HISTOLOGY

LECTURE # 14
INTRODUCTION TO TISSUE PROCESSING
Rationale:
Processing is a secondary step in the histology process. Tissue after fixation will
go through several steps that will allow some structural format to the tissue that will allow the
tissue to be sectioned and stain for diagnostic purposes.

Objective:
Once completed this lecture, the student should be able to:

a)Describe the different dehydrants, clearing and infiltrating agents

b)Learn the difference between processing options
c)Describe the proper and effective way of embedding tissues
d)Learn the terms open processor and closed processor.

TISSUE PROCESSING
INTRODUCTION
(Carson Book Pages 26 – 35)
All tissues must be adequately supported before they can be sectioned for microscopical
examination. The exceptions to this are frozen sections which are sectioned following a range of
preparatory freezing methods. Permanent tissues are more commonly taken through a series of
reagents and finally infiltrated and embedded in a stable medium which when hard, provides the
necessary support for microtomy. This treatment is termed tissue processing. Methods have
evolved for a range of embedding media and applications. The most widely used method for
routine preparation; sectioning, staining and subsequent storage of large numbers of tissue
samples is the paraffin wax method, which we will discuss in the embedding lecture.

Principles of tissue processing

Tissue processing is concerned with the diffusion of various substances into and out of stabilize
porous tissues. The diffusion process results from the thermodynamic tendency of processing
reagents to equalize concentrations inside and outside blocks of tissue, thus generally
conforming to Fick's Law: the rate of solution diffusion through tissues is proportional to the
concentration gradient (the difference between the concentrations of the fluids inside and outside
the tissue) as a multiple of temperature dependant constants for specific substances.
From this it can be seen that the significant variables in tissue processing are the operating
conditions, particularly temperature, the characteristics and concentrations of the reagents and
the properties of the tissue.

Dehydration (Removal of Water)

The first step in processing is dehydration. Water is present in tissues in free and bound
(molecular) forms. Tissues are processed to the embedding medium by removing some or all of
the free water. During this procedure various cellular components are dissolved by dehydrating
fluids. For example, certain lipids are extracted by anhydrous alcohols, and water soluble
proteins are dissolved in the lower aqueous alcohols.
Dehydration is effected as follows:

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Dilution dehydration, the most commonly used method. Specimens are transferred through
increasing concentrations of hydrophilic or water miscible fluids which dilute and eventually
replace free water in the tissues.

Chemical dehydration, where the dehydrant, acidified dimethoxypropane or diethoxypropane, is

hydrolyzed by free water present in tissues to form acetone and methanol 43-50 in an
endothermic reaction.Dehydration is necessary in all infiltration methods, except where tissues are
simply externallysupported by an aqueous embedding medium. Choice of a dehydrant is determined by
the natureof the task, the embedding medium, processing method, and economic factors. Dehydrants
differ in their capacity to cause tissue shrinkage. In the paraffin wax method, following any necessary
post fixation treatment, dehydration from aqueous fixatives is usually initiated in 60%-70%
ethanol, progressing through 90%-95% ethanol, then two or three changes of absolute ethanol
before proceeding to the clearing stage. While well fixed tissues can be transferred directly to
95% ethanol, incompletely fixed tissues may exhibit artifacts if placed directly in higher
alcohols. The dehydrant concentration at which processing is initiated depends largely upon the
fixative employed. Following fixation in anhydrous fixatives such as Carnoy's fluid, for example
dehydration is initiated in 100% ethanol.

To minimize tissue distortion from diffusion currents, delicate specimens are dehydrated in a
graded ethanol series from water through 10%-20%-50%-95%-100% ethanol.
Duration of dehydration should be kept to the minimum consistent with the tissues being
processed. Tissue blocks 1 mm thick should receive up to 30 minutes in each alcohol, blocks 5
mm thick require up to 90 minutes or longer in each change. Tissues may be held and stored
indefinitely in 70% ethanol without harm. Other dehydrants, including universal solvents, are
used in a similar manner to that described for ethanol, though generally in different concentration
increments.

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.
Ethanol is probably the most commonly used dehydrant in histology. It is supplied as 99.85%
ethanol (absolute ethanol, 100 High Grade or Standard Grade) and as special Methylated Spirits
(99.85% ethanol denatured with 2% methanol). Both are satisfactory for histological purposes.
Ethyl alcohol formulations differ in standards and nomenclature worldwide and it may be
necessary to consult various tables to ascertain the ethanol concentration.
Ethanol is a rapid, efficient and widely applicable dehydrant. It is normally a poor lipid solvent
except under microwave processing conditions. Ethanol 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

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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 available as 99.8% (absolute) isopropanol, slightly slower in
action and not as hydroscopic as ethanol, but a far superior lipid solvent. Isopropanol 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. Isopropanol 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. Isopropyl alcohol has also been
recommended as a xylene substitute. In microwave stimulated processing, though unsatisfactory
as a dehydrant, isopropanol is used as a transition solvent following ethanol dehydration.
Isopropanol only dissolves nitrocellulose in the presence of esters such as methyl benzoate or
methyl salicylate, and is used in methyl salicylate-based double-infiltration methods. It cannot be
used as a dehydrant in alcohol-ether-celloidin techniques. 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.

Normal and tertiary butanols are universal solvents mainly used for small-scale manual
processing of plant and animal tissues in teaching and research. Normal butanol is recommended
for processing lightly chitinized arthropods and rodent tissues. It causes less hardening and
shrinkage than ethanol, though this is offset by the prolonged processing schedules which may
result in tissue shrinkage. N-butanol is poorly miscible with water and only slowly miscible with
paraffin wax. It is flammable, with a penetrating camphor-like odor, and the vapors are eye
irritants. Iso-butanol, with similar properties and processing characteristics is a less costly
substitute for n-butanol. Tertiary-butanol is widely used in plant histology but rarely for animal
tissues. Below 26°C it is hygroscopic crystalline solid, a major disadvantage. In processing it is
used in a similar manner to n-butanol.

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 or oxitol is used as a dehydrant
preceding polyester wax embedding, for dehydration following dioxane-based fixation of hard
animal tissues, and in the agar-ester wax double embedding technique.
Ethoxyethanol is a colorless, nearly odorless flammable liquid, strongly hygroscopic, miscible
with water and most organic solvents. Cellosolve 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 years62. To avoid severe shrinkage, tissues are transferred from aqueous fixative
or washing via 60%-70% ethanol into full strength cellosolve.
Dioxane, 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. Dioxane dissolves
mercuric chloride, but precipitates potassium dichromate and other salts. It is cumulatively toxic
and a suspected carcinogen

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.
Dioxane is expensive and is normally reclaimed by drying over a
10-20 mm layer of calcium oxide or anhydrous cupric sulphate. Calcium chloride should not be
used as it reacts with dioxane and swells. Dioxane is also recovered by freezing hydrated solvent
in a spark-proofed refrigerator at 2-5°C. Water, which separates out, is decanted from the
crystalline dioxane which is then thawed, finally dried over a solid dehydrant and reused
Explosive peroxides form in dioxane exposed to air. They accumulate in recycled solvent which
should be periodically tested for the presence of peroxides.
Polyethylene glycols (PEG) are water miscible polymers used to dehydrate and embed
substances labile to the solvents and heat of 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.
Polyethylene glycol used for dehydration can be regenerated by heating at 104°C for 24 hours.

OTHER DEHYDRANTS
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 dehydrant though it may
cause tissue shrinkage; it may also act as a coagulant secondary fixative. Acetone is the best
dehydrant for processing fatty specimens. Tissues are dehydrated through four changes of
acetone, the last of which should always be fresh.
Tissues can be transferred directly from acetone to paraffin wax as the solvent boils off under
vacuum. However a transition solvent is normally interposed before the paraffin baths. 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.
Tetrahydrofuran can form explosive peroxides which renders solvent recovery distillation
dangerous
.
2,2 dimethoxypropane (DMP) and 2,2 diethoxypropane (DEP) are used for chemical
dehydration of tissues. They are flammable and form peroxides. DMP and DEP are miscible with
paraffin wax however methanol, one of the hydrolysis products, is not wax miscible and a post
dehydration rinse in acetone, a transition solvent such as methyl salicylate or toluene should
precede infiltration with wax. DMP shrinks tissues slightly less than DEP.

Chemical dehydration is suitable for rapid manual processing or machine processing, and is
comparable to conventional dehydration for tissue morphology and staining reactions.

Acidified DMP/DEP
can be reused several times, though dehydration times need to be extended. The reagent is stored
at 4°C in a spark-proofed refrigerator.
Phenol, beechwood creosote and aniline facilitate dehydration when mixed with transition

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solvents, as in Weigert's carbol-xylol (xylene 75 ml and phenol 25 ml). The coupling action
permits tissues and celloidin sections to be cleared from lower strength alcohols. Creosote and
aniline are used less commonly though in a similar manner to phenol. Phenol consists of clear
hygroscopic acicular crystals and is also available as 80% w/w liquefied phenol. It is soluble in
water, alcohol and most organic solvents except petroleum ethers. Concentrated solutions
coagulate nitrocellulose. On exposure to air and light, phenol and its solutions develop a pink to
reddish discoloration. Containers must be protected from light and tightly sealed. Phenol crystals
and 80% concentrate react violently with formaldehyde.

Clearing
(Removal of Alcohol)
Clearing is the transition step between dehydration and infiltration with the embedding medium.
Many dehydrants are immiscible with paraffin wax, and a solvent (transition solvent, ante
medium, or clearant) miscible with both the dehydrant and the embedding medium is used to
facilitate the transition between dehydration and infiltration steps. Shrinkage occurs when tissues
are transferred from the dehydrant to the transition solvent, and from transition solvent to wax. In
the final stage shrinkage may result from the extraction of fat by the transition solvent.
The term clearing arises because some solvents have high refractive indices (approaching that of
dehydrated fixed tissue protein) and, on immersion, anhydrous tissues are rendered transparent or
clear. This property is used to ascertain the endpoint and duration of the clearing step. The
presence of opaque areas indicates incomplete dehydration.
However, other solvents, notably chlorinated hydrocarbons, do not render tissues transparent and
the clearing endpoint (generally when the specimen sinks in the solvent) must then be
determined empirically. Transition solvents extract certain tissue substances such as lipids, but
otherwise do not alter tissue reactivity nor behave as secondary fixatives during processing.
Choice of a clearing agent depends upon the following:
• the type of tissues to be processed, and the type of processing to be undertaken
the processor system to be used
• intended processing conditions such as temperature, vacuum and pressure
• safety factors
• cost and convenience.

Transition solvents

HYDROCARBONS
These are odorless flammable liquids with characteristic petroleum or aromatic odors,
miscible with most organic solvents and with paraffin wax. They coagulate nitrocellulose.

Toluene and xylene clear rapidly and tissues are rendered transparent, facilitating clearing
endpoint determination. Concerns over the exposure of personnel to xylenerelate mainly to the
use of the solvent in coverslipping rather than in processing and xylene substitutes can be used in
these circumstances. Xylene hardens tissues fixed in non-protein coagulant fixatives and
prolonged clearing in the solvent should be avoided. Tissues stabilized in protein coagulant
fixatives (Bouin's or SUSA) are less affected. Benzene is more gentle and rapid than xylene and
toluene and is probably the best transition solvent, though toxicity and possible carcinogenicity
preclude its use in histology. Industrial grade xylene may contain nearly 25% of other solvents
such as ethyl benzene, with traces of benzene, odorous mercaptans and hydrogen sulphide. Only

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the sulphur and benzene-free solvent-grade xylene should be used for histological purposes.

Petroleum solvents have a gentle, non-hardening action on tissues, clear more slowly than
xylene and toluene, and do not render tissues transparent. Blends of aromatic, naphthenic and
aliphatic solvents (each with varying toxicity, flammability and solvent action) can be used as
xylene substitutes. Many of these solvents have a particularly strong petroleum odor which some
people find objectionable. Toxic effects of petroleum solvents are broadly similar to those of
pure hydrocarbons - skin degreasing, acute intoxication and narcosis in high concentrations.
Blends with high aromatic and naphthene but reduced paraffin content such as Shell X3B71, are
good, moderately toxic, high flash-point solvents. Those with high paraffin but little or no
naphthene and aromatic content often have low flammability and toxicity, and a slow and gentle
clearing action. Kerosene, some xylene substitutes and Shellsol 1626 have properties
intermediate between these two groups.

Chlorinated hydrocarbons are colorless solvents with sweet odors and are miscible with most
organic solvents and with paraffin wax. They are good lipid solvents and do not dissolve
nitrocellulose or render tissues transparent. Members of this group clear more slowly but harden
far less than xylene. Although non-flammable, solvents in this group decompose in the presence
of heat to form phosgene and hydrochloric acid. They are all narcotic and toxic to varying
degrees. Chlorinated hydrocarbons are ozone destroying chemicals, and from January 1996 1,1,1
trichloroethane and carbon tetrachloride are banned from use under the Montreal Protocol.

Chloroform is an expensive, heavy, highly volatile, slowly penetrating transition solvent. It

causes less brittleness than xylene and is often used on dense tissues such as uterus and muscle
which can be cleared overnight without undue hardening. Since chloroform attacks some plastics
and sealants its use may be restricted in certain closed system processors.

Carbon tetrachloride has similar properties, but because of its high toxicity is now rarely used
in histology.
Trichloroethane and other members of this group are commonly used as xylene and chloroform
substitutes. They include 1,1,1 trichloroethane (1,1,1 TCE), present in Inhibisol; 1,1,1 TCE and
perchloroethylene components of CNP30 and Histosol; and trichloroethylene. These solvents are
stable to light but tend to slowly liberate hydrochloric acid on contact with water. They contain
stabilizers to inhibit reactions with aluminum and its alloys. Although mildly toxic (except at
high concentrations) the decision to substitute them for more toxic solvents must be soundly
based. Because of their high volatility, members of this group may achieve and exceed maximum
allowable concentrations in poorly ventilated laboratories far more rapidly than xylene under the
same conditions.
ESTERS

These are colorless flammable solvents miscible with most organic solvents and with paraffin
wax.
n-Butyl acetate is used as a xylene substitute and nitrocellulose solvent.
Amyl acetate, methyl benzoate and methyl salicylate are chiefly used as nitrocellulose
solvents in double embedding techniques. They have low toxicity, but their strong penetrating
odors necessitate good laboratory ventilation. They are ideal for manual processing as tissues
may be left in them for extended periods without hardening. These esters are difficult to

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eliminate from paraffin wax and should be extracted from tissues with one or two brief changes
of toluene or similar solvent before passing through two or three changes of wax. Methyl
benzoate and methyl salicylate render tissues completely transparent and are used for clearing
helminthes parasites for examination and whole mounting. Methyl salicylate clears tissues from
96% ethanol, hardens less and has a more pleasant odor than methyl benzoate. It causes minimal
tissue shrinkage and hardening and tissues can remain in it indefinitely without harm. This ester
is one of the best though expensive transition solvents.

TERPENES
Terpenes are isoprene polymers found in essential oils originally derived from plants, though
some are now synthesized. They are the earliest transition solvents to be used in histology and
include turpentine and oils of bergamot, cedarwood, clove, lemon, origanum and sandalwood. In
general the natural oils are not highly pure compounds but contain several substances.
Many terpenes clear tissues and celloidin sections from 80%-95% alcohol, render tissues
transparent and have a slow gentle non-hardening action. Most are generally regarded as safe
though some have particularly strong odors which can be overpowering, requiring good
laboratory ventilation.
When used for processing hard, dense or chitinized non-mammalian tissues, terpenes may need
to be diluted with the anhydrous dehydrant and with wax in a series, with terpene:dehydrant or
wax ratios of 3:1, 1:1, 1:3 followed by three or four changes of pure wax. Tissue penetration is
aided and shrinkage minimized by diluting viscous terpenes.
Terpenes have low evaporation rates and are difficult to eliminate from paraffin wax,
necessitating one or two 30 minute changes of toluene or similar solvent to remove the terpene
before infiltration with wax. Brief immersion in toluene does not negate the effectiveness of the
terpene. Alternatively, tissues are given three, four or more changes of wax until the terpene has
been eliminated. Although biodegradable, terpenes are not water miscible and should not be
flushed away with water, but disposed of by recycling or incineration.

Cedarwood oil, largely composed of cedrene, rapidly clears tissues from 95% alcohol, hardens
tissues the least of all the transition solvents, but is difficult to eliminate from tissues during wax
infiltration. It is particularly useful for processing dense tissues such as uterus or scirrhous
carcinomas, and has a role in forensic histopathology in processing the hardened skin margins of
electrical burns and bullet wounds. Tissues can remain in cedarwood oil indefinitely without
harm. Low viscosity refined oil should be used for clearing. Formation of crystalline cedrol in
cedarwood oil can be overcome by the addition of 1 ml xylene or toluene to 80 ml cedarwood
oil. Cedarwood oil is expensive, but exhausted oil can be restored by filtering, then heating to
60°C under vacuum for 30-60 minutes.

Limonene (d+ limonene) is derived from citrus fruit and is a component of various proprietary
blends of transition solvents such as Clearene, Hemo-De and Histo-Clear marketed as xylene
substitutes. It is less viscous than cedarwood oil and is similar to the esters in clearing action and
in elimination from wax. Limonene may cause allergic skin reactions.

Terpineol is a clear almost colorless mixture of isomers with a faint pleasant odor and very low
evaporation rate. It clears tissues from 80%-90% alcohol with minimal hardening. Like the other

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terpenes it is difficult to eliminate from paraffin wax. It is a particularly useful substitute for
cedarwood oil in manual processing and is also used in open-dish microscopic examination of
cleared parasitic helminthes. Tissues may remain in it indefinitely without harm.

Infiltration and embedding media and methods

Ideally an infiltrating and embedding medium should be:
• soluble in processing fluids
• suitable for sectioning and ribboning
• molten between 30°C and 60°C
• translucent or transparent; colorless
• stable
• homogeneous
• capable of flattening after ribboning
• non-toxic
• odorless
• easy to handle
• inexpensive
In addition the properties of the medium should approach those of the tissues to be sectioned
with regard to density, elasticity, plasticity, viscosity and adhesion and should be harmless to the
embedded material.
Various substances have been used to infiltrate and embed tissues for microtomy. None
completely fulfill the foregoing criteria, and media are selected according to the nature of the
task for which they are required.

Embedding is the process by which tissues are surrounded by a medium such as agar, gelatin, or
wax which when solidified will provide sufficient external support during sectioning.

Infiltration (Interpenetration)
Is the saturation of tissue cavities and cells by a supporting substance which is generally, but not
always, the medium in which they are finally embedded. Tissues are infiltrated by immersion in
a substance such as a wax, which is fluid when hot and solid when cold. Alternatively, tissues can be
infiltrated with a solution of a substance dissolved in a solvent, for example nitrocellulose
in alcohol-ether, which solidifies on evaporation of the solvent to provide a firm mass suitable
for sectioning.

Double embedding is the process by which tissues are first embedded or fully infiltrated with a
supporting medium such as agar or nitrocellulose, then infiltrated a second time with wax in
which they are also embedded.

Investment generally refers to the practice of embedding wax infiltrated tissues in another wax,
such as Piccolyte-paraffin wax, modified to provide improved tissue support and sectioning
qualities.

Vacuum infiltration is the impregnation of tissues by a molten medium under reduced pressure.
The procedure assists the complete and rapid impregnation of tissues with wax, reduces the time
tissues are subjected to high temperatures thus minimizing heat-induced tissue hardening,
facilitates complete removal of transition solvents, and prolongs the life of wax by reducing

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solvent contamination. Vacuum infiltration requires a vacuum infiltrator or embedding oven,
consisting of wax baths, fluid trap and vacuum gauge, to which a vacuum of up to 760 mm Hg is
applied using a water or mechanical pump. Modern tissue processors are equipped to deliver
vacuum, or vacuum and pressure, to all or some reagent stations during the processing cycle.

Paraffin wax
PROPERTIES
Paraffin wax is a polycrystalline mixture of solid hydrocarbons produced during the refining of
coal and mineral oils. It is about two thirds the density and slightly more elastic than dried
protein.
Wax hardness (viscosity) depends upon the molecular weight of the components and the ambient
temperature. High molecular weight mixtures melt at higher temperatures than waxes comprised
of lower molecular weight fractions. Paraffin wax is traditionally marketed by its melting points
which range from 39°C to 68°C.
Tissue-wax adhesion depends upon crystal morphology of the embedding medium. Small,
uniform sized crystals provide better physical support for specimens through close packing.

Crystalline morphology of paraffin wax can be altered by incorporating additives which result in
a less brittle, more homogeneous wax with good cutting characteristics. There is consequently
less deformation during thin sectioning. Setting temperature does not appreciably affect crystal
size.

MODIFIED PARAFFIN WAXES

The properties of paraffin wax are improved for histological purposes by the inclusion of
substances added alone or in combination to the wax:
• improve ribboning: prolong heating of paraffin wax at high temperatures or use micro-
crystalline wax
• increase hardness: add stearic acid
• decrease melting point: add spermaceti or phenanthrene
• improve adhesion between specimen and wax (alter crystalline morphology): add 0.5%
ceresin, 0.1-5% beeswax, rubber, asphalt, bayberry wax, or phenanthrene.
Early histological wax formulationshave largely been replaced by uniform, high quality
proprietary blends of histological paraffin waxes. Additives recently incorporated in proprietary
waxes include the following:

Piccolyte 115, a thermoplastic terpene resin added at the rate of 5%-10% to the infiltrating wax,
or to the final investing paraffin wax to improve tissue support for thin sectioning and facilitate
flattening and expansion of sections on the waterbath. Piccolyte mixtures cannot be used in
certain models of fluid-transfer type tissue processors.

Plastic polymers such as polyethylene wax, added to improve adhesion, hardness and plasticity.
Polymer waxes are incorporated in the majority of proprietary histological paraffin wax blends
presently available.

Dimethyl sulphoxide (DMSO) added to proprietary blends of plastic polymer paraffin waxes
reduces infiltration times and facilitates thin sectioning. DMSO scavenges residual transition
solvent and probably alters tissue permeability by substituting for or removing bound water thus

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improving infiltration. Some individuals who handle DMSO-paraffin wax may experience an
unpleasant and annoying oyster or garlic taste probably caused by DMSO metabolites. Possible
health risks associated with the use of DMSO-paraffin wax are minimal if correct laboratory
hygiene is practiced.

Processing conditions
Temperature, pressure and agitation reduce the duration of tissue processing and improve the
quality of infiltration.

TEMPERATURE
At low temperatures structural elements of tissues are stabilized against the destructive effects of
solvent changes. This is possibly because of the stiffening and strengthening effect of cold upon
biopolymers resulting from diminution in thermal disruption of secondary bonds of the tissue
constituents. Unfortunately at low temperatures reagent viscosities increase and diffusion rates
decrease, resulting in prolonged processing times.
Isothermally processed mammalian tissues show finer detail and less artifacts than those
processed by the more practicable, common an-isothermic techniques. Heat increases the kinetic
energy of molecules and rate of diffusion, with a corresponding decrease in solution viscosity.

The application of mild heat within the range 37°C to 45°C, during the dehydration and clearing
steps considerably reduces processing times, but may concomitantly increase shrinkage. Tissue
shrinkage during infiltration in paraffin wax results mainly from the effect of heat on collagen.
High infiltration temperatures cause marked tissue shrinkage and hardeningwhich can be
avoided by maintaining embedding waxes 2-3°C above their melting points. Prolonged
immersion in paraffin wax at the correct temperature results in only slight tissue shrinkage
though tissues such as blood, muscle and yolk may harden and become brittle. The extent to
which tissues are affected during paraffin wax infiltration depends upon the combination of
fixative, dehydrant and transition solvent usedas well as the tissue type. Microwave stimulated
processing involves complex molecular interactions, the key element of which is internal
heating, with stimulation of diffusion, and concomitant reduction in the duration of tissue
processing.

PRESSURE AND VACUUM

High pressure facilitates infiltration of dense specimens with viscous resinous embedding media
at the block forming stage, but is rarely employed for biological specimens. Positive pressures
for fluid transfer that are encountered in closed system processors are probably too low to have a
significant influence on tissue infiltration.
Vacuum applied during dehydration, clearing and infiltration stages improves the quality of
processing. Tissues, particularly lung, are de-aerated, and the solvent boiling point is reduced,
thus facilitating evaporation of the reagent from the molten infiltration medium. Duration of wax
infiltration is dependent upon viscosity and is not reduced by the application of vacuum.

AGITATION
Fluid interchange between processing reagents and tissues is promoted by exposure of the
maximum tissue surface area to reagents. If tissues are allowed to settle on the bottom of a
container, remain static in the reagent, or are too tightly packed in the processor basket, tissue
surface area available for fluid exchange will be restricted and the concentration gradient

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between the fluid inside and outside the tissues will be low. Reagent diffusion time is therefore
increased and if the duration of processing is not correspondingly increased, inadequate
processing will result.
During processing, tissues should be loosely packed, suspended and agitated within the medium
to facilitate the exchange of dilute reagent from the tissues with the more concentrated reagent
replacing it. Agitation of tissues and fluids in manual processing is achieved using rotors or
magnetic stirrers. In automatic tissue processors, continual rotary or vertical motion of tissue
containers, or tidal action and flow of processing fluids ensures adequate fluid exchange. Ideally
tissue cassettes should be placed in processors so that the cassette perforations are perpendicular
to the fluid flow. For efficient and effective processing there should be a specimen volume to
processing fluid volume ratio of at least 1:50.

Alternate vacuum and positive pressure cycles during processing may provide some micro
agitation within tissues, but this has yet to be substantiated. In ultrasonic stimulated processing
tissues and fluids are subjected to high frequency agitation and associated phenomena, with
simultaneous reduction in processing time.

Processing methods and routine schedules

Tissues are usually more rapidly processed by machine than by manual methods, although the
latter can be accelerated by using microwave or ultrasonic stimulation. For routine purposes
tissues are most conveniently processed through dehydration, clearing and infiltration stages
automatically by machine. There are two broad types of automatic tissue processors - tissue-
transfer and fluid-transfer types.

Automated tissue processing

TISSUE-TRANSFER PROCESSORS
These processors are characterized by the transfer of tissues, contained within a basket, through a
series of stationary reagents arranged in-line or in a circular carousel plan. The rotary or carousel
is the most common model of automatic tissue processor, and was invented by Arendt in 1909. It
is provided with 9-10 reagent and 2-3 wax positions, with a capacity of 30-110 cassettes
depending upon the model. Fluid agitation is achieved by vertical oscillation or rotary motion of
the tissue basket. Processing schedules are card-notched, pin or touch pad programmed.
Tissue-transfer processors allow maximum flexibility in the choice of reagents and schedules
that can be run on them, in particular, metal-corrosive fixatives, a wide range of solvents, and
relatively viscous nitrocellulose solutions can all be accommodated. These machines have a
rapid turn-around time for day/night processing. In more recent models the tissue basket is
enclosed within an integrated fume hood during agitation and transfer cycles thus overcoming
the disadvantages of earlier styles.

FLUID-TRANSFER PROCESSORS
In fluid-transfer units, processing fluids are pumped to and from a retort in which the tissues
remain stationary. There are 10-12 reagent stations with temperatures adjustable between 30-
45°C, 3-4 paraffin wax stations with variable temperature settings between 48-68°C, and
vacuum-pressure options for each station. Depending upon the model these machines can

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process 100-300 cassettes at any one time. Agitation is achieved by tidal action. Schedules are
microprocessor programmed and controlled. Vacuum-pressure cycles coupled with heated
reagents allow effective reductions in processing times and improved infiltration of dense
tissues.

Fluid-transfer processors overcome the main drawbacks of the tissue-transfer machines. Tissues
are unable to dry out within the sealed retort and reagent vapors are vented through filters or
retained in a closed-loop system. Processors are provided with alert systems and diagnostic
programs for troubleshooting and maintenance. Some models are unable to accept mercury or
dichromate-based fixatives, certain solvents, for example chloroform, or wax additives such as
Piccolyte.

TISSUE RECOVERY PROCEDURES

Procedures for recovery of tissues that have air dried because of mechanical or electrical failure
of the processor are similar to those used for mummified specimens. Tissues accidentally
returned into fixative or alcohol following wax infiltration are recovered by methods outlined in

GENERAL CONSIDERATIONS
• Baskets and metal cassettes should be clean and wax-free.
• Tissues should not be packed too tightly in baskets so as to impede fluid exchange.
• Processors must be free of spilt fluids and wax accumulations to reduce hazards and to
ensure mechanical reliability.
• Fluid levels must be higher than the specimen containers.
• Timing and delay mechanism must be correctly set and checked against the appropriate
processing schedule.
• A processor log should be kept in which the number of specimens processed, processing
reagent changes, temperature checks on the wax baths and the completion of the routine
maintenance schedule, is recorded as an integral part of the laboratory quality assurance
program.

Manual tissue processing

Manual tissue processing is usually undertaken for the following reasons:
• power failure or breakdown of a tissue processor
• a requirement for a non-standard processing schedule as for:
• rapid processing of an urgent specimen
• delicate material
• very large or thick tissue blocks
• hard, dense tissues (nitrocellulose methods)
• special diagnostic, teaching or research applications
• small scale processing requirements
• resin embedding.

The main advantage of manual processing over automated methods lies in the flexibility of

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reagent selection, conditions and schedule design to provide optimum processing for small
batches of tissues. Exposure of tissues to the deleterious effects of some reagents can be carefully
monitored and regulated through observation and precise timing. There is usually considerable
latitude in the processing times given in schedules although maximum rather than minimum
times should be used, as it is better to extend processing rather than risk the problems of under
processed tissues. Manual processing is accelerated using microwave ovens or ultrasonics.
Universal solvents with particularly favorable attributes, normally precluded from routine
machine processing because of budgetary or safety constraints, can be successfully used in small
volumes under controlled conditions for manual processing.
Nonetheless manual processing can be time consuming and inconvenient. Care must be
exercised so that tissues are left overnight in reagents that will cause minimal harm. A permanent
series of solutions in wash bottles simplifies processing small single specimens. Tissues are
processed in tubes and agitated on a rotor. Reagents are pipetted, or decanted through a fine
sieve. Multiple specimens or large blocks are economically processed in large lidded jars of
processing fluids. The specimen to reagent volume ratio should be at least 1:50. Agitation is
provided by a magnetic-stirrer.

Dehydrated tissues float on the surface when transferred to higher density transition solvents
such as chloroform or cedarwood oil. However, if placed in lower density mixtures of dehydrant-
transition solvent before finally transferring to pure transition solvent, tissues will remain
submerged throughout the clearing stage. An alternative approach is to carefully layer the
dehydrant onto the transition solvent and introduce the tissue into the upper layer. The tissue
sinks as the dehydrant gradually replaces the transition solvent. Reagents are carefully decanted
and the specimen placed in a fresh change of transition solvent.

Microwave-stimulated processing
Rapid manual microwave-stimulated paraffin wax processing of small batches of tissues gives
excellent results which are comparable to tissues processed by longer automated non-microwave
methods.
Processing is undertaken in a dedicated microwave oven which is fitted with precise temperature
control and timer, and an interlocked fume extraction system to preclude accidental solvent
vapor ignition. Agitation is provided by an air-nitrogen system.

Domestic microwave ovens with a temperature probe and timer accurate to seconds are suitable
for tissue processing. A turntable or in-built radiation disperser facilitates even reagent heating.
Toxic and flammable solvent vapors generated during processing cannot always be adequately
vented from these ovens and present an ignition hazard if the electrical system is unprotected.
Ovens should therefore be used within a fume cupboard to minimize this problem. Calibration of
domestic ovens is essential for optimum results and the accuracy of the temperature probe,
duration of cycle time, and net power levels at various settings must be determined before the
oven is used to process tissues.

HINTS FOR MICROWAVE PROCESSING

• Tissue blocks should be as thin as possible. Length and width are not as important.
• Process blocks of similar thickness together.
• Reagent volumes should be at least 50 times that of specimen volume.
• The temperature probe should be placed centrally in processing baths.

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• Use a dummy load to check heat generation should reagents boil on minimum settings -
an equal volume of reagent irradiated together with the primary load effectively halves
the energy received by the primary load.
• Pre-heat paraffin wax baths in a conventional oven.
• An increase in the number of cassettes or fluid volumes will require a concomitant
increase in power and or time to achieve the correct processing temperature.

WATER-MISCIBLE MEDIA
Polyethylene glycols (PEG) are water soluble media used for investigation of heat and solvent-
labile lipids and proteins, and to overcome tissue shrinkage, damage and distortion inherent in
the paraffin wax technique. The polyethylene glycols, or Carbowaxes, are polymers of varying
length (the numerical suffix denotes molecular weight). At room temperature PEG 200 and PEG
600 are syrupy liquids, PEG 1000 is soft and slippery, PEG 1500 is hard, and PEG 4000 is hard
and brittle. In general they are less elastic, denser and somewhat harder than paraffin wax.
Crystal slip is a bigger problem than in paraffin and sectioning deformation is mainly non-
recoverable. Tissues are dehydrated by gradual infiltration through increasing concentrations of
aqueous PEG solutions, to pure PEG in which they are finally embedded. Sections are cut in a
low humidity environment, otherwise considerable difficulties arise. They are difficult to flatten
without loss of tissue and adhere poorly to slides, leading to the development of numerous
flotation fluids. Low viscosity nitrocellulose (LVN) or water insoluble polyvinyl acetate resin
incorporated into PEG dehydrating, infiltrating and embedding solutions allow water flotation of
sections. The PEG dissolves, leaving the tissue in a thin film of LVN or PVA which is mounted
on albumenized slides in the usual manner. These approaches surmount many of the previous
problems inherent with PEG. The nitrocellulose is removed from sections by immersing in PEG
200 for 15 minutes.
Problems with this method include the high viscosity of infiltrating media necessitating slow
agitation and uneven distribution of LVN in the final embedding mix which results in crazed
blocks. These can be overcome mostly by thorough blending of the LVN and PEG. With current
interest in immunohistochemistry, polyethylene glycols may warrant re-evaluation. However
considerable time and patience are required when using these waxes.

Polyethylene glycol monostearate (Nonex 63B), a water soluble synthetic wax is used in a
similar manner to polyethylene glycol and polyester waxes with application in histochemistry
and botanical histology.

WATER-TOLERANT MEDIA
Diethylene glycol distearate is a hard, brittle, water tolerant ester (m.p. 47-52°C). It has certain
deficiencies when used for routine embedding, unless combined with other substances as in ester
waxes. However it may be used unmodified for thin sectioning (0.5-2 µm) of freeze dried and
osmium tetroxide fixed tissues for high resolution light microscopy. Tissues are dehydrated and
cleared as in the paraffin wax method.

Ester waxes, developed by Steedman, and subsequently modifiedhave low melting points, are
hard at room temperature and have good adhesive properties. They are therefore ideal for
supporting and serially sectioning refractory hard, criticized material such as arthropods, and
tissues which heat-harden excessively. They are also used for simple investment of paraffin
blocks to be sectioned under hot conditions and in double embedding with agar. Ester waxes are

14
no longer commercially available and must be prepared from the basic ingredients.

Ester Wax 1960 (m.p. 48°C)

Polyester wax, developed by Steedman is a ribboning, low melting point wax which reduces
heat-induced artifacts. It is recommended for heat labile tissues, to minimize heat-induced
hardening in difficult tissues and is an ideal medium for combined light and scanning electron
microscopy of animal tissues. The properties of the wax facilitate immunohistochemical
investigations as antigenic determinants are well preserved. The main advantages of this medium
are low melting point and infiltration directly from 96% ethanol permitting a near isothermic
processing schedule for mammalian tissues. Polyester wax is no longer commercially available
and must be prepared from the basic ingredients.

HYDROPHOBIC MEDIA
Nitrocellulose
Celloidin (C) and Low Viscosity Nitrocellulose (LVN), mixtures of di- and tri-nitrocellulose, are
composed of yellowish-white matted filaments with the appearance of raw cotton. Nitrocellulose
is insoluble in water, but soluble in absolute ethanol-diethyl ether, amyl acetate, methyl
benzoate, methyl salicylate and 2-ethoxyethanol and is set by most hydrocarbon solvents. It is
highly flammable, and must be kept alcohol-damped with n-butanol or as 8% solutions in
ethanol-ether or 1% LVNC in methyl benzoate as it is explosive if detonated when dry. Celloidin
solutions have a low tolerance of water and dehydration must be thorough. LVN tolerates up to
6% of water, has superior penetration and final block hardness and is supplied in various grades
of viscosity and nitrogen content. Nitrocellulose tissue processing techniques are generally
employed for sectioning hard tissues such as bone, for topographical studies of central nervous
system tissues, or for delicate embryonic material. Tissues are processed at room temperature
producing minimal and shrinkage and hardening. Immunohistochemical investigations such as
immunophenotyping of lymphoid and non-lymphoid cells are possible on nitrocellulose
processed tissues.

Methods for difficult tissues

HARD DENSE TISSUES
Tissues largely comprised of thickened keratin, dense collagen, closely packed smooth muscle
fibers, colloid, areas of hemorrhage, thrombi or yolk, can be hardened excessively when
processed on routine schedules and consequently, sections may crumble or shatter. Ideally the
fixative, processing reagents, embedding medium and schedules should be selected to minimize
hardening in these tissues. Despite careful processing hard tissues frequently require treatment
with post-embedding adjuvant before microtomy.
Mammalian tissues such as uterus, scirrhous carcinoma, leiomyomas and keratinized tissues are
softened by fixing in 4% phenol in a mixture of absolute ethanol (75 ml), water (10 ml) and
chloroform (10 ml), or by treating fixed tissues using 4% aqueous phenol for 24-72 hours.
Similar results are obtained by dehydrating tissues using phenol in the first bath of absolute
ethanol, or in all dehydrant baths. Transition solvents such as chlorinated hydrocarbons and
terpenes are recommended as they do not exacerbate tissue hardness.

UNDERPROCESSED TISSUE
Carson Book, Page 26, Fig.2-1

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• Bone not dehydrated or cleared sufficiently
• Poor Infiltration
• Center of block is soft (White area), indication of
• Area not well infiltrated with paraffin
• Block cannot sectioned.

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