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HTL Test > Processing > Flashcards

Processing Flashcards

1
Q

Dilution Dehydration

A

Specimens are transferred through increasing concentrations of hydrophilic or water miscible fluids which dilute and eventually replace free water in the tissues

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2
Q

Chemical Dehydration

A

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

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3
Q

Steps in Processing

A

Dehydration, clearing, infiltration/embedding

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4
Q

Dehydrating Agents

A

Ethanol, Mmthanol, isopropanol, butanols, glycol ethers, acetone, tetrahydrofuran, dioxane

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5
Q

Alcohols

A

Clear, colorless, flammable, hydrophilic liquids, miscible with water and most organic solvents; alcohols also act as secondary coagulant fixatives during tissue processing

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6
Q

Ethanol

A

Most common used dehydrant
Poor lipid solvent except under microwave processing
Dissolves nitrocellulose slowly
Prolonged time in absolute causes hardening

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7
Q

Methanol

A

Good substitute to ethanol
Not used regularly due to volatility, flammability and cost
Poor lipid solvent
Only dissolve nitrocellulose when mixed with acetone

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8
Q

Isopropanol

A

Universal solvent
Good lipid solvent
Shrinks and hardens tissue less than ethanol
Used for hard, dense tissue
Used as transitional solvent after ethanol

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9
Q

Butanols

A

Universal solvent
Used for small-scale processing of plants
Normal butone used for lightly chitinized arthropods and rodent tissues

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10
Q

Glycol-ethers

A

Alcohol substitutes

Do not act as secondary fixatives, do not appear to alter tissue reactivity

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11
Q

Cellosolve (ethylene glycol monoethyl ether)

A

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

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12
Q

Dioxane

A

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

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13
Q

Polyethylene glycols

A

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

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14
Q

Acetone

A

Fast, effective ,may cause tissue shrinkage
It’s also a coagulant secondary fixative
Best for processing fatty specimens
Transitional solvent needed for paraffin baths

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15
Q

Tetrahydrofuran

A

Universal solvent

Dehydrates rapidly, causing little hardening or shrinkage

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16
Q

Universal Solvent

A

Can perform both dehydration and clearing

Miscible with water, organic solvents, paraffin wax

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17
Q

Universal solvent

A

Can perform both dehydration and clearing

Miscible with water, organic solvents, paraffin wax

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18
Q

Clearing

A

Transition step between dehydration and infiltration

Shrinkage may result from extraction of fat by the transition solvent

19
Q

Aromatic hydrocarbons

A

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

20
Q

Chlorinated hydrocarbons

A

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

21
Q

Chlorinated hydrocarbons

A

Chloroform, carbon tetrachloride
Good lipid solvents
Don’t dissolve nitrocellulose or make tissues transparent

22
Q

Limonene

A 
Xylene substitute
Overpowering citrus odor
Not water soluble, cannot be disposed of in the drain
Harden tissue less than xylene
Cause more paraffin contamination
23
Q

Aliphatic hydrocarbons

A

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

24
Q

Essential Oils

A

Oils of bergamot, cedarwood, clove, lemon, origanum and sandalwood
Slow gentle non-hardening action
Need to be cleared with xylene before infiltration

25
Q

Infiltrating and embedding medium should be:

A 
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
26
Q

Double Embedding

A

Process by which tissues are first embedded with supporting medium, gelatin or agar, then embedded a second time with paraffin

27
Q

Double Embedding

A

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

28
Q

Investment

A

Practice of embedding wax infiltrated tissues in another wax, modified to provide improved tissue support and sectioning qualities.

29
Q

Paraffin Wax

A

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

30
Q

Paraffin Wax

A

Polycrystalline mixture of solid hydrocarbons

Two thirds the density and slightly more elastic than dried protein

31
Q

Modifying Paraffin: improve ribboning

A

Prolong heating of paraffin wax at high temperatures or use micro-crystalline wax

32
Q

Modifying Paraffin: increase hardness

A

Add stearic acid

33
Q

Modifying Paraffin: decrease melting point

A

Add spermaceti or phenanthrene

34
Q

Celloidin

A

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

35
Q

Celloidin

A

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

36
Q

Gelatin/agar

A

Produce single block of friable or multiple tissue fragments
First step in double embedding
Used for frozen sections

37
Q

30% Sucrose

A

Best for frozen sections of formalin-fixed unprocessed tissue
Sucrose added before tissue is frozen

38
Q

Water soluble waxes

A 
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
39
Q

Glycol Methacrylate

A 
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
40
Q

Epoxy resins

A

Best for electron microscopy
Very thin sections obtained
Diamond knife: 60-90 nm thick
Glass knife: 0.5 microns thick

41
Q

Processing Temp

A

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

42
Q

Processing Temp

A

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

43
Q

Processing Pressure

A

Vacuum applied during dehydration, clearing and infiltration improves quality of processing

HTL Test (2 decks)

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