INTRODUCTION TO IMPREGNATION AND TISSUE EMBEDDING
The tissues, after fixation and dehydration process, are not sufficiently hard to cut into thin sections without a suitable support. Thus, tissues are first impregnated and then embedded in a suitable embedding medium to make a block which makes the tissue quite hard and provides a suitable support so that it can easily be cut into thin sections of few microns. The impregnation and embedding of tissues are done by using the same medium usually but sometimes two different mediums are used for these processes (Double embedding). The block may be made harden by cooling it at room temperature or in the refrigerator. There are various types of embedding medium used in the histopathology laboratory as per the properties of tissue and the tests to be done.
PROPERTIES OF AN IDEAL EMBEDDING MEDIUM
The embedding medium is considered as ideal if it bears the following qualities:
- The melting point of the embedding medium in the pure state should be below 65°C to avoid damage to the tissues by heating during impregnation.
- The embedding medium must be solidifying at room temperature.
- The embedding medium must penetrate the tissues replacing the fluid in which it is saturated, usually a clearing agent.
VARIOUS TYPES OF EMBEDDING MEDIUM
1.) Paraffin wax – Paraffin is solid at room temperature. The melting point of paraffin ranges from 40-60°C. For tropical countries hard wax having a melting point of 58-60°C is suitable. This is the most commonly used tissue embedding medium.
2.) Paraplast – This is a mixture of purified paraffin and plastic. It is easy to make the serial sections of 4 microns with this embedding medium due to its elasticity.
3.) Gelatin – Water soluble and so dehydration and Clearing is not necessary. It is used for delicate tissues and for the frozen sections. It has low melting point.
4.) Ester wax – It is harder than paraffin but has a low melting point of 46-48°C. It is soluble in alcohol so there is no need for the clearing step when using this tissue embedding medium.
5.) Tissue mate – It is the mixture of paraffin and rubber and has similar properties as that of Paraplast. The serial sections can easily be obtained using this embedding medium.
6.) Water-soluble waxes – These are Polyethylene glycol, having a melting point of 38-42°C. They cause less shrinkage than paraffin and no dehydration and clearing is needed. They do not make the tissues brittle & give better support than paraffin. They are used for demonstration of lipid and enzymes.
7.) Celloidin – This is purified nitrocellulose and is used for the hard and fragile specimens. Large specimens can easily be sectioned using this embedding medium. It takes 2-3 weeks to impregnate the tissues with Celloidin.
THE PARAFFIN WAX
Paraffin is a mixture of hydrocarbon produced in the cracking of paraffin (a mineral oil). The melting point of high-quality paraffin ranges 40-60°C.
Paraffin wax is the most popular embedding medium because of its properties and a large number of blocks can be prepared in a less amount of time. It also aids in the sectioning process and does not interfere with the staining process.
A series of sections cut from the wax block adhere to each other to form a ribbon, these sections are easier to handle than a single section. Paraffin Wax is on advantage here over other tissue embedding media like nitrocellulose.
As a precaution, the wax should be free from dust and impurities and the wax must be rapidly cooled to reduce the size of the crystals of the wax.
IMPREGNATION OF THE TISSUES
After Clearing, the tissues are infiltrated with a supporting medium which also replaces the clearing agent. The supporting medium will make the tissues firm, facilitates easy sectioning and keep the various components of the tissues in proper relation.
In this step, two processes take place simultaneously i.e. the Impregnation of the paraffin wax into the tissues and the Infiltration of Clearing agent out of the tissue into the surrounding. In this ways, the clearing agent is replaced by embedding medium.
This process is also called as the internal embedding of the tissues as the embedding medium penetrates the tissues and provides the support from inside of the tissue.
The Impregnation of tissues with molten paraffin is done in an embedding bath with a thermostat and a vacuum pump attached. The vacuum produced by vacuum pump increases the rate of wax impregnation and the paraffin wax penetrates the tissues more effectively. Paraffin of high melting point becomes hard and the paraffin of low melting point remains soft after cooling.
So it is necessary to use a high-quality wax in the histopathology laboratory if you don’t want to compromise on the quality of results.
THE TISSUE EMBEDDING OR BLOCK PREPARATION
After the impregnation of the tissues, the tissues are still cannot be cut into thin sections directly as mostly the size of tissues is less than 1 or 2 cm so they need a solid external support so that they can easily be cut into thin slices without getting distorted by the strokes of microtome knife/blade which is done by Tissue Embedding, also called as block preparation or external embedding.
In this, the wax impregnated tissues are placed in a mould in which molten paraffin wax is poured, which when get solidified provides a hard, solid support to the tissue so that it can easily be cut into the slices.
When performing the embedding of tissues, one should keep certain factors in his/her mind and the most important one is the choice of paraffin of the right degree of hardness, which depends on the following factors –
i.) Nature of the object/tissue (i.e. Hard or Soft)
ii.) Temperatures at which the tissues are sectioned.
iii.) Thickness of the section
A variety of moulds are used for embedding the tissues but the most popular one is Leuckhard L-shape embedding moulds, popularly known as the embedding L’s.
Nowadays, the inexpensive plastic moulds are replacing the other types of embedding containers. These plastic moulds support the blocks while it is being sectioned and are designed to fit according to the microtomes, eliminating the step of mounting the specimen on a block holder.
DOUBLE EMBEDDING OF TISSUES
As the name suggests, in this type of embedding two types of embedding medium are used. This term particularly refers to the embedding in which the tissues are first penetrated with nitrocellulose and then embedded in the Paraffin wax.
The goal of this is to produce a block that can be sectioned with a matrix of nitrocellulose in addition to paraffin. Artefacts which are peculiar to paraffin embedding are minimized by double embedding technique. This method is useful with bone, brain muscles and tissues obtained from the sacrificed animals.
After the proper impregnation and block preparation of the tissue, the most important step comes i.e. the section cutting which enables the making of thin sections of the tissues so that it can easily be examined under the microscope.