MT112 – Histopathologic and Cytologic

Techniques with General Pathology COMMONLY USED DEHYDRATING

DEHYDRATION AGENTS

 Alcohol (most commonly used)

DEHYDRATION  Acetone
 Dioxane (also a clearing agent)
 Removal of intracellular and
 Cellosolve
extracellular water from the fixed tissue
 Triethyl phosphate
in preparation for impregnation
 Tetrahydrofuran (also a clearing agent)
procedure

Why do we need to remove the water? A. ALCOHOL

> Because water is immiscible with the
clearing agent I. Ethyl Alcohol (ethanol)
- recommended for routine
 It uses “dehydrating agents” to remove dehydration of tissues
the water from the tissue - clear, colorless, flammable fluid
- it is the best dehydrating agent
Most widely used dehydrating agent in the lab: because it is fast acting
> Alcohol - mixes with water and many organic
> This alcohol would be at varying strengths solvents
and concentration - penetrates the tissues easily
- it is not poisonous
100% Alcohol is otherwise known as - not very expensive
absolute alcohol

 10 to 1 II. Isopropyl Alcohol

(Recommended ratio of Dehydrating agent - is a dehydrating and clearing agent
to Tissue)
- used as a substitute for Ethanol
10 parts dehydrating agent
1 part tissue sample
III. Methyl Alcohol
 Is done after fixation & prior to - a toxic dehydrating agent
impregnation - primarily used for blood and tissue
films
CHARACTERISTICS OF AN IDEAL - can also be used as a fixative for
DEHYDRATING SOLUTION smear preparations (e.g., bone
marrow smear & peripheral blood
Mentioned by Gregorios: smear)
 It should dehydrate fast without
producing considerable shrinkage or
distortion of the tissue sample IV. Butyl Alcohol
 It should not evaporate fast to ensure - utilized in plants and animal micro-
complete removal of intracellular and techniques
extracellular water - it is a slow dehydrating agent
 It should dehydrate even fatty tissues compared to ethanol
 It should not harden the tissues - produces less shrinkage and
excessively (just the right consistency) hardening compared to ethanol
 It should not remove stains - is recommended for non-urgent
 It should not be toxic to the body works
 It should not be a fire hazard
- optimum conc. for storage
How are these alcohol used as a - store not for a long time as it
dehydrating agent: would affect the staining process
of the specimen
 Subject the tissue to a series of
 Short period only for storage
progressively increasing
concentrations of alcohol.
 370C
(70% → 95% → 100%)
- hasten dehydrating time
It should not be the other way around.

X Not decreasing grades X

 To check for the complete dehydration:

When you start with higher grades of  ¼ inch thick layer of anhydrous
alcohol, it produces shrinkage and scopper sulfate at the bottom of the
hardening of the tissues which would container
lead to tissue distortion.  Cover it with filter paper
 Blue discoloration of crystals =
The deeper parts of the tissue would not indicates the full saturation of the
be penetrated completely resulting to dehydrating fluid with water
incomplete dehydration
This means that you have to change the
Incomplete dehydration  Unequal solution into a new/fresh solution and throw
impregnation  Poor cutting of sections the old solution. Then, continue the
because there’s still water in the tissue dehydration.
and it is still soft
Purpose of Anhydrous Copper Sulfate:
 The strength of the initial alcohol > Will hasten the dehydration by
required will depend upon the size, removing the water from the
nature of the tissue and the fixative dehydrating fluid
used.
B. ACETONE
Smaller and more delicate of tissues
require lower concentrations and shorter  Clear, colorless fluid that mixes with
intervals between changes of alcohol
water, ethanol, and most organic
solvents
Delicate tissue examples:
> Embryonic tissue  More miscible with epoxy resins than
-dehydration would start with 30% alcohol
alcohol [ethanol] (recommended)  Highly flammable and volatile (easily
evaporates)
You can store tissue samples in alcohol.  Cheap and rapid-acting but poor
If you store it at: penetration
 >80%  Could cause brittleness in tissues that is
- above 80% conc. placed with acetone for a long period of
- it would make the tissues hard, time
brittle and difficult to cut (too hard  Utilized for most urgent biopsies
to cut)  Not recommended for routine
dehydrating purposes
 < 70%
 Could remove lipids from the tissues
- less than/below 70% conc. in a
long period of time
 Dehydrates in 30 minutes to 2 hours
- causes tissue maceration
(destroyed tissue sample)

 70% - 80% C. DIOXANE

 Soluble in alcohol, water, ether,
 A.K.A. Diethylene Dioxide benzene, chloroform, acetone, and
 Both dehydrating and clearing agent xylene
 Readily miscible in water, melted
paraffin, alcohol and xylol (it mixes F. TETRAHYDROFURAN (THF)
immediately)
 Tissues may be placed directly in  Both dehydrating and clearing agent
dioxane after washing out  Could dissolve fat like acetone
 Tissues can be left for long periods of  Miscible w/ lower alcohols, ether,
time chloroform, acetone, benzene, and
 Tends to ribbon poorly xylene
 Expensive and extremely dangerous  May be used for refixing, clearing, and
- its vapor produces cumulative and highly dehydrating paraffin sections before and
toxic action in the body after staining
 Not should be used routinely  Will not dissolve out aniline dyes
 Gives improved results in most staining
- The lab room should be properly ventilated
procedures
when using dioxane
- All residues should be washed out in the sink  Is toxic when ingested or inhaled
- Should not be recycled (one time use only)  An eye and skin irritant
- really dangerous when there’s a
D. CELLOSOLVE contact

 A.K.A. Ethylene glycol monoethyl Prolonged exposure to tetrahydrofuran,

ether (e.g., up to 6 months) it may cause
 Dehydrates rapidly conjunctival irritation
 Tissues may be transferred from water
 Must be cautious when using
or normal saline to the cellosolve
chemicals in the laboratory
 Tissues can be stored in cellosolve for
months without producing hardening or Why are Dioxane and Tetrahydrofuran can be
distortion both dehydrating and clearing agent?
 Combustible at 110°F - 120°F (43.30C – > They are both miscible in water and
48.80C) paraffin wax
 Toxic by inhalation, skin contact and
ingestion
- Targets reproductive, fetal, urinary and
blood systems ADDITIVES

To avoid the toxic side-effects of cellosolve:  4% phenol

 Propylene-based glycol ethers could – added to each of the 95% ethanol
be used instead baths
- will act as a tissue softener during
the dehydration process for hard
tissues such as tendon, nail or dense
fibrous tissue
E. TRIETHYL PHOSPHATE - this is optional when using

 Removes water very readily (it is miscible

with water)
 Produces very little distortion and
hardening compared to alcohol