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
Dealcoholization
Translucent appearance
Must be miscible with paraffin to facilitate the penetration of the embedding medium
High index of refraction
Characteristics of a good clearing agent
Miscible with alcohol
Miscible with and easily removed by melted paraffin wax
Not produce shrinkage, hardening or damage to tissues
Not dissolve out aniline dyes
Not evaporate quickly
Make tissues transparent
Common clearing agents used
Chloroform
Toluene, Xylene, Benzene
Carbon Tetrachloride
Cedar Wood oil
Methyl Benzoate, Methyl Salicylate
Citrus Fruit oil
Xylene (Xylol)
Best clearing agent, routinely used, clearing time 1/2 to 1 hour
Xylene (Xylol)
Clears within 15-30 mins
Make tissues transparent
Miscible with absolute alcohol & paraffin
Not extract out aniline dyes
Not dissolve celloidin
Evaporates quickly in paraffin oven
Replaced by wax
Affordable
Highly inflammable
Prolonged usage makes tissues hard and brittle
Not suitable for nervous tissues & lymph nodes
Becomes milky
Toluene
Substitute for Xylene and Benzene, clearing time 1-2 hours
Toluene
Miscible with absolute alcohol & paraffin
Fairly rapid
Recommended for routine purposes
Tissues don't become hard & brittle even if left for 24 hours
Not carcinogenic
Slower than xylene & benzene
Tends to acidify
Highly conc. Solutions emit fumes that are toxic
More expensive
Benzene
Excellent but hazardous, recommended for urgent biopsies (15-60 mins)
Benzene
Volatilizes rapidly in paraffin oven
Miscible with absolute alcohol
Not make tissues hard & brittle
Min. shrinkage
Make tissues transparent
Highly inflammable
Extremely toxic to man
Carcinogenic (AML / aplastic anemia)
Chloroform
Not a true clearing agent, recommended for routine work (6-24 hours)
Chloroform
Miscible with absolute alcohol
Recommended for tough tissues
Suitable for large tissue specimens
Not inflammable
Hepatotoxic
Relatively slow
Do not make the tissues transparent
Not very volatile in paraffin oven
It evaporates quickly from a water bath
Cedarwood Oil
For CNS tissues, cytological studies (smooth muscles and skin), clearing time 2-3 days
Cedarwood Oil
Very penetrating
Miscible with 96% alcohol
Clears celloidin in 5-6 days
Minimum shrinkage & hardening without considerable damage & distortion
Not dissolve out aniline dyes
Make tissues transparent
Improves cutting of the sections
Slow
Hard to be eliminated from the tissues
Quality is not always uniform and good
May dry out before it is completely cleared
Milky upon prolonged storage
May produce crystals
Very expensive
Aniline Oil
Recommended for embryos, insects and very delicate specimens, can clear 70% alcohol
Clove Oil
Causes minimum shrinkage, can be adulterated, tissues become brittle, removes aniline dyes, dissolves celloidin, expensive
Carbon Tetrachloride
Its properties are very similar to that of chloroform, causes considerable tissue hardening, toxic