DEHYDRATION, CLEARING, AND INFILTRATION

• Dehydration


  The removal of water from the fixed tissue
• Dehydrating agent


  • It should dehydrate fast
  • It should not evaporate fast
  • It should be able to dehydrate even fatty tissues
  • It should not harden the tissues excessively
  • It should not remove stains
  • It should not be toxic
  • It should not be a fire hazard
• Dehydrating agents


  • Alcohol
  • Acetone
  • Dioxane
  • Cellosolve
  • Triethyl Phosphate
  • Tetrahydrofuran
• Alcohol


  The most commonly and widely used dehydrating agent in the laboratory
• Ethanol


  Recommended dehydrating agent for routine dehydration of tissues
• Isopropyl alcohol


  Dehydrating and clearing agent, substitute for ethanol
• Methyl alcohol


  A toxic dehydrating agent, utilized for blood and tissue films, also used as a fixative
• Butyl alcohol


  A slow dehydrating agent which produces less shrinkage and hardening than ethanol, for plant and animal micro-technique
• Using alcohol for dehydration

  1. Subject the tissue to a series of progressively increasing concentration of alcohol (70% -> 95% -> 100%)

  2. The progression of the concentration of alcohol should not be decreasing

  3. When 95% or absolute alcohol (100%) is used first, it would only harden the surface of the tissue and it cannot penetrate the deeper parts of the sample completely

  4. The strength of the initial alcohol required will depend upon the size, nature of the tissue and the fixative used

  5. 37°C – hasten dehydrating agent
• Clearing


  Removal of dehydrating agent, or alcohol, from the tissue and is replaced with a substance that will dissolve the wax with which the tissue is to be impregnated
• Characteristics of a good clearing agent

  • It should be miscible with alcohol, to promote rapid removal
  • It should be miscible with, and easily removed by, melted paraffin wax in impregnation and/or by mounting media to facilitate the impregnation and mounting of the sections
  • It should not produce excessive shrinkage, hardening or damage of tissue
  • It should not dissolve out aniline dyes
  • It should not evaporate quickly in a water bath
  • It should make the tissues transparent as the alcohol or dehydrating agent is removed
• Clearing agents


  • Xylene
  • Toluene
  • Benzene
  • Chloroform
  • Cedarwood Oil
  • Aniline Oil
  • Clove Oil
  • Carbon Tetrachloride
• Xylene


  The most commonly used clearing agent in the laboratory
• Impregnation


  The removal of clearing agent, a process whereby the clearing agent is completely removed from the tissue and is replaced by a medium that will fill all the tissue cavities
• Paraffin


  The simplest, most common, and the best infiltrating agent
• Equipment and materials


  • Forceps
  • Specimen label
  • Tissue capsule/cassette
  • Pencil
  • Tissue basket
  • 80% Alcohol
  • 95% Alcohol
  • 100% Alcohol
  • Clearing agent
  • Melted paraffin
• Specimen used


  Formalin-fixed tissue to be submitted to the histopathology laboratory or the histopathology department
• Procedure for dehydration, clearing and impregnation


  1. Note down the specimen number using a pencil

  2. Transfer the tissue in a tissue cassette with the label

  3. Place the tissue capsule/basket in 10% NBF until processing

  4. Dehydrate in 80% alcohol for 1 hour

  5. Dehydrate in 95% alcohol for 1 hour

  6. Dehydrate in 95% alcohol for 2 hours (2nd change)

  7. Dehydrate in 100% alcohol for 2 hours

  8. Dehydrate in 100% alcohol for 2 hours (2nd change) or overnight

  9. Clear in clearing agent of choice for 1 hour (e.g. Xylene)

  10. Clear in clearing agent of choice for 1 hour (2nd change)

  11. Impregnate w/ melted paraffin for 2 hours

  12. Impregnate w/ melted paraffin for 2 hours (2nd change)

  13. Impregnate w/ melted paraffin for 2 hours, solidify overnight (3rd change)

  14. Embed