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Leigh Winsor
INTRODUCTION
Stabilised tissues must be adequately supported before they can be sectioned for
microscopical examination. Whilst they may be sectioned following a range of
preparatory freezing methods, 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 (Table.1). Preeminent amongst these is the paraffin wax method, discussed here in detail, which is
considered to be the most suitable for routine preparation, sectioning, staining and
subsequent storage of large numbers of tissue samples.
The quality of structural preservation seen in the final stained and mounted section is
largely determined by the choice of fixative and embedding medium. During tissue
processing loss of cellular constituents and shrinkage or distortion should be minimal.
After fixation, post-fixation and preparatory procedures, the four main stages in the
paraffin method are dehydration, clearing, infiltration and embedding.
Tissue markers are applied to the surface of the specimen using disposable swabs and
allowed to dry.
India ink provides good black macro and microscopic marking, is resistant to processing,
but takes 15-30 minutes to dry, and may spread beyond the marked area. Silver and gold
inks are not recommended as they are solvent soluble4.
Silver nitrate (stick) provides a brown-black mark resistant to processing. Aqueous or
alcoholic silver nitrate solutions behave like India ink and are not recommended.
Artists' grade pigments are radio-opaque, processing resistant and provide good macro
and microscopic contrast. Prepare by finely grinding pigment (50% w/v) to a thin paste in
acetone4,and store in tightly stoppered containers. These markers dry in 15-30 minutes.
Particulate pigments, 8% pigment w/v in 24% gelatine solution3, dry in less than 5
minutes, or in about 10 seconds on chilled specimens. Paprika, turmeric, henna, India ink,
and Bismark brown are all inexpensive, strongly coloured processing-resistant pigments
with distinctive microscopic particle morphology.
Alcian blue, 1% aqueous solution, is a rapid and reliable stain for marking resection
margins of fixed breast5 and other biopsies. The specimen is dipped into the stain for a
few seconds then blotted dry. Sufficient dye remains to mark resection margins.
Eosin, Erythrosin and Rose Bengal, 1-2% aqueous, are used to stain small translucent
specimens. Tissues are stained for 5 minutes, rinsed in water then processed. Although
some dye is lost in the dehydration alcohols, sufficient remains to render the tissues
visible. Alternatively dye is incorporated in the 95% ethanol dehydrant, and tissues
stained during the routine dehydration step.
Tissue marking dyes are available commercially and have been favourably evaluated6.
Completion of fixation
Tissues should be fixed before processing is initiated. Poorly fixed tissues are
inadequately protected against the physical and chemical rigours of processing. Strategies
commonly employed to ensure complete fixation of tissues include:
CONCENTRATION
If the concentration gradient between fluid inside and outside the tissue is too high, rapid
reagent diffusion and accompanying strong diffusion currents have the potential to shrink
and disrupt tissues. For this reason specimens are almost always processed through a
graded series of reagents of increasing concentration, the more delicate the tissues, the
closer the gradations.
MISCIBILITY
Processing reagents which are miscible with water and with the embedding medium
reduce the number of processing stages and are termed universal solvents. To avoid
severe tissue shrinkage from concentration and polarity effects, they are often employed
in a graded series.
Many transition solvents, for example xylene, are extremely water-intolerant, and are
immiscible with hydrated alcohols. Couplers such as phenol mixed with a transition
solvent permit clearing from 70%-95% alcohols36. Originally couplers were employed to
overcome difficulties with hydrated higher alcohols. However they are now used in
processing yolky or blood-filled tissues which harden excessively if fully dehydrated in
absolute ethanol, and complement the use of tissue modifiers.
EVAPORATION RATE
The evaporation rate, rather than the vapour pressure or boiling point of a solvent, is the
best predictor of the rate of elimination of a substance from molten infiltrating wax.
Solvents with high evaporation rates are the most readily vaporised and are less likely to
contaminate the infiltration medium.
VISCOSITY
Viscosity is the internal friction of a particular substance which affects rate of flow
through tissues and is inversely proportional to temperature. It is particularly important in
the clearing and infiltration stages of processing. Substances with high molecular weight,
such as some transition solvents and waxes, have high viscosities and diffuse through
tissues more slowly than, for example, the lower molecular weight, lower viscosity
dehydrant alcohols.
If tissue shrinkage or swelling is to be avoided when the specimen moves from one
processing step to the next, the fluid already in the tissues must diffuse outward through
the tissue pores at the same rate as the fresh medium diffuses inwards. If the viscosity
differential between fluid inside and outside the tissue is too great, shrinkage will result.
Hence slow and gradual processing of tissues is necessary when viscous reagents are
used (for example in nitrocellulose embedding methods).
EMBEDDING MEDIA
Infiltrating and embedding media must fill all spaces within the tissue to support cellular
components adequately during microtomy. Density of the hardened medium should
approach that of the densest tissue component otherwise section deformation will result.
The matrix must be elastic enough to recover sectioning deformation, and plastic enough
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 stabilised against the destructive
effects of solvent changes35. 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 constituents35. 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 artefacts than those
processed by the more practicable, common an-isothermic techniques19. 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 37C to 45C, during the
dehydration and clearing steps considerably reduces processing times 18,36, but may
concomitantly increase shrinkage21. Tissue shrinkage during infiltration in paraffin wax
results mainly from the effect of heat on collagen16.
High infiltration temperatures cause marked tissue shrinkage and hardening17,21 which can
be avoided by maintaining embedding waxes 2-3C above their melting points21.
Prolonged immersion in paraffin wax at the correct temperature results in only slight
tissue shrinkage16,37 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 used17,22,23,38 as
well as the tissue type. Microwave stimulated processing involves complex molecular
interactions, the key element of which is internal heating, with stimulation of diffusion34,
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 stage19, 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 vacuum39.
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 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
processing40-41 tissues and fluids are subjected to high frequency agitation and associated
phenomena, with simultaneous reduction in processing time.
Dehydration
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 alcohols42.
Dehydration is effected as follows:
Dehydration is necessary in all infiltration methods, except where tissues are simply
externally supported by an aqueous embedding medium. Choice of a dehydrant is
determined by the nature of the task, the embedding medium, processing method, and
economic factors. Dehydrants differ in their capacity to cause tissue shrinkage (Fig. 3).
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.
Whilst well fixed tissues can be transferred directly to 95% ethanol,51 incompletely fixed
tissues may exhibit artefacts 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 minimise tissue distortion from diffusion
currents, delicate specimens are dehydrated in a graded ethanol series from water through
10%-20%-50%-95%-100% ethanol.36
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, colourless, 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 conditions34. Ethanol dissolves nitrocellulose
slowly unless combined in equal proportions (or better, 1:2)53 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 brittle19,22-23. Colloid, blood, collagen
and yolky tissues are particularly affected19. The problem is exacerbated by heat during
wax infiltration.
Anhydrous cupric sulphate added to the final absolute ethanol on a tissue processor
scavenges any water present. The salt is self-indicating: white when anhydrous, blue
when hydrated, and is only slightly soluble in ethanol. It is prepared by carefully heating
hydrated cupric sulphate until it turns white, on a hotplate at 250C. Cool in a desiccator.
Anhydrous calcium sulphate (Drierite) or molecular sieves act in a similar manner but are
non-indicating. Solid drying agents are placed in a 1-2 cm thick layer in the reagent
container, and covered with filter paper or fine sponge to avoid mixing with the
specimens during tissue agitation.
Methanol is a good ethanol substitute54 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 ethanol34.
Isopropanol was first suggested as an ethanol substitute during the prohibition era in the
United States54. It is a universal solvent available as 99.8% (absolute) isopropanol,
slightly slower in action and not as hygroscopic as ethanol, but a far superior lipid
solvent. Isopropanol is completely miscible with water and most organic solvents, is fully
miscible with melted paraffin wax55, and is readily expelled from tissues and wax baths.
Isopropanol shrinks and hardens tissues less than ethanol54-57 and is used to dehydrate
hard, dense tissues, which can remain in the solvent for extended periods without harm.
To minimise shrinkage, fixed tissues are transferred via 60%-70% isopropanol or ethanol
to absolute isopropanol.
Isopropyl alcohol has also been recommended as a xylene substitute58. In microwave
stimulated processing, though unsatisfactory as a dehydrant, isopropanol is used as a
transition solvent following ethanol dehydration34.
Isopropanol only dissolves nitrocellulose in the presence of esters59 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 chitinised arthropods38 and rodent tissues. It causes
less hardening and shrinkage than ethanol,38,60 though this is offset by the prolonged
processing schedules which may result in tissue shrinkage.18,22 N-butanol is poorly
miscible with water and only slowly miscible with paraffin wax. It is flammable, with a
penetrating camphor-like odour, and the vapours are eye irritants. Iso-butanol, with
similar properties and processing characteristics22,23 is a less costly substitute for nbutanol. Tertiary-butanol is widely used in plant histology61 but rarely for animal
tissues17,23. Below 26C it is hygroscopic crystalline solid, a major disadvantage. In
processing it is used in a similar manner to n-butanol.61
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.
Tissues can be transferred directly from acetone to paraffin wax as the solvent boils off
under vacuum68. However a transition solvent is normally interposed before the paraffin
baths. Acetone is not recommended for microwave processing as it causes excessive
nuclear shrinkage34.
Tetrahydrofuran is a colourless, highly volatile and flammable universal solvent with an
offensive ethereal odour. 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.69 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 dangerous66.
2,2 dimethoxypropane (DMP) and 2,2 diethoxypropane (DEP) are used for chemical
dehydration of tissues.43-51 They are flammable and form peroxides.50 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,48 a transition solvent such as methyl
salicylate49 or toluene should precede infiltration with wax. DMP shrinks tissues slightly
less than DEP.51 Chemical dehydration is suitable for rapid manual processing or machine
processing, and is comparable to conventional dehydration for tissue morphology and
staining reactions.51 Acidified DMP/DEP can be reused several times, though dehydration
times need to be extended. The reagent is stored at 4C in a spark-proofed refrigerator.
Phenol, beechwood creosote and aniline facilitate dehydration when mixed with
transition solvents, as in Weigert's carbol-xylol (xylene 75 ml and phenol 25 ml).36 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 discolouration. Containers
must be protected from light and tightly sealed. Phenol crystals and 80% concentrate
react violently with formaldehyde.
Clearing
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 wax17,18 (Fig 4). In the final stage shrinkage may
result from the extraction of fat by the transition solvent.18
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.70 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:
Transition solvents
HHYDROCARBONS
These are odourless flammable liquids with characteristic petroleum or aromatic odours,
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 xylene66
relate 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 stabilised in protein coagulant fixatives (Bouin's or SUSA) are less affected.17,23
Benzene is more gentle and rapid than xylene and toluene and is probably the best
transition solvent, though toxicity and possible carcinogenicity64 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 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.66 Many of these solvents have a particularly
strong petroleum odour 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 colourless solvents with sweet odours 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.72
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
muscle32 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 xylene73,74 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 stabilisers to inhibit reactions with
aluminium and its alloys.59 Although mildly toxic (except at high concentrations)64 the
decision to substitute them for more toxic solvents must be soundly based (Table 7.3)75.
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.75
ESTERS
These are colourless flammable solvents miscible with most organic solvents and with
paraffin wax.
n-Butyl acetate is used as a xylene substitute76 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 odours 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 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 helminth parasites for examination and whole
mounting. Methyl salicylate clears tissues from 96% ethanol, hardens less and has a more
pleasant odour than methyl benzoate. It causes minimal tissue shrinkage and hardening18
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,77
though some are now synthesised. They are the earliest transition solvents to be used in
histology78 and include turpentine and oils of bergamot, cedarwood, clove, lemon,
origanum and sandalwood.61,79 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 odours which can be overpowering, requiring
good laboratory ventilation.
When used for processing hard, dense or chitinised 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 minimised 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 oil65. Cedarwood oil is expensive,
but exhausted oil can be restored by filtering, then heating to 60C under vacuum for 3060 minutes80.
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.81,82
Terpineol is a clear almost colourless mixture of isomers with a faint pleasant odour and
very low evaporation rate. It clears tissues from 80%-90% alcohol with minimal
hardening. Like the other 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 helminths. Tissues may remain in
it indefinitely without harm.
Some of the specific safety requirements relating to processing solvents are summarised
in Table 3.
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 fulfil 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,
gelatine, or wax which when solidified will provide sufficient external support during
sectioning.
Infiltration 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 minimising heat-induced
tissue hardening, facilitates complete removal of transition solvents, and prolongs the life
of wax by reducing 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.
Parffin 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.33
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 39C to 68C.
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.84-86
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:
Early histological wax formulations36,88-89 have 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,90 or to the final investing paraffin wax to improve tissue support for thin
sectioning and facilitate flattening and expansion of sections on the waterbath.91-92
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.93 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.94 DMSO scavenges
residual transition solvent and probably alters tissue permeability by substituting for or
removing bound water thus improving infiltration. Some individuals who handle DMSOparaffin wax may experience an unpleasant and annoying oyster or garlic taste probably
caused by DMSO metabolites.95 Possible health risks associated with the use of DMSOparaffin wax66 are minimal if correct laboratory hygiene is practised.
a supply of clean, filtered paraffin wax held at 2-4C above its melting point.
a cold plate to rapidly cool the wax.
a supply of moulds in which to embed the tissues.
1 Open the tissue cassette, check against worksheet entry to ensure the correct number of
tissue pieces are present.
2 Select the mould, there should be sufficient room for the tissue with allowance for at
least a 2 mm surrounding margin of wax.
During cooling, paraffin wax shrinks up to 15%, causing compression in tissues.17 This
compression is almost fully recovered when sections are floated on a warm waterbath6;
compression resulting from microtomy is only partially recovered.
invented by Arendt in 1909.79 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 (Table 4)
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 (Fig.8) 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 (Fig.9) 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 30-45C, 3-4 paraffin wax stations with variable temperature settings
between 48-68C, and vacuum-pressure options for each station. Depending upon the
model these machines can 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 vapours 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. Representative schedules for rapid and
overnight processing are provided in Table 5.
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 Table 6.
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
The main advantage of manual processing over automated methods lies in the flexibility
of 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.
Schedules for rapid manual processing using chemical dehydration (Table 7), one and
two day routine processing (Table 8), and extended manual processing for large, thick, or
hard tissues (Table 9) are provided.
Universal solvents with particularly favourable 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.
Schedules are provided for manual processing using n-butanol (Table 10) and dioxane
(Table 11).
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.34,97-101
Processing is undertaken in a dedicated microwave oven (Fig. 10) which is fitted with
precise temperature control and timer, and an interlocked fume extraction system to
preclude accidental solvent vapour 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 vapours 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 minimise 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.34
EQUIPMENT
Tissues are processed in conventional plastic cassettes, including those with (provided the
metal lids lie below the fluid).9 Transparent glass or solvent-resistant plastic containers of
about 200 ml capacity are ideal for processing batches of up to 14 cassettes per container.
FIXATION
For rapid processing, tissues are fixed by microwave irradiation,9 or in 95% ethanol (600
ml)-polyethylene glycol PEG 400 (45 ml)102 from which specimens can be transferred
directly to dehydrant. Formaldehyde-fixed tissues must be rinsed in running tap water for
5 minutes before microwave processing and an extra dehydration change incorporated in
the schedule. Processing times for formaldehyde-fixed tissues need to be increased above
those provided for coagulant-fixed tissues.34 Picric acid fixed tissues should not be
microwave processed as there is an explosion risk even in well washed tissues.34
Processing schedules are provided in Table 12.
HINTS FOR MICROWAVE PROCESSING
- Tissue blocks should be as thin as possible. Length and width are not as important.34
Ultrasound-stimulated processing
Ultrasonics are used in histopathology to accelerate fixation, tissue processing for
electron microscopy, the decalcification of bone, tissue softening in post-embedding
adjuvants, improving the sensitivity of immunohistochemical reactions, conventional
staining and for accelerated tissue processing. Unfixed tissue blocks 1-2 mm thick can be
fixed and processed to paraffin wax using ultrasonic-stimulation in 1 hour 45 minutes.40-41
The most important effect of ultrasound at frequencies of 100 kHz-1 MHz is agitation. At
lower frequencies cavitation phenomena and attendant heat, pressure and streaming
effects may damage tissues and care must be exercised.
Processing is performed in reagent containers suspended in a detergent solution within
the transducer tank of an ultrasonic cleaner operated at 50 watts. An immersion heater is
used to elevate bath temperatures for paraffin wax infiltration. Tissues are placed in metal
or plastic cassettes for processing.
Coagulant fixatives provide optimal stabilisation for ultrasonic-stimulated processing.40
Tissues are dehydrated in ethanol and cleared in toluene, or preferably methyl benzoate
or methyl salicylate (Table 13). Cells and organelles such as cilia, microvilli and
desmosomes are all well preserved. Old and friable specimens sometimes exhibit
marginal distortion and erosion.
Resin embedding methods are now used for many of these applications. Non-resinous
embedding media include those listed below.
AQUEOUS MEDIA
Agar has a high melting point and low gelling temperature of agar make it ideal for
2 Glyceryl monostearate 30 g
3 300 polyethylene glycol distearate 10 g
METHOD
1 Melt the diethylene glycol distearate and heat it until clear. Add the glyceryl
monostearate and when dissolved add the polyethylene glycol distearate.
2 Filter through coarse filter paper supported in a ring rather than a filter funnel.
In Tropical Ester wax 1960119 (m.p. 50C) triethylene glycol monostearate 10 g is
substituted in the forgoing formula for the 300 polyethylene glycol distearate. This
modification permits sectioning and ribboning at room temperatures of 37.5C. Tissues
are infiltrated at 56-60C.
Ester waxes are soluble in 95% ethanol, n-butanol, cellosolve and xylene. Tissues are
usually dehydrated via 2-ethoxyethanol in which the waxes are soluble, obviating the
need for a transition solvent. Ester waxes do not charge with static electricity, and have
good ribboning, thin sectioning and glass adhesion properties.
Polyester wax, developed by Steedman120 is a ribboning, low melting point wax which
reduces heat-induced artefacts. It is recommended for heat labile tissues,121 to minimise
heat-induced hardening in difficult tissues and is an ideal medium for combined light and
scanning electron microscopy of animal tissues.122 The properties of the wax facilitate
immunohistochemical investigations as antigenic determinants are well preserved.123 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 (Table
15). Polyester wax is no longer commercially available and must be prepared from the
basic ingredients.
Polyester Wax 90/10 (m.p. 38C)
REAGENTS REQUIRED
1 400 polyethylene glycol distearate 90 g
2 1-hexadecanol (cetyl alcohol) 10 g
METHOD
1 Melt the polyethylene glycol at 60 C, then mix in the cetyl alcohol.
2 Cool to a solid from which working quantities are obtained.
The wax has good water tolerance and is soluble in most histological dehydrants and
transition solvents. Because of its low melting point, non-volatile transition solvents such
as cedarwood oil and other terpenes should not be used. Polyester wax adheres to metal
embedding moulds and paper-boat or plastic peel-a-way moulds are recommended.
Normally blocks are cut in cool room temperatures using a cooled knife. Polyester wax is
more conveniently sectioned on a cryotome at -5C to 22C. Sections are affixed to
gelatine subbed or aminoalkylsilane treated slides, or floated on amylopectin. 120 Blocks
and sections are stored at 4C. Polyester wax is dissolved from sections with absolute
ethanol. Steedman19 proposed simultaneous fixation and infiltration of tissues with picro-
4 For use melt the agar as previously indicated, cool to 50-60C, and hold in a 60C
oven. Loosen container caps before remelting agar.
5 Embed tissues by pipetting agar solution over tissue fragments correctly oriented on a
clean flat surface129 or membrane filter.105
TECHNICAL NOTES
1 Specimens can be embedded in silicon-rubber flat embedment moulds, moulds made
from cut-down plastic syringes,130 or in metal Tissue-Tek type moulds.132,135
2 Unstabilized agar embedments sometimes disintegrate during processing and must be
stabilised by the addition of 2.5%-4% formaldehyde to the medium, or by immersing
blocks in 70% ethanol or fixative for 1-3 hours.
Agar-Paraffin Wax Double Embedding For Fragments, Biopsies And Friable
Specimens135
REAGENT REQUIRED
Agar (prepared as above)
METHOD
1 Fill the appropriate sized Tissue-Tek base mould with agar medium cooled to
approximately 40C.
2 Number embedding cassette, cross checked with request slip and specimen container.
3 Place specimen in agar-filled mould and orientate.
4 Allow agar to solidify for 5 minutes. Place embedding cassette on top of the mould to
identify the tissue.
5 When the agar block is solid, detach the specimen from the mould by sliding a scalpel
down one side. Trim and notch the agar block as required, leaving a 3-5 mm width of
agar surrounding the tissue. Process normally.
Agar-Paraffin Wax Double Embedding For Bone Marrow Aspirates And Cell
Suspensions Using The Collodion Bag Technique137
REAGENT REQUIRED
Collodion (add 4% nitrocellulose to 25 ml absolute ethanol : 75 ml diethyl ether)
METHOD
1 Place 1-2 ml of collodion into a 10 ml glass centrifuge tube. Rotate the tube so that an
even layer of collodion coats and sets on the inside surface; gentle blowing into the tube
hastens the setting process.
2 Fill the tube with 70% ethanol and stand for 5 minutes to harden the collodion. Drain
the ethanol, rinse with water.
3 Add fixed aspirate or suspension to tube.
4 Centrifuge at 2000 rpm for 30 seconds.
5 Decant supernatant fluid.
6 Pipette 1-2 ml of agar, cooled to approximately 45C, into the centrifuge tube.
7 Rapidly resuspend the specimen and centrifuge at 2000 rpm for 1 minute.
8 Allow the mass to cool for 5 minutes.
9 Using fine forceps, carefully detach the collodion bag and contents from the tube.
10 Trim off unwanted collodion, and process as a normal specimen, taking care to orient
and embed the specimen correctly.
AGAR - ESTER WAX DOUBLE INFILTRATION
Double infiltration of tissues in agar and ester wax138-139 aids thin serial sectioning of
chitinised tissues at 0.5-1.0 m and is a possible alternative to pine resin-beeswax
paraffin wax used to support plastic vascular prostheses for sectioning.140 The fine
crystalline nature and hardness of ester wax improves tissue-wax adhesion and provides
adequate support for thin serial sectioning.
Agar-Ester Wax Double Infiltration138
REAGENTS REQUIRED
1 5% aqueous agar
Cellosolve (2-ethoxyethanol)
METHOD
1 Infiltrate fixed tissues in 5% aqueous agar solution for 1 hour at 50-60C.
2 Orientate tissues in an agar filled mould as indicated previously. Allow the agar to set.
Trim the block.
3 Pass the block through the following series, 30 minutes in each solution: 30%, 50%,
70% ethanol; 90% ethanol plus cellosolve, 2:1; the same 1:2; pure cellosolve, 3 changes;
cellosolve plus ester wax 1:1; pure ester wax, at least 2 changes, the last overnight.
4 Embed as usual and cool rapidly.
TECHNICAL NOTE
Buzzell141 dehydrates in dioxane, with amyl acetate as a transition solvent. Blocks are
best cut using a retracting microtome.
NITROCELLULOSE - PARAFFIN WAX DOUBLE INFILTRATION
Nitrocellulose-paraffin wax double embedding is mainly used for brain, friable tissues
and decalcified bone and is particularly useful for whole body sections of small animals
and chitinous tissues. It combines the plasticity and support provided by nitrocellulose
with convenient handling and microtomy of the paraffin technique.
Tissues may be (a)infiltrated with thick nitrocellulose solutions and the resulting block
trimmed, hardened in chloroform and infiltrated in paraffin wax, or (b)infiltrated with
thin low viscosity nitrocellulose (LVN) solutions which are integrated into a normal
processing schedule. Proprietary celloidin-ethanol-ether solutions provide a simple and
convenient double-embedding method (Table 16). The principle drawbacks of this
technique are prolonged exposure of tissues to absolute ethanol and the high flammability
and volatility of diethyl ether precluding machine processing. Use of methyl benzoate or
methyl salicylate as LVN solvents overcome these deficiencies.
In Peterfi's classic technique,142 tissues are dehydrated to absolute ethanol, infiltrated with
1% celloidin in methyl benzoate with 2-3 changes over 24-72 hours (until clear),
hardened in three changes of toluene for 8 hours, then infiltrated as usual in paraffin wax.
Molnar's variation143 (Table 17) is shorter and more logical, and is suitable for manual or
machine processing.
The n-butanol added to reduce the explosion hazard may contribute up to 30% of the
weight of the nitrocellulose127 and must be taken into consideration when preparing
solutions. Nitrocellulose dissolves slowly in methyl salicylate, and during preparation
mixtures should be periodically shaken to assist dissolution. In recent times LVN has
largely replaced celloidin.
Sections of double-embedded tissues may tend to wrinkle or curl on the waterbath.
Floating on 95% ethanol or Ruyter's fluid139 softens the LVN and facilitates section
flattening.