Dehydration

• Dehydration


  The process of removing intercellular and extracellular water from the tissues
• Dehydration process

  1. Placing the specimen in increasing or ascending grades/concentration of alcohol

  2. Done at room temperature (RT) only
• Tissue types

  • Delicate tissues (Embryonic tissue)
  • Routine tissues
• Initial alcohol concentration for delicate tissues

  Starts at 30% ethyl alcohol onwards in increasing or ascending grades of alcohol
• Initial alcohol concentration for routine tissues

  Starts at 70% ethyl alcohol to 95% ethyl alcohol to 100% ethyl alcohol
• Characteristics of a good dehydrating agent

  • Dehydrates rapidly
  • Does not evaporate very fast
  • Dehydrates fatty tissues
  • Does not harden tissues excessively
  • Does not remove stains
  • Not toxic to the body
  • Not a fire hazard
• Types of dehydrating agents

  • Alcohol
  • Acetone
  • Dioxane
  • Cellosolve
  • Triethyl phosphate
  • Tetrahydrofuran (THF)
• Alcohol dehydrating agents

  • Ethyl alcohol (Ethanol)
  • Methyl alcohol (Methanol)
  • Butyl alcohol (Butanol)
  • Tertiary butanol
  • Pentanol
  • Isopropyl alcohol (Isopropanol)
• Ethyl alcohol (Ethanol)


  • Best dehydrating agent, fast acting, clear, colorless, cheap, not poisonous but flammable, miscible with many solvents, penetrates tissue easily, recommended for routine tissue dehydration
• Methyl alcohol


  • Primarily used for blood tissue films and smear preparations, but toxic and can cause blindness and death
• Butyl alcohol (Butanol)


  • Used in plant and animal micro techniques, slow but produces less shrinkage of tissue, has a rancid, sweet odor
• Acetone


  • Cheap, rapid acting (30 minutes to 2 hours), clear, colorless, volatile, highly flammable, has poor penetration and can cause brittleness and shrinkage, only used for small pieces of tissue, capable of removing lipids
• Dioxane


  • Excellent universal solvent, compared to alcohol produces less shrinkage, miscible with water, paraffin, alcohol, and xylene, specimens tend to ribbon poorly, extremely dangerous and very expensive, but tissues can be stored for longer periods without affecting consistency and staining properties
• Cellosolve


  • Dehydrates rapidly, can be stored for months without producing tissue distortion or hardening, but highly toxic causing problems with reproductive, fetal, urinary, and blood systems
• Triethyl phosphate


  • Dehydrates or removes water very readily, produces very little distortion, shrinkage, and hardening, used to dehydrate sections and smears following certain stains
• Tetrahydrofuran (THF)


  • Universal solvent, miscible in both water and paraffin, dissolves many substances including fats, has an offensive ether-like odor, toxic and an eye and skin irritant
• Universal solvents

  • Tertiary butanol
  • Dioxane (Diethylene dioxide)
  • Tetrahydrofuran (THF)
• Anhydrous copper sulfate is used to ensure complete dehydration by removing water content from the dehydrating fluid
• Blue discoloration of anhydrous copper sulfate crystals indicates full saturation of dehydrating fluid with water
• Concentrations of alcohol above 80% make tissues hard, brittle, and difficult to cut, while concentrations below 70% macerate/soften the tissue
• Prolonged storage of tissues in alcohol can interfere with their staining properties
• Dehydration at 37°C speeds up or hastens the process
• Graupner's method uses pure dioxane and paraffin wax, Weiseberger's method uses dioxane and anhydrous calcium oxide (quicklime)