Histopathology - Impregnation & Embedding

Impregnation (Infiltration)

The process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will completely fill all the tissue cavities

Firm consistency

Easier handling

Cutting without damage

Advantages of Infiltration/Impregnation (3):

Embedding (Casting/Blocking)

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

Embedding medium

is the medium used to infiltrate the tissue

Paraffin wax

Celloidin

Gelatin

Plastic

4 types of tissue impregnation and embedding medium

Paraffin wax

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

prepared within 24 hours

Brittle

Overheated paraffin makes the specimen _______________

Shrinkage and hardening

Prolonged impregnation will cause excessive __________________

Retention

Inadequate impregnation will promote _______________ of the clearing agent

Oven or incubator

Tissue is submerged two or more changes of melted paraffin wax ---either in a praffin ______________ or ______________

55-60 C

Temperature regulated in oven or incubator:

56 C

Temperature for routine work:

20-24 C

Laboratory temperature:

54-58 C

If laboratory temperature is from 20-24 C the melting point is

50-54 C

If laboratory temperature is 15-18 C

Manual processing

Automated processing

Vacuum embedding

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

Manual processing

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

tissue is immersed in another solution for 3 hours

Automatic processing

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

more rapid diagnosis with less technicality

2-3 changes of wax

3 degrees above the melting point

Wax bath thermostats should be set at least ________________________

Vacuum embedding

involves the wax impregnation under negative atmospheric pressure

Recommended for urgent biopsies and delicate tissues

Lungs

Connective tissue

Decalcified bones

CNS

Eyes

Spleen

Tissues recommended for vacuum embedding

Vacuum embedding oven

flat bottomed heavy brass chamber covered with a heavy glass lid resting on a wide and thick rubber valve

500 mmHg

The degree of the vacuum should not exceed _____________

Vacuum impregnation

Has the fastest result

nature and size

Total impregnation time generally depends upon the _______________________________

Benzene and Xylene

Easily removed from the tissues

Chloroform and Cedarwood oil

More difficult to remove

2-5 C above the melting point

To avoid the shrinkage of the tissue paraffin oven must be maintained at a temperature __________________

Paraplast

Ester

Water soluble waxes

3 substitutes for paraffin wax:

Paraplast

a mixture of highly purified paraffin and synthetic plastic polymers with a melting point of 56-57 C

More elastic and resilient

Embeddol

56-58 C

less brittle and less compressible

Bioloid

recommended for embedding eyes

Tissue mat

A product of paraffin containing rubber

Ester wax

has a lower melting point (46-48 C)

Not soluble in water but soluble in 95% ethyl alcohol

can be used for impregnation without prior clearing

3-4 changes

polyethylene glycols

Water soluble waxes are mostly ____________________

38-42 C or 45-56 C

melting point of polyethylene glycols

Carbowax

most commonly used polyethylene glycols

does not require dehydration and clearing of tissues

does not remove neutral fats and lipids

suitable for histochemical studies

Celloidin impregnation

A purified form of nitrocellulose soluble

suitable for specimens with large hollow cavities which tend to collapse, for hard and dense tissues and for large tissue sections of the whole embryo

Low viscosity nitrocellulose

recommended for processing neurological tissues

Wet celloidin impregnation

Dry celloidin impregnation

2 methods for celloidin impregnation

Wet celloidin impregnation

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

12-24 hours

Dry celloidin impregnation

Preffered for processing of whole eye sections

Gilson's mixture

made up of equal parts of celloidin and cedarwood oil

Gelatin impregnation

rarely used except when dehydration is to be avoided

used as an embedding medium for delicate specimens and frozen sections

hardest to use

Orientation

process by which a tissue is arranged in precise positions

Leuckhart's embedding mold

Compoud embedding units

Plastic embedding ring and base mold

Disposable embedding molds

Types of blocking-out molds (4)

Leuckhart's embedding mold

consist of two L-shaped strips of heavy brass or metal

Compound embedding mold

Made up of a series of interlocking plates resting on a flat metal base

Plastic embedding ring and base mold

Consist of a special stainless base mold fitted with a plastic embedding ring

Tissue tek

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

Peel away

Plastic ice trays

Paper boats

Types of disposable embedding molds (3):

Peel away

disposable thin plastic embedding molds that are simply peeled off

Plastic ice trays

Busy routine laboratories

Paper boats

Utilized for embedding celloidin blocks but are equally useful for paraffin wax blocks

cheap and easy to make

Celloidin or nitrocellulose method

Used to be recommended for embedding hard tissues and for large sections of whole organs

Double embedding method

Process in which tissues are first infiltrated with celloidin and subsequently embedded in paraffin mass

Plastic impregnation

resin impregnation

Epoxy embedding plastics

made up of a carefully balanced mixture of epoxy plastic,catalysts, and accelerators

Polyester plastic

originally introduced for elctron microscopy and now seldomly used

Acrylic plastic

made up of acrylic or methacrylic acid, and are used extensively for light microscopy