CLEARING

• Dealcoholization


  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
• Methyl Benzoate & Methyl Salicylate


  Slow-acting, used in double embedding techniques