UND 362 Processing
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| name the four steps in processing | fixation, dehydration, clearing, infiltration
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| what will/can occur prior/post to processing | fixation (most important step), which one used or by what method depends on type, size and time. Post - a specialized techniques to remove material that can inhibit processing or staining
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| name 3 factors that can influence processing | agitation, heat, vacuum
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| How does agitation influence processing | increased surface area exposure (can be too slow or too fast) fast=breaking specs
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| how does heat affect processing | increases penetration (use caution because it can be flammable)
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| how does vacuum affect processing | (used during impregnation) it removes trapped air and reduces time for fatty tissue
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| What is the general principle behind dehydration in processing | removal of "unbound" water and fixative so wax can infiltrate)
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| when are the two times that dehydration occurs for tissue | during processing and staining
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| what are two problems that can be created during dehydration | over dehydration (removing "bound" water = hard and brittle tissue
under dehydration - incomplete removal of unbound water= soft and mushy blocks (more common than over dehydration)
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| what is the most common agent used in dehydration | ethanol (ethyl alcohol)
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| what are the they types of agents used in dehydration | alcohols, acetone, solvents
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| name the 4 alcohols used in dehydration | methanol, ethanol, isopropanol, butyl alcohol
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| what are 4 properties of alcohols as they relate to dehydration | clear colorless, flammable
hydrophilic
miscible w/h20 and organic solvents
second coagulent
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| give some common principles of ethanol pertaining to dehydration | most common,absolute (but gov. regulated) methalated alcohol not regulated, best to gradually increase % to reduce shrinkage, good for EM, drain disposal be less than 24%
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| If using phosphate formalin what % alcohol is best to use | 60-65%, if initial alcohol is 70%+ a precipitate will form causing cutting issues
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| what are three principles to methanol | rarely used alone, toxic/costly/volatile, substitute for ethanol, ABSORBED BY SKIN and liver breaks it into formaldehyde
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| give principles of isopropanol | ALWAYS contains 1% H20, substitute for ethanol, NOT used for staining (some are not soluble in isopropyl IE eosin), not for celloidin tech. (cellulose not dissolved), can be used in microwave
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| give 2 isopropanol facts pertaining to tissue | shrinks and hardens less than alcohol, good for dense/hard tissue, tissue can stay in isopropyl for a long time.
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| give 3 facts about butyl alcohol | UNIVERSAL solvent, for plant and animal tissue, less hardening than ethanol but takes a long time due to low dehydration rate
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| give 6 factors about acetone | UNIVERSAL solvent, miscible w/h20/ethanol/ organics, rapid dehydrant, absorbs h20 from air, can cause excessive shrinkage, removes lipids
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| give 1 adv. and 2 disadv to acetone | adv. works well with fatty tissue (use graded series of acetone and xylene) disadv. must use 20x volume to tissue size, extreme flammable
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| What are solvents not to be used with | delicate tissue
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| name three solvents for dehydration | dioxane, tertiary butanol, tetrahydrofuran
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| describe dioxane as a solvent | miscible with h20, xylene, paraffin
less shrinkage than ethanol
faster than ethanol but requires large volumes
CUMULATIVE toxicity
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| describe tertiary butanol as a solvent | miscible w/ alcohol/xylene/water
smelly, expensive
initial paraffin infiltration must be 1:1 tert. but. and paraffin
solidifies at room temp (25○ C)
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| describe tetrahydrofuran | miscible with MOST EVERYTHING
BEST universal solvent
rapid w/o excessive shrinkage
used for REprocessing
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| what is a universal solvent and name all four | does both the dehydrating and clearing steps dioxane, tertiary butanol, tetrahydrofuran, acetone
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| What does clearing do? | removal of dehyrating solutions IE alcohol (for paraffin entry) and makes tissue transparent
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| name a necessity for a clearant and two problems associated with them | must be miscible with dehydrant and infiltration medium
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| name the clearing agents 8, plus what else can be used | xylene, toluene, benzene, chloroform, acetone, essential oils, limonene, aliphatic hydrocarbons - also the univeral solvents (dioxane, tert. butanol, tetrahydrofuran)
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| what factors determine which clearant should be used | removed quickly, allow for complete infiltration, minimal damage to tissue, tissue type, processor used, safety factors, cost
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| give properties of xylene | most common, hardens w/o over exposure, displaces alcohol rapidly, not miscible with water, recylable, causes DEFATTING of skin!
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| give factors about limonene | citrus odor (clear and colorless),
clears slower than xylene but hardens less
will contaminate paraffin so paraf. will need to be changed
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| Alkane (aliphatic hydrocarbons) name 2 disadv. | hydro phobic (can't use with isopropanol since it has 1% h20), incompatible with mounting media
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| what are the 2 classes of alkanes | short and long (molecular chain length)
shorter chain is more effective, faster penetration, removes fat, allow cover slips to dry
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| how can you make xylene and alkanes work together | change more frequently
3 stations of clearant on processor, then 3 stations (3min each) to deparaffinize during staining, rotate alcohol carefully after eosin, keep anhydrous alcohol dry, do upside down coverslipping (ie FS line)
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| give 2 factors about toluene as a clearant | less hardening than xylene, tolerant of atmospheric water v xylene
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| name 3 adv and 3 disadv to benzene as clearant | adv. fast acting, evaporates rapidly from paraffin (less paraff. changes), doesn't overharden. Disadv. can overharden uterus, muscle, cn'at be used in open processors (evapo.), can only be used with abolute alcohol prior.
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| give 2 adv and 3 disadv to chloroform as clearant | less brittleness than xylene, good for connective tissue. Disadv. penetrates slower than xylene, NO TISSUE TRANPARENCY, absorbes moisture from air
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| give 1 adv/disadv to acetone as clearant (universal solvent) | low boiling point therefore replaced by paraffin at or greater than 58○C, GREATER shrinkage than xylene
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| give 4 factors about essential oils as clearants | plant derived (cedarwood most common), low evaporation rate, must be removed with a hydrocarbon before infiltration (ie xylene or toluene), used for special projects and tissue can stay in indefinitely
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| define infiltration | permeating tissue with support media (paraffin, waxes, celloidin, plastics)
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| what does (cable tax, im ted bat and C.G Space) releate to and what are each | clearing (cable tax)
infiltration (C.G. Space)
Dehydrants (I'm Ted Bat)
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| give the common props of paraffin | does large # of blocks, sections and staining easy, higher melting point = good support bad sectioning (lower melt pt opposite), temp 55-58○C, vacuum helps infil., baths must be monitored or rotated
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| carbowax has 4 particular properties name them | infiltrate directly from fixes (no dehydrate and clear), won't infiltrate large amts of fat, cen. ner. system takes extra time (3 chgs), softer than paraffin
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| what are 3 problems with carbowax | water soluble so the sections can disintegrate in regular h20 bath, all tissue must sink in carbowax prior to embedding, must be cooled in refridge and stored in bags w/ dessicant
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| what are the advantages to using celloidin | no heat required (therefore less hardening, shrinkage), maintains cellular relationships, works well with large specs
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| what are the disadv. to celloidin | takes long time, absorbs h20 readily, blocks must be stored in alcohol, requires ether for processing, sections are greater than 10 micrometers
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| name 2 plastics for infiltration | Glycol methacrylate (GMA), and epoxy
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| name some factors for using GMA | an acrylic that is miscible with h20, good for hard tisue and undecaldified bone (tissue usually dehydrated with 95% alcohol), microtomy difficult (must use glass knife), but can cut very thin sections (1-2 microns)
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| what are 2 drawbacks to using GMA | embedding medium is hard to remove, and it doesn't adher to slides well and will loosen in alcohol
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| give factors about epoxy infiltration | unless miscible with ethanel they require transitional fluid (eg clearing agents), GOOD for EM
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| what are 2 adv to using agar and gelatin | good for dbl medium technique (infiltration and embedding media are not the same), good for friable tissue embedding
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| what is the use for 30% sucrose in relation to infiltration | is a cryoprotectant (good for FS on formalin fixed, unprocessed tissue)
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| describe a closed processor and its adv | pumps reagent in and out of tissue
programmable, heat/agitation/vacuum in one, no spillage/fumes, alarm system
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| describe a microwave processor | closd system, shortens process, used on small specs, no graded reagents nor clearing reagents needed
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| if a processor has clogged lines what can be done to remedy it | caused by zinc/phosphate buffers being used with alcohol above 70% (to start), avoid by starting with 60-65% alcohol, keep ph below 7.0 (for zinc). clear lines with 5-20% acetic acid
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| what would cause poor processed tissue | water or fixative not removed, incomplete infiltration, clearing agent in paraffin, too much heat
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| what can be done to prevent or fix poor process tissues (in processor) | use anhydrous alcohol (keep water out of tissue), change solutions, use heat sparingly, make sure no contamination in processor
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| what would cause cell shrinkage | inadequate fixation, excessive dehydration, tissue drying out
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| what are some fixes for cell shrinkage (per processors) | complete fixation before processing, submerge completely
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| what are some reasons for incomplete fixation | fixation time not correct, inadequate fixative, thick sections, depleted fixative
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| what can be done to remedy incomplete fixation (per processors) | change solutions, more fixative time, thinner sections (3mm preferred)
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| what causes soft mushy tissue specs | thick specs.
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| what can be done to fix soft mushy tissue (steps) | shave tissue smaller, soak in xylene for 20-30 min. use tetrahydrofuran (universal) for 30-90min, xylene to clear, then use 3 changes of paraffin
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| what causes sponge artifact and how can it be prevented | when dry sponge is used on tissue, make sure sponge is presoaked in fixative
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| i'm Ted Bat |
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| C.G. Space |
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| Cable Tax |
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