Dehydration Flashcards
process of removing intercellular and extracellular water from the tissue following fixation and prior to wax impregnation
dehydration
___ involves slow substitution of the water in the tissue with an organic solvent.
___ removal of water by evaporation from a solid, semi-solid or liquid.
Dehydration
Drying
Characteristics of an Ideal Dehydrating Solution
- It should dehydrate rapidly without producing considerable shrinkage or distortion of tissues.
- It should not evaporate very fast.
- It should be able to dehydrate even fatty tissues.
4 It should not harden tissues excessively. 5. It should not remove stains. - It should not be toxic to the body.
- It should not be a fire hazard.
general rule in dehydration
whatever dehydrating agent is used, the amount in each step should not be less than 10 times the volume of the tissue in order to ensure complete penetration of the tissue by the dehydrating solution.
It is also important to keep the dehydration times as brief as possible to minimize the risk of extracting cellular constituents
Commonly Used Dehydrating Agents
- Alcohol (most common)
- Acetone
- Dioxane
- Cellosolve
- Triethyl phosphate
- Tetrahydrofuran
alcohol recommended for routine dehydration of tissues. It is a clear, colorless, flammable fluid
Ethyl Alcohol
- the alcohol recommended for routine dehydration of tissues
- It is a clear, colorless, flammable fluid.
- It is considered to be the best dehydrating agent because it is fast-acting, it mixes with water and many organic solvents, and it penetrates tissues easily
- It is not poisonous and not very expensive.
Ethyl Alcohol
toxic dehydrating agent, primarily employed for blood and tissue films and for smear preparations
Methyl alcohol
- utilized in plant and animal micro-techniques
- is a slow dehydrating agent
- producing less shrinkage and hardening than ethyl alcohol
- recommended for tissues which do not require rapid processing.
Butyl alcohol
Why is it not advisable to transfer fixed tissues directly from water or aqueous fixative directly into absolute ethanol?
causes a rapid removal of water which can distort the appearance of more delicate cells and structures.
Dehydration Process
1 - 70% ethyl alcohol
2 - 95% ethyl alcohol
3 - 100% ethyl alcohol
For delicate tissues, particularly embryonic tissues, dehydration starting with?
30% ethanol
what will happen to tissues directly transferred higher grades of alcohol?
Shrinkage and hardening leading to tissue distortion
very concentrated solution makes the tisssue ___
hard, brittle and difficult to cut
What will happen to prolonged storage in lower concentrations of alcohol (below 70%)?
will macerate the tissue and may later interfere with the staining properties of the specimen.
how will you ensure complete removal of water during dehydration?
use at least two changes of 100% ethanol of at least one half hour each.
What will happen if you leave tissues in 95% or 100% ethanol more than a total of 2 hours
tissues will harden
Tissues can be stored in ______ ethanol at any time during an interruption in the routine.
70%
A typical dehydration sequence for specimens not more than 4mm thick would be
70% ethanol 15 min
90% ethanol 15 min
100% ethanol 15 min
100% ethanol 15 min
100% ethanol 30 min
100% ethanol 45 min
What temperature will hasten dehydration time and is used for tissue sections that require urgent examinations such as fragmentary biopsies?
37°C
To insure complete dehydration, a layer of __________, about _____ deep is placed in the bottom of the container and covered with _______
anhydrous copper sulfate
1/4 inch
filter paper
What indicates a blue discoloration of copper sulfate crystals and what will you do after?
- full saturation of dehydrating fluids with water
- Alcohol is discarded and changed with a fresh solution.
Ethanol (ethyl alcohol) Boiling point
78.3° C
Butanol (butyl alcohol) Boiling point
117.7° C
Tertiary butanol (butyl alcohol) Boiling point
82.8° C
Isopropanol (isopropyl alcohol) Boiling point
82.3° C
Pentanol (amyl alcohol) Boiling point
128° C
ACETONE Boiling point
56° C
DIOXANE (Diethylene Dioxide) Refractive index; Boiling point
RI - 1.42
BP - 101.5°
CELLOSOLVE (Ethylene glycol monoethyl ether) Boiling point
156.4° C
TRIETHYLPHOSPHATE Boiling point
215° C
Advantages:
- Nontoxic
- Miscible in all proportions with water
- Little shrinkage if graded alcohols are used
- Can be used on eyes and embryos, if graded alcohols are used
- Fast acting
- Still considered best dehydrating solution
- Reliable
- Appears to cause less extraction of cellular components in general than other agents
- Inexpensive and easily obtained
Ethanol (ethyl alcohol)
Disadvantages:
- Long periods will cause excessive shrinkage and hardening
- May be difficult to obtain
- Extracts methylene blue and other thiazine dyes from sections
- Extracts more lipids than acetone
- May cause more shrinkage of specimen
- May react with an unreduced OsO4 remaining in specimen
- Only slightly miscible with most resins
Ethanol (ethyl alcohol)
Advantages:
- Less shrinkage and hardening than with ethyl
- Excellent for slow processing
- Miscible with paraffin
Butanol (butyl alcohol)
Disadvantages:
- Odorous
- Slow-acting
- Long periods of infiltration necessary
- Dehydrating power low
Butanol (butyl alcohol)
Advantages:
- Universal solvent—acts as dehydrating and clearing agent
- May be used in staining series as a dehydrating agent
- Mixes with water, ethanol, xylene, and paraffin in all
Tertiary butanol (butyl alcohol)
Disadvantages:
- Odorous
- More expensive than butanol
- Primary infiltration must be done in half tertiary butanol and half paraffin, prior to paraffin impregnation
- Reagent tends to solidify at room temperature or below 25° C
Tertiary butanol (butyl alcohol)
Advantages:
- Excellent substitute for ethanol
- Less shrinkage and hardening than ethanol
- No government restrictions on its use
- Sufficiently water-free to use in place of absolute ethanol
- Lillie considers it “the best all-around substitute for ethyl alcohol”
- Less expensive than tax-free alcohol
Isopropanol (isopropyl alcohol)
Disadvantages:
- Cannot be used in the celloidin technique since nitrocellulose is
insoluble in it
- Cannot be used for preparing staining solutions, since
dyes are not soluble in it
Isopropanol (isopropyl alcohol)
Advantages:
- Miscible with 90% alcohol, toluene and xylene
- Dissolves paraffin wax
Pentanol (amyl alcohol)
Disadvantages:
- Toxic
- Cannot be used in poorly ventilated rooms
- Not miscible with water
Pentanol (amyl alcohol)
- cheap, rapid-acting dehydrating agent utilized for most urgent
biopsies - a clear, colorless fluid that mixes with water, ethanol, and most organic solvents.
- more miscible with epoxy resins than alcohol, but is highly flammable
Acetone
How many hours does Acetone dehydrate?
1/2 to 2 hours
What happens when acetone is placed for a prolonged period of time?
Brittleness in tissue
Most lipids are removed from tissues with this dehydrating agent
Acetone
Why is acetone limited only to small pieces of tissues?
due to extreme volatility and inflammability
Advantages:
- Rapid dehydrating agent
- Less expensive than ethanol
- Does not extract methylene blue and other dyes from stained sections
- May cause less shrinkage of specimen than ethanol
- Not reactive with 0s04 remaining in specimen.
- Miscible with most embedding resins.
Acetone
Disadvantages:
- Requires a clearing agent
- Volume must be 20 times that of the tissue
- Best processing requires a graded series of a mixture of acetone and xylene before one can go into paraffin
- Needs good ventilation
- Evaporates rapidly
- Flammable
- Absolute acetone is easily contaminated with water, resulting in complete dehydration.
- Uranyl acetate and phosphotungstic acid are only soluble in dilute
solutions of acetone.
Acetone
- excellent dehydrating and clearing agent readily miscible in
water, melted paraffin, alcohol and xylol - produces less tissue shrinkage as compared to alcohol dehydration
- Its vapor produces a cumulative and highly toxic action in man; hence, it should not be used routinely.
Dioxane
Tissues can be left in this reagent for long
periods of time without affecting the consistency or staining properties of the specimen.
Dioxane
Graupner’s Dioxane Method
(1st) pure dioxane solution 1 hour
(2nd) pure dioxane solution 1 hour
(3rd) pure dioxane solution 2 hours
(1st) Paraffin wax 15 minutes
(2nd) Paraffin wax 45 minutes
(3rd) Paraffin wax 2 hours
Embed in mold and cool in water.
another method where the tissue is wrapped in a gauze
bag and suspended in a bottle containing dioxane and a little anhydrous calcium oxide. Water is displaced from the tissue by dioxane and in turn absorbed by calcium oxide or quicklime. Dehydration period ranges from 3-24 hours.
Weiseberger’s method
Advantages:
- Universal solvent—it dehydrates and clears
- Miscible with water, alcohol, xylene, and paraffin
- Does not harm tissue over long time periods
- Faster dehydrant than ethanol
Dioxane
Disadvantages:
- Needs large volume for dehydration
- Must be used in well-ventilated rooms
- Cumulatively toxic
- Odorous
- Distorts tissue-containing cavities
Dioxane
- dehydrates rapidly
- tissue may be transferred from water or normal saline directly and stored in it for months without producing hardening or distortion
CELLOSOLVE (Ethylene glycol monoethyl ether)
combustible at 110-120°F and are toxic by inhalation, skin contact, and ingestion.
Ethylene glycol ethers
What is the alternative for ethylene-based glycol ethers?
propylene-based glycol ethers
Advantages:
- Rapid dehydrating agent
- Tissue may remain in it for months without injury
- Avoids distortion and does not require graded dilutions
CELLOSOLVE (Ethylene glycol monoethyl ether)
Disadvantages:
- Expensive
- Rapidly absorbs water from the air
- Requires clearing agent
CELLOSOLVE (Ethylene glycol monoethyl ether)
- it removes water very readily and produces very little distortion and hardening of tissue.
- soluble in alcohol, water, ether, benzene, chloroform, acetone and xylene
- used to dehydrate sections and smears following certain stains and produces minimum shrinkage.
TRIETHYLPHOSPHATE
Advantages:
- May be used in routine paraffin technique
- Displaces water readily with slight distortion
- Does not harden tissue excessively
- May be used as a dehydrating solution in the staining sequence
- Soluble in alcohols, benzene, toluene, xylene, ether, chloroform
TRIETHYLPHOSPHATE
Dehydrating Agent without a disadvantage
TRIETHYLPHOSPHATE
- a reagent that both dehydrates and clears tissues since it is miscible in both water and paraffin.
- can dissolve many substances including fats and is in itself miscible with lower alcohols, ether, chloroform, acetone, benzene, and xylene
TETRAHYDOFURAN (THF)
- may be used for demixing, clearing, and dehydrating paraffin sections before and after staining
- causes less shrinkage and easier cutting of sections with fewer artifacts.
- does not dissolve out aniline dyes
- toxic if ingested or inhaled.
TETRAHYDOFURAN (THF)
- Vapors cause nausea, dizziness, headache and anesthesia. It is an eye and skin irritant
- prolonged exposure (up to 6 months) may cause conjunctival irritation
TETRAHYDOFURAN (THF)
Advantages:
- Miscible in all proportions with water, ether, chloroform, acetone, and the hydrocarbons xylene, toluene, and benzene
- Rapid without excessive shrinkage and hardening
- Low toxicity; low fire and explosion hazard
- Not toxic
- Better results than most universal solvents
- Solvents of mounting media
TETRAHYDOFURAN (THF)
Disadvantages:
- Odorous- should be used in well-ventilated room
- Evaporates rapidly
- Dyes are not soluble in tetrahydrofuran
TETRAHYDOFURAN (THF)
What is the dehydrating solvent and transition fluid in TEM?
ethanol and propylene oxide
a good substitute for propylene oxide
Acetonitrile
