HISTOPATH LAB FINALS TOPIC 1
CLEARING
• REMOVAL OF DEHYDRATING
SOLUTIONS, MAKING THE TISSUE
COMPONENTS RECEPTIVE TO THE
INFILTRATING MEDIUM
• AFTER STAINING → TRANSPARENT
TISSUE
•GLYCERIN AND GUM SYRUP→USED
WHEN THE TISSUE IS TO BE CLEARED
DIRECTLY FROM WATER→FROZEN
SECTION→NO DE- ALCOHOLIZATION
IN INVOLVED.
CHARACTERISTICS
OF
GOOD
CLEARING AGENTS
 Should be miscible with alcohol to
promote rapid removal of the
dehydrating agent from the tissue
 It should be miscible with and easily
removed by melted paraffin wax
and/or by mounting medium to
facilitate impregnation and mounting
of sections.
 Should not produce excessive
shrinkage, hardening or damage of
tissue Should not dissolve out aniline
dyes
 Should not evaporate quickly in a
water bath
 Should make tissues transparent
CLEARING AGENTS
• XYLENE
• TOLUENE
• BENZENE
• CHLOROFORM
•CEDARWOOD OIL
• ANILINE OIL
• CLOVE OIL
• CARBON TETRACHLORIDE
• METHYL BENZOATE
• METHYL SALICYLATE
• THF
XYLENE (XYLOL)
ADVANTAGES
• MOST RAPID
• MOST COMMON
• COLORLESS
• CHEAP
• MAKE TISSUE TRANSPARENT
•
MISCIBLE
WITH
ABSOLUTE
ALCOHOL AND PARAFFIN WAX
• CLEARING TIME → 30 MINUTES – 1
HOUR
DISADVANTAGES
• HIGHLY FLAMMABLE
• > 3HOURS → HARD/BRITTLE
TISSUES
•HARDENS/SHRINKS TISSUES → NOT
FOR NERVOUS SYSTEM & LYMPH
NODES
• CARCINOGENIC
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 HERE FOR 24 HOURS
• NOT CARCINOGENIC
• CLEARING TIME → 1 – 2 HOURS
DISADVANTAGES
•SLOWER
THAN
XYLENE AND
BENZENE
•HIGHLY CONCENTRATED SOLUTIONS
WILL EMIT FUMES THAT ARE VERY
TOXIC UPON PROLONGED EXPOSURE
• MORE EXPENSIVE
BENZENE
ADVANTAGES
• PENETRATES AND CLEARS TISSUES
RAPIDLY
• RAPID ACTING → 15 – 60 MINUTES
CLEARING TIME
• DOES NOT MAKE TISSUES HARD AND
BRITTLE
• VOLATILIZES RAPIDLY IN PARAFFIN
OVEN → EASILY ELIMINATED FROM
THE TISSUE
DISADVANTAGES
• HIGHLY FLAMMABLE
•TISSUE SHRINKAGE IF
LEFT FOR A LONG TIME
•CARCINOGENIC;
MAY
DAMAGE
BONE MARROW RESULTING TO
APLASTIC ANEMIA
CHLOROFORM
ADVANTAGES
• CLEARING TIME → 6 – 24 HOURS
• RECOMMENDED FOR TOUGH
TISSUES
• SUITABLE FOR LARGE TISSUE
SPECIMEN
• NOT INFLAMMABLE
DISADVANTAGES
• TOXIC TO LIVER → PROLONGED
INHALATION
• does not make tissue transparent
• NOT VERY VOLATILE IN PARAFFIN
OVEN→Difficult to remove FORM
PARAFFIN SECTIONS
• EVAPORATES QUICKLY FROM A
WATER BATH
CEDARWOOD OIL
ADVANTAGES
• USED TO CLEAR BOTH PARAFFIN
AND CELLOIDIN SECTIONS
• RECOMMENDED FOR CNS TISSUE,
CYTOLOGICAL STUDIES, SMOOTH
MUSCLE AND SKIN
• VERY PENETRATING
• CLEARS CELLOIDIN IN 5-6 DAYS
• CLEARING TIME → 2 – 3 DAYS
• DOES NOT DISSOLVE OUT ANILINE
DYES
DISADVANTAGE
• VERY SLOW CLEARING AGENT
• QUALITY IS NOT ALWAYS UNIFORM
AND GOOD
• BECOMES MILKY UPON PROLONGED
STORAGE AND SHOULD BE FILTERED
BEFORE USE.
• VERY EXPENSIVE
ANILINE OIL
 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 /
DIFFICULT
•TISSUES BECOME BRITTLE
•EXPENSIVE
CARBON TETRACHLORIDE (CCL4)
•PROPERTIES AND DISADVANTAGES
ARE LIKE
CHLOROFORM BUT CHEAPER
•HEPATOTOXIC
TETRAHYDROFURAN (THF)
•DEHYDRATING
AND
CLEARING
AGENT
•NON-TOXIC BUT WITH OFFENSIVE
ODOR AND SHOULD BE USED IN A
WELL-VENTILATED ROOM
METHYL
BENZOATE/
METHYL
SALICYLATE
•SLOW ACTING – USED FOR DOUBLE
EMBEDDING