Impregnation and Embedding Flashcards by Kyle Ancheta

process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will completely fill all the tissue cavities and give a firm consistency to the specimen

Impregnation (Infiltration)

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process by which the impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify

Embedding (Casting or Blocking)

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CHARACTERISTICS OF AN IDEAL INFILTRATING AND EMBEDDING MEDIUM

a. soluble in processing fluids
b. suitable for sectioning and ribboning
c. molten between 30°C and 60°C
d. translucent or transparent; colorless
e. stable
f. homogeneous
g. capable of flattening after ribboning
h. non-toxic, odorless, easy to handle, and inexpensive

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Four Types of Impregnation and Embedding Medium

Paraffin wax
Celloidin (collodion)
Gelatin
Plastic (Resin)

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simplest, most common and best embedding medium used for routine tissue processing

PARAFFIN WAX IMPREGNATION

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a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils

Paraffin wax

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Paraffin wax is solid at room temperature but melts at temperatures up to about _____ or ____

65°C or 70°C

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most common melting points for paraffin wax in histological use

56°C to 58°C
(56°C wax is normally used for routine work)

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Paraffin wax is use about ___ above its melting point

2°C above

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True or False: Tissue blocks and unstained mounted sections may be stored in paraffin for an indefinite period of time after impregnation without considerable tissue destruction

True

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True or False: Paraffin processing is recommended for fatty tissues

False (not recommended)

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Volume of paraffin wax to tissue

25x tissue volume

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duration and number of changes required for thorough impregnation of tissue depends on:

Size and type of tissues: longer time is required for thicker tissues

Use of vacuum imbedding: vacuum reduces the time required for complete impregnation.

Clearing agent employed

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True or False: hard tissues require wax with a lower melting point than soft tissues

False (higher melting point)

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Paraffin wax

lab temperature: ______; melting point: 54-58°C

lab temperature: 20-24°C; melting point: 54-58°C

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Paraffin wax

lab temperature: 20-24°C; melting point: _______

lab temperature: 20-24°C; melting point: 54-58°C

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Paraffin Wax

lab temperature: _______; melting point: 50-54°C

lab temperature: 15-18°C; melting point: 50-54°C

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Paraffin Wax

lab temperature: 15-18°C; melting point: _______

lab temperature: 15-18°C; melting point: 50-54°C

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True or False: larger and denser tissue blocks (e.g. bones, fibroids, brains) usually require longer periods but less frequent changes of wax

False (longer period and more frequent changes of wax)

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True or False: In paraffin wax, benzene and xylene are easily removed from the tissues while chloroform and cedarwood oil are more difficult to remove

True

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Three Methods of Paraffin Wax Impregnation and Embedding

Manual Processing
Automatic Processing
Vacuum Embedding

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In paraffin wax manual processing, at least ____ changes of wax are required at __ minutes intervals in order to insure complete removal of the clearing agent from the tissue

In paraffin wax manual processing, at least four changes of wax are required at 15 minutes intervals in order to insure complete removal of the clearing agent from the tissue

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Changes of wax required in paraffin wax automatic processing

2 to 3 changes

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True or False: presence of any odor in the clearing agent during final paraffin wax bath indicates that the paraffin wax is complete

False (paraffin wax needs to be changed)

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Most critical stage of tissue processing in automatic processor

Dehydration (that's why dehydrating fluids should be changed frequently and inadequate dehydration is difficult to correct once the tissue is in paraffin)

In automatic processing of paraffin wax, clearing agent and the dilute ethanol should be changed at least __________

once a week

In automatic processing of paraffin wax, wax bath thermostats should be set at least ________ above the melting point of the wax

at least 3 degrees above

Best embedding technique

Vacuum Embedding

A method of paraffin wax which involves wax impregnation under negative atmospheric pressure inside an embedding oven

Vacuum Embedding

Method of paraffin wax embedding which reduces the time when tissues are subjected to high temperatures thus minimizing heat-induced tissue hardening

Vacuum Embedding

A method that facilitates complete removal of transition solvents, and prolongs the life of wax by reducing solvent contamination

Vacuum Embedding

A method recommended for urgent biopsies, for delicate tissues such as lung, brain, connective tissues, decalcified bones, eyes, spleen and central nervous system

Vacuum Embedding

Advantage of this method is the time required for complete impregnation is reduced by 25% -75% of the normal time required for tissue processing

Vacuum Embedding

tissue should not be left in the paraffin oven for more than ______

4 hours

Paraffin oven must be maintained at a temperature _ to _°C above the melting point of paraffin

2 to 5°C

fresh wax should be filtered before use in a wax oven at a temperature _°C higher than its melting point

2°C

paraffin wax may be used only ______, after which, fresh wax must be utilized

twice

True or False: - fixed knife microtomes require relatively hard wax with a higher melting point - heavier microtome knives require harder paraffin wax than lighter ones

Both True

What are the substitutes for paraffin wax?

Paraplast Embeddol Ester Wax Water Soluble Waxes (Carbowax) Dimethyl Sulphoxide

A substitute of paraffin wax which is a mixture of highly purified paraffin and synthetic plastic polymers. It is also more elastic and resilient than paraffin wax thereby permitting large dense tissue blocks such as bones and brain to be cut easily with the same result as in double embedding.

Paraplast

Advantage of this substitute of paraffin wax includes: - blocks obtained are more uniform with better ribboning of sections - prevent formation of ice crystal artefacts - no deposit is left on the slide after staining, and no special processing schedule is required

Paraplast

recommended melting point of paraplast

56 to 58°C

synthetic wax substitute similar to Paraplast

Embeddol

melting point for embeddol

56 to 58°C (similar to paraplast)

A substitute of paraffin wax that is less brittle and less compressible than Paraplast

Embeddol

semisynthetic wax recommended for embedding eyes

Bio/Aid

product of paraffin, containing rubber, with the same property as Paraplast

Tissue Mat

2 types of embeddol

Bio/Aid and Tissue Mat

melting point for ester wax

46-48°C

A substitute of paraffin wax that is harder than paraffin

Ester Wax

A substitute of paraffin wax that is - not soluble in water but soluble in 95% ethyl Alcohol and other clearing agents

Ester Wax (hence, it can be used for impregnation without prior clearing of the tissue)

other name for cellosolve

ethylene glycol monoethyl ether

Clearing agents use in ester wax

cellosolve (ethylene glycol monoethyl ether) or xylene

Require changes of wax for ester wax to ensure complete tissue impregnation

3 to 4 changes of wax

Microtome use for ester wax-impregnated tissues due to its hardness

heavy-duty microtome (sliding or sledge type microtome)

Example of water soluble waxes

Carbowax

melting point for water soluble waxes

38-42°C or 45-56°C

most commonly used water soluble waxes

Carbowax

A polyethylene glycol containing 18 or more carbon atoms, which appears solid at room temperature

Carbowax

Carbowax does not require ____________ and _________ of the tissue

dehydration and clearing

True or False: Carbowax removes neutral fats and lipids which are soluble in reagents used

does not remove neutral fats and lipids

Changes of wax required for carbowax

four changes (one each in 70% and 90% and 2 times in 100% concentration)

True or False: Carbowax are hygroscopic (very easily dissolved in water)

True

It is added to proprietary blends of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning

Dimethyl sulphoxide (DMSO)

It scavenges residual transition solvent and probably alters tissue permeability by substituting for or removing bound water thus improving infiltration

Dimethyl sulphoxide (DMSO)

purified form of nitrocellulose soluble in many solvents, suitable for specimens with large hollow cavities which tend to collapse, for hard and dense tissues such as bones and teeth and for large tissue sections of the whole embryo

Celloidin (Collodion)

True or False: Celloidin requires heat

False (no heat is required, resultant block has a rubbery consistency which gives good support to the tissues)

A type of impregnation recommended for processing of neurological tissues

Celloidin

The _________________used in the dry celloidin technique helps to soften the brittle layers.

cedarwood oil

A method of impregnation recommended in cases when minimum shrinkage is required and when frozen section technique cannot be done

Celloidin

Methods for Celloidin Impregnation

Wet Celloidin and Dry Celloidin

Uses of wet celloidin

recommended for bones, teeth, large brain sections and whole organs

Use of dry celloidin

preferred for processing of whole eye sections

It is a mixture made up of equal parts of chloroform and cedarwood oil; added to the celloidin block before hardening to make the tissue transparent

Gilson's mixture

Gilson's mixture is made up of equal parts of ______________ and _________________

chloroform and cedarwood oil

True or False: dry celloidin does not use alcohol due to the presence of cedarwood oil in the block

True

another form of celloidin soluble in equal concentration of ether and alcohol, with a lower viscosity, allowing it to be used in higher concentrations and still penetrate tissues rapidly

Low Viscosity Nitrocellulose (L.V.N.)

Act to prevents tendency of tissues to crack when embedding chrome-mordanted tissues

Plasticizers

Examples of plasticizers

oleum ricini and castor oil

Impregnation method rarely used except when dehydration is to be avoided and when tissues are to be subjected to histochemical and enzyme studies

GELATIN IMPREGNATION

used as an embedding medium for delicate specimens and frozen tissue sections because it prevents fragmentation of tough and friable tissues when frozen sections are cut

Gelatin Impregnation

True or False: Gelatin are water-soluble and does not require decalcification and clearing

False (dehydration and clearing)

In gelatin impregnation, tissues should not be more than___ mm thick since gelatin-embedded specimens are harder to freeze than non-impregnated tissues

2-3

Addition of ___________ serves to prevent the growth of molds in gelatin impregnation

1% phenol

Process by which a tissue is arranged in precise positions in the mold during embedding, on the microtome before cutting, and on the slide before staining

Orientation

Types of Blocking-Out Molds

- Leuckhart’s Embedding Mold - Compound Embedding Unit - Plastic Embedding Rings and Base Mold - Disposable Embedding Molds (peel-away, plastic ice trays, and paper boats)

consists of two L-shaped strips of heavy brass or metal arranged on a flat metal plate and which can be moved to adjust the size of the mold to the size of the specimen

Leuckhart’s Embedding Mold

blocks produced are even, with parallel sides, and with a fairly shaped initial setting of the wax

Leuckhart’s Embedding Mold

A type of mold recommended for routine use

Leuckhart’s Embedding Mold (but too slow and cumbersome for use in a busy laboratory)

made up of a series of interlocking plates resting on a flat metal base, forming several compartments

Compound Embedding Unit

It has an advantage of embedding more specimens at a time reducing the time needed for blocking

Compound Embedding Unit

consist of a special stainless steel base mold fitted with a plastic embedding ring, which later serves as the block holder during cutting

Plastic Embedding Rings and Base Mold

equipped with a warm plate to manage the impregnated specimen, and a cold plate at -5°C for rapid solidification of the block

Tissue Tek

It is use to smear the inner mold of plastic ice trays before embedding

glycerin or liquid paraffin

normally utilized for embedding celloidin blocks but are equally useful for paraffin wax blocks

Paper Boats

A mold that has an advantage of being cheap and easy to make. It is also rapid embedding of small or large volume of individual specimen is possible.

Paper Boats

recommended for embedding hard tissues such as bones and teeth, and for large sections of whole organs like the eye, since the delicate layers of the eyeball are difficult to keep intact when other media are used

Celloidin or Nitrocellulose Embedding Method

process by which tissues are first embedded or fully infiltrated with a supporting medium such as agar or nitrocellulose, then infiltrated a second time with paraffin wax in which they are subsequently embedded

Double-Embedding

Medium use in double-embedding

agar or nitrocellulose

Process recommended for making small sections of celloidin blocks

Double-Embedding

Double-Embedding is use in ____________________

Electron microscopy

provide superior results for light microscopic studies, particularly in hard tissues such as undecalcified bone and for high resolution light microscopy of tissue sections thinner than the usual 4-6 µm, such as renal biopsies and bone marrow biopsy

PLASTIC (RESIN) EMBEDDING

Plastics Classification Based on Chemical Composition

Epoxy, polyester, and acrylic

Component of epoxy known to be carcinogenic

vinyl cyclohexane dioxide (VCD)

Three Types of Epoxy

Araldite Epon Spurr

A type of epoxy based on: Bisphenol A: Cyclohexane Dioxide: Glycerol:

A type of epoxy based on: Bisphenol A: Araldite Cyclohexane Dioxide: Spurr Glycerol: Epon

Originally introduced for electron microscopy in the mid- 1950s, but have been superseded by more superior epoxides, and are now seldom used

Polyester Plastics

Plastic used extensively for light microscopy

Acrylic Plastics

Plastic preferable to use when high resolution light microscopy is required

Acrylic plastics

Added to the plastic as a catalyst that decomposes to form phenyl radicals acting as an active site for the polymerization of acrylics

Benzoyl Peroxide

Two types of acrylic plastics

Polyglycol Methacrylate (GMA) Methyl Methacrylate (MMA)

A type of acrylic plastics that is hydrophilic media

Polyglycol Methacrylate (GMA)

A type of acrylic plastics that is hydrophobic media

Methyl Methacrylate (MMA)

Proved to be a popular embedding medium for light microscopy because it is extremely hydrophilic, allowing many staining methods to be applied

Polyglycol Methacrylate (GMA)

Widely used because of its hardness as the ideal embedding medium for undecalcified bone and is widely used for bone histomorphometry and bone marrow hematopathology

Methyl Methacrylate (MMA)

Impregnation and Embedding Flashcards

(115 cards)