3 Dehydration

• Dehydration is the removal of intracellular and extracellular water from the tissue

  • Done after fixation to allow the complete penetration of paraffin wax
• Increasing concentration of alcohol:

  • Routine – starts with 70% usually ethyl alcohol (always)
  • Embryonic tissues – starts with 30% of ethyl alcohol
• 10:1 = ratio of dehydrating agent and tissue
• Dehydration is the most critical step in automatic processing, while fixation in manual processing
• Less concentrated alcohol will produce lysis of cells
• 80% concentration of alcohol can be used to preserved photographic work
• An Ideal Dehydrating Solution should be:

  • Rapid acting w/no tissue shrinkage or distortion
  • Should not evaporate fast
  • Can dehydrate even fatty tissues
  • Non-toxic
  • Not fire hazard
  • Should not harden tissues excessively
• Alcohol is the most commonly used dehydrating Agent and is well recommended
• Ethyl Alcohol or Ethanol is recommended for routine dehydration

  • Best dehydrating agent
  • Fast acting
  • Clear, colorless flammable fluid
  • Mixes with water
  • Not poisonous
  • Not expensive
• 100% is an Absolute Ethyl Alcohol
• Methyl Alcohol is very toxic; used for blood and tissue films and smear preparation

  • Usually in hematology section, fixing smear
  • Causes occular toxicity
• Butyl Alcohol is used for plant and animal microtechnique

  • Very Slow
  • Producing less shrinkage
  • Hardening than ethyl alcohol
• Prolonged storage in 70% alcohol macerates tissues
• Recommended series of alcohol concentration is ascending grade of alcohol
• Acetone is a very cheap alcohol, but rapid acting

  • Utilized for most urgent biopsies (brain for rabies)
  • Not Recommended for routine tissue dehydration
  • causes tissue shrinkage
  • Clear, colorless
  • Mixes with water, ethanol and most organic solvents
  • Volatile
  • 30 mins – 2 hours
• Dioxane or Diethylene Dioxide is an excellent dehydrating and clearing agent

  • Less tissue shrinkage
  • Ribbon poorly (disadvantage)
  • Expensive
  • Dangerous
  • Uses: Graupner’s Method (dioxane, paraffin wax) and Weiseberger’s Method (gauze, Ca oxide)
• Cellosolve or Ethylene Glycol Monoethyl Ether dehydrates rapidly

  • Storage without producing hardening or distortion
  • Very combustible at 110-120°F
  • Toxic by inhalation, skin contact and ingestion
• Triethyl Phosphate removes water very readily and produces minimum distortion and hardening of tissues

  • Soluble in alcohol, water, ether, benzene, chloroform, acetone and xylene
  • Used to dehydrate sections and smears following certain stains
• Tetrahydrofuran acts both dehydrating agent and clearing agent; causes less shrinkage

  • Easier cutting w/ fewer artifacts
  • Gives improved staining procedure
  • Toxic (ingestion, inhalation)
  • Vapor causes nausea, dizziness, headache, and anesthesia
  • Prolonged exposure (up to 6 months) can cause conjunctival irritation
  • Very Offensive odor
• 4% phenol is an additive to dehydrating agent, which is added to 95% ethanol to soften hard tissues
• Hard tissues are immersed in glycerol/alcohol mixture or in Molliflex to soften tissue
• Method in Determining the Complete Dehydration:

  • Use Anhydrous Copper Sulfate method
  • Incomplete dehydration, clearing agent used is xyline = turn to milky appearance
• Dezenkerization is the removal of Mercuric chloride
• Isoprophanol is a type of alcohol; has clearing agents properties that can absorb microwaves and less toxic.
• 80% Grade alcohol in dehydration, photographic works; fixative for touch preparation