Histology: Clearing and Impregnation

Dealcoholization

The process whereby alcohol or a dehydrating agent is removed from the tissue and replaced by a fluid (clearing agent) that will dissolve the wax with which the tissue must be impregnated

In addition to removing alcohol, many of these substances have the property of making tissues transparent due to their high index of refraction (RI) approximately equal to that of tissues

Xylene/Xylol, Toluene/Toluol, Benzene, Chloroform, Cedarwood oil, Carbon disulfide, Oil of bergamot, Clove oil, Carbon tetrachloride, Aniline oil, Tetrahydrofuran, Petroleum ether, Cedar oil, Creosote

Terpineol, Phenol in alcohol, Dioxane, Carboxylol

Miscible with alcohol, miscible with melted paraffin wax/mounting medium, does not produce excessive shrinkage/hardening, does not dissolve aniline dyes, does not evaporate quickly in wax bath, makes tissues transparent

At least 10x the volume of the tissue

Gum syrup & Glycerin

½ to 2 hours (usual; 15-30 minutes for urgent biopsies); excellent and true clearing agent, most rapid clearing agent

Cheap, does not extract out aniline dyes, miscible with absolute alcohol and paraffin, can be used with celloidin sections

Highly flammable, becomes milky when dehydration is incomplete, NOT recommended for nervous tissues and lymph nodes (causes hardening and tissue shrinkage)

1-2 hours; substitute for xylene or benzene, acts slower than benzene and is expensive

Not carcinogenic, but highly concentrated fumes are toxic upon prolonged exposure

15-60 minutes; rapid acting, recommended for urgent biopsies and routine purposes, highly flammable

Carcinogenic or may damage bone marrow (Aplastic anemia)

6-24 hours; slower in action than xylene but causes less brittleness; best of traditional clearing agents for routine use, gives widest latitude

Recommended for tough (skin, fibroid, decalcified tissues), large tissue specimens, nervous tissue, lymph nodes, granulation tissue, and fetal/delicate specimens

Does not make tissues transparent, toxic to the liver after prolonged inhalation

2-3 days; clears both Paraffin and Celloidin sections, recommended for CNS tissues and cytological studies (especially smooth muscles and skin)

Very expensive, requires 2 changes, quality not always uniform, extremely slow, becomes milky on prolonged storage

Recommended for clearing embryos, insects, and very delicate specimens; clears 70% alcohol without excessive tissue shrinkage/hardening

Removes aniline dyes, dissolves Celloidin, tissues become brittle, quality not guaranteed (tends to be adulterated), not suitable for routine purposes (expensive)

Properties very similar to chloroform, but cheaper; toxic on prolonged exposure

Dehydrates and clears at the same time since it is miscible in both water and paraffin

Slow-acting clearing agents that can be used when double embedding techniques are required

Infiltration

The process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will fill all natural cavities, spaces, and interstices of the tissues, setting to a sufficiently firm consistency for cutting thin sections

At least 25x the volume of the tissue

1. Paraffin wax impregnation, 2. Celloidin (Collodion) impregnation, 3. Gelatin impregnation

Paraffin wax impregnation

54-58°C

Requires at least 4 changes of wax with 15 minutes interval

Makes use of an automatic tissue processing machine which fixes, dehydrates, clears, and infiltrates tissues, decreasing time and labor

Impregnation under negative atmospheric pressure using a vacuum of up to 760 mmHg applied via a water or mechanical pump

Recommended for urgent biopsies and delicate tissues such as lung, brain, connective tissues, decalcified bones, eyes, spleen, and CNS

Reduces 25-75% of impregnation time

MP: 56-57°C; mixture of highly purified paraffin wax and synthetic plastic polymers, more elastic and resilient than paraffin

MP: 56-58°C; synthetic wax substitute similar to paraplast, less brittle and less compressible than paraplast

MP: 38-42°C or 45-56°C (Ester wax: 46-48°C); semisynthetic wax recommended for embedding eyes

Harder than paraffin, not soluble in water, but soluble in 95% ethyl alcohol and other clearing agents

Mostly polyethylene glycols; solid at room temperature, soluble in water (does not require dehydration/clearing), suitable for enzyme histochemical studies

Produces shrinkage and hardening of tissues

Maintained at a temperature 2-5°C above the melting point of the wax

Impairs impregnating capacity and prevents formation of a good tissue block; remedied by heating wax to 100-105°C to evaporate water

Paraffin wax may be used only twice

Purified form of nitrocellulose soluble in many solvents

Specimens containing large cavities or hollow spaces which tend to collapse, hard/dense tissues (bones and teeth), large tissue sections of whole embryos, and whole organs like eyes

Causes much less shrinkage and distortion than paraffin wax

1. Slow and tedious, 2. Serial sections are difficult to prepare, 3. Very thin sections (<10 µm) are difficult to cut

Recommended for bones, teeth, large brain sections, and whole organs; tissues must be cut wet (knife & block kept moist with 70% alcohol)

Preferred for processing whole eye sections; uses Gilson's mixture (equal parts chloroform and cedarwood oil) in block, allowing cutting without alcohol

Soluble in equal concentration of Ether and Alcohol

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

Delicate specimens and frozen tissue sections (prevents fragmentation of tough and friable tissues)

Water soluble (does not require dehydration and clearing), low melting point so it does not overharden tissue