LESSON 5: CLEARING Flashcards
otherwise referred to as fluidity
VISCOSITY
benign overgrowth usually composed of
muscle tissue and fibers
FIBROID
benzene ring with two methyl groups
XYLENE
methyl benzene
TOLUENE
CHCl4
CHLOROFORM
CCl4
CARBONTETRACHLORIDE
C6H6
BENZENE
substances that cause malignancy
CARCINOGENIC
combustible
FLAMMABLE
cyclic organic compounds
AROMATIC
✓ Removal of dehydrating agent from the tissues and replacing it by a
solvent → transparent & translucent tissue
✓ Not all dealcoholizing agents act as clearing agents
✓ Clearing agents only: glycerin, gum syrup and Brun’s solution
✓ Dealcoholizing agents only: chloroform and carbon tetrachloride
CLEARING
➢ Done after dehydration & before infiltration
➢ Solvent: dealcoholize and act as solvent of paraffin
➢ Agents: xylene, toluene, dioxane and chloroform
Clearing in Embedding (TISSUE PROCESSING)
➢ Done after staining & before mounting
➢ microscopic preparations transparent (use of solvents with high refractive
index)
➢ Agents must be solvents of the Mounting media: xylene, toluene, terpineol,
carbol-xylene
Clearing in Mounting
Refractive index of clearing agents: approximately equal to that of the tissues
For the purpose of making the tissues transparent so that their internal structure is demonstrable to the naked eye.
✓ Should be miscible with dehydrating agent and either infiltrating medium or mounting
medium
✓ Should remove alcohol quickly & clear quickly without overhardening the tissues
✓ Should not dissolve out aniline dyes
✓ Should not evaporate quickly in the water baths
✓ Evaporates quickly in paraffin oven
✓ Could be used in amounts at least 10Xthe volume of tissue
Characteristics of an Ideal Clearing Agent
Agents with low BP are readily replaced by
paraffin (Except chloroform)
leads to slow penetration
Higher viscosity
does not affect the rate but affects the quality of cleared tissue
Refractive Index
▪ Special considerations should be noted depending on the clearing agent used
▪ Some reagents will not clear tissues
Reagent Used
✓ Most rapid (15 - 30 mins/ 30 min – 1 hr)
✓ Excellent clearing agent but tends to make tissues excessively hard & brittle.
✓ Turns milky when dehydration is not complete
Xylene/Xylol
✓ Rapid agent (15 – 60 mins)
✓ Carcinogenic, causes aplastic anemia
Benzene
✓ Similar to xylene but does NOT harden tissues nearly so much
✓ Slower than xylene or benzene (1 – 2 hrs)
✓ Not carcinogenic but emits toxic fumes
Toluene/Toluol
✓ For nervous tissues, lymph nodes & embryos
✓ Tissue do not become translucent
✓ Best for large specimens (up to 1cm thick) and tough tissues
✓ Toxic to the liver on prolonged inhalation
✓ Tissues tend to float: remedy → wrap tissues with absorbent cotton gauze
Chloroform
✓ Recommended for CNS, smooth muscles & skin
✓ Slow (2 – 3 days); minimal shrinkage
✓ For both celloidin and paraffin sections
✓ Tissue floats – use Absolute alcohol to prevent drying out of tissues
✓ Must be followed by immersion in xylene or benzene to remove oil from tissues
✓ Turns milky on prolonged storage
Cedarwood oil
✓ Clears 70% alcohol
✓ Recommended for embryos, insects and delicate specimens
Aniline oil
✓ Slow; may be adulterated; removes aniline dyes
Clove Oil
✓ Similar to chloroform
CCl4
Both dehydrating and clearing
Dioxane
For large pieces of tissues & embryonic materials
Amyl acetate
Substitute for cedarwood oil
Terpineol
Used in double embedding.
Methyl
Benzoate/Salicylate
when the tissue is to be cleared directly from water (no dealcoholization,
it merely improves the RI)
Glycerin, Gum
syrup & Brun’s
solution
✓ For materials that are difficult to clear (eg. Thick mucinous
Papanicolaou smears)
✓ Should be thoroughly rinsed in xylene prior to mounting
Carbo - Xylene
✓ Oil of bergamot
✓ Phenol in alcohol
✓ Creosote
Others USED FOR CLEARING
✓ Based on limonene: a volatile oil found in citrus peels
✓ Clearite - Long chain aliphatic HC
Newer Clearing agents
Most commonly used and most rapid clearing agent
Xylene
- Rapid-acting and clears tissues without causing hardness or brittleness
- Causes aplastic anemia
Benzene
Used as a substitute for xylene and benzene
Toluene
- Versatile reagents as they can be used for different types of tissues
- Used for delicate as well as tough tissue samples
CHCl3 and CCl4
Recommended for CNS, smooth muscle and skin tissues
Cedarwood oil
organ affected by chloroform toxicity
LIVER
factor that does not affect clearing rate
R.I.
turns milky if dehydration is incomplete
XYLENE
clearing in _______; reagent should be
miscible in the stain
MOUNTING
BP of chloroform compared to xylene
LOWER
anemia caused by benzene
APLASTIC
clearing agent that is prone to adulteration
CLOVE OIL
also known as artificial oil of lilac
TERPINEOL
turns milky on prolonged storage
CEDARWOOD OIL
Aromatic oil used for insects
ANILINE
