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UND 362 Processing

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Question
Answer
name the four steps in processing   fixation, dehydration, clearing, infiltration  
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  
name 3 factors that can influence processing   agitation, heat, vacuum  
How does agitation influence processing   increased surface area exposure (can be too slow or too fast) fast=breaking specs  
how does heat affect processing   increases penetration (use caution because it can be flammable)  
how does vacuum affect processing   (used during impregnation) it removes trapped air and reduces time for fatty tissue  
What is the general principle behind dehydration in processing   removal of "unbound" water and fixative so wax can infiltrate)  
when are the two times that dehydration occurs for tissue   during processing and staining  
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)  
what is the most common agent used in dehydration   ethanol (ethyl alcohol)  
what are the they types of agents used in dehydration   alcohols, acetone, solvents  
name the 4 alcohols used in dehydration   methanol, ethanol, isopropanol, butyl alcohol  
what are 4 properties of alcohols as they relate to dehydration   clear colorless, flammable hydrophilic miscible w/h20 and organic solvents second coagulent  
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%  
If using phosphate formalin what % alcohol is best to use   60-65%, if initial alcohol is 70%+ a precipitate will form causing cutting issues  
what are three principles to methanol   rarely used alone, toxic/costly/volatile, substitute for ethanol, ABSORBED BY SKIN and liver breaks it into formaldehyde  
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  
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.  
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  
give 6 factors about acetone   UNIVERSAL solvent, miscible w/h20/ethanol/ organics, rapid dehydrant, absorbs h20 from air, can cause excessive shrinkage, removes lipids  
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  
What are solvents not to be used with   delicate tissue  
name three solvents for dehydration   dioxane, tertiary butanol, tetrahydrofuran  
describe dioxane as a solvent   miscible with h20, xylene, paraffin less shrinkage than ethanol faster than ethanol but requires large volumes CUMULATIVE toxicity  
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)  
describe tetrahydrofuran   miscible with MOST EVERYTHING BEST universal solvent rapid w/o excessive shrinkage used for REprocessing  
what is a universal solvent and name all four   does both the dehydrating and clearing steps dioxane, tertiary butanol, tetrahydrofuran, acetone  
What does clearing do?   removal of dehyrating solutions IE alcohol (for paraffin entry) and makes tissue transparent  
name a necessity for a clearant and two problems associated with them   must be miscible with dehydrant and infiltration medium  
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)  
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  
give properties of xylene   most common, hardens w/o over exposure, displaces alcohol rapidly, not miscible with water, recylable, causes DEFATTING of skin!  
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  
Alkane (aliphatic hydrocarbons) name 2 disadv.   hydro phobic (can't use with isopropanol since it has 1% h20), incompatible with mounting media  
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  
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)  
give 2 factors about toluene as a clearant   less hardening than xylene, tolerant of atmospheric water v xylene  
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.  
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  
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  
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  
define infiltration   permeating tissue with support media (paraffin, waxes, celloidin, plastics)  
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)  
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  
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  
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  
what are the advantages to using celloidin   no heat required (therefore less hardening, shrinkage), maintains cellular relationships, works well with large specs  
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  
name 2 plastics for infiltration   Glycol methacrylate (GMA), and epoxy  
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)  
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  
give factors about epoxy infiltration   unless miscible with ethanel they require transitional fluid (eg clearing agents), GOOD for EM  
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  
what is the use for 30% sucrose in relation to infiltration   is a cryoprotectant (good for FS on formalin fixed, unprocessed tissue)  
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  
describe a microwave processor   closd system, shortens process, used on small specs, no graded reagents nor clearing reagents needed  
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  
what would cause poor processed tissue   water or fixative not removed, incomplete infiltration, clearing agent in paraffin, too much heat  
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  
what would cause cell shrinkage   inadequate fixation, excessive dehydration, tissue drying out  
what are some fixes for cell shrinkage (per processors)   complete fixation before processing, submerge completely  
what are some reasons for incomplete fixation   fixation time not correct, inadequate fixative, thick sections, depleted fixative  
what can be done to remedy incomplete fixation (per processors)   change solutions, more fixative time, thinner sections (3mm preferred)  
what causes soft mushy tissue specs   thick specs.  
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  
what causes sponge artifact and how can it be prevented   when dry sponge is used on tissue, make sure sponge is presoaked in fixative  
i'm Ted Bat    
C.G. Space    
Cable Tax    


   





 
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