Clearing (De- alcoholization)- MIDTERMS L9

CLEARING

• The process whereby alcohol or a dehydrating agent is removed from the tissue and replaced with a substance that will dissolve the wax with which the tissue is to be impregnated.

• Process of replacing the dehydrating fluid with a fluid that is miscible with both the dehydrating fluid and the impregnating/embedding medium.

MOST COMMONLY USED CLEARING AGENTS

• Xylene

• Dioxane

• Chloroform

• Cedarwood oil.

THE CLEARING AGENT

Will make microscopic tissue preparations transparent due to their high index of refraction.

CHARACTERISTICS OF A GOOD CLEARING AGENT

• Miscible with alcohol to promote rapid removal of the dehydrating agent

• Should be miscible with and easily removed by melted paraffin wax.

• Should not produce excessive shrinkage, hardening or damage of tissue

• Should not dissolve out aniline dyes

• Should not evaporate quickly in a water bath

• Should make tissues transparent

COMMON CLEARING AGENTS USED

• Xylene

• Toluene

• Benzene

• Chloroform

• Cedarwood oil

• Aniline oil

• Clove oil

• Carbon Tetrachloride

XYLENE

• Colorless clearing agent that is most commonly used. Most rapid clearing agent, suitable for urgent biopsies

• Clearing time: ½ to 1 hour

• Makes tissues transparent

• Does not extract aniline dye

• Can be used for celloidin sections because it does not dissolve celloidin

• It is cheap.

DISADVANTAGES OF XYLENE

• Highly flammable

• If used longer than 3 hours, it will make the tissues excessively hard and brittle

• Not suitable for nervous tissues and lymph nodes (causes considerable hardening and shrinkage of tissues)

• Xylene becomes milky when an incompletely dehydrated tissue is immersed in it.

TOLUENE

• May be used as a substitute for xylene or benzene

• Clearing time: 1 – 2 hours

• Acts fairly rapidly and is recommended for routine purposes

DISADVANTAGES OF TOLUENE

• Relatively slower than xylene and benzene

• Tends to acidify in a partially filled vessel

• More expensive.

BENZENE

• It is preferred as clearing agent in the embedding process of tissues because it penetrates and clears tissues rapidly

• Clearing time: 15 – 60 minutes

• Volatizes rapidly in paraffin oven, easily removed in the tissue

DISADVANTAGES OF BENZENE

• It is highly flammable

• If section is let in benzene for a long time, considerable tissue shrinkage may be observed

• Excessive exposure is toxic and carcinogenic to human. It may damage the bone marrow resulting in aplastic anemia.

CHLOROFORM

• For tough tissues, lymph nodes, nervous system, and embryo

• Not flammable

• Slower in action than xylene but causes less brittleness

• Suitable for large tissue specimens. Thicker tissue blocks (up to 1 cm) are can be processed

• Clearing time: 6 – 24 hours

DISADVANTAGES OF CHLOROFORM

• Relatively toxic to the liver after prolonged inhalation

• Wax impregnation after chloroform clearing is relatively slow

• Does not make tissues transparent

• Difficult to remove from paraffin sections because it is not very volatile

• Complete clearing is difficult to evaluate

CEDARWOOD OIL

• Used to clear both paraffin and celloidin sections during embedding process

• Recommended for central nervous system tissues and cytological studies.

• Clearing time: 2 – 3 days

• Very penetrating clearing agent

• Clears celloidin in 5 – 6 days

• Not dissolve aniline dyes and make tissue transparent

DISADVANTAGES OF CEDARWOOD OIL

• Extremely slow clearing agent, not recommended for routine purposes

• Becomes milky upon prolonged storage and should be filtered before use

• Very expensive

ANILINE OIL

• Not normally utilized as a routine clearing agent

• Recommended for clearing embryos, insects and very delicate specimens due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening

CLOVE OIL

• Causes minimum shrinkage of tissues

• Its quality is not guaranteed due to its tendency to become adulterated

• Wax impregnation after clearing with clove oil is slow and difficult

• Tissues become brittle, aniline dyes are removed and celloidin is dissolved

• EXPENSIVE- not suitable for routine clearing purposes

CARBON TETRACHLORIDE

• Its properties are similar to chloroform although it is relatively cheaper

• Same disadvantage of chloroform

• It produces considerable tissue hardening and dangerous to inhale on prolonged exposure due to its highly toxic effects

TETRAHYDROFURAN

Is superior to ordinary dehydrating and clearing agents due to its ability to perform two processes at the same time thereby shortening the total processing time and allowing more time for fixation.

METHYL BENZOATE AND METHYL SALICYLATE

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

GLYCERIN AND GUM SYRUP

• Are used when the tissue is to be cleared directly from water, as in a frozen section.

• No de-alcoholization is involved in this process.

• for Frozen section