clearing & dealcoholization

CLEARINGCLEARING

Jenivie Morillo JoseUniversity of Santo TomasNational Children’s Hospital

HISTOPATHOLOGYHISTOPATHOLOGY

& DEALCOHOLIZATION& DEALCOHOLIZATION

DEFINITION:

Process of removing alcohol from section of tissue by immersing them in ANTE MEDIUM

Tissue becomes transparent as a result of ante medium raising the REFRACTIVE INDEX

CLEARING / DEALCOHOLIZATION

MOST COMMON CLEARING AGENTS

CLEARING TIME: 15-60 minutes

BEST ROUTINE CLEARING AGENT for urgent

biopsies

Rapid in action

HIGHLY INFLAMMABLE, CARCINOGENIC

1. BENZENE

IDEAL CHARACTERISTICS:

Miscible with alcohol

Miscible with paraffin wax

Should not produce excessive tissue shrinkage and

hardening

Should not evaporate quickly in the water bath

Should make tissue transparent


CLEARING TIME: 6- 24 HOURS ONE BATH is needed Miscible with ABSOLUTE ALCOHOL Causes very little SHRINKAGE and hardening of tissues Suitable for LARGE TISSUE SPECIMENS, TOUGH TISSUES(skin, fibroid, decalcified tissues), for NERVOUS TISSUE, LYMPH NODES and EMBRYOS Slowly DISPLACES alcohol TOXIC to the LIVER

2. CHLOROFORM



ONE BATH is needed

Miscible with ABSOLUTE ALCOHOL

COLORLESS

For URGENT BIOPSIES

Rapid in action

Makes tissue TRANSPARENT

3. XYLENE(XYLOL)


NOT CARCINOGENIC

Cheap

HIGHLY INFLAMMABLE

VOLATILIZED rapidly in PARAFFIN OVEN

EXCESSIVE tissue shrinkage and

hardening

MILKY if INCOMPLETELY DEHYDRATED

3. XYLENE(XYLOL) *continuation*



ONE BATH is needed

Miscible with ABSOLUTE ALCOHOL

Rapid in action

NOT CARCINOGENIC

HIGHLY INFLAMMABLE

Shrinkage of tissues

4. TOULENE


For CENTRAL NERVOUS SYSTEM, SKIN and SMOOTH MUSCLE and CYTOLOGICAL WORKCLEARING TIME: Indefinite until clearTWO BATHS Miscible with 95% alcoholBEST CLEARING AGENTMinimal shrinkage and hardening of the tissuesExtremely slowExpensive

5. CEDAR OIL


For SKIN and SMOOTH MUSCLE

CLEARING TIME: Indefinite

TWO BATHS

Miscible with 95% alcohol

LITTLE shrinkage

Wax impregnation is SLOW and DIFFICULT

Makes tissue BRITTLE

6. CLOVE OIL


For SKIN and SMOOTH MUSCLE


TWO BATHS


NOT EXTRACT aniline dyes

Used with CELLOIDIN PREPARATION

Expensive

7. OIL OF BERGAMOT


Spanish hop oil or thyme oil or mixtures of these two, with or without turpentineFor SKINCLEARING TIME: IndefiniteTWO BATHS miscible with 95% alcoholDOESN’T DISSOLVE celloidinDISSOLVES ANILINE DYESExpensive

8. OIL OF ORIGANUM


methyl salicylate; artificial oil

For DELICATE TISSUES


TWO BATHS


9. OIL OF WINTERGREEN


HARDENING EFFECT OF ALCOHOL can

be avoided

Mainly for CELLOIDIN SECTIONS but must

be soaked by CHLOROFORM or XYLENE

before mounting

POOR DEHYDRANT

OXIDIZES easily on STANDING

9. OIL OF WINTERGREEN *continuation*


For SMOOTH MUSCLE


TWO BATHS

Minimal shrinkage of tissue

VERY INFLAMMABLE, with FOUL ODOR

Wax impregnation is SLOW and DIFFICULT

10. CARBON DISULFIDE


For ROUTINE work

Similar to CHLOROFORM

11. CARBON TETRACHLORIDE


For FRIABLE TISSUES

CLEARING TIME: up to 1 hour

Made by adding ANHYDROUS CRYSTALS

of PHENOL to XYLENE until no more

dissolves

Good for MOIST and HUMID CLIMATES

CORROSIVE

12. CARBOL XYLENE


For CELLULAR ORGANS, FRIABLE TISSUES, SKIN, ATHERMATOUS ARTERIES, HARD COLLAGENOUS SPECIMENS CLEARING TIME: 2-4 hours THREE BATHS Colorless liquid with 65°C (boiling point), -108.5°C (freezing point) and 0.8894 (density) Wide range solvent (including fats) Soluble in most solvents (lower alcohols, ketones, esters, chloroform, ether, acetone, benzene, zylene, dioxane, creosote)

13. TETRAHYDROFURAN


SUPERIOR to ORDINARY DEHYDRATING

CLEARING AGENTS (can perform 2 processes at

the same time)

SHORTENS the TOTAL PROCESSING TIME and

allowing MORE time for fixation

Causes CONJUNCTIVAL IRRITATION for about 6

MONTHS

OFFENSIVE ODOR

13. TETRAHYDROFURAN *continuation*


EXCELLENT for DELICATE MATERIAL

especially EYES

CLEARING TIME: 2-4 hours

TWO BATHS


Solution consists of 1 part XYLENE and 4

parts TERPINEOL

Expensive

14. TERPINEOL


For SMOOTH MUSCLE

CLEARING TIME: 2 hours

TWO BATHS

Good for PARAFFIN and CELLOIDIN

WORK

DEHYDRATES and CLEARS very quickly

VOLATILE

15. PHENOL


EXCELLENT CLEARING AND

DEALCOHOLIZATION AGENT

Cedar oil, gasoline and petroleum ether

cause the LEAST HARDENING of tissues

16. HIGH TEST”AVIATION LEAD FREE GASOLINE”

APPLICATIONS OF CLEARING IN HISTOPATHOLOGY:

1. To make tissue, embryos and parasite

transparent

2. Dealcoholization of tissue preparatory to

wax impregnation

3. Dealcoholization of stained sections prior

to mounting in PERMOUNT, CLARITE or

CANADA BALSAM

clearing & dealcoholization

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