Clearing, aka Dealcoholization, is the removal of dehydrating solutions, making the tissue components receptive to the infiltrating medium
removes components of alcohol on tissue for the paraffin wax to penetrate, because paraffin wax is not freely miscible
Xylene or Xylol makes microscopic tissue preparation transparent due to their high refractive index of refraction
Glycerin and Gum Syrup are used when the tissue is to be cleared directly from water
A good clearing agent should be miscible with alcohol to promote rapid removal of the dehydrating agent from the tissue
A good clearing agent should be miscible with and easily removed by melted paraffin wax and/or by mounting medium to facilitate impregnation and mounting of sections.
A good clearing agent should:
not dissolve out aniline dyes.
not evaporate quickly in a water bath.
make tissues transparent.
Dealcoholization can remove all kinds of alcohol, especially absolute alcohol
Deparaffinization is to dissolve paraffin wax on tissue.
Clearing agent and paraffin wax are freely miscible.
High Refractive Index
On mounting always soak stained slide on xylene, then put mounting media Canada balsam then cover slip to make tissue transparent.
Milky appearance = Incomplete Dehydration
Xylene/Xylol
ADVANTAGES:
Most rapid
Most common
Colorless
Cheap
Make tissue transparent
Miscible with absolute alcohol and paraffin wax
30 minutes – 1 hour
DISADVANTAGES:
Highly inflammable
Hardens/ Shrinks
Carcinogenic
In xylene/xylol, >3 Hours = hard or brittle tissues
Xylene/xylol hardens and Shrinks, which is why it is NOT for nervous systems and lymph nodes
Toluene
ADVANTAGES:
Substitute for xylene and benzene
Miscible both absolute alcohol and paraffin wax
Tissues do not become excessively hard and brittle even if left for 24hrs
Not carcinogenic
1-2 hrs
DISADVANTAGES:
Slower than xylene and benzene
Highly concentrated solution will emit fumes that are very TOXIC upon prolonged exposure
More Expensive
Benzene
ADVANTAGES:
Penetrates and clears tissues RAPIDLY
Rapid Acting = 15-60 minutes
Does not make tissues hard and brittle
DISADVANTAGES:
Highly flammable
Tissue shrinkage if left for a long time
Carcinogenic
Benzene volatilizes rapidly in paraffin oven = easily eliminated from tissue
Benzene is highly flammable, which is why it is stored in close cabinet, in a well-ventilated room
Benzene is carcinogenic, which may damage bone marrow resulting to Aplastic Anemia
Chloroform
ADVANTAGES:
6-24 hours
Recommended for tough tissues
Suitable for large tissue specimen
Not inflammable
DISADVANTAGES:
Does not make tissue transparent
Not very volatile in paraffin oven
Evaporates quickly from a water bath
Chloroform is an ingredient for making metamphetamine/ drugs
Cedarwood oil
ADVANTAGES:
Used to clear both paraffin and celloidin sections
Recommended for CNS tissue, cytological studies, smooth muscle, and skin
Very penetrating
Does not dissolve aniline dyes
DISADVANTAGES:
Very slow clearing agent
Quality is not always uniform and good
Very expensive
Cedarwood oil
Clears celloidin in 5-6 days
Paraffin wax clears in 2-3 days
Cedarwood oil becomes milky upon prolonged storage and should be filtered before use
Aniline oil is 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
wax impregnation here
slow and difficult
tissues become brittle
expensive
Carbon tetrachloride (CCL4) have properties and disadvantages like chloroform, but cheaper
Hepatotoxic and may cause damage to liver
Tetrahydrofuran is both a dehydrating and clearing agent; it is non-toxic but with offensive odor and should be used in a well-ventilated room
Tetrahydrofuran is much effective as a dehydrating agent than clearing because it has weak properties
Methyl benzoate or Methyl salicylate is slow acting and is used for double embedding
In Frozen section, no dealcoholization is involved
Chloroform is toxic to Liver because of prolonged inhalation