LESSON 5: CLEARING Flashcards
Removal of dehydrating agent from the tissues and replacing it by a solvent → transparent & translucent tissue
CLEARING
what are the clearing agents only?
glycerin, gum syrup and Brun’s solution
What are the dealcoholizing agents only?
chloroform and carbon tetrachloride
dealcoholize and act as solvent of paraffin
Solvent
Done after dehydration & before infiltration
Clearing in Embedding
Done after staining & before mounting
Clearing in Mounting
Complete the following:
Characteristics of an Ideal Clearing Agent
- Should be __________ with ______________ and either infiltrating medium or mounting medium
- Should ____________ & clear quickly without overhardening the tissues
miscible; dehydrating agent
remove alcohol quickly
Complete the following:
Characteristics of an Ideal Clearing
✓ Should not dissolve out _________
✓ Should not evaporate quickly in the _________
✓ Evaporates quickly in _________
✓ Could be used in amounts at least __________________
aniline dyes
water baths
paraffin oven
10Xthe volume of tissue
does not affect the rate but affects the quality of cleared tissue
Refractive Index
This clearing agents is the most rapid (15 - 30 mins/ 30 min – 1 hr)
Xylene/Xylol
✓ 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
Toluene/Toluol
✓ 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
Cedarwood oil
✓ 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 de-alcoholization, it merely improves the RI)
Glycerin, Gum syrup & Brun’s solution
For materials that are difficult to clear (eg. Thick mucinous Papanicolaou smears)
Carbo - Xylene
Should be thoroughly rinsed in xylene prior to mounting
Carbo - Xylene
a volatile oil found in citrus peels
Based on limonene:
Long chain aliphatic HC
Clearite
