Lec 3: Clearing & infiltration Flashcards
It is the appearance of tissue after it has been treated with clearing solutions to remove the dehydrating agent
Clearing
A process of tissue preparation wherein the dehydrating agent is replaced with a clearing agent
Clearing
Characteristics of a clearing agent (6)
- removes alcohol
- high refractive index
- miscible with wax & mounting medium
- miscible with ethanol & wax
- removes fat
- NOT less than 10 times the volume of the tissue
The clearing agent must be _____ times the volume of the tissue
NOT be less than 10 times
ADVANTAGE:
• most rapid
• makes tissue transparent
• miscible with abs. alcohol & paraffin
• does NOT extract aniline dyes
• doesn NOT dissolve celloidin (mounting)
• evaporates quickly in paraffin oven
DISADVANTAGE:
• flammable
• toxic & narcotic (high conc.)
XYLENE
- most common
XYLENE
1. What causes it to become milky?
2. How long does it take to brittle and harden the tissue?
3. Clearing time.
4. TRUE or FALSE: Xylene can be used to replace wax.
5. TRUE or FALSE: Xylene can be used for celloidin sections because it dissolves celloidin.
- When an incompletely dehydrated tissue is immersed in Xylene.
- Longer than 3 hours
- 15 to 30 minutes
- TRUE
- FALSE
ADVANTAGE
• miscible with absolute alcohol and paraffin
• NOT carcinogenic
• clears overnight
• for routine purposes
DISADVANTAGES
• tends to acidify (partially filled vessel)
• emit toxic fumes at high conc.
Toluene
ADVANTAGES
• miscible with absolute alcohol
• makes tissues transparent
• does NOT brittle & harden
DISADVANTAGE
• flammable
• toxic & carcinogenic
Benzene
BENZENE
1. Clearing time
2. Why is it easily eliminated from the tissue?
- 15 to 60 minutes
- Benzene volatilizes rapidly in paraffin oven
• miscible with absolute alcohol
• for tough tissues
• for nervous tissues, lymph nodes, embryos
• for large tissue specimen
• NOT flammable
• tissues tend to float
Chloroform
CHLOROFORM
1. Clearing time
2. It is toxic to what organ after prolonged inhalation?
3. How to avoid tissues from floating?
4. TRUE or FALSE: Chloroform is volatile in paraffin oven.
5. TRUE or FALSE: Chloroform evaporates quickly in a water bath
- 6 to 24 hours
- Toxic to Liver
- Wrap tissue with absorbent cotton gauze to facilitate sinking.
- FALSE.
» Chloroform is NOT volatile in paraffin oven. - TRUE.
• miscible with 96% absolute alcohol
• does NOT dissolve aniline dyes
• extremely slow
• very expensive
• milky
• hard to eliminate in paraffin bath
• causes drying out of tissue
• produces crystals at 35°C
Cedarwood Oil
CEDARWOOD OIL
1. How long does it take to clear celloidin?
2. What to do when Cedarwood oil is hard to eliminate in paraffin bath?
3. How to prevent drying out of tissue?
4. What to do when crystals are produced?
5. The reasons it becomes milky.
- 5 to 6 days
- Transfer tissue from oil to benzene for 1½ hour, then place tissue in wax.
- Superimpose absolute alcohol on clearing agent’s surface. Once saturated, transfer specimen to fresh Cedarwood.
- Heat solution up to 200°C to dissolve crystals
-
a. Cedarwood was not filtered before used
b. Prolonged storage
- aniline dyes are removed
- celloidin is dissolved
Clove oil
- similar to Chloroform
- tissue hardening
- dangerous to inhale & prolonged exposure
Carbon Tetrachloride
- dehydrating and clearing agent
- non toxic
- offensive odor
Tetrahydrofuran
- miscible with water & paraffin
- changed 3 times within 4 hours
- toxic to liver
- maybe embedded with paraffin within 4 hours after fixation
- maybe transferred to dioxane straight from Bouin’s fluid or a formalin fixative
Dioxane
The complete removal of clearing agent by substitution of impregnating solution
Impregnation/Infiltration
Characteristics of an Impregnating Reagent
- empty spaces of tissue and cells
- hardens tissue
- at least 25 times the volume of the tissue
Types of Tissue Impregnation
- paraffin wax
- celloidin wax
- gelatin
- cut with ease without distortion
- nucleic acids may be recovered decades after fixation
- many staining procedures are permitted
- overheating makes specimen brittle
- inadequate impregnation makes tissue soft & shrunken
- not recommended for fatty tissues
Paraffin wax
• simplest, most common, best routinely used
Ways of paraffin wax impregnation (3)
- manual processing
- automatic processing
- vacuum embedding
PARAFFIN WAX IMPREGNATION
1. Automatic processing example
2. Vacuum embedding example
- Autotechnicon
- Vacuum oven, vacuum fume
PARAFFIN WAX IMPREGNATION
1. in vacuum embedding, complete impregnation is reduced by ___%.
2. This hastens the removal of air bubbles & clearing agent
3. Used for the complete removal of solvents, and for urgent biopsies & delicate tissues.
- 25% to 75%
- Vacuum
- Vacuum embedding
An automated system to process tissue specimen from fixation down to infiltration
Autotechnicon
A purified form of nitrocellulose
Celloidin
- for specimens with large hollow cavities
- for hard and dense tissues (bones and teeth)
- for large tissue sections of whole embryos
- for neurological tissues
Celloidin
- does not require heat
- very slow
- unable to cut tissue into very thin sections
- flammable vapor
- very volatile
Celloidin
- recommended when dehydration is to be avoided
- water soluble
- for delicate specimens & frozen tissue sections
- does NOT require clearing
Gelatin
GELATIN
1. Function of phenol
2. Tissue should not be ____ thick.
- Prevents mold formation
- NOT be more than 2-3 mm
GELATIN: Steps
After the fixative has been washed out, the tissue is placed in:
- 10% gelatin with 1% phenol for 24 hours
- Transfer it to 20% gelatin with 1% phenol for 12 hours
- Transfer it to 20% gelatin with 1% phenol
- Cooled in refrigerator until impregnation and embedding are completed
- Recommended for embryos, insects, and very delicate specimens
- can clear 70% alcohol
Aniline Oil
