CLEARING

• Clearing


  The process whereby alcohol or a dehydrating agent is removed from the tissue and replaced with a substance that will dissolve the wax with which the tissue is to be impregnated or used as the medium on which the tissue is to be mounted
• Clearing agent


  • Miscible with alcohol to promote rapid removal of the dehydrating agent from the tissue
  • Miscible with, and easily removed by melted paraffin wax and/or by mounting medium to facilitate impregnation and mounting of sections
  • Does not produce excessive shrinkage, hardening or damage of tissue
  • Does not dissolve out aniline dyes
  • Does not evaporate quickly in a water bath
  • Makes tissues transparent
• Factors affecting choice of clearing agent

  • Type of tissues to be processed, and the type of processing to be undertaken
  • Processor system to be used
  • Intended processing conditions such as temperature, vacuum and pressure
  • Safety factors
  • Cost and convenience
  • Speedy removal of dehydrating agent
  • Ease of removal by molten paraffin wax
  • Minimal tissue damage
• Commonly used clearing agents

  • Xylene
  • Toluene
  • Benzene
  • Chloroform
  • Cedarwood oil
  • Aniline oil
  • Clove oil
• Xylene


  • Colorless clearing agent most commonly used in histology laboratories
  • Clearing time is usually 1/2 to 1 hour
  • Used for clearing, both for embedding and mounting procedures
  • Generally suitable for most routine histologic processing schedules of less than 24 hours, and when the tissue block size is less than 5 mm. in thickness
• Advantages of xylene


  • Most rapid clearing agent, suitable for urgent biopsies
  • Makes tissues transparent
  • Miscible with absolute alcohol and paraffin
  • Does not extract out aniline dyes
  • Can be used for celloidin sections
  • Evaporates quickly in paraffin oven and can be readily replaced by wax
  • Cheap
• Disadvantages of xylene


  • Highly inflammable and should be appropriately stored
  • Makes tissues excessively hard and brittle if used longer than 3 hours
  • Causes considerable hardening and shrinkage of tissues, not suitable for nervous tissues and lymph nodes
  • Becomes milky when an incompletely dehydrated tissue is immersed in it
  • Irritates eyes, nose and respiratory tract, can be absorbed through skin and cause dermatitis, toxic and narcotic at high concentrations
• Special handling procedures and storage requirements for xylene

  • Keep container tightly closed to prevent xylene from subliming and entering the atmosphere
  • Only non-sparking tools may be used to handle xylene
  • Store in a cool and dry area away from incompatible substances
  • Store xylene in a flammable liquid storage cabinet
  • Wash hands thoroughly after handling xylene
  • Remove contaminated clothing and wash before reuse
  • Keep away from heat, sparks, flames, sources of ignition
  • Transport chemicals in closed containers, in the smallest amounts possible, and use aids such as carts, chemical transport carriers
  • Store chemicals below eye level
• Toluene


  • Better at preserving tissue structure and more tolerant of small amounts of water left behind in the tissues than xylene
  • Time recommended for clearing is 1-2 hours
• Advantages of toluene


  • Miscible with both absolute alcohol and paraffin
  • Acts fairly rapidly and recommended for routine purposes
  • Tissues do not become excessively hard and brittle even if left in toluene for 24 hours
  • Clears overnight
  • Not carcinogenic
• Disadvantages of toluene


  • Slower than xylene and benzene
  • Tends to acidify in a partially filled vessel
  • Highly concentrated solutions will emit toxic fumes upon prolonged exposure
  • More expensive
• Benzene


  • Preferred by some as clearing agent in the embedding process of tissues because it penetrates and clears tissues rapidly
  • Used to be a popular routine clearing agent until its highly carcinogenic properties were recognized, its use is now strongly discouraged
• Advantages of benzene


  • Rapid acting, hence recommended for urgent biopsies (15-60 minutes) and routine purposes
  • Volatilizes rapidly in paraffin oven and is easily eliminated from the tissue
  • Miscible with absolute alcohol
  • Does not make tissues hard and brittle
  • Causes minimum shrinkage
  • Makes tissues transparent
  • Clears overnight
• Disadvantages of benzene


  • Highly flammable
  • Excessive exposure may be extremely toxic and carcinogenic, can damage bone marrow resulting in aplastic anemia
  • If a section is left in benzene for a long time, considerable tissue shrinkage may be observed
• Chloroform


  • Slower in action than xylene, but causes less brittleness
  • Thicker tissue blocks, even up to 1 cm. in thickness, can be processed
  • Tissues placed in chloroform do not become translucent
• Advantages of chloroform


  • Recommended for routine work (6-24 hours)
  • Miscible with absolute alcohol
  • Recommended for tough tissues, nervous tissues, lymph nodes and embryos because it causes minimum shrinkage and hardening
  • Suitable for large tissue specimens
  • Not inflammable
• Disadvantages of chloroform


  • Relatively toxic to the liver after prolonged inhalation
  • Wax impregnation after chloroform clearing is relatively slow
  • Does not make tissues transparent
  • Difficult to remove from paraffin sections, may deteriorate the wax
  • Attacks the rubber seal used in vacuum impregnating bath
  • Complete clearing is difficult to evaluate
  • Tissues tend to float in chloroform
• Cedarwood oil


  • Used to clear both paraffin and celloidin sections during the embedding process
  • Especially recommended for central nervous system tissues and cytological studies
  • Requires two changes in clearing solution, clearing is usually complete in 2-3 days
• Advantages of cedarwood oil

  • Very penetrating
  • Miscible with 96% alcohol which it removes readily
  • Clears celloidin in 5-6 days
  • Causes minimal shrinkage of tissues
  • Tissues may be left in oil indefinitely without considerable damage and distortion
  • Does not dissolve out aniline dyes
  • Makes tissues transparent
  • Does not harden tissues
  • Does not interfere too seriously with paraffin penetration if not completely removed
  • Clearing often improves cutting of the sections
• Disadvantages of cedarwood oil

  • Extremely slow clearing agent, not recommended for routine purposes
  • Slightly slower in penetrating than benzene
  • Hard to eliminate from the tissues in paraffin bath, making the wax impregnation process very slow
  • Quality not always uniform, tissues may float and dry out before clearing is complete
  • Becomes milky upon prolonged storage and should be filtered before use
  • Crystals may form if used to clear acetic-alcohol fixed tissues
  • Very expensive
• Aniline oil


  Not normally utilized as a routine clearing agent, recommended for clearing embryos, insects and very delicate specimens due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening
• Clove oil


  • Causes minimum shrinkage of tissues, but quality not guaranteed due to tendency to become adulterated
  • Wax impregnation after clearing with clove oil is slow and difficult
• Cedarwood oil


  • Becomes milky upon prolonged storage and should be filtered before use
  • May produce crystals with a melting point of approximately 35°C when used to clear acetic-alcohol fixed tissues, which must be heated to 200°C to dissolve
• Cedarwood oil is very expensive
• Aniline oil


  Not normally utilized as a routine clearing agent but recommended for clearing embryos, insects and very delicate specimens, due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening
• Clove oil


  • Causes minimum shrinkage of tissues but its quality is not guaranteed due to its tendency to become adulterated
  • Wax impregnation after clearing with clove oil is slow and difficult
  • Tissues become brittle, aniline dyes are removed, and celloidin is dissolved
  • Expensive, making it unsuitable for routine clearing purposes
• Carbon tetrachloride


  • May be used in clearing tissues for embedding
  • Properties are very similar to that of chloroform
  • Produces considerable tissue hardening and is dangerous to inhale on prolonged exposure due to its highly toxic effects
• Tetrahydrofuran


  • Superior to ordinary dehydrating and clearing agents due to its ability to perform two processes at the same time, thereby shortening the total processing time and allowing more time for fixation
  • Non-toxic but has offensive odor and should be used in a well-ventilated room
• Dioxane


  • Miscible both with water and paraffin
  • Used primarily when time is important because the tissues may be embedded with paraffin within 4 hours after fixation
  • Causes greater shrinkage than xylene
  • Dangerous, with toxic fumes that are harmful to the liver
• Xylene substitutes


  • Limonene reagents
  • Mineral oil mixtures
  • 1.7% dish washing solution
  • Vegetable oils
  • Coconut oil
• Terpenes


  • Isoprene polymers found in essential oils originally derived from plants, though some are now synthesized
  • The earliest transition solvents to be used in histology and include turpentine and oils of bergamot, cedarwood, clove, lemon, oreganum and sandalwood
  • Generally regarded as safe though some have particularly strong odors which can be overpowering, requiring good laboratory ventilation
  • Moderately effective solvents, but considered toxic
  • Dry slowly, leave an oily residue on slides and are relatively expensive
• Limonene


  • A volatile oil found in citrus peels which goes by several trade names
  • A natural oil found in the skins of citrus fruits, such as lemons or oranges
  • Obtained industrially by the steam distillation of orange peel which is a byproduct of the orange juice industry
  • A clear, colorless fluid with a distinctly citrus aroma, not unpleasant to most people, although some do not like it
  • Often sold as a xylene replacement, but not universally successful as a clearant immediately prior to cover slipping due to compatibility issues with mounting media
• Orange oil based clearing agents

  • Offer the clearing action with the lowest hazard rating of all xylene alternatives
  • Excellent for preserving fine tissue structure, and can often be used in place of xylene with no alteration of protocol
  • Important to use a product which has been rigorously purified then stabilized, as orange oils that are neither pure nor stable can break down to produce compounds which will interfere with staining procedures
• Chlorinated hydrocarbons


  • Can be effective solvents, but are considered toxic chemicals, posing serious health risks
  • Government regulations have restricted most of the effective solvents in this class
• Coconut oil


  • An efficient substitute for xylene, as it is non-hazardous, less expensive and causes less shrinkage of the tissue
  • Can be used as a de-alcoholization agent in the histopathological laboratory, without losing the quality of the histological details
  • The only drawback is its tendency to get solidified at a lower temperature, which can be overcome by performing the clearing procedure in an incubator, maintaining the required temperature
• Bleached palm oil


  • An effective substitute for xylene as a clearing agent during tissue processing and as a dewaxing agent during staining
  • Nontoxic, nonhazardous, nonflammable, bio-degradable, economic, easy to handle, and readily available