4 Clearing

Cards (31)

  • 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