!

CLEARNING:
CLEARING
AGENT

DESCRIPTION

1. Benzene

" Routine work

2. Chloroform

" Routine work

" For nervous tissues, lymph
nodes and embryos

3. Xylene
(Xylol)

" Colorless
" For urgent biopsies

4. Toluene

" Routine work

5. Cedar Oil

" For CNS, skin, smooth muscle

and cytological work

6. Clove Oil

MISCIBILITY BATH
______

___

TIME
15-60 min.

6-24 hours
Absolute
alcohol
(74 O.P. spirit)

1
15-30 min.

Absolute
alcohol

15-60 min.
Indefinite
until clear

" For skin and smooth muscle

7. Oil of Bergamot
8. Oil of Origanum

" Spanish hop oil/Thyme oil or

both (with or without turpentine)
" For skin

9. Oil of
Wintergreen

" Methyl salicylate

" Artificial Oil
" For delicate tissues

10. Carbon disulfide

11. Carbon
tetrachloride

" For smooth muscles

" Routine work

95% alcohol

______

______

2
Indefinite

ADVANTAGES
"
"
"
"
"

Best for urgent biopsies

Rapid
Little shrinkage and hardening
For large tissue specimens
For tough tissues (e.g. skin,
fibroid and decalcified tissues)

"
"
"
"

Rapid
Makes tissue transparent
Not carcinogenic
Cheap

"
"
"
"
"

Rapid
Not carcinogenic
Penetrates very well
Best clearing agent
Minimal shrinkage and
hardening

DISADVANTAGES
" Highly flammable
" Carcinogenic
" Displaces the alcohol
" Toxic to liver (prolonged
inhalation)
" Highly flammable
" Volatilized rapidly in paraffin
" Excessive shrinkage and
hardening
" Milky: incomplete dehydrated
tissue
" Highly flammable
" Shrinkage
" Extremely slow
" Expensive

" Little shrinkage

" Wax impregnation: slow and

difficult
" Brittle tissue (prevention: mix
oil of bergamot)

" Does not extract aniline dyes

" Used with celloidin preparation

" Expensive

" Does not dissolve celloidin

" Dissolves aniline dyes

" Expensive

" Hardening is avoided

" Celloidin sections: must be
soaked by chloroform/xylene
before mounting

" Poor dehydrant (split by small

amounts of water)
" Oxidize on standing

" Minimal shrinkage

" Very flammable

" Foul odor
" Wax impregnation: slow and
difficult after use

" Little shrinkage and hardening

" Displaces the alcohol
" For large tissue specimens
" Toxic to liver (prolonged
" For tough tissues (e.g. skin,
inhalation)
fibroid and decalcified tissues)

A. Jimenez

!
12. Carbol-xylene

13. Tetrahydrofuran
14. Terpineol

" For friable tissues

" Anhydrous phenol crystals
+ xylene (until dissolved)
" For cellular organs, friable
tissues, skin, athermatous
arteries and hard collagenous
specimens
" 1 part xylene + 4 parts
terpineol

______

___

Up to 1
hour

_______

2-4 hours

95% alcohol

2-4 hours
2

15. Phenol

" For smooth muscles

_______

16. High Test

Aviaion Lead
Free Gasoline

" Excellent clearing agent

________

2 hours
___

DECOLORIZATION/CLEARING
- Process of removing alcohol from section of tissue by immersing in
ante-medium
CHARACTERISTICS OF A GOOD CLEARING AGENT:
- Miscible with alcohol
o Rapid removal of the dehydrating agent from tissue
- Miscible with paraffin wax and/or mounting medium
o Facilitate impregnation and mounting of sections
- Do not produce excessive tissue shrinkage and hardening
- Do not evaporate quickly in water bath
- Make tissue transparent
o Higher refractive index
CEDAR OIL, GASOLINE & PETROLEUM
" Least hardening of tissues

______

" Moist and humid climates

" Corrosive

" Dehydrating and clearing agent

" Shortens total processing time
(more for fixation)

" Fumes: conjunctival irritation

(after 6 months)
" Offensive odor

" Clears from 95% alcohol

" For delicate material (e.g. eyes)
" Good for paraffin and celloidin
work
" Dehydrates and clears quickly
_______________________

" Expensive
" Volatile
_______________________

TETRAHYDROFURAN (THF)
- Colorless liquid
- Boiling point:: 65 C
- Freezing point: -108.5 C
- Density: 0.8894
- Wide range solvent (inc. fats)
- Soluble in most solvents
o Lower alcohols
o Acetone
o Ketones
o Benzene
o Esters
o Xylene
o Chloroform
o Dioxane
o Ether
o Creosote
" Mixes with water! in all proportions forming: azeotrope
o Constant boiling point mixture (64 C)
! 9% THF
! 6% water
" Mixes with paraffin wax in all proportion

APPLICATIONS OF CLEARING IN HISTOPATHOLOGY:

" To make tissue, embryos and parasites transparent
" For dealcoholization of tissue preparatory to wax impregnation
" For dealcoholization of stained sections prior to mounting in:
o Permount
o Clarite
o Canada Calsam

A. Jimenez