Dehydration

• DEHYDRATION


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


  is the removal of water by evaporation from a solid, semi-solid or liquid.
• Solid tissues should NEVER be allowed to air dry
• Dehydration


  involves slow substitution of the water in the tissue with an organic solvent
• whatever dehydrating agent is used, the amount in each step should not be less than 10 times the volume of the tissue in order to ensure complete penetration of the tissue by the dehydrating solution.
• Ethyl alcohol


  he alcohol recommended for routine dehydration of tissues.
• Ethyl alcohol


  considered to be the best dehydrating agent because it is fast-acting, it mixes with water and many organic solvents, and it penetrates tissues easily. It is not poisonous and not very expensive.
• Methyl alcohol


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


  utilized in plant and animal micro-techniques, is a slow dehydrating agent, producing less shrinkage and hardening than ethyl alcohol and is recommended for tissues which do not require rapid processing.
• Tissues with a high water content such as embryo tissue would require a much shorter time
• Ethanol (ethyl alcohol)

  Boiling point 78.3° C
• Ethanol (ethyl alcohol)

  Still considered best dehydrating solution

  Can be used on eyes and embryos, if graded alcohols are used
• Butanol (butyl alcohol)

  Boiling point 117.7° C
• Butanol (butyl alcohol)


  Less shrinkage and hardening than with ethyl

  Miscible with paraffin
• Tertiary butanol (butyl alcohol)

  Boiling point 82.8° C
• Tertiary butanol


  Universal solvent—acts as dehydrating and clearing agent
• Isopropanol (isopropyl alcohol)


  Boiling point 82.3° C
• Isopropanol (isopropyl alcohol)


  Lillie considers it “the best all- around substitute for ethyl alcohol”
• Isopropanol (isopropyl alcohol)


  -Cannot be used in the celloidin technic since nitrocellulose is insoluble in it

  -Cannot be used for preparing staining solutions, since dyes are not soluble in it
• Pentanol (amyl alcohol)

  Boiling point 128° C
• Pentanol (amyl alcohol)


  Miscible with 90% alcohol, toluene and xylene

  Dissolves paraffin wax
• ACETONE

  Boiling point 56° C
• ACETONE


  cheap, rapid-acting dehydrating agent utilized for most urgent biopsies which it dehydrates in 1/2 to 2 hours
• Acetone is more miscible with epoxy resins than alcohol, but is highly flammable and requires considerable care in handling
• ACETONE


  Does not extract methylene blue and other dyes from stained sections
• DIOXANE (Diethylene Dioxide)

  Refractive index 1.42
• DIOXANE (Diethylene Dioxide)

  Boiling point 101.5° C
• DIOXANE


  excellent dehydrating and clearing agent readily miscible in water, melted paraffin, alcohol and xylol
• Graupner's Method

  (1st) pure dioxane solution 1 hour

  (2nd) pure dioxane solution 1 hour

  (3rd) pure dioxane solution 2 hours

  (1st) Paraffin wax 15 minutes

  (2nd) Paraffin wax 45 minutes

  (3rd) Paraffin wax 2 hours Embed in mold and cool in water.
• Weiseberger's method


  tissue is wrapped in a gauze bag and suspended in a bottle containing dioxane and a little anhydrous calcium oxide. Water is displaced from the tissue by dioxane and in turn absorbed by calcium oxide or quicklime. Dehydration period ranges from 3-24 hours.
• CELLOSOLVE (Ethylene glycol monoethyl ether)


  Boiling point 156.4° C
• Ethylene glycol ethers are combustible at 110-120°F and are toxic by inhalation, skin contact and ingestion
• TRIETHYLPHOSPHATE

  Boiling point 215° C
• TRIETHYLPHOSPHATE


  removes water very readily and produces very little distortion and hardening of tissue. It is soluble in alcohol, water, ether, benzene, chloroform, acetone and xylene. It is used to dehydrate sections and smears following certain stains and produces minimum shrinkage.
• TETRAHYDOFURAN


  reagent that both dehydrates and clears tissues since it is miscible in both water and paraffin

  can dissolve many substances including fats and is in itself miscible with lower alcohols, ether, chloroform, acetone, benzene and xylene.
• TETRAHYDOFURAN


  It may be used for demixing, clearing and dehydrating paraffin sections before and after staining