HTMLE TFST #1: Impregnation, Embedding, Trimming, and Sectioning

Impregnation

"Infiltration"

Embedding

"Casting" or "Blocking"

Impregnation

▪︎ 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.

▪︎ Allows easier handling and cutting of suitably thin sections without any damage or distortion to the tissue and its cellular components.

Embedding

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

Soluble in processing fluids

Suitable for sectioning and ribboning

Molten between 30℃ and 60℃

Translucent or transparent; colorless

Stable

Homogenous

Capable of flattening after ribboning

Non-toxic

Odorless

Easy to handle

Inexpensive

Qualities of ideal infiltrating and embedding medium.

Paraffin wax

Celloidin

Gelatin

Plastic

Four types of impregnation and embedding medium.

Paraffin wax

▪︎ Simplest, most common and best embedding medium used for routine tissue processing.

▪︎ Polycrystalline mixture of solid hydrocarbons produced during refining of coal and mineral oils

▪︎ Solid at room temperature but melts at temperatures up to about 65℃ or 70℃

56℃ to 58℃

Most common melting point of paraffin wax for histological use.

Paraffin Wax

Should be used about 2℃ above its melting point.

Longer

_______ time is required for impregnation and embedding of thicker tissues.

Vacuum

This reduces the time required for complete impregnation.

Size and type of tissues

Use of vacuum embedding

Clearing agent employed

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

Manual processing

Automatic processing

Vacuum embedding

Three ways by which paraffin wax impregnation and embedding of tissues may be performed:

Manual Processing

▪︎ Atleast 4 changes of wax are required at 15 minutes intervals in order to ensure complete removal of the clearing agent from the tissue.

▪︎ Specimen is then immersed in another fresh solution of melted paraffin for approx. 3 hours to insure complete embedding or casting of tissue.

Automatic processing

▪︎ Makes use of automatic tissue processing machine which fixes, dehydrates, clears, and infiltrates tissues.

▪︎ More rapid diagnosis with less technicality.

▪︎ 2-3 changes of wax

▪︎ Constant tissue agitation which accelerates and improves tissue penetration giving rise to more consistent results.

Vacuum Embedding

▪︎ Involves wax impregnation under negative atmospheric pressure inside an embedding oven.

▪︎ Reduces the time when tissues are subjected to high temperatures thus minimizing heat-induced tissue hardening.

▪︎ Facilitates complete removal of transition solvents, and prolongs the life of wax by reducing solvent contamination.

Vacuum Embedding

▪︎ Hastens the removal of air bubbles and clearing agent from the tissue block, thereby promoting a more rapid wax penetration of the tissue.

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

Vacuum embedding oven

Consists of a flat-bottomed heavy brass chamber covered with a heavy glass lid resting on a wide and thick rubber valve which produces an airtight seal when the chamber is being used.

4 hours

Tissue should not be left in the paraffin oven for more than?

Paraplast

▪︎ Mixture of highly purified paraffin and synthetic plastic polymers with a melting point of 56-57℃

▪︎ More elastic and more resilient － permits large dense tissue blocks (e.g. bones, brain) to be cut easily with the same result as in double embedding.

▪︎ Blocks obtained are more uniform than with any other medium, with better ribboning of sections.

Paraplast

▪︎ Serial sections may be cut with ease, without cooling the tissue block, thereby preventing the formation of ice crystal artefacts.

▪︎ No deposit is left on the slide after staining, and no special processing schedule is required.

▪︎ Soluble in common clearing agents and follows the same time schedule for paraffin impregnation.

▪︎ DOES NOT TEND TO CRACK like other paraffin wax substitutes.

56 to 58℃

Recommended melting point for paraplast

Embeddol

▪︎ Synthetic wax substitute similar to Paraplast with a melting point of 56 to 58℃

▪︎ Less brittle and less comprehensible than Paraplast

Bio/aid

Semisynthetic wax recommended for embedding eyes.

Tissue Mat

Product of paraffin, containing rubber, with the same property as Paraplast.

Ester Wax

▪︎ Lower melting point (46-48℃) but is harder than paraffin.

▪︎ Not soluble in water but is soluble in 95% Ethyl Alcohol and other clearing agents

▪︎ Can be used for impregnation WITHOUT prior clearing of the tissue.

Heavy duty microtome

Sectioning of ester wax-impregnated tissues should be done on a _______________ due to the relative hardness of the wax.

Water soluble waxes

▪︎ Plastic polymers, mostly polyethylene glycols with melting points of 38-42 C or 45-56 C.

▪︎ Polymer waxes are incorporated to improve adhesion, hardness, and plasticity.

Carbowax

▪︎ Polyethylene glycol containing 18 or more C atoms which appears solid at room temp

▪︎ Most commonly used water soluble wax

▪︎ Soluble in and miscible with water

▪︎ Processing time is reduced with the special advantage that harmful effects produced by ordinary dehydrating agents are consequently avoided.

Carbowax

▪︎ Water soluble wax that does not remove neutral fats and lipids, and allows them to be demonstrated in thin sections.

▪︎ Tissues are not exposed to too much heat so that excessive hardening, shrinkage and brittleness of tissue is avoided.

▪︎ Suitable for many enzyme histochemical studies

▪︎ Cytological details are excellently preserved.

Carbowax

▪︎ Due to its hygroscopic nature, this water soluble wax is very easily dissolved in water

▪︎ Must avoid contact of the block with water or ice.

▪︎ Tissue sections are very difficult to float out and mount due to its extreme solubility in water, dehydrating, and clearing agents.

Dimethyl sulphoxide (DMSO)

▪︎ Added to proprietary blends of plastic polymer paraffin waxes and reduces infiltration times and facilitates thin sectioning.

▪︎ Scavenges residual transition solvent and probably alters tissue permeability by substituting for or removing bound water thus improving infiltration.

▪︎ Metabolites can cause unpleasant and annoying oyster/garlic taste

Celloidin

▪︎ Purified form of nitrocellulose soluble in many solvents

▪︎ Suitable for specimens with large hollow cavities which tend to collapse, for hard and dense tissues (e.g., bones and teeth) and for large tissue sections of the whole embryo.

Celloidin

▪︎ "Colloidon"

▪︎ Supplied in thin (2%), medium (4%), or thick (8%) solutions of cellulose dissolved in equal parts of ether and alcohol.

▪︎ Used mainly for preparing soft tissue sections of mixed consistency (e.g., eyes and brain)

▪︎ No heat is required

▪︎ Resultant block has a rubbery consistency which gives good support to tissues.

Inability to cut thin sections

Storage of blocks in alcohol

Speed of technique (several weeks or months)

Disadvantages of celloidin

Paraffin

When eye sections are embedded through this method, the retina may be detached from the harder tissues (e.g. sclera and choroid) that encircle it.

Wet Celloidin Method

Method for celloidin impregnation of tissue recommended for bones, teeth, large brain sections, and whole organs.

Dry Celloidin Method

Celloidin impregnation method preferred for the processing of whole eye sections.

Low Viscosity Nitrocellulose (LVN)

▪︎ Another form of celloidin soluble in equal concentration of ether and alcohol, with a lower viscosity.

▪︎ Can be used in higher concentrations and still penetrates tissues rapidly.

▪︎ Preferred than ordinary celloidin

▪︎ Forms a harder tissue block and makes cutting of thinner sections possible.

Plasticizers

Tendency of tissues to crack when using LVN may be prevented by adding?

Oleum ricini

Castor oil

Examples of plasticizers.

Low Viscosity Nitrocellulose

▪︎ More explosive than celloidin, should be handled with care

▪︎ Usually marketed while wet with alcohol.

▪︎ When no longer needed for future use, it should be carefully destroyed.

Gelatin Impregnation

▪︎ Rarely used except when dehydration is to be avoided and when tissues are to be subjected to histochemical and enzyme studies.

▪︎ Embedding medium for delicate specimens and frozen tissue sections because it prevents fragmentation of tough and friable tissues when frozen sections are cut.

▪︎ Water soluble, does not require dehydration and clearing.

▪︎ Has a low melting point and does not cause over-hardening of tissues by heating.

Too soft

If the embedding medium is ____________ for the material, the tissue will not be supported and sections will be torn or shredded.

Too hard

If the embedding medium is _____________ for the tissue, sections will be brittle and will shatter.

Orientation

Tissue is arranged in precise positions in the mold during embedding, on the microtome before cutting, and on the slide before staining.

Leuckhart's Embedding Mold

▪︎ Blocking-out mold that 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.

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

▪︎ Mold is adjustable to give a wide variety of sizes to fit the size of the tissue block for casting.

▪︎ Recommended for routine use, although too slow and cumbersome.

Compound Embedding Unit

▪︎ Made up of a series of interlocking plates resting on a flat metal base, forming several compartments.

▪︎ Can embed more specimens at a time, reducing the time needed for blocking.

Plastic Embedding Rings and Base Mold

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

Tissue Tek

▪︎ Equipped with a warm plate to manage the impregnated specimen and a cold plate at 5C for rapid solidification of the block.

▪︎ Consists of a white plastic cassette mold with a detachable, perforated steel hinge, and a Snap-On lid, used to hold the tissue specimen throughout fixation, dehydration, clearing and wax impregnation.

Tissue Tek

▪︎ Easy to use, less paraffin wax needed, faster embedding, firmly attached tissue and holder, and permanent identification.

▪︎ Produces easier orientation when re-sectioning of tissue is required.

▪︎ Blocks can be filed immediately after sectioning.

Peel-Away

Plastic Ice Trays

Paper Boats

3 Disposable Embedding Molds.

Peel-Away

▪︎ Disposable thin plastic embedding molds

▪︎ Simply peeled off one at a time, as soon as the wax has solidified

▪︎ Gives perfect even block without trimming

▪︎ May be placed directly in the chuck or block holder of the microtome.

Plastic Ice Trays

▪︎ Used in ordinary refrigerators but may be recommended for busy routine laboratories.

▪︎ Each compartment may be utilized for embedding one tissue block, which may then be removed by bending the plastic tray once the wax has solidified or by smearing the inner mold with glycerin or liquid paraffin before embedding.

Paper boats

▪︎ Normally used for embedding celloidin blocks but are equally useful for paraffin wax blocks.

▪︎ Cheap and easy to make

▪︎ Provide easy and accurate identification of specimen, avoids confusion and interchange of tissue blocks.

▪︎ Rapid embedding of small or large volume of individual specimen is possible, since they can be made to suit any size of tissue.

Celloidin or Nitrocellulose Embedding Method

Used to be 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.

Double-Embedding

▪︎ Process by which tissues are first embedded or fully infiltrated with a supporting medium (e.g. agar or nitrocellulose) then infiltrated a second time with paraffin wax in which they are subsequently embedded.

▪︎ Used to facilitate cutting of large blocks of dense firm tissues (e.g. brain)

▪︎ Recommended for making small sections of celloidin blocks.

Plastic (resin) embedding

Provided superior results for light microscopic studies, particularly in hard tissues such as undecalcified bone and for high resolution light micrsocopy of tissue sections thinner than the usual 4-6 um such as renal biopsies, and bone marrow biopsies.

Epoxy

Polyester

Acrylic

Classification of plastics based on their chemical composition.

Epoxy

▪︎ Embedding plastics made up of a carefully balanced mixture of epoxy plastic, catalysts and accelerators.

▪︎ Hydrophobic and subsequent oxidation by peroxide to correct this may produce tissue damage

▪︎ May reduce antigenicity of embedded tissue and may compromise result of immunohistochemical staining

▪︎ May cause sensitization if absorbed by skin or inhalation

Araldite

▪︎ Epoxy plastic based on bisphenol A

▪︎ Infiltration is slow, partly because the epoxy plastic itself is a large molecule.

Epon

▪︎ Epoxy plastic based on glycerol

▪︎ Have lower viscosity but are often sold as mixtures of isomers

Spurr

▪︎ Epoxy plastic based on cyclohexene dioxide

▪︎ Can be obtained pure, have very low viscosity, and infiltrate the fastest

Vinyl cyclohexane dioxide

Carcinogenic component of epoxy plastics.

Polyester plastics

Originally introduced for electron microscopy but is now seldom used.

Acrylic plastics

▪︎ Made up of esters of acrylic or methacrylic acid and used extensively for light microscopy.

Sectioning

Process whereby tissues are cut into uniformly thin slices or "sections" with the aid of a microtome, to facilitate the studies under the microscope.

Paraffin Sections

Celloidin Sections

Frozen Sections

Three general types of tissue sections

Trimming

Process wherein after the wax block is removed from the mold, the identification number is noted and the excess wax is cut off from the block to expose the tissue surface in preparation for actual cutting.

Coarse facing

Done on the microtome at approx. 30 microns at a time until the entire tissue surface is exposed.

Heated spatula

After coarse trimming, a _____________ is held between the tissue block and the block holder until the wax begins to melt.

Freezer

Placing blocks in a _____________ can cause surface cracking, where the friable tissue separates from the surrounding wax cohesive sections become difficult to obtain.

Fine Trimming

May be done by either setting the thickness adjuster at 15mm or by advancing the block using the coarse feed mechanism.

4-6

Sections are between how many microns in thickness for routine histologic procedures?

Cold wax

Provides better support for the harder elements in a specimen allowing thinner sections to be obtained.

Floatation

Should expand the section to its original dimensions and ensure that it is completely flat.

Butchlii

Man who introduced paraffin wax embedding

Microtomy

Process by which processed tissue is trimmed and cut into uniformly thin slices or "sections" to facilitate studies under the microscope.

Microtome

Instrument capable of cutting a section at a predetermined thickness by sliding the block into a cutting tool, usually a steel knife, glass or diamond blade, which is fixed and attached to the machine.

Block holder

Knife carrier and knife

Pawl, ratchet feed wheel, and adjustment screws

Three essential parts of a microtome

Block holder

Where the tissue is held in position

Knife carrier and knife

For actual cutting of tissue sections

Pawl, ratchet feed wheel and adjustment screws

To line up the tissue block in proper position with the knife, adjusting the proper thickness of the tissue for successive sections.

Rocking microtome

Rotary microtome

Sliding microtome

Freezing microtome

Cryostat or cold microtome

Ultrathin microtome

Kinds of microtome

Rocking microtome

For cutting serial sections of large blocks of paraffin embedded tissues.

Rotary microtome

For cutting paraffin embedded sections.

Sliding microtome

For cutting celloidin embedded sections.

Freezing microtome

For cutting unembedded frozen sections

Cryostat or cold microtome

For cutting frozen sections

Ultrathin microtome

For cutting sections for electron microscopy.

Rocking Microtome

▪︎ "Cambridge Microtome"

▪︎ Simplest

▪︎ Has been used to cut small and large blocks of paraffin tissues.

▪︎ Theoretically not recommended for serial sections since tissues are cut in slightly curved planes

▪︎ Currently not favored by most laboratories because of restrictions in size of tissue block that can be cut, and the difficulty of reorienting the block.

Paldwell Trefall

Invented the Rocking Microtome in 1881

Minot

Invented the Rotary Microtome in 1885-86

Rotary Microtome

▪︎ Used to cut paraffin embedded tissues

▪︎ Most common type used for both routine and research laboratories, especially for sectioning paraffin-embedded tissues.

▪︎ Knife and the block holder are brought together by upward and vertical motions, cutting sections in a perfectly flat plane, thereby allowing excellent serial sections to be cut.

Rotary Microtome

▪︎ "Minot Microtome"

▪︎ Heavier and more stable than rocking microtome, more complex in design and construction, and more expensive.

▪︎ Knife is placed in a blade-up position (dangerous)

▪︎ Heavier knife is used, so there is less vibration.

▪︎ The cutting angle (tilt) of the knife is adjustable so it can cut harder tissue.

▪︎ Can cut celloidin-embedded sections by using a special holder to set the knife obliquely.

Adams

Developed the sliding microtome in 1789

Base-Sledge Microtome

Standard Sliding Microtome

Two types of sliding microtome

Base-Sledge Microtome

▪︎ Consists of two movable pillars holding the adjustable knife clamps, allowing the knife to be set at an angle for cutting celloidin sections.

▪︎ Chuck/block holder is set on a heavy metal base which can be moved backwards and forwards under the knife.

Base-Sledge Microtome

▪︎ Favored in labs where very hard tissue or large blocks are usually sectioned

▪︎ Suited for sectioning specimens embedded in all forms of media, especially for cutting sections from tough tissue blocks which may offer great resistance to the knife.

▪︎ Originally designed for cutting sections of very large blocks (whole brain)

24cm

How long is the knife used for base-sledge microtome?