4 Clearing

• Clearing, aka Dealcoholization, is the removal of dehydrating solutions, making the tissue components receptive to the infiltrating medium

  • removes components of alcohol on tissue for the paraffin wax to penetrate, because paraffin wax is not freely miscible
• Xylene or Xylol makes microscopic tissue preparation transparent due to their high refractive index of refraction
• Glycerin and Gum Syrup are used when the tissue is to be cleared directly from water
• A good clearing agent should be miscible with alcohol to promote rapid removal of the dehydrating agent from the tissue
• A good clearing agent should be miscible with and easily removed by melted paraffin wax and/or by mounting medium to facilitate impregnation and mounting of sections.
• A good clearing agent should:

  • not dissolve out aniline dyes.
  • not evaporate quickly in a water bath.
  • make tissues transparent.
• Dealcoholization can remove all kinds of alcohol, especially absolute alcohol
• Deparaffinization is to dissolve paraffin wax on tissue.

  • Clearing agent and paraffin wax are freely miscible.
• High Refractive Index

  • On mounting always soak stained slide on xylene, then put mounting media Canada balsam then cover slip to make tissue transparent.
• Milky appearance = Incomplete Dehydration
• Xylene/Xylol

  ADVANTAGES:

  • Most rapid
  • Most common
  • Colorless
  • Cheap
  • Make tissue transparent
  • Miscible with absolute alcohol and paraffin wax
  • 30 minutes – 1 hour

  DISADVANTAGES:

  • Highly inflammable
  • Hardens/ Shrinks
  • Carcinogenic
• In xylene/xylol, >3 Hours = hard or brittle tissues
• Xylene/xylol hardens and Shrinks, which is why it is NOT for nervous systems and lymph nodes
• Toluene

  ADVANTAGES:

  • Substitute for xylene and benzene
  • Miscible both absolute alcohol and paraffin wax
  • Tissues do not become excessively hard and brittle even if left for 24hrs
  • Not carcinogenic
  • 1-2 hrs

  DISADVANTAGES:

  • Slower than xylene and benzene
  • Highly concentrated solution will emit fumes that are very TOXIC upon prolonged exposure
  • More Expensive
• Benzene

  ADVANTAGES:

  • Penetrates and clears tissues RAPIDLY
  • Rapid Acting = 15-60 minutes
  • Does not make tissues hard and brittle

  DISADVANTAGES:

  • Highly flammable
  • Tissue shrinkage if left for a long time
  • Carcinogenic
• Benzene volatilizes rapidly in paraffin oven = easily eliminated from tissue
• Benzene is highly flammable, which is why it is stored in close cabinet, in a well-ventilated room
• Benzene is carcinogenic, which may damage bone marrow resulting to Aplastic Anemia
• Chloroform

  ADVANTAGES:

  • 6-24 hours
  • Recommended for tough tissues
  • Suitable for large tissue specimen
  • Not inflammable

  DISADVANTAGES:

  • Does not make tissue transparent
  • Not very volatile in paraffin oven
  • Evaporates quickly from a water bath
• Chloroform is an ingredient for making metamphetamine/ drugs
• Cedarwood oil

  ADVANTAGES:

  • Used to clear both paraffin and celloidin sections
  • Recommended for CNS tissue, cytological studies, smooth muscle, and skin
  • Very penetrating
  • Does not dissolve aniline dyes

  DISADVANTAGES:

  • Very slow clearing agent
  • Quality is not always uniform and good
  • Very expensive
• Cedarwood oil

  • Clears celloidin in 5-6 days
  • Paraffin wax clears in 2-3 days
• Cedarwood oil becomes milky upon prolonged storage and should be filtered before use
• Aniline oil is 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
  • wax impregnation here
  • slow and difficult
  • tissues become brittle
  • expensive
• Carbon tetrachloride (CCL4) have properties and disadvantages like chloroform, but cheaper

  • Hepatotoxic and may cause damage to liver
• Tetrahydrofuran is both a dehydrating and clearing agent; it is non-toxic but with offensive odor and should be used in a well-ventilated room
• Tetrahydrofuran is much effective as a dehydrating agent than clearing because it has weak properties
• Methyl benzoate or Methyl salicylate is slow acting and is used for double embedding
• In Frozen section, no dealcoholization is involved
• Chloroform is toxic to Liver because of prolonged inhalation