Clearing Flashcards
process whereby alcohol or a dehydrating agent is removed from the tissue and replaced with a substance that will dissolve the wax with which the tissue is to be impregnated (e.g. paraffin) or used as the medium on which the tissue is to be mounted (e.g. Canada balsam).
Clearing
most commonly used clearing agent
xylene
are used when the tissue is to be cleared directly from water, as in
a frozen section.
Glycerin and gum syrup
has a lower boiling point than xylene but takes longer than the latter to clear
chloroform
affects the speed of penetration of the clearing agent
Viscosity
What will happen to prolonged exposure of most clearing agents?
tissue become brittle therefore more difficult to cut
- colorless clearing agent that is most commonly used in histology
laboratories. - Clearing time is usually 1/2 to 1 hour
- used for clearing, both for embedding and mounting procedures
Xylene (Xylol)
example of toxicities believed to be caused by intermediate product of xylene metabolism
methyl benzaldehyde
What will happen if xylene is used longer than 3 hours?
makes tissue excessively hard and brittle
What happens to xylene when an incomplete dehydrated tissue is immersed?
milky
better at preserving tissue structure and is more tolerant of small amounts of water left behind in the tissues than xylene
Toluene
Why is xylene more commonly used than toluene
Toluene is expensive and more toxic
What is the time recommended for clearing Toluene?
1-2 hours
What are the different Clearing Agents?
- Xylene
- Toluene
- Benzene
- Chloroform
- Cedarwood Oil
- Aniline Oil
- Clove Oil
- Carbon Tetrachloride
- Tetrahydrofuran
- Dioxane
What are the other xylene substitutes?
- Terpenes
- Limonene
- Orange oil based clearing agents
- Chlorinated hydrocarbons
- Coconut oil
- Bleached palm oil
- preferred by some as clearing agent in the embedding process of
tissues because it penetrates and clears tissues rapidly - used to be a popular routine clearing agent until recently when its highly carcinogenic properties were recognized.
Benzene
How many minutes does benzene clear the tissue in urgent biopsies?
15-60 minutes
Benzene is carcinogenic and may damage the bone marrow resulting in?
Aplastic anemia
- slower in action than xylene, but causes less brittleness
- Thicker tissue blocks, even those up to 1 cm in thickness, can be processed.
- tissues do not become translucent
Chloroform
How to avoid tissues floating in chloroform?
wrapping the tissues with absorbent cotton gauze to facilitate sinking of the section in solution
- used to clear both paraffin and celloidin sections during the embedding process
- recommended for central nervous system
tissues and cytological studies, particularly of smooth muscles and skin. - requires two changes in clearing solution
Cedarwood oil
How long does it usually take to complete the clearing process in Cedarwood oil?
2-3 days
How long does Cedarwood oil clears celloidin?
5-6 days
How to improve or hasten the slow process of impregnation in using Cedarwood oil?
transferring the specimen from oil to benzene for 1/2 hour before finally placing the tissue in wax
Tissues cleared in cedarwood oil initially float before gradually staying to the bottom as clearing proceeds hence tissue may dry out before it is completely cleared. How to prevent this?
superimposing absolute alcohol on the surface of the clearing agent, once saturated, the specimen should then be transferred
to a fresh solution of cedarwood oil.
What happens to Cedarwood oil if prolonged storage?
milky
Cedarwood oil that has been previously used to clear acetic-alcohol fixed tissues may produce crystals with a melting point of approximately 35°C and therefore interfere with adequate clearing of tissue. What can you do to restore the solution to its normal state?
The solution must be heated to 200°C in order to dissolve the crystals
not normally utilized as a routine clearing agent but it is recommended for clearing embryos, insects and very delicate specimens, due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening.
Aniline oil
- causes minimum shrinkage of tissues
- its quality is not guaranteed due to its tendency to become adulterated.
- Wax impregnation after clearing is slow and difficult
- Tissues become brittle, aniline dyes are removed, and celloidin is dissolved
Clove oil
- its properties are very similar to that of chloroform although it is relatively cheaper
- its disadvantage is the same as that of chloroform
- produces considerable tissue hardening, and is dangerous to inhale on prolonged exposure due to its highly toxic effects.
Carbon tetrachloride
- superior to ordinary dehydrating and clearing agents due to its ability to perform two processes at the same time, thereby shortening the total processing time and allowing more time for fixation
- It is non-toxic but has offensive odor and should be used in a well-ventilated room.
Tetrahydrofuran
- miscible both with water and paraffin
- used primarily when time is important because the tissues may be embedded with paraffin within 4 hours after fixation
- is changed 3 times within 4 hours and the tissues are transferred directly to paraffin (3 changes are made in a total of 90 minutes).
- causes greater shrinkage than xylene does
- dangerous (fumes are toxic to human especially to the liver)
Dioxane
- are isoprene polymers found in essential oils originally derived from plants, though some are now synthesized.
- clear tissues and celloidin sections from 80%-95% alcohol, render tissues transparent and have a slow gentle non-hardening action
- safe but have strong odors
- moderately effective solvents, but considered toxic
- dry slowly, leave an oily residue on slides, and expensive
Terpenes
- one of the recommended xylene substitutes from the terpene family
- a volatile oil found in citrus peels
- natural oil found in the skins of citrus fruits, such as lemons or oranges, and in cooking is usually referred to as lemon or orange zest
- obtained industrially by the steam distillation of orange peel
- a clear, colorless fluid with a distinctly citrus aroma
- does not mix well with mounting medium dissolved in xylene or toluene
Limonene
- offer the clearing action with the lowest hazard rating of all xylene alternatives
- excellent for preserving fine tissue structure, and can often be used in place of xylene with no alteration of protocol
- if neither pure nor stable can break down to produce compounds which will interfere with staining procedures
Orange oil based clearing agents
- can be effective solvents, but they are considered toxic chemicals, posing serious health risks
- Government regulations have restricted most of the effective solvents in this class
Chlorinated hydrocarbons
- an efficient substitute for xylene, as it is non-hazardous, less
expensive and causes less shrinkage of the tissue - can be used as a dealcoholization agent without losing the
quality - have the tendency to get solidified at a lower temperature but can be overcome by performing cleaning procedure in an incubator, maintaining the required temperature
Coconut oil
- gives good tissues, sections and histological slides
- nontoxic, nonhazardous, nonflammable, bio-degradable, economic, easy to handle, and readily available
Bleached palm oil
