IMPREGNATION AND EMBEDDING Midterms Lec 6

Infiltration (aka impregnation)

Tissues are placed in a substance that will completely fill all cavities and
interstices
Gives firm consistency to tissue
Facilitate handling and cutting

Embedding (aka casting or blocking)

Impregnated tissue is placed into a precisely arranged position in a mold
containing a medium
*medium used to infiltrate the tissue is usually the same medium utilized for
impregnation = embedding medium

4 Types of Tissue Impregnation and Embedding Media

Paraffin wax
Celloidin (Collodion)
Gelatin
Platsic

Paraffin wax simplest, most common and best embedding used

Advantages
o Thin individual serial sections may be cut ease from majority of tissues
without undue distortion
o Very rapid = within 24 hours
Disadvantages
o Overheated paraffin = brittle specimen
o Prolonged impregnation = excessive tissue shrinkage and hardening =
difficult cutting of sections
o Not recommended for fatty tissues

Paraffin Wax Infiltration

Polycrystalline mixture of solid hydrocarbons
56C
*lab with temp 20-24C, 54-58C melting point of paraffin wax
*lab with temp 15-18C, melting point should be 50-54C
*paraffin oven or an incubator = 55-60C
More blocks are processed in a short time
Serial sections are easily obtained
Routine and special staining can be easily done

3 Ways by which Paraffin Wax Impregnation and Embedding of Tissues may be

performed
By Manual Processing
 At least 4 changes are requires at 15 minutes intervals
Approximately 3 hours
By Automatic Processing
Makes use of an automatic tissue processing machine
o Autotechnicon
Fixes, dehydrates, clears and infiltrates tissues
2-3 changes of wax to remove the clearing agent
With constant tissue agitation
*Elliot Bench-Type Processor mounted on rollers to permit the
turning of platforms and easy access to beakers and wax baths
By Vacuum Embedding
Under negative atmospheric pressure inside an embedding oven to
hasten removal of air bubbles and clearing agent from the tissue block
= more rapid wax penetration of tissues
*maintained at a temperature of 2-4C above melting point of wax
*degree of vacuum should not exceed 500mm

Modified Paraffin Waxes

Better properties and additional substances to improve ribboning
Stearic acid ( hardness)
Phenanthrene or spermaceti ( melting point)
Ceresin, beeswax (improve adhesion)
Examples
o Piccolyte 115 (thermoplastic terpene resin)
o Plastic polymers (ex. Polyethylene wax)
o Dimethyl sulfoxide (DMSO)

Subsitute for Paraffin Wax

1) Paraplast
Mixture of highlt purified paraffin and synthetic plastic polymers
Melting point (MP): 56-57C
o Embeddol
Synthetic wax similar to paraplast
MP: 56-58C
o Bioloid
Semisynthetetic; for embedding eyes
o Tissue mat
Product of paraffin containing rubber
2) Ester Wax
Lower melting point: 46-48C
Harder than paraffin
Soluble in 95% ethanol (not in water)
Sliding or sledge type microtome
3) Water Soluble Waxes
 Mostly polyethylene glycols
Melting point: 38-42C or 45-56C
Carbowax (most common)

Types of Tissue Impregnation

A. Celloidin Impregnation
Colloidon
Purified form of nitrocellulose soluble
Suitable for specimens
o With large cavities/hollow spaces which tend to collapse
o Large embryos
2 methods
a. Wet Celloidin Method
Recommended for bones, teeth, large brain sections and
whole organs
b. Dry Celloidin Method
Preferred for processing of whole eye sections
Nitrocellulose Method
o Low Viscosity Nitrocellulose (L.V.N)
Soluble in equal concentrations of ether and alcohol
Forms harder tissue block and makes cutting thinner sections
possible
B. Gelatin Impregnation
Rarely used
Except when
o Dehydration is to be avoided
o Tissues are to be subjected to histochemical and enzyme studies

Aqueous Media
Agar
Gelatin
Sodium Carboxymethyl Cellulose
Polyvinyl alcohol
o Highly polar, water soluble
o Histochemical studies of lipids and enzymes

Water Miscible Media

PEGs
Heat and solvent-labile lipids and protein
Prevents tissue shrinkage and damage
Less elastic, denser

Alternative Embedding Media

Resins
 Heat or reagent labile tissues hard or dense tissues
Tissues with poor adhesion with wax
Very thick/thin sections are needed
Sectioning whole organs
o Epoxy
o Polyester
o Acrylic

Factors Affecting Paraffin Wax Impregnation

1) Nature and size of the tissues
Brain
Skin
Dense bone
Blocks from female breast with large amount of retained secretions in dilated
ducts
2) Clearing agent used prior to impregnation
Example
o Benzene and Xylene
Easily removed from tissues
o Chloroform and Cedarwood Oil
Difficult to remove

Precautions Observed in Paraffin Wax Impregnation

1) Avoid prolonged treatment of tissues in melted paraffin
2) Avoid infiltration in overheated paraffin (above 60C)
3) Oven must be maintained at 2-5C above its melting point of wax
4) Wax must be pure, free from dust, foreign matter
5) Fresh wax should be filtered before use in a wax oven
6) Paraffin wax should be used only twice
7) Hard wax with higher melting point requires a heavy duty type of microtome
8) Hard and dense tissues require wax with higher melting point than soft ones