INTRODUCTION TO CLEARING OF TISSUES
After dehydrating the tissues, the alcohol present in them must be removed before they are impregnated and embedded with paraffin. The Paraffin wax used for embedding is immiscible in both alcohol and water. Thus the alcohol must be replaced by some medium in which both alcohol and paraffin are soluble. The medium needs to be eliminated during wax impregnation. This treatment is known as Clearing. Clearing agent also increases the refractive index of the tissues and thereby makes the tissues relatively more transparent.
CLEARING AGENTS
1.) Xylene: It is the most commonly used clearing agent in histopathology laboratory. It is colorless watery liquid with a characteristic aromatic odor. It is insoluble in water but soluble in organic solvents like ethanol, benzene, acetone etc.
2.) Benzene: Benzene is rarely used as a clearing agent as it is a carcinogen and potentially causes cancer. It is a colorless, flammable liquid with a characteristic sweet and gasoline-like odor. It is slightly soluble in water and completely soluble in organic solvents like acetone, ethanol, chloroform etc.
3.) Chloroform: Besides its various uses in Pharmaceuticals, Dyes & Pesticides industries and in Refrigerant industries, it can also be used as a clearing agent in histopathology laboratory. It is a Colorless, volatile liquid with a characteristic ether-like odor. It is slightly soluble in water but completely soluble in organic solvents like ethanol, benzene, acetone etc.
DEHYDRATION OF TISSUES – TECHNIQUE OF REMOVING WATER FROM THE TISSUES
4.) Toluene: It is also a good clearing agent but less commonly used in histopathology laboratory. It is a colorless, flammable and clear liquid with a characteristic aromatic odor. It is insoluble in water but soluble in Organic solvents like Acetone, Chloroform, Ethanol, Benzene etc.
5.) Cedarwood oil: It is an excellent clearing agent for tissues but less commonly used due to its slow penetrating rate. It is light yellow to pale brown colored viscous fluid with a characteristic woody odor. It is insoluble in water but soluble in organic solvents like ethanol, acetone, etc.
Here is the list of popular clearing agents, their Advantages, and Disadvantages:
| S. No. | CLEARING AGENTS | ADVANTAGES | DISADVANTAGES |
|---|---|---|---|
| 1.) | XYLENE | i.) Its action is very rapid. ii.) Cheap & slightly inflammable. iii.) Readily eliminate in the paraffin oven. | i.) Prolonged treatment with this reagent makes the soft tissues like Brain & Spleen quite brittle. |
| 2.) | BENZENE | i.) It penetrates the tissues rapidly. ii.) It causes minimum shrinkage. iii.) It is a Cheap clearing agent. | i.) It is a carcinogen and potentially causes cancer. ii.) It is a flammable liquid. |
| 3.) | CHLOROFORM | i.) It is widely used for its hardening effect. ii.) Ideal for hard & delicate tissues like Bone and the Brain. | i.) Its action is slower than other clearing agents. ii.) It can cause faintness if inhaled. |
| 4.) | TOLUENE | i.) Tissues can be kept in this for a longer period. ii.) Its action is similar to benzene but it is less toxic. | i.) It causes irritation if accidentally inhaled or come into contact with skin or eyes. |
| 5.) | CEDAR WOOD OIL | i.) It has a gentle action on the tissues. ii.) It is the excellent Clearing agent for tissues. | i.) It is very expensive. ii.) It slowly penetrates the tissues. |
THE OTHER CLEARING AGENTS WHICH ARE LESS COMMONLY USED INCLUDES:
- Carbon Disulphide
- Methyl benzoate
- Methyl salicylate
- Paraffin-xylol mixture
- Carbon tetrachloride
- Clove oil
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what happens when tissues specimen after alcohol immersion is placed in xylene and left overnight in xylene ?
A deep research based article. Thank you.