MIDTERMS [HCT LEC] MT_QUIZ25 - Y3T1 PDF

● ● ● Should not evaporate fast ○ Most used are more alcohol to water ratio ■ water is added to reduce dehydration Can dehydrate even tatty tissues ○ Bakit mo lalagyan chicharon ng alcohol it would turn turbid ○ Bula bula na milky miscibility ○ like dissolves like; non polar si alcohol ■ mix with...

● ● ● Should not evaporate fast ○ Most used are more alcohol to water ratio ■ water is added to reduce dehydration Can dehydrate even tatty tissues ○ Bakit mo lalagyan chicharon ng alcohol it would turn turbid ○ Bula bula na milky miscibility ○ like dissolves like; non polar si alcohol ■ mix with water ■ miscible with water ■ non poisonous ■ penetrates easily ○ Si chicharon needs gasoline ether ○ Slightly miscible Non toxic ○ most important (BAKIT MO GAGAMITIN? -prof) Not fire hazard Should not harden tissues excessively ● ● ● ● ● ● Alcohol - most common, least expensive Acetone - bigger tendency to evaporate rapidly Dioxane Cellosolve Triethyl phosphate Tetrahydrofuran (THF) - least common, most expensive ● ● ALCOHOL - PROGRESSIVELY INCREASING ● ● ● ● ● ● ● ● ● ● ● minimum ● ● ● Minimum shrinkage Minimum distortion and hardening of tissue Used to dehydrate sections and smears following certain stains papanicolau smear or vaginal swab increase solution we utilize triethy phosphate as dehydrant TETRAHYDROFURAN - THF ● ● ● ● Prolonged storage in 70 % alcohol - macerates tissues ○ slowly penetrates tissues Directly placed in high grade alcohol - shrinkage & hardening of tissues ○ Because it does not only remove the water content, but a lot of the cellular components/ part of the cytoplasm or the cells ○ Hardening of tissue will result to difficulty in tissue sectioning Dehydrates and clears Less shrinkage Easier cutting w/ fewer artifacts Non toxic; 6 months exposure – conjunctival irritation (like sore eyes) Offensive odor (use well ventilated room) anesthsia Called someone on lab histopathology Formaldehyde used to from the laboratory 1 is to 20 parts a liter vs @37 percent in a bucket of water Do not forget to label Date is indicated the person and specimen as all of them appear the same grayish in color even if it is a liver….. WEEK 8.2: CLEARING CLEARING / DEALCOHOLIZATION ● Readily available because it is used to remove stains or paints Cheap Rapid acting Volatile Tissue shrinkage ○ not recommended because of the last two bullets ● ● ● ● ● DIOXIDE (DIETHYLENE DIOXIDE) ● ● ● ● Ethylene Glycol Monoethyl Ether - other name Dehydrates rapidly Storage without producing hardening or distortion ○ Rarely used in dehydration TRIETHYL PHOSPHATE ACETONE ● w/ CELLOSOLVE ETHYL ○ best, fast, mixes with water and penetrates easily, not poisonous, cheap ○ Considered the fastest ● ○ Reacts automatically ADDITIVES TO DEHYDRATING AGENTS ○ Mixes with water and penetrates easily ● 4 % phenol added to 95 % ethanol – softens hard tissues 490 phenol ○ Readily available in pharmaceuticals ● Hard tissues – immerse in glycerol/alcohol mixture or in 95W ethanol Molliflex ○ Widely utilized as a disinfectant ○ A must have in everyone’s pocket Most chemicals used in decalcification is an acidic in a certain ○ magsisimula sa mababa then pataas ng pataas concentration mol (CASINO tubo sugar cane) Miss x bile sludge vs neoplasm on process METHYL Attending physician is not not normal neoplasm in progress surgeon ct scan 95% accurate 5% inaccurate ○ Toxic, for blood films 2.5 cm mass ○ Recommended for blood films Allowable mass is 1 cm only BUTYL Consultation with relatives to diagnose Operation close or open surgery ○ Slow action, does not dehydrate rapidly Close holed out then specialzed instruments laparoscomy ○ Utilized for Plant and Animal microtechnique Forcep 3 strings only ○ Utilized for the organic substance retained Open open cut then whole organ is observed similar to autopsy but under ALCOHOL ● ● Expensive Dangerous - vapor - cumulative ○ Dangerous because once inhaled, the vapor is carcinogenic –it will cause cumulative effects in the human body Graupner' s method Dioxane w/ paraffin wax ○ Dioxane in combination with paraffin wax Weiseberger' s method ○ Dioxane in combination with Ca oxide placed in a gauze Dioxane ( a oxide tylertk-DAAC.TT DEHYDRATING AGENTS ● ● ● Excellent dehydrating & clearing agent Less tissue shrinkage Tissues can be left here for a long time Ribbons poorly ○ Ribbons - tissue sections that are cut ○ Ribbons are poorly stained Process where we try to remove excess alcohol and excess water in order for the histopathologist to see the tissues clearly without any interference or any debris that will interfere with microscopy Removal of dehydrating solutions, making the tissue components receptive to the infiltrating medium After staining - transparent tissues Improve refractive index of the tissue ○ Refractive index is the ability of the tissues to be closely monitored or closely seen if it is focused under HPO or LPO fililinaw CHARACTERISTICS OF GOOD CLEARING AGENTS ● ● Miscible with alcohol and paraffin &/or mounting medium Should not produce excessive tissue shrinkage & hardening BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 9 ● ● Should not evaporate quickly in a waterbath Should make tissues transparent ○ Important because clearing agents remove unwanted debris so the tissues will appear naturally without any artifacts or debris which will interfere with microscopy CLEARING AGENTS ● ● ● ● ● Xylene Toluene Benzene Chloroform Cedarwood oil ● ● ● ● ● ● Aniline oil Clove oil Carbon Tetrachloride Methyl benzoate Methyl salicylate THF XYLENE (XYLOL) ● ● ● ● ● ● ● Most common, colorless Most rapid Cheap Milky if incomplete dehydration Hardens/shrinks tissues ○ Not recommended for ■ Nervous system & lymph nodes ■ Because it is highly volatile and carcinogenic Hard/brittle tissues if > 3 hours Banned - difficult for the laboratory to secure xylene in the market CLOVE OIL ● ● ● ● CARBON TETRACHLORIDE (CCL4) ● ● ● ● ● ● ● ● Not carcinogenic: but will emit fumes that are toxic upon prolonged exposure Rapid acting Tissues do not become excessively hard and brittle even if left here for 24 hours Slower than xylene & benzene expensive ● ● ● ● ● Totally banned - organic chemical that is no longer available commercially Rapid acting Volatilizes rapidly in paraffin oven Tissue shrinkage if left for a long time Carcinogenic; may damage bone marrow resulting to aplastic anemia ● ● Dehydrating and clearing agent Non toxic but with offensive odor and should be used in a well-ventilated room ● ● Permeating the tissue with a support medium Clearing medium is completely removed from the tissue and replaced by a medium that will completely fill all the tissue cavities ○ Lahat ng maliliit na spaces within the tissue ○ This helps in the tissue processing in the context of the tissue being hard enough for it to be cut ■ Easier to cut ■ hindi madaling masira during the process of cutting or tissue ribbon WEEK 8: INFILTRATION/ IMPREGNATION ● ● ● CHLOROFORM ● ● ● ● ● For tough tissues – decalcified, nervous, lymph nodes, embryos – minimum shrinkage and hardening For large specimens Toxic to liver (prolonged inhalation) Does not make tissues transparent Tissues tend to float CEDARWOOD OIL ● ● ● ● ● ● ● Very penetrating Extremely slow For CNS, smooth muscles, skin, cytology Milky on prolonged storage (filter) May produce crystals – heat to 200 C Very expensive Maybe used for paraffin and celloidin sections ANILINE OIL ● ● For clearing embryos, insects and delicate specimens due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening Preserves egg of insects Slow acting – used for double embedding TETRAHYDROFURAN (THF) BENZENE ● Totally banned Carcinogenic Properties and disadvantages are like chloroform but cheaper Hepatotoxic toxic to the liver METHYL BENZOATE/METHYL SALICYLATE TOLUENE ● Causes minimum shrinkage of tissues Wax impregnation here – slow / difficult ○ If it is used for a longer period of time, it will be difficult to remove Tissues become brittle - longer period of time Expensive NOTE: Usually during the process of clearing, dehydration, and infiltration, there are some leftover clearing materials that fill up the gaps of the tissues o We need to remove them for us to fill the gaps with other materials that will solidify at a lower temperature During the tissue processing procedure, when clearing is done, the vacant areas were usually left behind by the clearing agent o The purpose of clearing is to allow tissues to become transparent o The purpose of dehydration is to remove the water content ■ combined with alcohol, (alcohol and water) will then evaporate thus leaving vacant tissue areas Waxes or any semi solid materials that eventually solidify, will be allowed to permeate/penetrate the inner cavity of the tissues to establish solid materials which will then facilitate easy sectioning/cutting TYPES OF TISSUE IMPREGNATION/EMBEDDING ● ● ● ● Paraffin Wax ○ Manual – 4 changes of wax 15 mins interval ○ Automatic – Autotechnicon, Elliott Bench type ○ Vacuum – fastest, negative atmospheric pressure, gives the fastest result Celloidin (Collodion) ○ Wet – bones, teeth, large brain sections ○ Dry – whole eye sections Gelatin Plastic PARAFFIN WAX IMPREGNATION ● ● ● ● ● ● Melting Point: 54°C – 58°C 1- 9 Remains solid or slowly solidifies at 20°C – 24°C 4 Simplest, most common, best Melting point: 54c-58c at 20c-24c Ease in cutting or Eary cuttig ○ More malleable Permanent paraffin blocks ○ Can be stored for several years BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 10 ● ● ● ● ○ becomes permanent once impregnated with paraffin Good staining results ○ can be viewed under microscope under Hematoxylin & Eosin staining procedure Not recommended for fatty tissues ○ Madaling nadudurog when using paraffin wax ○ Madaling nadudurog when using paraffin wax ○ Eventually, fatty tissues when mixed with paraffin, will combine easily because they are highly miscible— it doesn’t serve its purpose of filling in the parts which are then cleared by the clearing agents Overheated paraffin -> brittle specimen Inadequate impregnation -> clearing agent retained ○ Usually visible if the clearing agent is not completely removed during clearing procedure ■ Saline (clearing agent) was still left during the procedure hence, once combined with paraffin, paraffin will not impregnate/penetrate the gaps of the tissue since the clearing agent is still present SUBSTITUTE PARAFFIN WAX ● PE Biti ● ● Paraplast – more elastic / resilient (56-57C) ○ Embedded – less brittle (56-58C) ○ Bioloid – for eyes ○ Tissue Mat – with rubber Ester Wax – low melting point (46-48C) ○ Harder than paraffin; water insoluble, can be used for impregnation without prior clearing ○ Known organic substances that form an esterified ○ wax, usual byproducts of aldehydes and ketones ○ Harder than paraffin; water insoluble, can be used for impregnation without prior clearing ○ Assuming that there is leftover saline during The clearing procedure, we recommend the use of ester wax for impregnation Water Soluble Wax ○ Recommended for enzyme histochemistry (special staining/procedures in Histopath) ■ Because it is highly hygroscopic ■ Water combined with wax, is said to be miscible ○ Carbowax – water soluble (no dehydration/clearing); for enzyme histochemistry; hygroscopic ● ● ● ● ● ● WEEK 8.2: EMBEDDING ● ● ● ● ● ● ● ● ● ● DOUBLE EMBEDDING 2% Celloidin for 3 days and subsequent paraffin for large blocks of dense firm tissues – brain Obsolete – due to paraffin waxes with different types of resins ● ● ● ● Epoxy, polyester, acrylic For hard tissues, renal and bone marrow biopsies For ultrathin sections requiring minimal distortion With VCD – vinylcyclohexene dioxide-carcinogenic PLASTIC (RESIN) EMBEDDING WEEK 9.1: IMPREGNATION AND EMBEDDING Therese R. Suratos, RMT DEFINITION IMPREGNATION process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will completely fill all the tissue cavities and give a firm consistency to the specimen. Permits cutting of thicker tissue sections Crumbling of tissues avoided Very slow Very volatile; it easily evaporates ○ esp. if not kept properly Thin sections difficult to cut Serial sections difficult to prepare - difficult to prepare Photomicrographs - difficult to obtain Very volatile Suitable for specimens with large hollow cavities which tend to collapse Recommended for osseus tissues or calcified tissues like bones, teeth, large brain sections and whole organs placed in celloidin materials + DRY CELLOIDIN METHODS Dry eyes " ● Preferred for whole eye section ● ● ● ● ● LVN – low viscosity nitrocellulose soft to harder A form of celloidin but more expensive y Used for softer tissues since it forms harder tissue blocks Forms harder tissue block; makes thinner sections When dry, striking or dropping the container will cause explosion. ● The use of polysaccharide agar material NITROCELLULOSE METHOD GELATIN IMPREGNATION Leuckhart’s Embedding Mold Compound Embedding Unit Plastic Embedding Rings & Base Mold ○ Tissue Tek – machine w/ warm plate Disposable Embedding Molds ○ Peel Away ○ Plastic Ice Trays ○ Paper boats ○ TIMS tissue processing and embedding system ● ● ● - WET CELLOIDIN METHODS Casting /Blocking Place tissue in a precisely arranged position in a mold with a medium which is then allowed to solidify (orientation) Orientation – process by which tissue is arranged in precise positions in the mold during embedding, on the microtome before cutting, and on the slide before staining BLOCKING OUT MOLDS ● ● ● CELLOIDIN ● ● ● ● Rarely used – when dehydration is avoided; for histochemical and enzyme studies Recommended for special occasions/procedures only: ○ histochemical and enzyme studies Does not require dehydration & clearing Embedding medium for delicate specimen and frozen sections Low melting point Can be used as substitute for paraffin wax EMBEDDING process by which the impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify. QUALITIES OF A GOOD INFILTRATING AND EMBEDDING MEDIUM ● ● ● ● ● ● ● Soluble in processing fluids Suitable for sectioning and ribboning molten between 30°C and 60°C translucent or transparent; colorless stable Homogeneous Capable of flattening after ribboning non-toxic Odorless Easy to handle Inexpensive EMBEDDING MEDIUM ● ● The medium used to infiltrate the tissue is usually the same medium utilized for impregnation, and for general purposes There are generally four types of impregnation and embedding medium, namely: 1. Paraffin wax 2. Celloidin (colloidion) 3. Gelatin 4. Plastic BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 11 PARAFFIN WAX IMPREGNATION PARAFFIN - simplest, most common, and best embedding medium used for routine tissue processing. PARAFFIN WAX - a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils. ● It is solid at room temperature but melts at temperatures up to about 65°C or 70°C. ● Paraffin wax can be purchased with melting points at different temperatures,the most common for histological use being about 56°C to 58°C ● At its melting point, it tends to be slightly viscous, but this decreases as the temperature is increased. ● The traditional advice with paraffin wax is to use this about 2°C above its melting point. ● Wax hardness (viscosity) depends upon the molecular weight of the components and the ambient temperature. ● To decrease viscosity and improve infiltration of the tissue, technologists often increase the temperature to above 60°C or 65°C. ○ ↑ temperature = ↓ viscosity ● High molecular weight mixtures melt at higher temperatures than waxes composed of lower molecular weight fractions. ○ ↑ MW mixtures will met at ↑ temperature. ● Paraffin wax is traditionally marketed by its melting points which range from 39°C to 68°C ● Tissue-wax adhesion depends upon the crystal morphology of the embedding medium. ● Small, uniform sized crystals provide better physical support for specimens through close packing. ● Crystalline morphology of paraffin wax can be altered by incorporating additives which result in a less brittle, more homogeneous wax with good cutting characteristics. ● There is consequently less deformation during thin sectioning. Make a list for temperatures ADVANTAGES AND DISADVANTAGES OF PARAFFIN WAX IMPREGNATION ADVANTAGES 1. 2. 3. 4. 5. ● ● ● Thin individual serial sections may be cut with ease from the majority of tissues without distortion The process is very rapid, allowing sections to be prepared within 24 hours Tissue blocks and unstained mounted sections may be stored in paraffin for an indefinite period of time after impregnation without considerable tissue destruction Because formalin-fixed, paraffin-embedded tissues may be stored indefinitely at room temperature, and nucleic acids (both DNA and RNA) may be recovered from them decades after fixation, they are an important resource for historical studies in medicine Many staining procedures are permitted with good results. DISADVANTAGES 1. 2. 3. 4. 5. Overheated paraffin makes the specimen brittle. Prolonged impregnation will cause excessive tissue shrinkage and hardening, making the cutting of sections difficult. Inadequate impregnation will promote retention of the clearing agent. Tissues become soft and shrunken, and tissue blocks crumble when sectioned and break up when floated out in a water bath. Tissues that are difficult to infiltrate, e.g. bones, teeth, brains and eyes, need long immersion for proper support; otherwise, they will crumble on sectioning. Prolonged immersion in paraffin, on the other hand, is not advisable Paraffin processing is not recommended for fatty tissues. The dehydrating and clearing agents used in the process dissolve and remove fat from the tissues NOTE: 10 years na nakatago sa cabinet, merong drawers for each block in case need ulit i-test etc. pwede sya i-cut ulit. Once gumamit na ng formalin, indefinite time na yung pag-use nya In every hosp, there are certain protocols for histopath technologies, 1 hour each thenusually 12 stations w diff reagents para mabuo ung pagprocess sa tissues PARAFFIN WAX IMPREGNATION ● ● ● ● ● ● ● 1. MANUAL PROCESSING ● ● At least four changes of wax are required at 15 minutes intervals in order to ensure complete removal of the clearing agent from the tissue. The specimen is then immersed in another fresh solution of melted paraffin for approximately 3 hours to ensure complete embedding or casting of tissue. SAMPLE TIME SCHEDULE FOR MANUAL PROCESSING OF TISSUES (about 3 mm) FIXATION 10% Buffered Formalin 24 hours DEHYDRATION NOTE: ● ● paraffin oven or in an incubator which has been regulated at 55-60°C The duration and number of changes required for thorough impregnation of tissue depends on: ○ Size and type of tissues: Longer time is required for thicker tissues. ○ Use of vacuum embedding: Vacuum reduces the time required for complete impregnation. ○ Clearing agent employed Common waxes have melting points of 45°C, 52°C, 56°C and 58°C. The 56°C wax is normally used for routine work. (most commonly used in the laboratory) In a laboratory with temperature ranging from 20-24°C, paraffin wax with a melting point of 54-58°C is indicated. If the laboratory temperature is between 15-18°C, the melting point of wax to be used should be between 50 and 54°C. Hard tissues require wax with a higher melting point than soft tissues. There are three ways by which paraffin wax impregnation and embedding of tissues may be performed: 1. By manual processing 2. By automatic processing 3. By vacuum embedding After being completely cleared, the tissue is submerged in two or more changes of melted paraffin wax, either in a 70% alcohol 95% alcohol 100% alcohol 100% alcohol 100% alcohol 6 hours 12 hours 2 hours 1 hour 1 hour CLEARING Xylene or Toluene Xylene or Toluene 1 hour 1 hour IMPREGNATION Paraffin wax Paraffin wax Paraffin wax Paraffin wax 15 minutes 15 minutes 15 mins 15 mins EMBEDDING Paraffin wax 3 hours 2. AUTOMATIC PROCESSING ● ● ● ● ● ● This method makes use of an automatic tissue processing machine (i.e. Autotechnicon) which fixes, dehydrates, clears and infiltrates tissues, thereby decreasing the time and labor needed during the processing of tissues. This results in a more rapid diagnosis with less technicality. Usually, only 2- 3 changes of wax are required to remove the clearing agent and properly impregnate the specimen. This is made possible due to constant tissue agitation which accelerates and improves tissue penetration giving rise to more consistent results. One example of an automatic tissue processing machine is the Elliott Bench-Type Processor. The machine is mounted on rollers to permit the turning of platforms and easy access to beakers and wax baths. It makes use of ○ 12 individual processing steps, with ten 1-liter capacity glass beakers and; BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 12 ○ two thermostatically controlled wax baths with a safety device cut-out switch to protect the wax against overheating A transfer arm controlled by electrical current moves the tissues from one processing reagent to another (by clock schedules). It can be removed by raising a spring-loaded plunger in the center of the cover plate, thereby allowing the tissue to be arranged manually anytime during the processing. Agitation of fluid is accompanied by a continuous vertical movement or rotation of the specimen carrier by a mechanism connected to the transfer arm. An electrical clock connected to a metal disc notched in positions of 15 minutes or more, serves to control the time needed for each processing step. The clock rotates and sets the transfer arm and mechanism into motion, moving the tissue to the next position. A delay mechanism is provided in instances where processing time may exceed 24 hours. ● ● ● ● ● ● SAMPLE TIME TABLE FOR THICK AUTOMATIC PROCESSING OF TISSUES (3-5 mm) REAGENT 10% buffered formalin 10% buffered formalin 70% alcohol Absolute alcohol (1) Absolute alcohol (2) Absolute alcohol (3) Xylene or Toluene (1) Xylene or Toluene (2) Benzene (1) Benzene (2) Chloroform (1) Chloroform (2) Chloroform (3) Wax (1) Wax (2) Schedule 1 2 hrs. 2 hrs. 2 hrs. 1 hr. 1 hr. 1 hr. 1 hr. 1 hr. PROCESSING TIME Schedule 2 Schedule 3 2 hrs. 2 hrs. 2 hrs. 2 hrs. 2 hrs. 2 hrs. 3 hrs. 2 hrs. 3 hrs. 2 hrs. 3 hrs. 2 hrs. 30 mins. 1 hr. 2 hrs. 2 hrs. 3 hrs. 3 hrs. 2 hrs. 2 hrs. 2 hrs. 2 hrs. 2 hrs. NOTE: ● No need to memorize ● 3. VACUUM EMBEDDING ● ● ● ● ● ● ● ● ● ● ● PRECAUTIONS WITH AUTOMATIC PROCESSING ● ● ● ● ● ● ● ● ● ● The frequency with which fluids are changed depends on the number and sizes of the tissues processed. The presence of any odor in the clearing agent during the final paraffin wax bath indicates that the paraffin wax needs to be changed. Dehydrating fluids should be changed frequently since dehydration is the most critical stage of tissue processing and inadequate dehydration is difficult to correct once the tissue is in paraffin. The first 100% ethanol bath should be discarded, and the others moved down, so that the final bath has fresh 100% ethanol after two complete processing runs of loads of at least three-quarters capacity. The clearing agent and the dilute ethanols should be changed at least once a week. To avoid spillage, fluid and wax containers must be filled to the appropriate level and correctly located in the machine. Wax accumulating on Wax accumulating on any surface or beaker leads must be removed and any spillage should be wiped away. Wax bath thermostats should be set at least 3 degrees above the melting point of the wax, and timing should be checked when loading the machine, especially if the machine is equipped with a delay mechanism. Due to the viscosity of molten paraffin wax, some form of gentle agitation is highly desirable. If the processor is to be run overnight it should be programmed to hold on the first ethanol bath and not finish until the next morning so the specimens do not sit in hot paraffin longer than the time indicated. If specimens are fresh they may incubate in formalin in the first stage on the machine. It is important to not keep the tissues in hot paraffin too long or else they become hard and brittle. Processed tissues can be stored in the cassettes at room temperature indefinitely ● ● ● Involves wax impregnation under negative atmospheric pressure inside an embedding oven. It reduces the time when tissues are subjected to high temperatures thus minimizing heat-induced tissue hardening. It facilitates complete removal of transition solvents, and prolongs the life of wax by reducing solvent contamination. Vacuum hastens the removal of air bubbles and clearing agent from the tissue block, thereby promoting a more rapid wax penetration of the tissue. This technique is particularly recommended for urgent biopsies, for delicate tissues such as lung, brain, connective tissues, decalcified bones, eyes, spleen and central nervous system. Vacuum infiltration requires a vacuum infiltrator or embedding oven, consisting of wax baths, fluid trap and vacuum gauge, to which a vacuum of up to 760 mm Hg is applied using a water or mechanical pump. Modern tissue processors are equipped to deliver vacuum, or vacuum and pressure, to all or some reagent stations during the processing cycle. The time required for complete impregnation is reduced by 25%-75% of the normal time required for tissue processing. The tissue is not over-exposed to heat; brittleness, shrinkage and hardening of tissues consequent to overheating is therefore prevented. The tissue can also be transferred after clearing to a heated bath of paraffin wax from which air can be evacuated. The vacuum embedding oven consists of a flat-bottomed heavy brass chamber covered with a heavy glass lid resting on a wide and thick rubber valve which produces an airtight seal when the chamber is being used. The vacuum chamber is enclosed in a thermostatically controlled water-jacket, usually maintained at a temperature of 2-4°C above the melting point of the wax. The degree of the vacuum should not exceed 500 mm Hg. A stopcock is provided to prevent water from being sucked back into the trap bottle and vacuum chamber when the water or suction pump is closed AFTER FIXATION AND DEHYDRATION, PROCEED AS FOLLOWS: 1. Clear in two changes of xylene, for 1 hour each. 2. Place the tissue in molten wax, in a vacuum chamber and make the oven airtight. Exhaust the air slowly by means of a vacuum pump or Venture suction pump until there is a negative pressure of 400 to 500 mm. Mercury. 3. Leave for 15 minutes, then slowly readmit air until normal atmospheric pressure is reached. 4. Place the tissue in fresh wax. 5. Repeat steps 3 and 4. 6. Place the tissue into fresh wax. 7. Repeat step 3 and leave for 30 to 45 minutes. 8. Bring to normal atmospheric pressure and embed the tissue. ● ● NOTE: The exhaustion and readmission of air must be gradual or the specimen may be ruined. Of the three methods of paraffin wax impregnation, VACUUM IMPREGNATION gives the fastest result. BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 13 ● ● ● ● Total impregnation time, however, generally depends upon the nature and size of the tissues to be processed, and the type of clearing agents to be used. Larger and denser tissue blocks (e.g. bones, fibroids, brains) usually require longer periods and more frequent changes of wax. Benzene and xylene are easily removed from the tissues while chloroform and cedarwood oil are more difficult to remove and require more frequent wax changes. Addition of benzene may hasten displacement of cedarwood oil with less tissue shrinkage ● ● ● ● PRACTICAL CONSIDERATIONS ● ● ● ● ● ● ● ● ● ● ● Since prolonged treatment in melted paraffin causes shrinkage and hardening of tissues, making cutting difficult, the tissue should not be left in the paraffin oven for more than 4 hours. The shorter the time in the hot oven with adequate paraffin impregnation and evaporation of the clearing agent, the better it is for the tissue. Tissues become increasingly harder and more brittle as they are heated. Infiltration in overheated paraffin (above 60°C) will also produce shrinkage and hardening of tissues and destroying lymphoid tissues completely. To avoid this, the paraffin oven must be maintained at a temperature 2 to 5°C above the melting point of paraffin to be used for impregnation. Paraffin wax must be pure, i.e. free from dust, water droplets and other foreign matter. Fresh wax should be filtered before use in a wax oven at a temperature 2°C higher than its melting point. Wax that has been trimmed away from the impregnated tissue may be melted and filtered for future use, with a coarse filter paper, e.g. Green's No. 904. When wax has been reused, some amount of water inevitably is mixed with it. If excessive, this may impair the impregnating capacity of the medium and prevent formation of a good tissue block. Water must therefore be removed by heating the wax to 100 -105°C, thereby raising its melting point. Paraffin wax may be used only twice, after which, fresh wax must be utilized. When using an automatic tissue processing machine, wax usually becomes admixed with the clearing agent, especially in the first beaker; hence, water must be discarded. For fixed knife microtomes, a relatively hard wax with a higher melting point is recommended. Heavier microtome knives require harder paraffin wax than lighter ones. SUBSTITUTES FOR PARAFFIN WAX 1. Paraplast 2. Embeddol 3. Ester wax 4. Water soluble wax 5. Dimethyl sulfoxide (DMSO) 2. EMBEDDOL ● ● ● ● ● ● ● Paraplast is a mixture of highly purified paraffin and synthetic plastic polymers, with a melting point of 56-57°C. It is more elastic and resilient than paraffin wax thereby permitting large dense tissue blocks such as bones and brain to be cut easily with the same result as in double embedding. Blocks obtained are more uniform than with any other medium, with better ribboning of sections. Serial sections may be cut with ease, without cooling the tissue block, thereby preventing the formation of ice crystal artifacts. No deposit is left on the slide after staining, and no special processing schedule is required. It is soluble in common Embeddol is a synthetic wax substitute similar to Paraplast with a melting point of 56-58°C. It is less brittle and less compressible than Paraplast. Bio/aid is a semisynthetic wax recommended for embedding eyes. Tissue Mat is a product of paraffin, containing rubber, with the same property as Paraplast. 3. ESTER WAX ● ● ● ● ● ● Ester Wax has a lower melting point (46-48°C), but it is harder than paraffin. It is not soluble in water, but is soluble in 95% Ethyl Alcohol and other clearing agents; hence, it can be used for impregnation without prior clearing of the tissue. Cellosolve (ethylene glycol monoethyl ether) or xylene may be used as clearing agents, if indicated. In such instances, removal of the clearing agent must be gradual; that is, the tissue must be placed in a solution containing equal proportion of clearing agent and ester wax for 3- 6 hours before finally transferring it to pure wax. Three to four changes of wax are required to ensure complete tissue impregnation. Sectioning of ester wax-impregnated tissues should be done on a heavy duty microtome (e.g. sliding or sledge type microtome) due to the relative hardness of the wax. 4. WATER SOLUBLE WAX ● ● ● ● ● ● ● 1. PARAPLAST ● clearing agents and follows the same time schedule for paraffin impregnation, and does not tend to crack like other paraffin wax substitutes. Generally, Paraplast with a melting point of 56°C to 58°C is recommended. During the winter, 54°C to 56°C Paraplast may be used if the tissue is cut in a cool room. During the summer it may be necessary to use 60°C to 63°C, although this is to be avoided if possible in order to not to "cook" the tissue. "Cooked" tissue does not section well or, if it does, it does not stain well and most details are destroyed. ● ● ● Water Soluble Waxes are plastic polymers, mostly polyethylene glycols with melting points of 38-42°C or 45-56°C. Polymer waxes are incorporated in the majority of proprietary histological paraffin wax blends presently available to improve adhesion, hardness and plasticity. CARBOWAX - The most commonly used, a polyethylene glycol containing 18 or more carbon atoms, which appears solid at room temperature. It is soluble in and miscible with water; hence does not require dehydration and clearing of the tissue. It is soluble in and miscible with water; hence does not require dehydration and clearing of the tissue. The tissues are fixed, washed out and transferred directly into the melted Carbowax. Processing time is reduced with the special advantage that harmful effects produced by ordinary dehydrating agents are consequently avoided. It does not remove neutral fats and lipids which are soluble in reagents used for routine processing with paraffin, hence, allowing these substances to be demonstrated in thin sections. Tissues are not exposed to too much heat so that excessive hardening, shrinkage and brittleness of tissue is avoided; hence, making Carbowax technique suitable for many enzyme histochemical studies. Cytologic details are excellently preserved. For routine processing, four changes of Carbowax, one each in 70% and 90% and 2 times in I00% concentration, at a temperature of 56°C are used, at 30 minutes, 45 minutes and 1 hour (with agitation), respectively. BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 14 ● Specimens are then embedded in fresh Carbowax at 50°C and rapidly cooled in a refrigerator. Due to its hygroscopic nature, Carbowax is very easily dissolved in water. Hence care must be taken to avoid contact of the block with water or ice. Tissue sections are very difficult to float out and mount due to its extreme solubility in water, dehydrating and clearing agents. Adding soap to water or using 10% Polyethylene Glycol 900 in water will reduce tissue distortion and promote flattening and "floating out" of sections. ● ● ● WET CELLOIDIN METHOD ● ● ● ● ● 5. DIMETHYL SULPHOXIDE (DMSO) ● DMSO added to proprietary blends of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning. DMSO scavenges residual transition solvent and probably alters tissue permeability by substituting for or removing bound water thus improving infiltration. Some individuals who handle DMSO-paraffin wax may experience an unpleasant and annoying oyster or garlic taste probably caused by DMSO metabolites. Possible health risks associated with the use of DMSO-paraffin wax are minimal if correct laboratory hygiene is observed. Not commonly used ● ● ● ● ● ● DRY CELLOIDIN METHOD ● ● CELLOIDIN IMPREGNATION ● Celloidin (Collodion) is a purified form of nitrocellulose soluble in many solvents, suitable for specimens with large hollow cavities which tend to collapse, for hard and dense tissues such as bones and teeth and for large tissue sections of the whole embryo. It is supplied in thin (2%), medium (4%) or thick (8%) solutions of cellulose dissolved in equal parts of ether and alcohol. This is used mainly for preparing soft tissue sections of mixed consistency such as eyes and brain. No heat is required, and the resultant block has a rubbery consistency which gives good support to the tissues. Disadvantages: include inability to cut thin sections, storage of blocks in alcohol and speed of technique (which can take several weeks or months). ● ● ● ● ADVANTAGES AND DISADVANTAGES OF CELLOIDIN IMPREGNATION ADVANTAGES 1. 2. 3. 4. It permits cutting of tissue sections which are thicker than in paraffin wax, and is recommended for processing of neurological tissues. Its rubbery consistency allows tissue blocks that are either very hard or of varying consistency, to be cut without undue distortion. Dense tissues which are hard to infiltrate (e.g. bones and brain) and specimens which tend to collapse easily due to air spaces (e.g. eyes) are supported better, thereby avoiding the crumbling of tissues during sectioning. When eye sections are embedded by the paraffin method, the retina may be detached from the harder tissues (e.g. sclera and choroid) that encircle it. The cedarwood oil used in the dry celloidin technique helps to soften the brittle layers. It does not require heat during processing; hence, producing minimum shrinkage and tissue distortion especially for cutting large bone sections. It is, therefore, recommended in cases when minimum shrinkage is required and when frozen section technique cannot be done. DISADVANTAGES 1. 2. 3. 4. 5. Celloidin impregnation is very slow (lasting for several days or weeks). Very thin sections (less than I 0 μ) are difficult to cut. Serial sections are difficult to prepare. Vapor of the ether solvent is very flammable; hence, it should never be used near an open flame. Photomicrographs are difficult to obtain CELLOIDIN IMPREGNATION METHODS ● Two methods are used for celloidin impregnation of tissue: ● ● Recommended for bones, teeth, large brain sections and whole organs. After the usual fixation and dehydration of the tissue, it is placed in equal parts of ether and alcohol for 12-24 hours. The tissue is then placed in thin celloidin (2-4%) for 5-7 days, transferred to medium celloidin (4-6%) for another 5-7 days, drained off and poured with thick celloidin (8-12%) until the specimen has become impregnated, usually between 3-5 days. The specimen is removed from the celloidin, transferred to an embedding medium containing freshly poured thick celloidin and kept in a tightly covered jar or dessicator in order to evaporate the alcohol-ether solvent. The dessicator top is removed for a few seconds, time and again, to admit fresh air and harden the tissue block. Evaporation must be gradual to achieve a consistent, uniform degree of hardness throughout the block and prevent the formation of air bubbles. When the ball of the finger leaves no mark on the surface of the tissue block, evaporation and consequently, embedding, is considered to be complete. The tissue block is then stored in 70-80% alcohol until ready for cutting. This is done to avoid dehydration and shrinkage of tissues. Preferred for processing whole eye sections. The principle and procedure of this method is similar to wet celloidin method, except that 70% alcohol is not used for storage before cutting. Instead, GILSON’S MIXTURE - made up of equal parts of chloroform and cedarwood oil, is added to the celloidin block before hardening, to make the tissue transparent. The dry method does not make use of alcohol due to the presence of cedarwood oil in the block NITROCELLULOSE ● ● ● ● ● ● ● ● ● Low Viscosity Nitrocellulose (L.V.N.) is another form of celloidin soluble in equal concentration of ether and alcohol, with a lower viscosity, allowing it to be used in higher concentrations and still penetrate tissues rapidly. Because of this, many workers prefer L.V.N. to the ordinary celloidin for impregnation and embedding. It forms a harder tissue block and makes cutting of thinner sections possible. The tendency of tissues to crack may be prevented by adding plasticizers (e.g. oleum ricini or castor oil) when embedding chrome-mordanted tissues. Low viscosity nitrocellulose is more explosive than celloidin and should therefore be handled with care. When dry, striking or dropping the container will cause the substance to explode. It is usually marketed while wet with alcohol. The container must be kept tightly covered and protected from sunlight to avoid evaporation of alcohol. When no longer needed for future use, the nitrocellulose should be carefully destroyed, since the material becomes increasingly dangerous as the alcohol continues to evaporate GELATIN IMPREGNATION ● ● ● ● Gelatin impregnation is rarely used except when dehydration is to be avoided and when tissues are to be subjected to histochemical and enzyme studies. It is used as an embedding medium for delicate specimens and frozen tissue sections because it prevents fragmentation of tough and friable tissues when frozen sections are cut. It is water-soluble, and does not require dehydration and clearing, although fixatives (such as 10% formalin) should still be washed out by running water whenever indicated. It has a low melting point and does not cause over-hardening of tissues by heating. After the fixative has been completely washed out, the tissue is placed in 10% gelatin with 1% phenol for 24 hours, transferred to 20% gelatin with 1% phenol for the next 12 hours, and finally to another fresh solution of 20% gelatin with 1% phenol which is then allowed to cool in a BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 15 refrigerator until impregnation and embedding are completed. Gelatin-embedded tissues are then transferred to 10% formalin for 12-24 hours in order to harden the tissue. Tissues should not be more than 2-3 mm thick since gelatin-embedded specimens are harder to freeze than non-impregnated tissues. The 1% phenol serves to prevent the growth of molds. Excess gelatin may be removed by floating the sections oh to paper and trimming them with scissors. The volume of the impregnating medium should be at least 25 times the volume of the tissue. ● ● ● ● ● ● ● ● ● ● ● ● ● ● After impregnation, the tissue is placed into a mold containing the embedding medium and this medium is allowed to solidify. Ideally the embedding medium should match the tissue type in strength and hardness. If the embedding medium is too soft for the material, the tissue will not be supported and sections will be torn or shredded. If the medium is too hard for the tissue, sections will be brittle and will shatter. To infiltrate the tissues with supporting embedding medium, tissues must be free of all water (since usually embedding medium is not miscible with water). Paraffin embedded tissues are arranged at the bottom of the mold together with their proper labels and immersed in melted paraffin at a temperature between 5- 10°C above its melting point and then cooled rapidly in a refrigerator at -5°C or immersed in cold water to solidify. This will allow hardening of tissues, giving them a firmer consistency and better support, thereby facilitating the cutting of sections The process by which a tissue is arranged in precise positions in the mold during embedding, on the microtome before cutting, and on the slide before staining, is known as Orientation. Generally speaking, the surface of the section to be cut should be placed parallel to the bottom of the mold in which it is oriented TYPES OF BLOCKING-OUT MOLDS LEUCKHART’S EMBEDDING MOLD ● ● ● ● ● NOTE: Automatic tissue processor/Tissue processing in Histopathology video: https://www.youtube.com/watch?si=ySu4RBvwiE2_lqe2&v=90eKJQJcc XA&feature=youtu.be (lullaby ) EMBEDDING ● TISSUE TEK ● Consists of two L-shaped strips of heavy brass or metal arranged on a flat metal plate and which can be moved to adjust the size of the mold to the size of the specimen. Blocks produced are even, with parallel sides, and with a fairly shaped initial setting of the wax. The mold is adjustable to give a wide variety of sizes to fit the size of the tissue block for casting. It is recommended for routine use, although, too slow and cumbersome for use in a busy laboratory. ● ● ● ● ● DISPOSABLE EMBEDDING MOLDS PEEL-AWAY DISPOSABLE THIN PLASTIC EMBEDDING MOLDS ● ● ● Is made up of a series of interlocking plates resting on a flat metal base, forming several compartments. It has the advantage of embedding more specimens at a time, thereby reducing the time needed for blocking. PLASTIC EMBEDDING RINGS AND BASE MOLD ● Consist of a special stainless steel base mold fitted with a plastic embedding ring, which later serves as the block holder during cutting. Available in 3 different sizes, are simply peeled off one at a time, as soon as the wax has solidified, giving perfect even block without trimming. It may be placed directly in the chuck or block holder of the microtome PLASTIC ICE TRAYS ● ● Such as those used in ordinary refrigerators may be recommended for busy routine laboratories. Each compartment may be utilized for embedding one tissue block, which may then be removed by bending the plastic tray once the wax has solidified or by smearing the inner mold with glycerin or liquid paraffin before embedding. PAPER BOATS ● ● ● COMPOUND EMBEDDING UNIT ● Model that is equipped with a warm plate to manage the impregnated specimen, and a cold plate at -5°C for rapid solidification of the block. It consists of: ○ A white plastic cassette mold with detachable perforated stainless steel hinge ○ Snap-On lid ■ used to hold the tissue specimen throughout fixation, dehydration, clearing and wax impregnation With the Tissue Tek system, the specimen is placed on the base mold, the plastic embedding ring is placed in position, filled up with wax, and then placed on a small cool area to allow the wax in the base of the mold to semi-harden. This will allow easy orientation of the block. Once the tissue has been properly oriented, the base of the cassette is placed on top and together, they are placed on the cold plate so that the paraffin wax can cool and harden quickly. After the paraffin wax has solidified (usually 5 minutes), the block is taken out together with the embedding ring and is immediately ready for cutting without having to undergo trimming or mounting, thereby saving time and effort. The advantages of Tissue Tek include ease of use, less paraffin wax needed, faster embedding, firmly attached tissue and holder, and permanent identification. It produces easier orientation when resectioning of tissue is required, and blocks can be filed immediately after sectioning. ● Are normally utilized for embedding celloidin blocks but are equally useful for paraffin wax blocks. They have the advantage of being cheap and easy to make. They provide easy and accurate identification of specimen, thereby avoiding confusion and interchange of tissue blocks. Rapid embedding of small or large volume of individual specimen is possible, since paper molds can be made to suit any size of tissue BASIC METHOD THE BASIC METHOD IS THE SAME FOR EACH 1. Open cassette to view tissue sample and choose a mold that best corresponds to the size of the tissue. A margin of at least 2 mm of paraffin surrounding all sides of the tissue gives best cutting support. Discard cassette lid. 2. Put small amount of molten paraffin in mold, dispensing from paraffin reservoir BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 16 3. 4. 5. 6. 7. 8. 9. Using warm forceps, transfer tissue into mold, placing cut side down, as it was placed in the cassette. When the tissue is in the desired orientation, add the labeled tissue cassette on top of the mold as a backing. Press firmly. Hot paraffin is added to the mold from the paraffin dispenser. Be sure there is enough paraffin to cover the face of the plastic cassette. Cool the top surface of the Paraplast by blowing gently on it. Tissues at this stage are very brittle and should be handled with care. If necessary, fill cassette with paraffin while cooling, keeping the mold full until solid Cool thoroughly in cold running tap water. If you use ice water for the final cooling, you may split the block owing to too rapid shrinkage. Paraplast naturally splits in the line of least resistance-right through the tissue Paraffin should solidify in 30 minutes. When the wax is completely cooled and hardened (30 minutes) the paraffin block can be easily popped out of the mold; the wax blocks should not stick. If the wax cracks or the tissues are not aligned well, simply melt them again and start over If you use plastic cups, the Paraplast block can be removed as soon as it is cooled. The stainless steel mold should slip off easily when cool and can be used again. EMBEDDING ● ● It is important to work quickly while transferring specimens or wax from the oven because wax hardens quickly. Always remember to put the wax container back into the oven immediately and close the oven door between transfers. Orientation of tissues in the Paraplast block is important for tissues such as an artery or fallopian tube so that they can be properly placed in a predetermined plane, such as cross sections. Also, trimming is excessively difficult in a block embedded with two or more tissues if they are not carefully lined up before the Paraplast is cooled particularly in hard tissues such as undecalcified bone and for high resolution light microscopy of tissue sections thinner than the usual 4-6 μm, such as renal biopsies and bone marrow biopsies. Plastics are classified into epoxy, polyester, or acrylic, based on their chemical composition. EMBEDDING ● ● ● ● ● ● ● ● ● ● ● ● CELLOIDIN OR NITROCELLULOSE EMBEDDING METHOD ● ● ● ● Used to be recommended for embedding hard tissues such as bones and teeth, and for large sections of whole organs like the eye, since the delicate layers of the eyeball are difficult to keep intact when other media are used. Tissues are embedded in shallow tins of enamel pans which are covered by sheets of weighted glass. Bell jars can be used to control the rate or evaporation of the solvent. The use of celloidin is discouraged now because of the special requirements needed for processing and the limited use of these types of sections in neuropathology. DOUBLE-EMBEDDING ● ● ● ● ● Is the process by which tissues are first embedded or fully infiltrated with a supporting medium such as agar or nitrocellulose, then infiltrated a second time with paraffin wax in which they are subsequently embedded. This is used to facilitate cutting of large blocks of dense firm tissues like the brain. They are also recommended for making small sections of celloidin blocks. A shortcoming of using agar as the pre-embedding media is that certain tissues shrink during the embedding process, and the agar-based pre-embedding media limits tissue expansion during slide mounting, resulting in difficulties with the tissue sample adhering to the microscope slide. The availability of paraffin waxes containing different types of resins has made this technique obsolete. ● ● ● ● ● ● ● ● PLASTIC (RESIN) EMBEDDING ● The introduction of plastic resin embedding media has provided superior results for light microscopic studies, ● Epoxy embedding plastics are made up of a carefully balanced mixture of epoxy plastic, catalysts and accelerators. Three types of epoxy plastics are used in microscopy, i.e., those based on either bisphenol A (Araldite), or glycerol (Epon), or cyclohexene dioxide (Spurr). Infiltration by Araldite is slow, partly because the epoxy plastic itself is a large molecule. The glycerol-based epoxy plastics (Epon) have a lower viscosity but are often sold as mixtures of isomers. Cyclohexene dioxide-based plastics (Spurr) can be obtained pure, have very low viscosity, and infiltrate fastest. Spurr’s Resin is a Low Viscosity mixture which provides rapid infiltration of tissues. It's easy to prepare and mixes rapidly. This resin is compatible with ethanol so no change to propylene oxide is needed prior to infiltration. Polymerization at 60°C is recommended. Epoxy plastics have several disadvantages. They are hydrophobic and subsequent oxidation by peroxide to correct this may produce tissue damage. Epoxy plastics have several disadvantages: ○ They are hydrophobic and subsequent oxidation by peroxide to correct this may produce tissue damage. ○ Epoxide groups may reduce antigenicity of embedded tissue, and may compromise the result of immunohistochemical staining. ○ More importantly, epoxy resins may cause sensitization if absorbed by skin or inhalation. ○ The components of many epoxy plastics are toxic and one of its components, vinyl cyclohexane dioxide (VCD) is known to be carcinogenic. For protection, gloves should always be worn when handling these plastics, and adequate facilities including an operational fume hood must be provided to remove the toxic vapors and properly dispose of toxic waste. Polyester plastics were originally introduced for electron microscopy in the mid- 1950s, but have been superseded by more superior epoxides, and are now seldom used. Acrylic plastics are made up of esters of acrylic or methacrylic acid, and are used extensively for light microscopy. Polyglycol methacrylate (GMA) has proved to be a popular embedding medium for light microscopy because it is extremely hydrophilic, allowing many staining methods to be applied, yet tough enough when dehydrated to section well on most microtomes. The polar water soluble, 2-hydroxyethyl methacrylate, commonly known as "glycol methacrylate", or GMA, has found an increasing number of applications for the embedding of biological tissue for transmission electron microscopy (TEM), for the preservation and observation of fine structure not previously subjected to solvent dehydration. GMA forms only non-crosslinked straight chains on polymerization and therefore requires no hardener. GMA and specifically low acid GMA offers a number of advantages over other systems: Dehydration of tissues can be made directly in aqueous dilutions of GMA or optionally in organic solvents. NOTE: Mga nice to know nalang ung mga next part; read and familiarize BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 17 LOW ACID GMA ADVANTAGES ● Low acid GMA offers a number of advantages over other systems: ○ Dehydration of tissues can be made directly in aqueous dilutions of GMA or optionally in organic solvents. ○ GMA does not need to be water-free and indeed it works best with at least some water present. ○ Infiltration of tissue with monomer can be performed at room temperature or lower. ○ Polymerization of GMA can be performed at ambient temperature of 0°C with UV radiation to 60°C in an oven. ○ Thin sections of polymerized GMA can be cut with glass or diamond knives. ○ Sections from Low Acid GMA, unlike ordinary technical grade GMA, resist the uptake of stain, thereby greatly reducing the occurrence of non-specific background staining. ○ Enzyme digestion, a variety of stains and immunological localizations may be performed on thick sections of GMA without removal of the plastic. BENZOYL PEROXIDE ● ● ● Is added to the plastic as a catalyst that decomposes to form phenyl radicals acting as an active site for the polymerization of acrylics. Unlike epoxy plastics, the viscosity of acrylic plastic is low so that short infiltration times are possible, although the size and nature of tissue, along with processing and embedding temperature will affect the times required for infiltration and embedding. radicals can be produced spontaneously by heat or light, so acrylic plastics and their monomers should be stored in dark bottles in a cool place to prevent premature polymerization. ● The alternative use of hydrophobic methyl methacrylate permits the plastic to be dissolved, and for certain techniques, this may be a very useful property ● Specimens should only be processed under an operational fume hood. Processing is best achieved if the specimen is agitated continuously on a roller mixer. Small aliquots of benzoyl peroxide should be dried carefully away from direct heat and sunlight as it is potentially explosive. It is important that no water is present before dissolving the catalyst (2 minutes) in the infiltrating solution. It must be completely dissolved in the infiltrating solution, and this may take up to 30 minutes. The acrylic plastic mixes are best prepared only in the quantity required, preferably using a large glass vial. It is advisable to measure the quantities volume by weight. Any waste solutions containing plastic components must be handled and discarded in accordance with local and legal requirements PRACTICAL CONSIDERATIONS ● ● ● ● ● RECITATION: 1. 2. 3. ● EMBEDDING ● ● ● ● ● ● ● ● Acrylic plastics based on methyl methacrylate (MMA) are also widely used because of its hardness as the ideal embedding medium for undecalcified bone and is widely used for bone histomorphometry and bone marrow hematopathology. Compared to water-soluble methacrylates (e.g., glycol methacrylate, GMA), MMA offers a variety of advantages. MMA penetrates tissues better than GMA and the histological quality of bone sections is generally higher in MMA-embedded bone samples compared to GMA-embedded specimens. GMA forms crosslinks during polymerization of the plastic so that removal of the resin from tissue sections is not possible for GMA-embedded material. MMA, on the other hand, can easily and completely be removed from tissue sections. This results in superior staining characteristics and excellent morphological detail. However, conventional MMA embedding causes almost complete loss of enzyme activity and protein antigenicity in the tissues, and therefore precludes the use of histochemical and immunohistological methods. In general, it is preferable to use acrylic plastic sections when high resolution light microscopy is required, because of their ease of handling and the quality of staining achieved. However, all acrylic hydrophilic media (including glycol methacrylate) are insoluble so that all staining occurs with the plastic in situ. Because of this, the embedding medium itself may become stained, or the matrix may act as a physical barrier to particular molecules causing problems during immunohistochemical staining. What is the recommended melting point of Paraplast? ○ 56-58 deg C TRUE/FALSE. Due to its hygroscopic nature, Carbowax is very easily dissolved in water ○ TRUE TRUE/FALSE. Wet Celloidin Method is recommended for bones, teeth, large brain sections and whole organs ○ TRUE NOTE: His