San Pedro College

Department of Medical Laboratory Science

MANUAL OF HISTOPATHOLOGIC TECHNIQUES

Learning Activity No. 5

The Appropriate Dehydration of Tissue Samples

SPECIFIC LEARNING OBJECTIVES:

At the end of the activity, the Medical Laboratory Science student should
be able to:

1. Explain the basic principle of dehydrating tissues for histologic evaluation.

2. Value the importance of dehydration in processing tissue specimen.
3. Enumerate the different types of dehydrating agents.
4. Appreciate the advantages of each dehydrating agent
5. Acknowledge the disadvantages of each dehydrating agent.
6. Perform the process of dehydration properly.

LEARNING CONTENT

Because melted paraffin wax is hydrophobic (immiscible with water), most

of the water in a specimen must be removed before it can be infiltrated with
wax. This process is known as dehydration. The most common manner of
carrying it out is by immersing specimens in a series of ethanol (alcohol)
solutions of increasing concentration until pure, water-free alcohol is reached.
Ethanol is miscible with water in all proportions so that the water in the
specimen is progressively replaced by the alcohol. A series of increasing
concentrations is used to avoid excessive distortion of the tissue.
As a general rule, it is relevant to keep contact time with chemicals as brief
as possible to limit tissue distortion and to minimize the risk of extracting cellular
constituents. The ideal amount of dehydrating agent, whatever type it may
be, should also be maintained at a fluid to tissue ratio of 10:1 to achieve
adequate dehydration
In choosing the dehydrating agent to be used, one should keep the
following characteristics in mind: A dehydrating agent should:
a. Dehydrate rapidly without producing considerable tissue shrinkage;
b. Not evaporate too fast;
c. Not harden tissues excessively;
d. Not remove stains;
e. Not be a fire hazard;
f. Not be toxic to the handler (e.g. histotechnologist);
g. Be able to dehydrate a wide range of tissue types.

DEHYDRATING AGENTS

ALCOHOLS - These are clear, colorless, flammable, hydrophilic liquids,

miscible with water and, when anhydrous, with most organic solvents. In
addition to their role as dehydrants, alcohols also act as secondary
coagulant fixatives during tissue processing. Examples include:
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 Ethanol is probably the most commonly used dehydrating agent in
histology. It is a rapid, efficient and widely applicable agent. It is
normally a poor lipid solvent except under microwave processing
conditions. It dissolves nitrocellulose slowly unless combined in equal
proportions (or better, 1:2) with diethyl ether. Processing times in
absolute ethanol should be minimal. Progressive removal of bound
water from carbohydrates and proteins during prolonged immersion
in absolute ethanol causes tissues to harden excessively and
become brittle. Colloid, blood, collagen and yolky tissues are
particularly affected. The problem is exacerbated by heat during
wax infiltration.

Methanol is a good ethanol substitute but rarely used for routine

processing because of its volatility, flammability and cost. It is a poor
lipid solvent, and will not dissolve nitrocellulose unless mixed with
acetone. In microwave processing it tends to harden tissues more
than ethanol.

Isopropanol was first suggested as an ethanol substitute during the

prohibition era in the United States. It is a universal solvent, slightly
slower in action and not as hydroscopic as ethanol, but a far superior
lipid solvent. It is completely miscible with water and most organic
solvents, is fully miscible with melted paraffin wax, and is readily
expelled from tissues and wax baths. It shrinks and hardens tissues less
than ethanol and is used to dehydrate hard, dense tissues, which can
remain in the solvent for extended periods without harm. To minimize
shrinkage, fixed tissues are transferred via 60%-70% isopropanol or
ethanol to absolute isopropanol. Isopropanol is a solvent for some
lipid-soluble dyes, but is not used in staining work stations as many
other dyes are insoluble in this solvent.

GLYCOL-ETHERS Unlike the alcohols, these reagents do not act as

secondary fixatives, and apart from solvent effects do not appear to
alter tissue reactivity.

2-Ethoxyethanol, Ethylene glycol monoethyl ether, cellosolve is used

as a dehydrating agent preceding polyester wax embedding, for
dehydration following dioxane-based fixation of hard animal tissues,
and in the agar-ester wax double embedding technique. is a
colorless, nearly odorless flammable liquid, strongly hygroscopic,
miscible with water and most organic solvents. It dissolves
nitrocellulose and tends to decompose on exposure to sunlight. It is
rapid but non-hardening in action, and tissues can remain in it for
years.

Dioxane or 1,4 diethylene dioxide causes less tissue shrinkage and

hardening than ethanol and is excellent for tissues excessively
hardened by ethanol-xylene processing. It has a rapid but gentle
action, and is best used in a graded series. Tissues may remain in it for
long periods without harm. It is a colorless, flammable universal
solvent with an odor similar to butanol, freezes at 12°C, and is miscible
with water, most organic solvents and paraffin wax. It is
.
cumulatively toxic and a suspected carcinogen.
Polyethylene glycols (PEG) are water miscible polymers used to
dehydrate and embed substances labile to the solvents and heat of
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 the paraffin wax method. They are clear, viscous, slightly hydroscopic
liquids or solids of low toxicity. Polyethylene glycols are miscible with
most organic solvents and dissolve nitrocellulose. Dehydration is
initiated in the low molecular weight liquid glycols. Tissues pass
through glycols of increasing molecular weight and viscosity, and are
finally embedded in a high molecular weight PEG which is solid at
room temperature.

ACETONE is a colorless flammable liquid with sharp characteristic

ketonic odor, low toxicity and is freely miscible with water and organic
solvents. It is a fast, effective dehydrating agent though it may cause
tissue shrinkage; it may also act as a coagulant secondary fixative. It is
the best for processing fatty specimens. Tissues are dehydrated through
four changes of acetone, the last of which should always be fresh.
Acetone is not recommended for microwave processing as it causes
excessive nuclear shrinkage.

TETRAHYDROFURAN is a colorless, highly volatile and flammable universal

solvent with an offensive ethereal odor. It is completely miscible with
water, most organic solvents, paraffin wax and mounting media. It
dissolves mountants, but not most dyes. The solvent dehydrates rapidly
causing little shrinkage or hardening, and is possibly the best of the
universal solvents. It is less toxic than dioxane for which it can be
substituted. Tissues are processed as in dioxane method.
.

TIME ALLOTMENT: 3 hours

LEARNING RESOURCES:

500 ml beakers 70% ethanol

Forceps 90% ethanol
Alcohol lamps Absolute ethanol

LEARNING STRATEGIES: Pre-lab discussion, Instructor-guided performance

PROCEDURE:

1. Below is a typical dehydration sequence for specimens not more than 4mm
thick. Label and fill your beakers with the chemicals listed below. Then
immerse your samples into them in the same order. Note the time intervals.

70% ethanol 15 min

90% ethanol 15 min
100% ethanol 15 min
100% ethanol 15 min
100% ethanol 30 min
100% ethanol 45 min
2. At this point all but a tiny residue of tightly bound (molecular) water should
have been removed from the specimen. Wrap in foil and store your sample.

LEARNING ACTIVITY NO. 5

STUDENT LEARNING EVALUATION

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 The Appropriate Dehydration of Tissue Samples

Name : _______________________ Score: ________

Course/Yr./Sec.:_______________ Date: _________

Draw the set up for the dehydration process. (10pts)

Guide Questions:

1. What makes ethanol the most common choice for a dehydrating

agent?(5pts)
_____________________________________________________________________
_____________________________________________________________________
____________________________________________________________________

2. Among the dehydrating agents mentioned, which do you think would

perform better than ethanol? Why? (5pts)
_____________________________________________________________________
_____________________________________________________________________
____________________________________________________________________

3. What is the purpose of subjecting formalin-fixed tissues to an ascending

order of graded alcohol?(5pts)
_____________________________________________________________________
_____________________________________________________________________
____________________________________________________________________

4. Identify the advantages and disadvantages of the dehydrating agents

listed in the table below. Fill in your answers to complete the table.
DEHYDRATING ADVANTAGE (1pt each) DISADVANTAGE (1pt each)
AGENT
Isopropanol

Cellosolve

Acetone

Rubrics for Scoring Guide Questions

5 pts 4 pts 3pts 2 pts 1 pts

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Excellent Good Average Fair Needs
The answer The answer The answer The answer improvement
meets all of meets most of meets some of meets only a The answer
the criteria: the criteria: the criteria: few of the does not meet
1.Relevance 1. Relevance 1. Relevance criteria: any of the
2. Clarity 2. Clarity 2. Clarity 1. Relevance following
3.Conciseness 3.Conciseness 3.Conciseness 2. Clarity criteria:
4. Grammar 4. Grammar 4. Grammar 3.Conciseness 1. Relevance
4. Grammar 2. Clarity
3. Conciseness
4. Grammar

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