DEHYDRATION

• Dehydration


  Process of removing intercellular and extracellular water from the tissue following fixation and prior to wax impregnation
• Dehydration process


  1. Place fixed specimen in 70% ethyl alcohol in water

  2. Progress through 95% ethyl alcohol to 100% ethyl alcohol

  3. For delicate tissues, start with 30% ethanol
• It is important to keep the dehydration times as brief as possible to minimize the risk of extracting cellular constituents
• Characteristics of an ideal dehydrating solution

  • Dehydrate rapidly without producing considerable shrinkage or distortion of tissues
  • Not evaporate fast
  • Able to dehydrate even fatty tissues
  • Not harden tissues exclusively
  • Not remove stains
  • Not toxic to the body
  • Not a fire hazard
• Commonly used dehydrating agents

  • Alcohol (most common)
  • Acetone
  • Dioxane
  • 4-cellosolve
  • Triethyle phosphate
  • Tetrahydrofuran
• Alcohol


  Ethyl alcohol (ethanol) is the alcohol recommended for routine dehydration of tissues
• Methyl alcohol


  A toxic dehydrating agent, primarily employed for blood and tissue films and smear preparations
• Butyl alcohol


  A slow dehydrating agent, producing less shrinkage and hardening than ethyl alcohol, recommended for tissues which do not require rapid processing
• Alcohol dehydration process


  1. Tissue is passed through a series of progressively increasing concentrations of alcohol

  2. Concentrated alcohols (95% or absolute) tend to harden only the surface of the tissue

  3. 70% or lower concentrations of alcohol, gradually increased to 95%, is used to avoid unequal impregnation
• The strength of initial alcohol required will depend upon the size, and nature of each tissue and fixative used
• Smaller and more delicate tissues require lower concentrations and shorter intervals between changes of succeeding ascending grades of alcohol
• General schedule (time in hours) for alcohol dehydration

  • 10% formolsaline, Zenker or Helly's, Bouin's fluid, Susa, Carnoy/formol sublimate, Flemming's fluid, running water: 1-12 hours
  • Alcohol 30%: 1-6 hours
  • Alcohol 50%: 1-6 hours
  • Alcohol 70%: 1-12 hours
  • Alcohol 90%: 1-6 hours
  • Absolute alcohol: 1-6 hours
• A temperature of 37°C will hasten dehydration time and is especially used for tissue sections that require urgent examinations
• Anhydrous copper sulfate placed in the bottom of the container and covered with filter paper will accelerate dehydration by removing water from the dehydrating fluid
• Acetone


  A cheap, rapid-acting dehydrating agent utilized for most urgent biopses, dehydrating in 1/2 to 2 hours
• Acetone penetrates tissues poorly and causes brittleness in tissues that are placed in it for a prolonged period of time
• Most lipids are removed from tissues with acetone
• Acetone is not recommended for routine dehydration processes due to considerable tissue shrinkage
• Dioxane


  An excellent dehydrating and clearing agent, readily miscible in water, melted paraffin, alcohol and xylol, producing less tissue shrinkage than alcohol
• Dioxane is extremely dangerous, producing a cumulative and highly toxic action in humans, so it should not be used routinely
• Dioxane dehydration process


  1. 1st pure dioxane solution: 1 hour

  2. 2nd pure dioxane solution: 1 hour

  3. 3rd pure dioxane solution: 2 hours

  4. 1st paraffin wax: 15 minutes

  5. 2nd paraffin wax: 45 minutes

  6. 3rd paraffin wax: 2 hours

  7. Embed in mold and cool in water
• Tissues treated with a chromate fixative should be thoroughly washed in running tap water prior to treatment with dioxane to remove the chromate
• Cellosolve (ethylene glycol monoethyl ether)

  Dehydrates rapidly, tissue may be transferred from water to normal saline directly to cellosolve and stored in it for months without producing hardening or distortion
• Ethylene glycol ethers are combustible and toxic, so propylene-based glycol ethers should be used instead if possible
• Triethyl phosphate


  Removes water very readily and produces very little distortion and hardening of tissue, used to dehydrate sections and smears following certain stains
• Tetrahydrofuran (THF)


  A reagent that both dehydrates and clears tissues, causing less shrinkage and easier cutting of sections with fewer artifacts
• THF is toxic if ingested or inhaled, and vapors cause nausea, dizziness, headache and anesthesia, so it should be used in a well-ventilated room
• When phenol (4%) is added to each of the 95% ethanol baths, it acts as a softener for hard tissues such as tendon, nail, or dense fibrous tissue
• Hard tissues can also be immersed in a glycerol/alcohol mixture or in "molliflex"