Dehydration, and Clearing Flashcards
Process of removing intercellular & extracellular water from the tissue following fixation & prior to wax impregnation
dehydration
charac of an ideal dehydrating soln
- must dehydrate rapidly
- not evaporate very fast
- be able to dehydrate fatty tissues
- should not harden the tissue
- not toxic
- not be a fire hazard
- should not remove stains
commonly used dehydrating agents
Alcohol
Acetone
Dioxane
Cellosolve
Triethyl Phosphate
Tetrahydrofuran
ü for routine dehydration of tissues
ü Best dehydrating agent - fast acting
ü Not poisonous
ü Not expensive
Ethyl
ü toxic
ü Primarily employed for blood and tissue films and for smear preparation
Methyl
ü Utilized in Plant and Animal
Micro technique
ü Slow d.a.
ü recommend for tissues which do not require rapid processing
Butyl
Factors to considered in dehydration
- Size and nature of tissue
-30% - Type of Fixative used
- Temperature
- 370C - hasten dehydration time - Ratio
- not be less than 10X
ü Producing less shrinkage and hardening than ethyl alcohol
butyl
Effect Alcohol-Concentration
Ø 85%-95%
Ø above 80% -
Ø 95% or absolute alcohol
Ø Prolonged storage in lower concentration (below 70%)
- liable to produce considerable shrinkage and hardening of tissues leading to distortion
Ø 85%-95%
- make tissues hard brittle and difficultto cut
above 80%
tend to harden only the surface of the tissue while the deeper parts are not completely penetrated
95% or absolute alcohol
Rapid in action but penetrates tissues poorly and causes brittleness in tissues that are prolonged dehydrated.
acetone
Prolonged storage in lower concentration tends to macerate the tissue
(below 70%)
Tissue is passed through a series of progressively increasing concentrations of alcohol.
70% Alcohol 6 hours
95% Alcohol. 12 hours
100% Alcohol 2 hours
100% alcohol 1 hour
100% alcohol 1 hour
A typical dehydration sequence for specimens not more than 4mm thick
70% ethanol 15 min
90% ethanol 15 min
100% ethanol 15 min
100% ethanol 15 min
100% ethanol 30 min
100% ethanol 45 min
Cheap, rapid acting dehydrating agent.
Ø Dehydrates in __
Ø Clear, colorless highly flammable and extremely volatile fluid.
Ø Produces considerable tissue shrinkage
Ø Not recommended for routine dehydration purposes.
Acetone
1⁄2 to 2 hours
Excellent dehydrating and clearing agent
Ø Produces less tissue shrinkage
Ø tend to ribbon poorly
Ø Expensive and extremely dangerous
Dioxane (Diethyl dioxide)
Dehydrates rapidly
The tissue may be transferred from__ or __ directly to it and stored in it for
months without producing hardening or distortion.
Ø CAUTION: is combustible at __ and is toxic.
Ø __ should be used instead.
cellosolve ( Ethylene glycol Monoethyl ether)
water or normal saline
- 110°Fto120°F
- Propylene based glycol ether
Removes water
Ø Produces very little distortion and hardening of tissues
Ø Soluble in __,__,__,__,__,__
triethyl phosphate
- Alcohol, Water, Ether, Benzene, Chloroform Acetone and Xylene
Ø lt may be used for , _ and _ paraffin sections before and after staining.
Ø It causes less shrinkage and easier cutting of sections with fewer artefacts.
Ø It does not dissolve __
Ø It is toxic if ingested or inhaled
Ø Vapors causes ,,,_
tetrahydrofuran
demixing, clearing and dehydrating
- aniline dyes
nausea, dizziness, headache and anesthesia
Additives to Dehydrating Agents
4% Phenol
Glycerol alcohol mixture or in “Molliflex
is added to each 95% ethanol baths as part of dehydration process
- it acts as a softener for hard tissues
4% Phenol
Ø is commonly accomplished using __ as a dehydrating solvent and __ as a transition fluid.
Ø Both solvents have some undesirable properties:
Ø __ is a good substitute for propylene
Ø non-carcinogenic, less toxic and not as flammable as propylene oxide
Ø excellent dehydrating agent
Tissue processing for transmission electron microscopy (TEM)
- ethanol
- propylene oxide
- Acetonitrile
is the transition step between dehydration and infiltration with the
embedding medium.
Clearing
Process whereby alcohol is removed from the tissue & replaced with a substance that will dissolve the wax with which the tissue is impregnated (__) or the medium on which the tissue is to be mounted. (_)
De-alcoholization
Paraffin
Canada Balsam
Most commonly used clearing agents are __,__,__,__
The clearing agent will make microscopic tissue preparations transparent due to their high index refraction.
xylene, dioxane, chloroform and cedarwood oil
charac of good clearing agents
Miscible with alcohol to promote rapid removal for the dehydrating agent
Should be miscible with and easily removed by melted paraffin wax. Should not produce excessive shrinkage, hardening or damage of tissue
Should not dissolve out aniline dyes
Should not evaporate quickly in a water bath
Should make tissues transparent
common clearing agents used
v Xylene (30 mins – 1 hr)
v Toluene
v Benzene
v Chloroform
v Cedarwood oil
v Aniline oil
v Clove oil
v Carbon Tetrachloride
Ø Colorless clearing agent that is most commonly used. Most rapid clearing agent, suitable for urgent biopsies
Ø Clearing time: __
Ø Makes tissues transparent
Ø Does not extract _
Ø Can be used for_ because it does not dissolve it
Ø It is cheap.
xylene
1⁄2 to 1 hour
aniline dye
celloidin sections
Ø Clearing time:__
Ø Acts fairly rapidly and is recommended for routine purposes
Ø Tissues do not become excessively hard and brittle even if left for 24 hrs
toluene
1 – 2 hours
Ø Clearing time: __
Ø Does not make tissues hard and brittle but it causes minimum shrinkage
Ø It makes tissues transparent.
Ø It is highly flammable
Ø If section is let in it for a long time, considerable tissue shrinkage
may be observed
Ø Excessive exposure is toxic and carcinogenic to human.
Ø It may damage the bone marrow resulting in _
Benzene
15 – 60 minutes
aplastic anemia
Ø Slower in action than xylene but causes less brittleness
Ø Suitable for large tissue specimens. Thicker tissue blocks (up to 1 cm) are can be processed
Ø Clearing time: __
Ø Recommended for __,__,__,__
Ø Not flammable
chloroform
- 6 – 24 hours
-tough tissues, nervous tissues, lymph nodes and embryos
Ø Used to clear both paraffin and celloidin sections during embedding process
Ø Recommended for __ and __.
Ø Clearing time: __
Ø Very penetrating clearing agent
Ø Clears __ in 5 – 6 days
Ø Does not dissolve aniline dyes
Ø Makes tissues transparent`
Ø Extremely slow clearing agent, not recommended for routine purposes
Ø Becomes milky upon prolonged storage and should be filtered before use
Ø Very expensive
cedar wood oil
central nervous system tissues and cytological studies
2 – 3 days
celloidin
Ø Not normally utilized as a routine clearing agent
Ø Recommended for clearing_,_ and _
due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening
aniline oil
embryos, insects and very
delicate specimens
Ø Causes minimum shrinkage of tissues
Ø Its quality is not guaranteed due to its tendency to become adulterated
Ø Wax impregnation after clearing with it is slow and difficult
Ø Tissues become brittle, _ are removed and _ is dissolved
Ø Expensive solution
Ø Unsuitable for routine clearing purposes
clove oil
aniline dyes
celloidin
Ø Its properties are similar to chloroform
Ø Same disadvantage of chloroform
Ø It produces considerable tissue hardening and dangerous to
inhale on prolonged exposure due to its highly toxic effects
carbon tetrachloride
Ø Slow-acting clearing agents that can be used when double embedding techniques are required.
Methyl Benzoate and Methyl Salicylate
Ø Tissues can be left for long periods of time without affecting the
consistency or staining properties of the specimen
dioxane (diethyl dioxide)
Used to dehydrate sections and smears
triethyl phosphate
It both dehydrates and clears tissues since it is miscible in water and paraffin.
tetrahydrofuran
Ø Highly flammable
Ø If used longer than 3 hours, it will make the tissues excessively hard and brittle
Ø Not suitable for _ and _
Ø becomes milky when an incompletely dehydrated tissue is immersed in it.
xylene
nervous tissues and lymph nodes
May be used as a substitute for xylene or benzene
Ø It is not carcinogenic
Ø Relatively slower than xylene and benzene
Ø More expensive.
toluene
It is preferred as clearing agent in the embedding process of tissues because it penetrates and clears tissues rapidly
benzene
Ø Relatively toxic to the _ after prolonged inhalation
Ø Wax impregnation after clearing is relatively slow
Ø Does not make tissues transparent
Ø Difficult to remove from paraffin sections because it is not very volatile
Ø Complete clearing is difficult to evaluate
chloroform
liver
