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HTL processing

define dehydration removal of water to allow embedding in nonaqueous media
define clearing removal of dehydration agents to make tissue receptive to infiltration agents, renders tissue translucent due to high refractive index
define infiltration permeating (flowing throughout) tissue. Technically all solutions in processing infiltrate tissue. The infiltration step refers specifically to the infiltration of paraffin or other support media
define embedding enclosing tissue in the infiltration medium and allowing it to harden
define universal solvent A solution that performs the actions of two agents (e.g. clearing and dehydrating)
define carbowax a water soluble wax, solid polyethylene glycol
define cross-section cutting a sample such that all layers of tissue can be seen in a single face.
define decalcification removal of calcium from tissues to facilitate routine paraffin processing and microtomy
define ion exchange calcium ions are extracted by standard decal solution, and exchanged in the solution by a gel that is included in the container. Reduced the need to change decal solutions due to calcium saturation, and speeds the process.
define chelating agent organic compounds capable of binding certain metals.
define miscible capable of mixing or being mixed. Water and alcohol are miscible, water and xylene are not.
special precautions properties and actions of alcohols (ethyl methyl butyl isopropyl) Ethyl: hydrophilic, flammable-- Methyl: toxic (liver turns it to formaldehyde) rarely used alone, primarily for blood smears/touch prep-- butyl: good for plants, takes longer, less shrinking/hardening-- Isopropyl: can't be used with celloidin/eosin
special precautions properties and actions of xylene neurotoxic, defatting, miscible with alcohol and paraffin, does not mix with even small quantities of water; becomes cloudy. overhardens tissue, especially fibrous, muscular, and CNS tissue
special precautions properties and actions of toluene less hardening than xylene, more tolerant to atmospheric water contamination
special precautions properties and actions of benzene very fast acting, very volatile and toxic (carcinogen, effects blood/marrow)
special precautions properties and actions of acetone very fast acting dehydrating agent. also fixes tissue, used for rabies diagnosis and cell surface antigen IHC, acid/alkaline phoshatase enzyme demonstration. Can also boil off to be replaced by paraffin as pseudo-clearing agent
special precautions properties and actions of chloroform penetraes very slowly, does not render tissue transparent, carcinogenic, very volitile
special precautions properties and actions of cedarwood oil expensive and only used in special occasions. does not shrink or harden tissue, can clear following 95% alcohol. can store tissue indefinitely without harm
special precautions properties and actions of limonene derivatives less hardening than xylene, more contamination of paraffin less toxic than other clearing agents.
special precautions properties and actions of aliphatic hydrocarbons penetrate quickly, remove fat effectively. Not compatible with some mounting media, difficult to use in high humidity
why is there reason for caution using alcohol to immediately follow fixation in phosphate buffered formaldehyde? phosphates in the fixative will precipitate in the tissue if alcohol is greater than 70%, causing difficulty in microtomy. It is recommended to start dehydration with one station of 50-60% alcohol for this reason
what should be used following cedarwood oil clearing? why? an aromatic hydrocarbon should be used to ensure complete removal of oil from tissue, or sectioning will be difficult
list 3 universal solvents Dioxane -- tertiary butanol -- tetrahydrofuran
what happens to H&E if water is in the clearing agent during processing staining will be blotchy and uneven with poor chromatin patterns
compare carbowax to paraffin carbowax is water soluble, and can infiltrate directly from aqueous fixative. does not require dehydration or clearing. does not remove fat, preserves some enzyme activity, softer tissue
compare celloidin to paraffin tedious, processing and staining. only used rarely, especially for CNS samples. Thin sections are difficult.
compare glycol methacrylate to paraffin GMA is water miscible, provides support for exremely hard tissue like calcified bone, glass (Ralph) knives 1-2┬Ám sections. media is not usually removed from sections, making staining difficult
compare epoxy resin to paraffin tisue must be dehydrated, and cleared with a "transitional fluid" most commonly propylene oxide. Allow EM sections of 60-90nm with diamond knives. common epoxies: araldite, epon, spurr
compare agar and gelatin to paraffin can be used for friable tissue. Tissue can be embedded in gelatin, fixed in 5% formalin, and routinely processed for double embedded in paraffin/
tissues should be processed by ________ for electron microscopy Epoxy resins. Dehydration, clearing with propylene oxide, infiltrated with resin, and hardened by polymerization
tissues should be processed by ________ for enzyme histochemistry frozen, maybe GMA,
tissues should be processed by ________ for undecalcefied bone plastic (glycol methacrylate)
tissues should be processed by ________ for fat demonstration frozen, osmium post fixation, or carbowax
how does paraffin melting point influence section thickness, ribboning, and hardness? higher melting point (harder, more support, thinner sections, poor ribboning) Lower melting point (softer, less support, thicker sections, better ribbons)
what problem arises from overheated infiltration paraffin tissue will become hardened, wax should be kept 2-4 degrees over melting point
what problem arises from overheated embedding paraffin section quality may be affected but tissue is not exposed for prolonged periods, so will not be meaningfully affected
what problem arises from inadequate dehydration clearing and infiltrating reagents will not penetrate tissue with water present, leaving tissue soft and impossible to section properly
what problem arises from excessive dehydration Bound water is removed from tissue, resulting in hard, brittle, tough to section tissue: microchatter can be solved by soaking in water
what problem arises from excessive or inadequate clearing over or under clearing will result in similar issues to over/under dehydration, as tissue will either be over hardened, or non-receptive to infiltrating reagent
identify differences in microwave vs closed processing microvave processing heats all reagents, does not utilize vacuum or pressure, and is much much faster
what are 2 ways to decrease processing time to paraffin microwave process. use smaller pieces of tissue
give two QC procedures for paraffin processing ensure adequate reagent levels are maintained, and ensure reagents are changed regularly
How does one validate a new processing program tissue of the same type, size, and fixation should be run on old and new programs, and sectioning, firmness, and staining quality should be compared directly. The new program should yeild as good or better results
what effect does temperature have on paraffin while it cools into a bock? why is this important the faster it freezes, the smaller the crystals, yielding better support for tissue, and smoother sectioning
what are some ways that grossing can aid in proper embedding orientation? marking with ink or notching the side to face "up" at the grossing station will help ensure proper orientation
describe simple acid decal calcium is soluble at H ~4.5, so adding an acid (5-10% nitric, formic, or hydrochloric acids) will solubilize Ca ions, that then diffuse out of tissue.
describe ion echange resin decal a layer of resin covered by formic acid is used. The resin absorbs free calcium ions from tissue, replacing them with other ions.
describe electrolytic decal mix formic and hydrochloric acid with cathode (negative) and anode (positive). Tissue is attached to anode, and positive calcium ions are rapidly pulled toward cathode. decal can be complete in 4-6hr, but may severely damage tissue.
describe chelation decal EDTA ca bind to calcium. It is very slow, acting on the outer layers of the hydroxyapatite crystal.
how does one determine decal endpoint? mechanical: bend or scrape tissue to determine pliability: not ideal chemical: mix decal fluid with ammonium hydroxide and ammonium oxalate to precipitate calcium. Replace solution following each positive reaction. radiography: x-ray the sample
how can one decrease decal time? use diamond saws to trim very thin sections of bone, include ion exchange media, or use electrolytic decal.
describe consequences of over/under decal under decal= calcium deposits remain that are unsectionable in paraffin Over decal= reduced staining, especially in nuclear basophilia (may depend on type of acid decal used)
give effects of decal on fixed/unfixed tissue if unfixed tissue is decaled, morphology of tissue may be affected/altered. Decal can also be combined with fixatives
what can be done when calcification is found in the finished block during microtomy face the block, and place in decal fluid to remove surface calcium deposits. Use the first section possible as decal wil penetrate only a short way into block.
why would one look at undecalcefied bone if diagnosis of metabolic bone disease is required
identify 3 methods for freezing tissue for cryostat sections cryostat freezing -- allows large ice crystals to form; worst in muscle enzyme studies isopentane/liquid nitrogen liquid chlorofluorocarbons dry ice alone or with an acetone slurry
how should one freeze tissue for muscle enzyme studies isopentane and liquid nitrogen
what are some problems in frozen sections if unfixed sections are used, they can be biohazardous freeze artifact if sections are thawed and refrozen, or samples are frozen slowly,
give a problem associated with eosin as a colorant in dehydration increased tissue autoflorescence, giving difficulty with FISH procedures
what is paraffin a mixture of hydrocarbons produced by cracking petroleum.
what are the additives for embedding paraffin beeswax (reduces crystal size, increases stickyness) rubber (promotes ribboning, reduces brittleness. increases stickyness) other waxes (produce smooth texture and smaller crystals) plastics (increase hardness and support)
list the water soluble embedding media Agar, Gelatin, carbowax, OCT
Why is celloidin preferable for delicate tissues and structures/ It does not require heating at any step, so there is minimal shrinkage and distortion
Created by: joshuajohnson7



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