Fresh Tissue Examination/Fixation PDF

Introduction to Fresh Tissue Examination/Fixation MLS 113 – HISTOPATH Ms. Harley Rose B. Bautista, RMT M1 L2 PROCESSING OF TISSUE FORMIC ACI...

Introduction to Fresh Tissue Examination/Fixation MLS 113 – HISTOPATH Ms. Harley Rose B. Bautista, RMT M1 L2 PROCESSING OF TISSUE FORMIC ACID-SODIUM CITRATE SOLUTION DECALCIFICATION − recommended for autopsy materials, bone − Procedure whereby calcium or lime salts are marrow, cartilage. removed from tissues following fixations. 4. TRICHLOROACETIC ACID − Recommended ratio of fluid to tissue volume for − very slow acting; weak decalcifying agent. decalcification is 20:1 − suitable for small spicule of bone. Calcium may be removed by the following acid: ✓ Chelating agents 5. SULFUROUS ACID ✓ Ion exchange resins − Is a very weak decalcifying solution suitable only ✓ Electrical ionization for MINUTE PIECES OF BONE METHODS OF DECALCIFICATION 6. CHROMIC ACID (FLEMMING’S FLUID) ACID DECALCIFYING AGENT − may be used as both fixative and decalcifying 1. NITRIC ACID agent. − most common and the fastest decalcifying agent − suggest for minute bone specimens. used so far. However, it inhibits nuclear stain and − consider as carcinogenic. destroying tissues especially in concentrated solutions. 7. CITRIC ACID – CITRATE BUFFER SOLUTION − pH 4.5 a) AQUEOUS NITRIC ADD SOLUTION 10% − excellent nuclear and cytoplasmic staining. − recommended for urgent biopsies, for needle and − Too slow small biopsy specimens to permit rapid diagnosis within 24 hrs. or less − Contains chloroform as preservatives. b) FORMOL-NITRIC ACID − Rapid acting CHELATING AGENTS − recommended for urgent biopsies. 1. EDTA (VERSENE) Produce yellow color − most common chelating agent Remedy: neutralizing tissue in 5% sodium sulfate − recommended for detailed microscopic studies and washing in running tap water for at least 12 hrs. − very slow decalcifying agent Addition of 0.1% urea to pure concentrated nitric acid will − for small specimens: 1-3 weeks also make discoloration disappear without affecting the − 6-8 weeks or longer totally decalcify dense cortical efficiency of the decalcifying agent bone. − excellent for immunohistochemistry or enzyme c) PERENYI’S FLUID staining and electron microscopy − recommended for routine purposes − inactivates ALP; to restored add magnesium − nuclear and cytoplasmic staining is good. chloride Maceration is avoided due to the presence of chromic acid and alcohol. ION EXCHANGE RESIN − not recommended for urgent work. − not recommended for fluids containing nitric acid − composed of chromic acid, ethyl alcohol and nitric or hydrochloric acid. acid. − the decalcifying agent is then added, usually 20- 30 times of the volume of the tissue d) PHLOROGLUCIN NITRIC ACID − the tissue will stay in solution 1-14 days most rapid decalcifying agent ELECTROPHORESIS 2. HYDROCHLORIC ACID - most rapid method VON EBNER’S FLUID − moderately rapid decalcifying agent POST DECALCIFICATION − does not require washing out before dehydration − Involves lithium carbonate to wash the tissue after the − recommended for teeth and small pieces of bone decalcification is complete. − Also decalcified tissue can rinse in running tap water 3. FORMIC ACID to re moved acids. − may be used as both fixative and decalcifying agent. TISSUE SOFTENER’S − recommended for routine decalcification of 1. Perenyi’s fluid – may act as both decalcifying and postmortem research tissues. tissue softners 2. 4% aqueous sol TRANSCRIBED BY: Bucad, Shermella T. BSMT3B REFERENCES: Lecture Videos Psalm 63: 1-8 Introduction to Fresh Tissue Examination/Fixation MLS 113 – HISTOPATH Ms. Harley Rose B. Bautista, RMT M1 L2 3. Molliflex – tissue immersed in molliflex appear − Limited for only small specimen swollen and soapy. − Volatility and inflammable − Most lipids are removed from tissues with this FACTORS INFLUENCE OF DECALCIFICATION dehydrating agent. 1. Concentration and volume − More concentrated, more rapidly; more harmful 3. DIOXANE (DIETHYL DIOXIDE) − Excellent dehydrating agent and clearing agent 2. Structure and temperature − Readily miscible with water and paraffin − Ratio: 20:1 − Tends tissue ribbon poorly − higher concentration and greater amount of fluid will − Highly toxic in man and expensive increase the speed of the process. 3. Heat 4. CELLOSOLVE (EHTYLENE GLYCOL 4. Mechanical Agitation MONOETHYLETHER) 5. Accelerates the rate of diffusion and speeds up − Cellosolve dehydrates rapidly. The tissue may be the decalcification process. transferred from water or normal saline directly to cellosolve and stored in it for months without MEASURING EXTENT OF DECALCIFICATION producing hardening or distortion. 1. Physical or mechanical test – Touching or bending the tissue with the fingers. Inaccurate method. 5. TRIETHYL PHOSPHATE 2. X – ray or radiological method – Very expensive; − Used to dehydrate sections and smears following most ideal; most sensitive and most reliable method. certain stains and produces minimum shrinkage 3. Chemical method (calcium oxalate test) − Simple, reliable and convenient method 6. TETRAHYDROFURAN (THF) − Cloudiness will signify incomplete decalcification − Both dehydrates and clears tissue since it is miscible in both water and paraffin DEHYDRATION Starts by placing the fixed specimen in 70% to 95% to CLEARING 100% ethyl alcohol. De-alcoholization; process whereby alcohol or a For delicate tissues, particularly embryonic tissues dehydrating agent is removed from the tissue and replaced starting 30% ethyl alcohol with a substance that will dissolve the wax with which the The amount of dehydrating agent is should not be less tissue to be impregnated. than 10 times the volume of the tissue to ensure Must be miscible with water, paraffin and mounting medium. complete penetration of the tissue by the dehydrating COMMON CLEARING AGENTS agent. 1. XYLENE (XYLOL) − Colorless clearing agent, commonly used in 1. ALCOHOL histology laboratories. − Anhydrous copper sulfate will accelerate − Miscible with absolute alcohol and paraffin dehydration by removing water from dehydrating − It is cheap; used for celloidin sections fluid. A blue discoloration will indicate a complete − Highly inflammable dehydration. − Not suitable for nervous tissues and lymph nodes A. ETHYL ALCOHOL − Xylene turns milky when an incompletely − recommend for routine dehydration of dehydrated tissue is immersed in it. tissues − best dehydrating agent 2. TOLUENE B. METHYL ALCOHOL − May be used as a substitute for xylene and − Toxic dehydrating agent benzene. − Used for blood and tissue film for smear − More expensive preparations − Miscible with absolute alcohol and paraffin C. BUTYL ALCOHOL − For plant and animal microtechniques 3. BENZENE − Slow dehydrating agent − Preferred by some as clearing agent in the embedding process of tissues because it penetrates 2. ACETONE and clears tissue rapidly. − Cheap, rapid acting dehydrating agent − Carcinogenic, may damage the bone marrow − For urgent biopsies which dehydrates 30 mins to 2 resulting aplastic anemia hrs − Miscible with absolute alcohol TRANSCRIBED BY: Bucad, Shermella T. BSMT3B REFERENCES: Lecture Videos Psalm 63: 1-8 Introduction to Fresh Tissue Examination/Fixation MLS 113 – HISTOPATH Ms. Harley Rose B. Bautista, RMT M1 L2 4. CHLOROFORM METHODS OF PARAFFIN WAX IMPREGNATION − does not make tissue translucent A. MANUAL PROCESSING − recommended for tough tissues (skin, fibroid, − Four changes of wax are required at 15 minutes decalcified tissues) interval. − toxic to liver − tissue tend to float in chloroform to avoid this B. AUTOMATIC PROCESSING wrapped the tissues with absorbent cotton gauze to − Use autotechnicon which fixes, dehydrates, clears facilitate sinking of the section in solution and infiltrates tissues − Miscible with absolute alcohol − 2 -3 changes of wax are required. 5. CEDARWOOD OIL C. VACCUM EMBEDDING − Recommended for central nervous system tissues − Involves the wax impregnation under negative and cytological studies (particularly smooth atmospheric pressure inside an embedding oven to muscles) hasten removal of air bubbles and clearing agent − Requires two changes in clearing solution − Most rapid − It makes tissue transparent − Recommended in urgent biopsies − Extremely slow clearing agent SUBSTITUTES FOR PARAFFIN WAX 6. ANILINE OIL 1. PARAPLAST − Recommended for clearing embryos, insects and − Mixture of highly purified paraffin and synthetic very delicate specimens plastic polymers − Melting point: 56-57 C 7. CLOVE OIL − More elastic and resilient − Unsuitable for routine clearing process − For bones and brains 2. EMBEDDOL 8. CARBON TETRACHLORIDE − Synthetic wax substitute similar to paraplast − Properties are very similar to chloroform − Melting point is: 56-58 C 9. METHYL BENZOATE AND METHYL SALICYLATE 3. TISSUE MAT – contains rubber − Double embedding technique is needed 4. ESTER WAX − Has a lowering melting point 46-48 C IMPREGNATION − Harder than paraffin Process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will 5. WATER SOLUBLE WAX (CARBO WAX) completely fill the cavities, thereby giving a firm consistency − Melting point: 38-42 C or 45-46 C to the specimen and allowing easier handling and cutting suitably thin sections without any damage to the tissue and 2. CELLOIDIN IMPREGNATION its cellular components. Purified form of nitrocellulose soluble in many solvents → Paraffin impregnation Suitable for specimens with large hollow cavities → Celloidin impregnation which tend to collapse for hard and dense tissues → Gelatin impregnation such as bones teeth and for large tissue sections such as whole embryo. 1. PARAFFIN WAX IMPREGNATION Recommended for processing neurological tissue Simplest, most common and best embedding Very slow medium, used for routine processing TWO METHODS FOR CELLOIDIN IMPREGNATION Very rapid WET CELLOIDIN METHOD Prolong impregnation will cause excessive − Recommended for bones teeth large brain and whole shrinkage and hardening making cutting of sections organs difficult Not recommended for fatty tissues DRY CELLOIDIN METHOD ✓ For routine work melting point is: 56-58 C − Recommended for whole eye sections ✓ For the lab temp between: 15-18 C − Does not use alcohol due to the presence of Melting point is: 50-54 C cedarwood oil in the block. ✓ For the lab temp between 20-24 C Melting point is 54-58 C TRANSCRIBED BY: Bucad, Shermella T. BSMT3B REFERENCES: Lecture Videos Psalm 63: 1-8 Introduction to Fresh Tissue Examination/Fixation MLS 113 – HISTOPATH Ms. Harley Rose B. Bautista, RMT M1 L2 3. GELATIN IMPREGNATION MICROTOMY Rarely used except for histochem and enzyme Process by which processed tissue, most commonly a studies paraffin embedded tissue, is trimmed and cut into uniformly Used for delicate specimens and frozen tissue thin slices or sections to facilitate studies under microscope Water soluble Tissue should not be more than 2-3mm thick; add ESSENTIAL PARTS OF MICROTOME 1% phenol to prevent molds 1. BLOCK HOLDER – Tissue held in position. Excess gelatin may remove by floating the sections 2. KNIFE CARRIER AND KNIFE – for actual cutting of to paper and trimming them with scissors tissue sections. 3. PAWL, RATCHET FEED WHEEL AND ADJUSTMENT EMBEDDING SCREWS – line up the proper position with the knife. After impregnation, the tissue placed into a mold containing the embedding medium and these media allow solidifying. 5 KINDS OF MICROTOME → In this process orientation is very important. 1. ROCKING (CAMBRIDGE) MICROTOME → Temperature of melted paraffin is 5-10 deg C above the − For cutting large blocks of paraffin embedded melting point tissues. → Immersed in cold or ref temp to solidify − Invented by paldwelltrefall in 1881 − Simplest among the different types of microtomes 1. Leukhart’s embedding mold – Contains two L- Cut the tissue passes to the knife edge in slightly shaped strips of heavy brass curved plane in 10-12 u thickness 2. Compound Embedding unit – Made up of a series of interlocking plates resting on a flat metal base 2. ROTARY (MINOT) MICROTOME 3. Plastic embedding rings and base molds – Consist − For cutting paraffin embedded sections. of special stainless steel base mold fitted with a plastic − Invented by Minot in 1885-1886 embedding ring, which later serves as the block holder − Most common type used for both routine and during cutting research laboratories. 4. Tissue-tek – Equipped with a warm plate to manage − Cut the tissue in 4-6 u thickness. the impregnated specimen. 5. Disposable embedding molds 3. SLIDING MICROTOME → Peel-away − For cutting celloidin embedded sections Developed → Plastic ice trays by Adams in 1789 → Paper boats − Most dangerous type of microtome. REMEMBER BASE SLEDGE MICROTOME- consists of two movable ✓ Celloidin or nitrocellulose method – recommended pillars holding the adjustable knife clamps. for embedding hard tissues. ✓ Double-embedding method-process; in which tissue STANDARD SLIDING – the blocks remain stationary, first infiltrate by celloidin and embedded in paraffin. the knife is move backward and forward during the process of sectioning. It is for cutting celloidin sections. 4. FREEZING MICROTOME − For cutting unembedded frozen sections. Invented by Queckett in 1848 − Used a carbon dioxide to freeze the block holder and tissue evenly. 5. CRYOSTAT OR COLD MICROTOME − Used in fresh tissue microtomy − Consist of rotary microtome − For rapid diagnosis Maintained a temperature between -5 to -30 degcel average is -20 degcel. 6. ULTRATHIN MICROTOME − Cut tissue at 0.5 micra − For electron microscopy TRANSCRIBED BY: Bucad, Shermella T. BSMT3B REFERENCES: Lecture Videos Psalm 63: 1-8 Introduction to Fresh Tissue Examination/Fixation MLS 113 – HISTOPATH Ms. Harley Rose B. Bautista, RMT M1 L2 MICROTOME KNIVES *Sections are floated out to a water bath set at 45-50 degcel or 6-10 degcel lower than the melting point of the wax used PLANE-CONCAVE One side of the knife 25 mm in for embedding tissue. KNIFE is flat while other is length *Sections should not be left on the water bath for a long time concave. Less 30 secs will be enough to avoid distortion concave are for cutting celloidin DRYING TECHNIQUES embedded tissue PARAFFIN OVEN – maintained a temperature 2-5 blocks. More concave degcel above the melting point of the paraffin used. is for paraffin sections Thermostatically controlled incubators may be used at 37 degcel and 45-55 degcel BICONCAVE Both sides concave, 120 mm in KNIFE recommended for length ADHESIVES cutting paraffin 1. MAYER’S RGG ALBUMIN embedded sections − Most commonly used ; it is very easy to make; convenient and inexpensive PLANE-WEDGE Both sides are 100 mm in − Equal amount of egg white and glycerin KNIFE straight, for frozen length sections. 2. DRIED ALBUMIN − DRIED ALBUMIN+nacl= dried albumin HONING − Addition of thymol to prevent growth of molds Removal of gross nicks on the knife edge 10-20 strokes; 20-30 strokes; heel to toe movement or direction; edge 3. GELATIN 1% first. − Gelatin+ distilled water+glycerol+phenol crystals OIL STONES: 1. BELGIUM YELLOW- for manual sharpening; usually 4. GELATIN FORMALDEHYDE MIXTURE gives the best result. − 1% gelatin + 2% formaldehyde 2. ARKANSAS – gives more polishing effects. 3. FINE CARBORUNDUM - used only for badly nicked 5. STARCH PASTE knives. − Powdered starch+ distilled water+ hydrochloric acid+ thymol LUBRICANTS: 1. MINERAL 6. PLASMA 2. CLOVE OIL − Readily available from outdated blood storeed 3. XYLENE in blood banks or hematology. Dispensed it 4. LIQUID PARAFFIN into sterile tubes of 0.5 ml each. *Use to remove scattered small particles of stones and metals 7. POLY-L-LYSINE − Must be used within a few days after they STROPPING prepared, since its effectiveness slowly Process whereby the burr formed during honing is decreases in time. removed and the cutting edge of the knife is polished and sharpen. 8. APES (3-aminopropytriethylxysilane) Toe to heel direction; 40-120 double strokes are − Better than poly-l-lysine, can be stored for a required. long time without losing their adhesiveness. Use paddle strop made of horse leather that usually treated with vegetable oil. Mineral oil is not STAINING recommended and should never touched or in contact Applying dyes on the section to see and study the in strope it will tend to blister and destroy the leather. architectural pattern of the tissue and physical characteristics of the cells. ✓ TRIMMING – cutting of excess wax from the block. ✓ CLEARANCE ANGLE – between the facet and tissue MAJOR GROUPS OF STAINING block; 0-15 C 1. HISTOLOGICAL STAINING ✓ CAMEL HAIR BRUSH- Use to pick up the complete − process whereby tissue constituents are ribbons. demonstrated in sections by direct interaction with a dye. TRANSCRIBED BY: Bucad, Shermella T. BSMT3B REFERENCES: Lecture Videos Psalm 63: 1-8 Introduction to Fresh Tissue Examination/Fixation MLS 113 – HISTOPATH Ms. Harley Rose B. Bautista, RMT M1 L2 − Used to demonstrate the general relationship of MOST COMMON COUNTER STAINS tissues and cells with differentiation of nucleus and cytoplasm. CYTOPLASMIC STAINS RED YELLOW GREEN 2. HISTOCHEMICAL STAINING (HISTOCHEMISTRY) Eosin Y Picric acid Light green SF − process whereby various constituents of tissues Eosin B Orange G Lissamine are studied thru chemical reactions. green Phloxine B Rose Bengal ✓ PERL’S PRUSSIAN BLUE – reaction for hgb NUCLEAR STAINS ✓ PAS – for carbohydrates RED BLUE Neutral red Methylene blue 3. IMMUNOHISTOCHEMICAL STAINING Safranin O Toluidine blue − immunologic and histochemical techniques that Carmine Celestine blue allow phenotypic markers to be detected using a Hematoxylin enzyme labeled antibodies. 8. METALLIC IMPREGNATION – process where specific METHODS OF STAINING tissue elements are demonstrated not by stains but by 1. DIRECT STAINING colorless solutions of metallic salts which are thereby − giving color to the sections by using aqueous or reduced by the tissue, producing an opaque usually alcoholic dye solutions. black deposit on the surface of the tissue or bacteria. Example: methylene blue; eosin 9. VITAL STAINING – selective staining of living cell constituents, demonstrating cytoplasmic structures by 2. INDIRECT STAINING phagocytosis of the dye particle; nucleus is resistant to − whereby the action of the dye is intensified by vital stain therefore it is not demonstrated. adding another agent or mordant. Demonstration of nucleus in vital staining indicates of MORDANT- serves as a link or bridge between the death cells. tissue and dye. Intravital staining – staining of living cells is done Example: Potash alum/potassium alum in by injecting the dye top any part of the animal erlichshematoxylin. body. Common dyes used are lithium, carmine and ACCENTUATOR – accelerates or hastens the india ink speed of staining reaction. Supravital staining – used to stain living cells Example: potassium hydroxide in loeffler’s immediately after removal from the living body. methylene blue and phenol in carbolthionine and Common dyes used are: carbolfuchsin. ✓ Neutral red- best vital dye ✓ Janus green- recommended for mitochondria 3. PROGRESSIVE STAINING ROUTINE H AND E STAINING (REGRESSIVE − tissue elements are stained in definite sequence STAINING) and staining. No decolorization required. 1. First xylene bath for 3 mins; for decolorization removal of excess paraffin wax 4. REGRESSIVE STAINING 2. Transfer to second xylene bath for 2-3 mins − tissue is first overstained and the excess stain is 3. Immerse in the first bath of absolute ethyl alcohol for 2 removed or decolorized from unwanted parts of the minutes tissue until the desire color is obtained. 4. Transfer to a bath of 95% ethyl alcohol for 1 to 2 minutes for hydration descending grades of alcohol 5. DIFFERENTIATION/DECOLORIZATION 5. Rinse in running water for 1 minute − removal of excess stain from the tissue during 6. Stain with harris alum hematoxylin for 5 mins. (primary regressive staining. dye) 7. Wash in running tap water to remove excess stain 6. METZCHROMATIC STAINING 8. Differentiate in 1% acid alcohol for 10-30 secs. − use of specific dyes which differentiate particular 9. Rinse in tap water substances by staining them with a color that is 10. Blue in ammonia water average of 5mins; to intensify different from that of the stain itself. the color of the nucleus 11. Wash in running tap water for 5 mins. 7. COUNTERSTAINING 12. Counterstain with 5% aqueous eosin for 5 mins. If − application of a different color stain to provide alcoholic stain is used time is reduced to 30 secs or 1 contrast and ack ground to the staining of the minute. structural components to be demonstrated. 13. Dehydrate, clear and mount. TRANSCRIBED BY: Bucad, Shermella T. BSMT3B REFERENCES: Lecture Videos Psalm 63: 1-8 Introduction to Fresh Tissue Examination/Fixation MLS 113 – HISTOPATH Ms. Harley Rose B. Bautista, RMT M1 L2 RESULT: ✓ SUDAN IV– recommended for staining neutral Nuclei: blue to black lipids or triglycerides. Karyosome- dark blue ✓ SUDAN III – first sudan dye introduced in Cytoplasm- pale pink immunohistochemistry; good stain for central RBC, keratin- bright orange-red nervous system. Cartilage- pink Calcium and calcified bones-purplish blue MOUNTING Muscle fibers- deep pink To preserve and support a stained section for light microscopy, it is mounted on a clear glass slide, and 1. NATURAL DYES – obtained from plants and animals , covered with a thin glass coverslip. The slide and coverslip previously utilized from dyeing wool and cotton. must be free of optical distortions, to avoid viewing artifacts. HEMATOXYLIN- derived by extraction from the core CHARACTERISTICS OF GOOD MOUNTING MEDIUM or the heartwood of a Mexican tree known as 1. A refractive index should be as near as possible to hematoxylincampechianum. that of the glass which is 1.518. a) ALUM HEMATOXYLIN – recommended for 2. Should be freely miscible with xylene and toluene progressive staining of tissues, stain nuclei but 3. Should not dry quickly leave the background tissue unstained. It is also 4. Should not crack or produce artefactual granularity use alum or potassium aluminum as mordant. upon drying. ✓ Ehrlich’s hematoxylin – ripened by sodium 5. Should not dissolve out or fade tissue sections. iodate 6. Should not cause shrinkage and distortion of ✓ Harris hematoxylin – ripened by: Mercuric tissues oxide 7. Should not leach out any stain or affect staining ✓ Cole’s hematoxylin- ripened by an alcoholic 8. Should not change in color or pH solution ✓ Mayer’s hematoxylin- ripened by sodium AQUEOUS MOUNTING MEDIA iodate 1. WATER b) IRON HEMATOXYLIN - it used for − has a low refractive index, moderately photomicrography transparent ✓ Weigerts solution- use ferric ammonium − good for temporary mounting. chloride as mordants 2. GLYCERIN ✓ Heidenhain’s solution – use ferric ammonium − also be used as a preservative sulfate (iron alum) as mordants − has a high refractive index c) PHOSPOTUNGSTIC ACID HEMATOXYLIN − a very suitable for semi-permanent mounting (PTAH) – demonstrates structures in paraffin as medium with a refractive index of 1.46 well as celloidin and frozen sections. Staining is 3. FARRANT’S MEDIUM usually 12-24 hours. Use 1% aqueous − does not need heated before use phosphotungstic acid as mordant. − require ringing − Refractive index of 1.43 COCHINEAL DYES – is an old dye extracted from the 4. APATHY’S MEDIUM female cochineal bug (coccuscacii). − does not require ringing ✓ Picrocarmine- used in neuropathological studies − recommended for methylene blue stained ✓ Best’s carmine- used for demonstration of nerve preparations and as a general purpose glycogen aqueous mountant − Refractive index of 1.52 ORCEIN – vegetable dye extracted from certain 5. BRUN’S FLUID lichens. It is used mainly in staining of elastic fibers. − for mounting of frozen sections. 2. SYNTHETIC DYES – known as “coal tar dyes” derived from hydrocarbon benzene collectively known as aniline dyes. 3. LYSOCHROMES – oil soluble dyes. ✓ SUDAN BLACK – most sensitive of the oil soluble dyes. It has a much greater affinity for phospholipids than other lysochromees. TRANSCRIBED BY: Bucad, Shermella T. BSMT3B REFERENCES: Lecture Videos Psalm 63: 1-8 Introduction to Fresh Tissue Examination/Fixation MLS 113 – HISTOPATH Ms. Harley Rose B. Bautista, RMT M1 L2 RESINOUS MOUNTING MEIDA 1. CANADA BALSAM − natural resin extracted from the canabian tree, (ABUS BALSAMEA). − Recommended for whole mounts and for thick sections because it does not shrink much. − It is miscible with xylene. − Refractive index of 1.524 2. DPX − recommended for small tissue sections but not for whole mounts because of shrinkage produced on drying. − Refractive index of 1.532 3. XAM − synthetic resin mixture − dries quickly without retraction and preserves stains well − Refractive index of 1.52 4. CLARITE − soluble in xylene − Refractive index of 1.544 TRANSCRIBED BY: Bucad, Shermella T. BSMT3B REFERENCES: Lecture Videos Psalm 63: 1-8