a thin section of fresh frozen tissues especially for fluorescence antibody staining technique.5. Ultrathin- Cutting sections for electron microscope - celloidin- Specimen used should be small- Should be fixed in osmium tetroxide and embedded in plastics.Microtome knives:1. Plane concave knife- 25mm- one side flat, other concave Flat – recommended for cutting celloidin embedded tissue blocks using a sliding microtome. Concave – used for cutting paraffin embedded tissues. Using its Base Sledge microtome, Rotary, Rocking2. Biconcave knife- 120mm- both sides are concave Concave – cutting paraffin embedded tissues using a rotary microtome.3. Plane wedge knife- 100 mm- Both sides are straight Straight – recommended for frozen section or for cutting extremely hard/tough specimen embedded in a paraffin block.- Using a base sledge sliding microtome.Sectioning- Cutting thinly the specimen with the use of microtome- Thin materials – sections3 types of tissue sections:1. Paraffin Section- Leaded in a paraffin block- Cut using the rotary microtome/Rocky microtome2. Celloidin section- Embedded in a celloidin- Cut by sliding microtome3. Frozen Section- Fixed with carbon dioxide or cryostat
  8. 8. General Histology and Histotechnique (1st Semester; SY 2012-2013)Faults occurring during tissue processing FAULTS REASONS REMEDY1. Brittle or hard tissue. - Prolong fixation, dehydration - Softened by soaking in a small clearing, and infiltration of dish containing water with paraffin in overheated paraffin detergent or phenol. oven. - Dry out of tissue before actual fixation.2. Clearing agent turns milky as - Water is not completely - Repeat dehydration with soon as tissue is placed in it. removed due to incomplete absolute alcohol then clear dehydration. again.3. On trimming tissue smells of - Clearing agent not completely - Repeat paraffin impregnation. clearing agent. removed due to insufficient impregnation.4. Tissue is opaque, section- - Insufficient clearing. - Repeat clearing and if the cutting is difficult due to object has been embedded, presence of alcohol prolong clearing up to 12 hrs. And embed it again.5. Tissue shrinks away from wax - Insufficient dehydration so - Repeat the whole process. when trimmed. there is incomplete clearing and impregnation.6. Tissue is soft when block is - Incomplete fixation - Repeat fixation procedure trimmed7. Air holes found in tissue during - Incomplete infiltration or - Repeat impregnation. trimming. impregnation.8. On trimming, wax appears - Contaminated wax - Re-embed in a freshly filtered crystalline. - The bluff Nat cold rapidly wax.9. Paraffin block after cooling is - there is insufficient paraffin - Repeat impregnation and re- moist and crumbles impregnation embed.Faults observed during section-cutting: Faults Reasons Remedy1. Sections fail to form ribbons. - Surfaces and edges of the bluff - Re-trim the block are not parallel - Re-adjust the block - Horizontal surface of the block - Reduce the tilt of the knife is not parallel to the knife - Re-adjust the thickness of the - Knife is tilted too much section - Sections are too thick - Re-sharpen the knife - Knife is dull2. Sections roll up on cutting so - Knife is blunt - Re-sharpen the knife that they adhere and get - The tilt of the knife is great - Reduce the tilt broken against the knife edge. - The knife edge is dirty - Clean the knife edge3. Ribbon is crooked, curved and - The knife is blunt or dull; there - Adjust the knife so that knife uneven instead of straight. is a dull spot on the knife edge will present a uniformly producing an irregular knife sharp edge to the block. edge. - The knife is not parallel to the - Re-adjust the knife and the block block.4. Sections are wrinkled, - The paraffin block is warm and - Cool the paraffin on ice water
  9. 9. General Histology and Histotechnique (1st Semester; SY 2012-2013) compressed or jammed. soft until firmed. - The knife edge is coated with - Clean the knife edge paraffin - Re-adjust the thickness of - The section are too thin section - Microtome set screw is loose. - Tighten the screw - The tilt of knife is too vertical. - Reduce the tilt5. Sections are torn and crumble - Incomplete dehydration, - Repeat dehydration, clearing when cut. clearing and impregnation and impregnation - Paraffin is warmed and soft. - Cool and harden the paraffin in ice water for ¼ to ½ hour.6. A hole is formed in the section. - Bubble or dirt is formed in the - Re-embed in freshly filtered embedding medium wax.7. Sections adhere to the knife or - There is a static electrically due - Breathe out or blow gently in other parts of the machine. to low atmospheric humidity. the block and knife to break-up static electricity. Boil water - Knife edge is dirty inside the laboratory. - Knife edge is dull - Clean the knife edge - Knife tilt is great - Re-sharpen the knife - Reduce the tilt8. Sections are lifted from the - Knife tilt is too great - Reduce tilt knife on upstrokes - Knife is dull - Re-sharpen the knife - Paraffin is soft - Cool the paraffin in ice water9. Sections cut is sometimes thin, - Knife is blunt - Re-sharpen the knife and sometimes thick - Knife is not completely clamp - Re-adjust the knife - The tilt of knife is great - Reduce the tilt - The knife or block holder is - Tighten adjusting and locking loose. screws.Paraffin Section- Sections are usually cut between 4-6 micron in thickness.- Micrometer gauge is set to the required thickness and the knife is positioned in such a way that the center of the blade is in line with the block and the knife has been securely clamped in place.- Cutting is then started until complete sections come out of the block.- A regular cutting rhythm is required- The knife is usually tilted at 0-15 degrees angulations’ on a microtome to allow a clearance angle between the cutting facet and tissue block- Sections are removed in ribbons of ten, to allow easy location of serial sections- The sections are then floated out in water bath set at 45-50°C to flatten the sectionsStaining- To impart a color to the tissues- Parts to be visible under microscopeTwo Categories of dyes:1. Natural Dyes- Obtained from plants and animalsHematoxilyn- Derived by either extraction from the heartwood of Mexican tree known a Hematoxilyn campechianum.- Most valuable stain in histology used by cytologist
  10. 10. General Histology and Histotechnique (1st Semester; SY 2012-2013)- Powerful nuclear and chromatin staining capacity- With striking polychrome qualitiesHematin – active coloring agents2. Synthetic Dyes- Known as Coal Tar Dyes- Hydrocarbon benzene derivation- Aniline dyesExamples: Picric acid- Ability to form salts with alkaline- Fix and stain at the same time by itself- Used as a fixative- Decalcifying agen- Tissue softener Eosin- a valuable stain in Histotechnique, important stain in connective tissue and cytoplasm.- Histopathology – counter-stain after hematoxilyn and before methylene blue- Background for contrasting stains because it gives a pleasing and colorful contrast to nuclear stain.Red acid dye available in 2 shades:-a. Bluish- Deepen red color Ex. Eosin Bb. Yellowish- Most usable- Readily soluble in water and less in alcohol- Available in both aqueous and alcoholic solutions- Slows a green fluorescence especially in alcoholic medium. Ex. Eosin Y3. Basic dyes- coloring substance is found in the basic components- Acid radical is taken from sulfuric, acetic and hydrochloric acid.Ex. Methylene blue*Note: On a chemical basis certain parts of cells and tissues that are acidic in character (e.g. nucleus) have greateraffinity for basic dyes, while basic constituents (e.g. cytoplasm) take more of the acid stains. Fixation of tissues with mercuric chloride and formaldehyde usually favors staining with basic dyes. Picric acid and Chromium – fixed tissues usually take an acidic dyes Ethyl alcohol or acetic acid – fixed tissues readily take in both basic and acidic dyes.4. Neutral Dyes- Combining aqueous solution of acid and basic dyes.- Capable of staining cytoplasm and nucleus simultaneously and differentially- Soluble in alcohol but not in water.
  11. 11. General Histology and Histotechnique (1st Semester; SY 2012-2013)Ex. WBC differentiation Giemsa’s stain Leishman’s stain3 main groups of tissue staining1. Histological Staining- process by the tissue staining constituents are demonstrated in section by direct interaction with a dye or staining solution producing coloration of the active tissue components.Example:- Mircroanatomical stains- Bacterial stains- Specific tissues stains – muscle, connective tissue, neurological stains.2. Histochemical staining (Histochemistry)- A process whereby tissue constituents are studied through chemical reactions that will permit microscopy localization of a specific tissue substance.3. Immunohistochemistry- Combination of 1st and 2nd classification- Allow phenotypic markers to be detected using fluorescent labeled or enzyme labeled.Staining Technique1. Direct Staining- A process of giving color to the section by using aqueous or alcoholic dye solution.Ex. Methylene blue, Eosin2. Indirect Staining- Whereby the action of the dye is intensified by adding another agent or mordant.- Mordant – serve as a bridge or link between the tissue and the dye.- Mordant + dye -> Lake + Tissue = “Tissue mordant dye complex3. Progressive Staining- The process whereby tissue elements are stained in a definite sequence.- Definite sequence? - to have a satisfactory differential coloration of tissues- Once the dye is taken by the tissue it is not wash anymore.4. Regressive Staining- The tissue is first over-stained then decolorized- Over-stained – to obliterate the cellular details decolorized until the desired color is achieved.- Differentiation – the selective removal of excess stain.5. Metachromatic Staining- Entails the use of specific dyes which differentiate particular substances by staining them with a color that is different from that of the stain itself.Where is this used?- Employ in staining cartilages, connective tissues, epithelial tissues- Belong to basic dyes
  12. 12. General Histology and Histotechnique (1st Semester; SY 2012-2013)Example: Methylene blue, Safranin.6. Counterstaining- Provide contrast and backgroundExample: Safranin7. Microanatomical Staining- General differentiation of nucleus and cytoplasm.8. Metallic Impregnation- Where specific tissue elements are demonstrated by colorless solutions of metallic salts- Metallic salts – reduced by the tissue ; reduced also by bacteria- It’s not absorbed by the tissues but it is held physically on the surface as precipitates- Valuable metals: Ag, Chloride, Silver nitrate9. Vital Staining- Selective staining of living cell constituents- Demonstrates cytoplasmic structures by phagocytosis of the dye particle.10. Intravital Staining- By injecting the dye into any part of animal body.- Purpose: To produce different coloration of cells- Most specially in reticuloendothelial systemExamples: Lithium, Indian11. Supravital Staining- To stain living cells immediately after removal from the living body.Ex. Neutral redStaining of Paraffin Section Paraffin wax is poorly permeable to most staining solutions and should therefore remove from the section prior to staining. This is usually done by immersing the paraffin section in a solvent (xylene) two times, at 1-2 minutes duration. Xylene is not miscible with aqueous solutions and low graded alcohol and therefore be subsequently removed with absolute alcohol followed by descending grades of alcohol to prevent damage and detachment of sections due to possible production of diffusion currents. The alcohol is then finally replaced with water, before actual staining of section is performed After deparaffinization with xylene, the section is subjected to decreasing grades of alcohol. After staining, the section is again dehydrated with increasing grades of alcohol and cleared with two changes of xylene to prepare the section for mounting, since most mountants are miscible with xylene. The second change of xylene will raise the refractive index of the glass slide, thereby reducing light refraction during microscopic examination.*Note: If section float off the slide during staining, fix section in a Bunsen flameSlides must not be dirty of greasy.
  13. 13. General Histology and Histotechnique (1st Semester; SY 2012-2013)Mounting- A process of rendering slides permanency- Used to keep the slide to its permanent position- Seraphy Fluid – applied between the tissue and cover slip.Mounting media importance: It protects the stained sections from getting scratched and from bleaching or deterioration due to oxidation, thereby preserving the slides for permanent keeping. Facilitate easy handling and storage Prevent the damage of sections which may lead to distortion of image during microscopic examination.Characteristics of a good mounting medium: It should not dry quickly It should not dissolve out of fade tissue sections It should not cause shrinkage and distortion of tissues It should set hard, thereby producing permanent mounting of sections.2 main groups of mounting media:1. Aqueous media- Design to mount water miscible preparationsExamples: Glycerin, water2. Resinous media- Used for preparations that has been dehydrated and cleared in xylene and are recommended for majority of staining method.Examples: Balsan, Colorless nail polishTypes of Mounting1. Dry Mount- It requires no water- Usually used in inanimate objects2. Wet mount- It requires water- Used to prepare slides to cold living organisms whether they are motile or not.Ringing- A process of sealing the margins of cover slip.Purpose:1. To prevent the escape of fluid or semi-fluid mounts2. To prevent evaporation of mounts3. To immobilize the cover slip4. To prevent sticking of the slides upon storage