CLEARING Flashcards
Is the process whereby alcohol or dehydrating agent is removed from the tissue and replaced with a substance that will dissolve the wax with which the tissue is impregnated or the medium on which the tissue is mounted (canada balsam)
CLEARING
appearance of tissue in clearing
translucent
the internal structures become visible to the naked eyes because..
Because of high refractive indices embryos and parasites become transparent, internal structures become visible to the naked eye.
what causes the tissue to become brittle and difficult to cut
prolonged exposure
____ are readily replaced by melted paraffin
low boiling points
affects the speed of penetration of clearing agent
viscocity
clearing characteristics
Miscible with alcohol, for rapid removal of dehydrating agent
Miscible and easily removed with melted paraffin wax for impregnation and mounting
Should not produce excessive shrinkage, hardening or damage of tissue
Should not dissolve out aniline dyes
Should not evaporate quickly in a water bath
Make tissue transparent
COMMON CLEARING AGENTS
Xylene
Toluene
Benzene
Chloroform
Cedarwood oil
Aniline oil
Clove oil
Carbon tetrachloride
Colorless, most commonly used
Time ½ - 1 hour
Most rapid, cheap
Urgent biopsies 15-30 minutes
Tissue transparent
Miscible with absolute alcohol and paraffin
Does not extract out aniline dyes
Does not dissolve celloidin
Evaporates quickly in paraffin oven, readily replaced by wax during impregnation and embedding
xylene
xylene disadvantage
Highly flammable,
Longer than 3 hours make tissue excessively brittle
Not suitable for nervous tissues and lymp nodes
Becomes milky when incomplete dehydrated tissue is immersed
TIME- 1-2 hours, for routine, NOT carcinogenic
Miscible with absolute alcohol and paraffin
Tissues do not become excessively hard and brittle even if left for 24 hours
Toluene
toluene disadvantage
Slower than xylene n benzene
More expensive
Acidify the partially filled vessels
Emit toxic fumes if high conc.
Clears 15-60 minutes, routine, makes tissue transparent
Rapid acting, urgent biopsies
Miscible with abs alcohol
Volatile, Easily eliminated
Minimum shrinkage and does not tissues hard and brittle
benzene
benzene disadvantage
Highly flammable, well ventilated room needed
Toxic, carcinogenic, may damage bone marrow (aplastic anemia)
Routine (6-24 hours)
Miscible with abs alcohol, large tissue specimens, not flammable
Recommended for tough and dense tissues (skin, uterus, fibroid and decalcified)
Nervous, lymph nodes, embryos (due to minimum shrinkage and hardening of tissues
Slow penetrating transition solvent
chloroform
chloroform disadvantage
TOXIC TO LIVER, expensive
WAX IMPREgnation is slow
Does not make tissue transparent
Difficult to remove from paraffin section, not volatile
Vapor attacks the rubber seal used in vacuum impregnating bath
Evaporates quickly from water bath
Tissue floats; to avoid-wrap it with absorbent cotton gauze
Requires 2 changes
Clears tissu from 95% alcohol
Recommended for dense tissues (uterus) CNS, cytologic studies, smooth muscles and skin
Tissues can remain indefinitely2-3 days
Clears celloidin 5-6 days
Does not dissolve out aniline dyes
Makes tissue transparent
cedar wood oil
cedar wood oil disadvantage
SLOW, becomes milky upon prolonged storage, very expensive, no uniform quality, turns milky upon prolonged storage
Expensive
Used oil can be restored by filtering then heating to 60 oC under vacuum for 30-60 minutes
For embryos, insects, very delicate specimens
aniline oil
Wax impregnation is slow, expensive
cause sminimum shrinkage
Tissues become brittle aniline dye is removed, celloidin wax is dissolve
clove oil
Properties similar to chloroform
Cheaper
Highly toxic
carbon tetrachloride
Slow-acting
Double embedding techniques
Chiefly used as nitrocellulose solvents in double embedding techniques
Amyl acetate
METHYL BENZOATE AND
METHYL SALYCILATE
Are isoprene polymers (natural plants & synthetic)
Biodegradable, not water soluble
Disposed by recycling or incineration
terpenes
Colorless, flammable reagents
Miscible with most organic solvents and paraffin
esters
Used as xylene substitute and nitrocellulose solvent
n-butyl acetate
Derived from citrus fruit,
Similar to the esters in clearing action and eliminating from wax
limonene
Clear, almost colorless mixture of isomers
Very low evaporation rate
Substitute for cedarwood oil
terpineol
