🫀MIDTERM HPCT DEHYDRATION AND CLEARING

• to remove fixative and water from the tissue


  aim of dehydration
• ➢ 65-75% EtOH -> 95% EtOH -> 100% Absolute Alc


  Increasing strength of alcohol
• Ethanol


  for routine dehydration of tissues.

  ▪ BEST DEHYDRATING AGENT
• Methyl alcohol


  employed for blood and tissue films

  ▪ can cause blindness (optic blindness)
• Butyl alcohol


  utilized in plant & animal micro techniques
• Industrial methylated spirit

  (denatured alcohol

  ethanol + small amount of methanol
• Industrial methylated spirit

  (denatured alcohol


  used in the same way as ethanol
• Acetone

  ​

  BOTH fixative and dehydrating agent.
• Acetone


  for: Brain or CNS
• Dioxane (Diethylene dioxide)

  Cellosolve (Ethylene glycol monoethyl ether)

  THF (Tetrahydrofuran)

  BOTH dehydrating and clearing agent
• Triethyl phosphate


  Used to dehydrate smears producing minimal shrinkage
• Cellosolve (Ethylene-glycol-monoethyl-ether)


  Fast dehydrating

  - Disadvantage: combustible at 110-120F
• GAUPNER'S METHOD:


  3x changes (both dehydration and clearing)
• WEISEBERG METHOD


  wrap the tissue with gauze (add dioxane +

  anhydrous calcium oxide)
• Dioxane (Diethylene dioxide)


  excellent dehydrating and clearing agent
• Dioxane (Diethylene dioxide)


  Tissues can be left for long periods of time without affecting the

  staining properties of the specimen
• THF (Tetrahydrofuran)


  Eye and skin irritants and has offensive odor
• 4% phenol (tissue softener) + each 95% ethanol baths


  acts as a tissue softener for hard tissues such as tendons, nails, or

  dense fibrous tissues.
• Anhydrous copper sulfate


  can act as BOTH dehydrating agent and an indicator of water

  content of the last bath (100% ETOH).
• Anhydrous copper sulfate and xylene


  indicator for dehydration/ pressence of water
• Xylene


  - Clearing agent

  - Indicator for dehydration
• CLEARING AGENT


  should be miscible with alcohol to remove dehydrating agent from the

  tissue
• (Xylene and Benzene)


  Carcinogenic
• XYLENE/XYLOL


  MOST COMMONLY USED clearing agent in routine procedures
• XYLENE/XYLOL


  Used for embedding and mounting procedure
• XYLENE/XYLOL


  clearing Used for urgent biopsies
• 30 - 60 MINUTES


  XYLENE/XYLOL

  Time for clearing
• 1-2 hours


  Time for clearing

  TOLUENE

  ​
• TOLUENE

  ​

  Substitute for xylene or benzene
• BENZENE

  ​

  Recommended for URGENT BIOPSIES
• CHLOROFORM

  ​

  May cause HEPATOTOXICITY
• BENZENE

  ​

  Most Rapid clearing agent
• TOLUENE

  ​

  Not carcinogenic
• CEDARWOOD OIL


  Recommended for CNS TISSUE AND CYTOLOGY
• 6-24 hours


  Clearing time: CHLOROFORM

  ​
• (15-60 minutes)


  Clearing time: BENZENE

  ​
• 2-3 days


  Clearing time: CEDARWOOD DOIL
• ANILINE OIL


  Recommended for embryos, insects, and VERY DELICATE TISSUES
• CLOVE OIL


  Becomes adulterated
• CARBON TETRACHLORIDE


  Highly toxic upon prolonged exposure