Processing Flashcards
Dilution Dehydration
Specimens are transferred through increasing concentrations of hydrophilic or water miscible fluids which dilute and eventually replace free water in the tissues
Chemical Dehydration
Where the dehydrant, acidified dimethoxypropane or diethoxypropane, is hydrolyzed by free water present in tissues to form acetone and methanol 43-50 in an endothermic reaction
Steps in Processing
Dehydration, clearing, infiltration/embedding
Dehydrating Agents
Ethanol, Mmthanol, isopropanol, butanols, glycol ethers, acetone, tetrahydrofuran, dioxane
Alcohols
Clear, colorless, flammable, hydrophilic liquids, miscible with water and most organic solvents; alcohols also act as secondary coagulant fixatives during tissue processing
Ethanol
Most common used dehydrant
Poor lipid solvent except under microwave processing
Dissolves nitrocellulose slowly
Prolonged time in absolute causes hardening
Methanol
Good substitute to ethanol
Not used regularly due to volatility, flammability and cost
Poor lipid solvent
Only dissolve nitrocellulose when mixed with acetone
Isopropanol
Universal solvent
Good lipid solvent
Shrinks and hardens tissue less than ethanol
Used for hard, dense tissue
Used as transitional solvent after ethanol
Butanols
Universal solvent
Used for small-scale processing of plants
Normal butone used for lightly chitinized arthropods and rodent tissues
Glycol-ethers
Alcohol substitutes
Do not act as secondary fixatives, do not appear to alter tissue reactivity
Cellosolve (ethylene glycol monoethyl ether)
Used for:
polyester wax embedding
following dioxanefixation of hard tissues
in agar ester wax double embedding technique
Dissolves nitrocellulose
Decomposes in sunlight
Doesn’t harden or shrink
Dioxane
Universal solvent
Causes less tissue shrinkage & hardening than ethanol
Excellent for tissues hardened by ethanol-xylene processing
Dissolves mercuric chloride, but precipitates potassium dichromate and other salts
Polyethylene glycols
Used to dehydrate and embed substances that can change in the solvents and heat of the paraffin wax method
Dissolve nitrocellulose
Start with low molecular weight liquid glycols, pass through glycols of increasing MW and viscosity, and embedded in a high MW PEG
Acetone
Fast, effective ,may cause tissue shrinkage
It’s also a coagulant secondary fixative
Best for processing fatty specimens
Transitional solvent needed for paraffin baths
Tetrahydrofuran
Universal solvent
Dehydrates rapidly, causing little hardening or shrinkage
Universal Solvent
Can perform both dehydration and clearing
Miscible with water, organic solvents, paraffin wax
Universal solvent
Can perform both dehydration and clearing
Miscible with water, organic solvents, paraffin wax
Clearing
Transition step between dehydration and infiltration
Shrinkage may result from extraction of fat by the transition solvent
Aromatic hydrocarbons
Xylene, toluene, benzene
Clear rapidly and makes tissues transparent
Hardens tissues fixed in non-protein coagulant fixatives
Coagulate nitrocellulose
Benzene most gentle but a carcinogen
Chlorinated hydrocarbons
Chloroform, carbon tetrachloride
Good lipid solvents
Don’t dissolve nitrocellulose or make tissues transparent
Clear slower but harden less then xylene
Chloroform better for uterus, muscle, tendon
Desiccates connective tissue
Chlorinated hydrocarbons
Chloroform, carbon tetrachloride
Good lipid solvents
Don’t dissolve nitrocellulose or make tissues transparent
Limonene
Xylene substitute Overpowering citrus odor Not water soluble, cannot be disposed of in the drain Harden tissue less than xylene Cause more paraffin contamination
Aliphatic hydrocarbons
Low in reactivity and toxicity
Penetrate tissue rapidly, remove fat more effectively, allow coverslips to dry normally
Intolerant to water, incompatible with some mounting media
Essential Oils
Oils of bergamot, cedarwood, clove, lemon, origanum and sandalwood
Slow gentle non-hardening action
Need to be cleared with xylene before infiltration
Infiltrating and embedding medium should be:
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 Nnon-toxic Odorless Easy to handle
Double Embedding
Process by which tissues are first embedded with supporting medium, gelatin or agar, then embedded a second time with paraffin
Double Embedding
Process by which tissues are first embedded or fully infiltrated with a supporting medium such as agar or nitrocellulose, then infiltrated/embedded a second time with wax
Investment
Practice of embedding wax infiltrated tissues in another wax, modified to provide improved tissue support and sectioning qualities.
Paraffin Wax
Polycrystalline mixture of solid hydrocarbons
Two thirds the density and slightly more elastic than dried protein
Melting points which range from 39°C to 68°C
Processed in short time
Serial sections easily obtained
Most staining done easily
Paraffin Wax
Polycrystalline mixture of solid hydrocarbons
Two thirds the density and slightly more elastic than dried protein
Modifying Paraffin: improve ribboning
Prolong heating of paraffin wax at high temperatures or use micro-crystalline wax
Modifying Paraffin: increase hardness
Add stearic acid
Modifying Paraffin: decrease melting point
Add spermaceti or phenanthrene
Celloidin
Slow process, takes weeks
Doesn’t produce sections as thin as paraffin embedding
Section cutting is done wet
Sections stored in ethanol
Staining done on free floating sections
Processing done without heat, avoid heat produced artifacts
Immunophenotyping of lymphoid and non-lymphoid cells
Celloidin
Slow process, takes weeks
Doesn’t produce sections as thin as paraffin embedding
Section cutting is done wet
Sections stored in ethanol
Staining done on free floating sections
Processing done without heat, avoid heat produced artifacts
Gelatin/agar
Produce single block of friable or multiple tissue fragments
First step in double embedding
Used for frozen sections
30% Sucrose
Best for frozen sections of formalin-fixed unprocessed tissue
Sucrose added before tissue is frozen
Water soluble waxes
Dehydration and clearing not needed Sections cannot be placed in water bath Lipids are not removed, requires long periods of infiltration Some enzymes will remain active Tissue blocks must be chilled in fridge
Glycol Methacrylate
Best for undecalcified bone Infiltration done after dehydration Cut with glass knives Can be used with some enzyme stains Do not adhere to glass slides well
Epoxy resins
Best for electron microscopy
Very thin sections obtained
Diamond knife: 60-90 nm thick
Glass knife: 0.5 microns thick
Processing Temp
Low temp:
-structural elements of tissue stabilized
-prolonged processing times due to high viscosity and low diffusion
High temp:
-cause tissue shrinkage and hardening
-may produce artifacts
Mild heat, 37°C to 45°C, during dehydration and clearing reduces processing times, but may increase shrinkage
Processing Temp
Low temp:
-structural elements of tissue stabilized
-prolonged processing times due to high viscosity and low diffusion
High temp:
Mild heat, 37°C to 45°C, during dehydration and clearing reduces processing times, but may increase shrinkage
Processing Pressure
Vacuum applied during dehydration, clearing and infiltration improves quality of processing
