4. Infiltration, Embedding, Microtomy and Mounting

Infiltration

Also known as impregnation

Infiltration

Replaces the clearing agent with a medium that fil the tissue cavities producing firm consistency and easy handling and cutting

Embedding

Also known as casting or blocking

Embedding

Impregnated tissue is placed in an embedding mold and involves preparing tissue blocks and putting wax

Orientation

Tissue is arranged in a precise position within the mold

Leuckhart’s Embedding Mold

Embedding mold with 2 L-shaped strips of heavy brass or metal; adjustable to give shape

Compound Embedding Unit

Embedding mold with compartments to embed many tissues at the same time

Plastic Embedding Rings and Base Mold

Most common embedding mold with a handle which will be attached with the microtome

Plastic Embedding Rings and Base Mold

Embedding mold with plastic ring on top in which the tissue block will stick to the plastic ring. The square will be inserted into the microtome.

Disposable Molds

Tissue block adopts the shape of this mold; not stable and the end product is prone to deformities

Paraffix Wax

Simplest and most common wax for embedding which provides easy cut without distortion

<24 hrs

Duration using paraffin wax

56 deg C

Melting point of paraffin wax

100-105 deg C

Water in wax

Manual Processing

Most common method of paraffix wax impregnationin histology specimen preparation.

Vacuum Processing

Impregnation under negative atmospheric pressure inside an embedding oven; fastest method

Double Embedding

Wax used in infiltration is different from the wax used in embedding; Used for additional stability but not usually practiced

Paraplast

Paraffin + synthetic plastic polymer; Melting point at 56-57 deg C

Embeddol

Similar to paraplast

Bioloid

Semi-synthetic wax for the embedding of eyes

Tissue Mat

Wax containing rubber

Ester Wax

Harder than paraffin and insoluble in water but soluble in 95% ethanol; used in a sliding microtome; Melting point at 46-48 deg C

Polyethylene Glycol

Wax that is miscible and soluble in water; No need for dehydration and clearing; Used for enzyme histochemistry

Carbowax

Most common polyethylene glycol wax

Celloidin

Purified method of nitrocellulose; very slow; For specimens with large hollow cavities, dense and hard tissues

Wet Celloidin

Celloidin method for bones, brain sections, and teeth

Dry Celloidin

Celloidin method for whole eye sections

Nitrocellulose Method

Celloidin methon using low viscosity nitrocellulose; Explosive and the preferred method for harder tissue blocks and thinner sections

Plastic (Resin)

Wax used for electron microscopy to allow tissue sectioning of 80 nm

Plastic (Resin)

Wax used for high resolution light microscopy for renal biopsies and hematopoietic tissues

Double Embedding

Tissue is first infiltrated with an infiltrating media and embedded using a different media; For external support

Trimming

Cutting away extra wax above or below the embedded tissue using a used blade

Microtome

An instrument used for cutting

Sharp Razor

Earliest form of microtome

Rotary Microtome

Most commonly used microtome

Block Holder or Chuck’s

Placed to the tissue block

Knife Carrier and Knife

Has a lock for safety feature

Rocking or Cambridge Microtome

Invented by Paldwell Trefall; simplest microtome used for large paraffin section

10-12 um

Thickness using rocking/Cambridge microtome

Rotary Microtome

Invented by Minot; Routinely used for paraffin embedded section

4-6 um

Thickness using rotary microtome

Sliding Microtome

Invented by Adams; For celloidin embedded section and dense tissues; Most dangerous type of microtome

Freezing Microtome

Invented by Queckett; For frozen sections and urgent biopsies

Carbon Dioxide

Propellant used in freezing microtome

Cryostat or Cold Microtome

Commonly used for rush frozen sections with temperature of -20 deg C; rotary microtome embedded inside a cryostat

Ultrathin Microtome

Used for electron microscopy; Tissues are usually embedded in resinous plastic

Diamond Knife

Special knife used in ultrathin microtome

0.5 um

Thickness using ultrathin microtome

Plane Concave

Microtome knife for both celloidin and paraffin section

25 mm

Length of plane concave knife

Celloidin

Used a less concave knife

Paraffin

Uses a more concave knife

Plane Wedge

Knife used for frozen section or very hard tissues

100 mm

Length of plane wedge knife

Diamond or Glass Knife

Knife used for ultrathin microtome

Disposable Knife

More commonly used knife coated with tetrafluoroethylene

Automatic Machines

Tend to remove more metal during sharpening so that nives become worn quickly

Manual Methods

Remove far less metal but require more skill, experience and time to produce a satisfactory edge

Honing

Sharpening the knife; removal of gross nicks or irregularities

Heel to Toe

Movement in honing

10-20

Stroke per surface in honing

• Soapy water

• Mineral oil

• Clove oil

Lubricants used for honing

Xylene

Cleaner for honed knives

Belgium Yellow

Hone that yields best results

Arkansas

Hone that produces the most polishing effect

Fine Carborundum

Hone used for badly nicked knives

Plate Glass

Hone that is the excellent substitute for stoneR

Stropping

Removal of burrs or irregularities after honing

Toe to Heel

Movement in stropping

40-120

Number of strokes for stropping

Horse Leather

Strope that is used

Adhesion

After sectioning, tissue sections are floated-out on a water bath to be placed on a glass slide.

Adhesion

Achieved through drying or use of adhesive agents

Adhesion

Process of attaching tissue ribbons to your tissue slides

Adhesive

A substance which can be smeared to the slides so that the sections stick well to the slide

Mayer’s Egg Albumin

Most commonly used adhesive agent; Easy to make, convenient and inexpensive

Thymol Crystals

Present in Mayer’s Egg Albumin to prevent the growth of molds

• Egg white

• Glycerin

• Crystals of Thymol

Formula for Mayer’s Egg Albumin

Dried Albumin

Dried and stored in 70% alcohol until it is ready for staining

• Dried albumin

• Sodium chloride

• Distilled water

• Crystals of thymol

Formula for Dried Albumin

1% Aqueous Gelatin

Adhesive agent which is thick in consistency added to floatation bath as a mixture instead of directly coating the slide

• Gelatin

• Distilled water

• Glycerol

• Phenol crystals

Formula for 1% Aqueous Gelatin

Poly-L-Lysine

Widely used adhesive agent used as a section adhesive in immunohistochemistry but the effectiveness slowly decreases in time

• Aqueous detergent

• Distilled water

Formula for Poly-L-Lysine

APES (3-aminopropyl-triethoxysilane)

Very useful in cytospin preparations of proteinaceous or blood material and better than poly-L-lysine because they can be stored for a long time without losing their adhesiveness

Mounting

A process of securing the tissue specimen with a coverslip using a syrupy fluid or mounting medium

Aqueous Media

Used for mounting sections from distilled water when stains would be decolorized or removed by alcohol and xylene

• Gelatin

• Glycerin jelly or hum Arabic

• Glycerol

• Sugar

• Preservative

Aqueous media is usually made up of:

Water

Low refractive index; moderately transparent and evaporates easily; Good only for temporary mounting; Does not allow tissues to be examined under the oil immersion lens

1.46

Refractive index of glycerin

Glycerin

May also be used as a preservative; Has a high RI and provides greater visibility if slightly diluted with water

Glycerin

Standard mountant for fat stains

1.47

Refractive index of glycerin jelly

Glycerin Jelly

Kaiser’s 1880; standard mounting medium used when dehydration and clearing with xylene cannot be made

1.43

Refractive index of Farrant’s medium

Farrant’s medium

Mounting media that does not solidify upon storage; Does not need to be heated before use; Takes longer time to harden

1.52

Refractive index of Apathy’s medium

Apathy’s medium

Used for methylene blue-stained nerve preparations; General purpose aqueous mountant; One of the most useful aqueous mountants for fluorescent microscopy

1.47

Refractive index of Brun’s fluid

Brun’s fluid

Recommended for mounting frozen sections from water