Tissue Processing (1)

7/27/2019 Tissue Processing (1)

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Tissue processing Leigh Winsor

INTRODUCTIONStabilised 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 ofreagents and finally infiltrated and embedded in a stable medium which when hard,

provides the necessary support for microtomy. This treatment is termed tissue processing.

ethods have evolved for a range of embedding media and applications !Table."#. $re%

eminent amongst these is the paraffin wax method, discussed here in detail, which isconsidered 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 islargely determined by the choice of fixative and embedding medium. &uring tissue

processing loss of cellular constituents and shrinkage or distortion should be minimal.

'fter fixation, post%fixation and preparatory procedures, the four main stages in the

paraffin method are dehydration, clearing, infiltration and embedding.

Tissue sampling and identificationTissue sampling generally follows standard protocols",( established by each laboratory for particular species and categories of specimens. Tissue blocks for processing should be as

thin as is consistent with the purpose for which they are required, usually "%( mm thick

for urgent specimens and rapid processing) *%+ mm for routine material processedovernight. Specimens should not be tightly packed into processing cassettes or

containers, but should have sufficient free space to facilitate fluid exchange. Small

specimens and tissue fragments are processed in fine mesh containers, wrapped in lens

tissue, sandwiched between sponge biopsy pads or more safely, double embedded inagar%paraffin wax.

Specimens are generally identified by a numbering system that is not bleached by

subsequent fluid and solvent treatment. xamples include !a# a numbered card labelgenerated by computer%printer, or handwritten in soft lead pencil or waterproof ink !b#

colour coded plastic cassettes, machine or manually labelled.

Tissue marking and orientation

arking facilitates identification and correct orientation of particular tissue pieces orsurfaces during embedding and subsequent microscopical examination. Tissue blocks are

simply marked by cutting a notch on the reverse side of the block face to be sectioned, or

by trimming the block to a particular shape. -owever dye marking is preferred for certain

surgical specimens, small tissue pieces, and for serial sectioning orientation.

TISSUE MARKIN SU!STANCES

riteria* for the selection of a suitable tissue marker are/

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• the marking substance must be relatively insoluble in fixative, processing reagents

and embedding medium.

• it must survive fixation and processing and not result in unacceptable

contamination of the reagents and other tissues processed simultaneously.

• it must remain on the surface of the specimen and not penetrate tissue.

•it should not react unfavourably with histological stains and must be clearlyidentifiable both macroscopically and microscopically.

• for some purposes it may be important that the marker is either radiolucent or

radio%opaque.

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 "+%*0 minutes to dry, and may spread beyond the marked area. Silver and goldinks are not recommended as they are solvent soluble1.

Sil"er nitrate !stick# provides a brown%black mark resistant to processing. 'queous oralcoholic silver nitrate solutions behave like 2ndia ink and are not recommended.

Artists# grade pigments are radio%opaque, processing resistant and provide good macro

and microscopic contrast. $repare by finely grinding pigment !+03 w4v# to a thin paste inacetone1,and store in tightly stoppered containers. These markers dry in "+%*0 minutes.

$articulate pigments, 53 pigment w4v in (13 gelatine solution*, dry in less than +minutes, or in about "0 seconds on chilled specimens. $aprika, turmeric, henna, 2ndia ink,

and 6ismark brown are all inexpensive, strongly coloured processing%resistant pigments

with distinctive microscopic particle morphology.

Alcian %lue, "3 aqueous solution, is a rapid and reliable stain for marking resection

margins of fixed breast+ and other biopsies. The specimen is dipped into the stain for afew seconds then blotted dry. Sufficient dye remains to mark resection margins.

Eosin& Er't(rosin and Rose !engal, "%(3 aqueous, are used to stain small translucent

specimens. Tissues are stained for + minutes, rinsed in water then processed. 'lthoughsome dye is lost in the dehydration alcohols, sufficient remains to render the tissues

visible. 'lternatively dye is incorporated in the 7+3 ethanol dehydrant, and tissues

stained during the routine dehydration step.

Tissue marking dyes are available commercially and have been favourably evaluated8.

Completion of fi)ationTissues should be fixed before processing is initiated. $oorly fixed tissues areinadequately protected against the physical and chemical rigours of processing. Strategies

commonly employed to ensure complete fixation of tissues include/



• microwave irradiation of biopsy specimens in normal saline.9%7

• continuing fixation on the tissue processor with one or more changes of the

routine fixative, often at elevated temperatures of 10:%80:.

• secondary fixation of tissues in formol sublimate on the tissue processor "0, or in

an alcoholic fixative which will complete fixation whilst initiating dehydration.

•fixing in buffered phenol%formaldehyde p- 9.0 and p- +.+ sequence at 10:

""

.

$ost fi)ation procedures;n completion of fixation, tissues fixed in certain reagents must undergo special

treatment.

*i)ati"es containing dic(romate and c(romium trio)ide

&ichromates and chromium trioxide are reduced to insoluble green%brown chromic oxide

in the higher alcohols and in dioxane. Tissues must be washed for 5%"( hours in running

water before transferring to 803%903 ethanol or dioxane.

*i)ati"es containing p(osp(ate$hosphate salts precipitate in alcoholic solutions stronger than 903 ethanol, in dimethoxy

propane, and in diethoxy propane. 2f they are deposited within tissues the precipitate can

cause sectioning difficulties"(. Tissues are rinsed free of fixative with water and processing initiated in 803%903 ethanol.

*i)ati"es containing picric acid

Tissues fixed in non%alcoholic picric acid%based fixatives are washed in repeated "%*

hourly changes of +03%903 ethanol until the supernatant is faintly yellowish or clear.This may take (%* days. Specimens fixed in alcoholic picric acid fluids are washed in

503%703 ethanol, as anhydrous conditions must be maintained. $icric acid retained in

tissues can impede wax infiltration and exacerbate static electrification of ribbons duringsectioning. 2t also has an adverse affect on stored wax embedded tissues"*.

*i)ati"es containing urea

Tissues fixed in urea containing fluids are washed overnight before transfer to 13

formaldehyde solution for storage. <rea complexes with formaldehyde to form insolubleurea%polymer pigments"*.

Specific fi)ati"e re+uirements

arnoy fixed tissues are near%anhydrous and are placed directly in absolute ethanol or in

alcohol%transition solvent.

-eidenhain=s S<S' fixed tissues are transferred directly to 7+3 ethanol, as

trichloroacetic acid fixed collagen swells in aqueous solutions.

Tissues fixed in osmium tetroxide%based fixatives are washed for + hours in running

water and dehydration initiated in *03 ethanol. ;smium tetroxide is reduced to blackosmium in ethanol.



$rinciples of tissue processingTissue processing is concerned with the diffusion of various substances into and out of

stabilised porous tissues. The diffusion process results from the thermodynamic tendency

of processing reagents to equalise concentrations inside and outside blocks of tissue, thusgenerally conforming to >ick=s ?aw/ the rate of solution diffusion through tissues is

proportional to the concentration gradient !the difference between the concentrations ofthe fluids inside and outside the tissue# as a multiple of temperature dependant constants

for specific substances.

>rom 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.

T(e tissue

Tissue porosity has a ma@or impact on processing, subsequent microtomy and staining.

$orosity at an ultrastructural level is determined by the nature and composition of the

tissues, and the effects of fixatives, modifiers, and processing reagents to which thetissues are sub@ected. Tissue porosity involves natural and artefactual pores, and the

swelling and shrinkage of the biopolymer matrix !>ig. "#"1%"+. ven after fixation cell

surfaces continue to act as osmotic membranes"+. 2rrespective of the fixative used, all

tissues undergo limited shrinkage and hardening during dehydration, clearing andinfiltration"+%"5 as well as staining and mounting"+. -ardening generally results from tissue

shrinkage, accompanied in most cases by decreased tissue porosity"7. >atty tissues usually

require extended processing as lipids, such as myelin in brain tissues and general bodyfats, inhibit the diffusion of processing reagents.

*I,ATION

2n general there is a tendency for tissues fixed in reagents that cause little initialshrinkage to undergo a greater degree of shrinkage during dehydration. onverselytissues fixed in substances which cause considerable initial shrinkage, contract less

during dehydration"9,(0,(".

Aon%protein coagulant fixatives such as formaldehyde, result in little ultrastructural tissue

damage"1. -owever these agents tend to swell tissues"+, and generally fail to giveadequate protection from shrinkage and hardening during subsequent processing to

paraffin wax !>ig. (#"+,"9,(0%(*. Aon%fixative salts, such as calcium chloride, incorporated in

formaldehyde fixatives further stabilise tissues and as a consequence reduce processing%induced tissue shrinkage"+,"8.

$rotein%coagulant fixatives such as ethanol, mercuric chloride or picric acid, shatter tissue

ultrastructure"1 but this may increase porosity. Tissues may shrink during fixation but are

protected against further significant contraction and hardening during processing !>ig.(#"+,"8,"9,(0%(*. Secondary fixation of formaldehyde fixed tissues with coagulant fixative

mixtures including formal sublimate, -elly=s or 6ouin=s fluids, enhances the response of

these tissues to processing, sectioning and staining"0.

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&uration of fixation determines the extent of tissue stabilisation and consequently

porosity and influences reactivity in histological and immunohistochemical procedures.

<nderfixed tissues are inadequately protected against processing reagents and exhibit arange of artefacts"*, including those associated with secondary fixation by the dehydrant.

$rolonged exposure to primary and secondary fixatives during processing may impair

tissue reactivity, particularly in immunohistochemical investigations.

SO-.ENT E**ECTS

?oss of certain substances from tissues during processing may also indirectly affect tissue

porosity and result in shrinkage(1. ven where the ultrastructure has not undergone

disruption, tissue porosity can be increased for example, by the dissolution of lipid%richstructures such as membranes and fat droplets by processing solvents, which can then

result in shrinkage(+.

Some processing fluids such as glycerol for example, also affect tissues by increasing

softness but decreasing porosity"7 resulting in protracted clearing and infiltration times

thus negating the original softening action. ;ther reagents including cedarwood oil"7maintain both softness and porosity and facilitate subsequent processing steps.

TISSUE MODI*IERS

Tissue modifiers such as phenol swell unfixed collagen and elastic fibres"1, enhance protein polymer formation in formaldehyde fixed tissues"",(8 coagulate proteins, and

probably maintain or promote tissue porosity. These phenomena underlie the use of

phenol and other surfactants to stabilise and soften hard tissues during fixation"",(8%*0 and processing"",(8,(7,*"%*(.

DENSIT/ AND T0ICKNESS

Bariable tissue density affects infiltration and subsequent microtomy

**

. Spongy, parenchymatous tissues are usually more rapidly infiltrated than hard and dense tissues.6lock thickness also influences the rate of reagent diffusion and hence processing time.

Tissue thickness should be optimised for particular processing schedules, or alternatively

processing times are ad@usted to accommodate thick, thin or large tissue blocks.

$rocessing reagents

The chemical and physical properties of reagents which influence processing include

polarity, concentration, miscibility with water, solvents and embedding media,

evaporation rate, and viscosity !Tables (, *#. Thermal conductivity, heat capacity, boiling point and the electromagnetic conductivity of reagents are particularly important in

microwave%stimulated processing*1.

$O-ARIT/

To minimise tissue distortion there should be a gradual change in polarity of the processing fluid from highly polar aqueous fixatives and solutions to the embedding

medium which is usually non%polar !hydrophobic#*+. Tissues generally shrink when

transferred to a fluid of relatively lower polarity*+.





CONCENTRATION

2f 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. >or 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.

MISCI!I-IT/

$rocessing 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 employedin a graded series.

any transition solvents, for example xylene, are extremely water%intolerant, and are

immiscible with hydrated alcohols. ouplers such as phenol mixed with a transition

solvent permit clearing from 903%7+3 alcohols*8. ;riginally couplers were employed to

overcome difficulties with hydrated higher alcohols. -owever 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.

E.A$ORATION 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 tocontaminate the infiltration medium.

.ISCOSIT/

Biscosity is the internal friction of a particular substance which affects rate of flowthrough tissues and is inversely proportional to temperature. 2t is particularly important inthe 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 viscositydehydrant alcohols.

2f 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. 2f the viscositydifferential between fluid inside and outside the tissue is too great, shrinkage will result.

-ence slow and gradual processing of tissues is necessary when viscous reagents are

used !for example in nitrocellulose embedding methods#.

EM!EDDIN MEDIA

2nfiltrating and embedding media must fill all spaces within the tissue to support cellular

components adequately during microtomy. &ensity 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



to facilitate thin sectioning*0. Tissue%medium adhesion is enhanced if the embedding

matrix has a fine uniform crystalline morphology which intimately contacts the tissue.

Biscosity and melting point of the infiltration medium partly determine the duration andtemperature of processing conditions.

$rocessing conditionsTemperature, pressure and agitation reduce the duration of tissue processing and improve

the quality of infiltration.

TEM$ERATURE

't low temperatures structural elements of tissues are stabilised against the destructiveeffects 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*+. <nfortunately at low temperatures reagentviscosities increase and diffusion rates decrease, resulting in prolonged processing times.

2sothermally processed mammalian tissues show finer detail and less artefacts than those processed by the more practicable, common an%isothermic techniques"7. -eat increases

the kinetic energy of molecules and rate of diffusion, with a corresponding decrease insolution viscosity. The application of mild heat within the range *9: to 1+:, during the

dehydration and clearing steps considerably reduces processing times"5,*8, but may

concomitantly increase shrinkage(". Tissue shrinkage during infiltration in paraffin waxresults mainly from the effect of heat on collagen"8.

-igh infiltration temperatures cause marked tissue shrinkage and hardening"9,(" which can

be avoided by maintaining embedding waxes (%*: above their melting points(".

$rolonged immersion in paraffin wax at the correct temperature results in only slight

tissue shrinkage"8,*9

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 used"9,((,(*,*5 aswell as the tissue type. icrowave stimulated processing involves complex molecular

interactions, the key element of which is internal heating, with stimulation of diffusion*1,

and concomitant reduction in the duration of tissue processing.

$RESSURE AND .ACUUM

-igh pressure facilitates infiltration of dense specimens with viscous resinous embedding

media at the block forming stage"7, but is rarely employed for biological specimens.

$ositive pressures for fluid transfer that are encountered in closed system processors are

probably too low to have a significant influence on tissue infiltration.

Bacuum 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.&uration of wax infiltration is dependent upon viscosity and is not reduced by the

application of vacuum*7.



AITATION

>luid interchange between processing reagents and tissues is promoted by exposure of the

maximum tissue surface area to reagents. 2f tissues are allowed to settle on the bottom ofa 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. Ceagent diffusiontime is therefore increased and if the duration of processing is not correspondingly

increased, inadequate processing will result.

&uring processing, tissues should be loosely packed, suspended and agitated within the

medium to facilitate the exchange of dilute reagent from the tissues with the moreconcentrated reagent replacing it. 'gitation of tissues and fluids in manual processing is

achieved using rotors or magnetic stirrers. 2n automatic tissue processors, continual rotary

or vertical motion of tissue containers, or tidal action and flow of processing fluidsensures adequate fluid exchange. 2deally tissue cassettes should be placed in processors

so that the cassette perforations are perpendicular to the fluid flow. >or efficient and

effective processing there should be a specimen volume to processing fluid volume ratioof at least "/+0.

'lternate vacuum and positive pressure cycles during processing may provide some

micro agitation within tissues, but this has yet to be substantiated. 2n ultrasonic stimulated

processing10%1" tissues and fluids are sub@ected to high frequency agitation and associated phenomena, with simultaneous reduction in processing time.

De('drationThe 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. &uring this procedure various cellular components are dissolved bydehydrating fluids. >or example, certain lipids are extracted by anhydrous alcohols, andwater soluble proteins are dissolved in the lower aqueous alcohols1(.

&ehydration is effected as follows/

• &ilution dehydration, the most commonly used method. Specimens are

transferred through increasing concentrations of hydrophilic or water misciblefluids which dilute and eventually replace free water in the tissues.

• hemical dehydration, where the dehydrant, acidified dimethoxypropane or

diethoxypropane, is hydrolysed by free water present in tissues to form acetone

and methanol1*%+0 in an endothermic reaction.

&ehydration is necessary in all infiltration methods, except where tissues are simplyexternally supported by an aqueous embedding medium. hoice of a dehydrant is

determined by the nature of the task, the embedding medium, processing method, and

economic factors. &ehydrants differ in their capacity to cause tissue shrinkage !>ig. *#.





2n the paraffin wax method, following any necessary post fixation treatment, dehydration

from aqueous fixatives is usually initiated in 803%903 ethanol, progressing through

703%7+3 ethanol, then two or three changes of absolute ethanol before proceeding to theclearing stage.

Whilst well fixed tissues can be transferred directly to 7+3 ethanol,+"

incompletely fixedtissues may exhibit artefacts if placed directly in higher alcohols. The dehydrant

concentration at which processing is initiated depends largely upon the fixativeemployed. >ollowing fixation in anhydrous fixatives such as arnoy=s fluid, for example

dehydration is initiated in "003 ethanol. To minimise tissue distortion from diffusion

currents, delicate specimens are dehydrated in a graded ethanol series from water through"03%(03%+03%7+3%"003 ethanol.*8

&uration of dehydration should be kept to the minimum consistent with the tissues being

processed. Tissue blocks " mm thick should receive up to *0 minutes in each alcohol,

blocks + mm thick require up to 70 minutes or longer in each change. Tissues may be

held and stored indefinitely in 903 ethanol without harm.

;ther dehydrants, including universal solvents, are used in a similar manner to that

described for ethanol, though generally in different concentration increments.

De('drating agentsA-CO0O-S

These are clear, colourless, flammable, hydrophilic liquids, miscible with water and,when anhydrous, with most organic solvents. 2n addition to their role as dehydrants,

alcohols also act as secondary coagulant fixatives during tissue processing.

Et(anol is probably the most commonly used dehydrant in histology. 2t is supplied as77.5+3 ethanol !absolute ethanol, "00 -igh Drade or Standard Drade# and as specialethylated Spirits !77.5+3 ethanol denatured with (3 methanol#. 6oth are satisfactory

for histological purposes. thyl alcohol formulations differ in standards and nomenclature

worldwide and it may be necessary to consult various tables to ascertain the ethanolconcentration.

thanol is a rapid, efficient and widely applicable dehydrant. 2t is normally a poor lipid

solvent except under microwave processing conditions*1. thanol dissolves nitrocellulose

slowly unless combined in equal proportions !or better, "/(#+* with diethyl ether.$rocessing times in absolute ethanol should be minimal. $rogressive removal of bound

water from carbohydrates and proteins during prolonged immersion in absolute ethanolcauses tissues to harden excessively and become brittle"7,((%(*. olloid, blood, collagenand yolky tissues are particularly affected"7. The problem is exacerbated by heat during

wax infiltration.

'nhydrous cupric sulphate added to the final absolute ethanol on a tissue processor

scavenges any water present. The salt is self%indicating/ white when anhydrous, bluewhen hydrated, and is only slightly soluble in ethanol. 2t is prepared by carefully heating



hydrated cupric sulphate until it turns white, on a hotplate at (+0:. ool in a desiccator.

'nhydrous calcium sulphate !&rierite# or molecular sieves act in a similar manner but are

non%indicating. Solid drying agents are placed in a "%( cm thick layer in the reagentcontainer, and covered with filter paper or fine sponge to avoid mixing with the

specimens during tissue agitation.

Met(anol is a good ethanol substitute+1 but rarely used for routine processing because of

its volatility, flammability and cost. 2t is a poor lipid solvent, and will not dissolvenitrocellulose unless mixed with acetone. 2n microwave processing it tends to harden

tissues more than ethanol*1.

Isopropanol was first suggested as an ethanol substitute during the prohibition era in the<nited States+1. 2t is a universal solvent available as 77.53 !absolute# isopropanol,

slightly slower in action and not as hygroscopic as ethanol, but a far superior lipid

solvent. 2sopropanol 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.

2sopropanol shrinks and hardens tissues less than ethanol

+1%+9

and is used to dehydratehard, dense tissues, which can remain in the solvent for extended periods without harm.

To minimise shrinkage, fixed tissues are transferred via 803%903 isopropanol or ethanolto absolute isopropanol.

2sopropyl alcohol has also been recommended as a xylene substitute+5. 2n microwave

stimulated processing, though unsatisfactory as a dehydrant, isopropanol is used as a

transition solvent following ethanol dehydration*1.

2sopropanol only dissolves nitrocellulose in the presence of esters+7 such as methyl

benzoate or methyl salicylate, and is used in methyl salicylate%based double%infiltration

methods. 2t cannot be used as a dehydrant in alcohol%ether%celloidin techniques.2sopropanol is a solvent for some lipid%soluble dyes, but is not used in staining workstations as many other dyes are insoluble in this solvent.

Normal and tertiar' %utanols are universal solvents mainly used for small%scale manual

processing of plant and animal tissues in teaching and research. Aormal butanol is

recommended for processing lightly chitinised arthropods*5 and rodent tissues. 2t causesless hardening and shrinkage than ethanol,*5,80 though this is offset by the prolonged

processing schedules which may result in tissue shrinkage."5,(( A%butanol is poorly

miscible with water and only slowly miscible with paraffin wax. 2t is flammable, with a penetrating camphor%like odour, and the vapours are eye irritants. 2so%butanol, with

similar properties and processing characteristics((,(* is a less costly substitute for n%

butanol. Tertiary%butanol is widely used in plant histology8" but rarely for animaltissues"9,(*. 6elow (8: it is hygroscopic crystalline solid, a ma@or disadvantage. 2n

processing it is used in a similar manner to n%butanol.8"

-/CO-1ET0ERS

<nlike the alcohols, these reagents do not act as secondary fixatives, and apart fromsolvent effects do not appear to alter tissue reactivity.



21Et(o)'et(anol, ethylene glycol monoethyl ether, cellosolve or oxitol is used as a

dehydrant preceding polyester wax embedding,"7 for dehydration following dioxane%

based fixation of hard animal tissues8(, and in the agar%ester wax double embeddingtechnique.

thoxyethanol is a colourless, nearly odourless flammable liquid, strongly hygroscopic,miscible with water and most organic solvents. ellosolve dissolves nitrocellulose and

tends to decompose on exposure to sunlight. 2t is rapid but non%hardening in action, andtissues can remain in it for years8(. To avoid severe shrinkage, tissues are transferred

from aqueous fixative or washing via 803%903 ethanol into full strength cellosolve.

Dio)ane, ",1 diethylene dioxide causes less tissue shrinkage and hardening thanethanol"9,((%(*,8(%8* and is excellent for tissues excessively hardened by ethanol%xylene

processing. 2t has a rapid but gentle action, and is best used in a graded series. Tissues

may remain in it for long periods without harm. 2t is a colourless, flammable universal

solvent with an odour similar to butanol, freezes at "(:, and is miscible with water,

most organic solvents and paraffin wax. &ioxane dissolves mercuric chloride, but precipitates potassium dichromate and other salts. 2t is cumulatively toxic and a suspected

carcinogen81.

&ioxane is expensive and is normally reclaimed by drying over a "0%(0 mm layer ofcalcium oxide or anhydrous cupric sulphate. alcium chloride should not be used as it

reacts with dioxane and swells8*. &ioxane is also recovered by freezing hydrated solvent

in a spark%proofed refrigerator at (%+:. Water, which separates out, is decanted from thecrystalline dioxane which is then thawed, finally dried over a solid dehydrant and

reused8+.

xplosive peroxides form in dioxane exposed to air. They accumulate in recycled solventwhich should be periodically tested for the presence of peroxides. 88

$ol'et('lene gl'cols !$D# 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. $olyethylene glycols are

miscible with most organic solvents and dissolve nitrocellulose. &ehydration is initiatedin 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

$D which is solid at room temperature. $olyethylene glycol used for dehydration can beregenerated by heating at "01: for (1 hours89.

OT0ER DE0/DRANTS

Acetone is a colourless flammable liquid with sharp characteristic ketonic odour, low

toxicity and is freely miscible with water and organic solvents. 2t is a fast, effectivedehydrant though it may cause tissue shrinkage) it may also act as a coagulant secondary

fixative. 'cetone 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 vacuum85. -owever a transition solvent is normally interposed before the paraffin

baths. 'cetone is not recommended for microwave processing as it causes excessivenuclear shrinkage*1.

Tetra('drofuran is a colourless, highly volatile and flammable universal solvent with anoffensive ethereal odour. 2t is completely miscible with water, most organic solvents,

paraffin wax and mounting media. 2t dissolves mountants, but not most dyes. The solventdehydrates rapidly causing little shrinkage or hardening, and is possibly the best of the

universal solvents.87 2t is less toxic than dioxane for which it can be substituted. Tissues

are processed as in dioxane method. Tetrahydrofuran can form explosive peroxides whichrenders solvent recovery distillation dangerous88.

2&2 dimet(o)'propane !&$# and (,( diethoxypropane !&$# are used for chemical

dehydration of tissues.1*%+" They are flammable and form peroxides.+0 &$ and &$ are

miscible with paraffin wax however methanol, one of the hydrolysis products, is not wax

miscible and a post dehydration rinse in acetone,

15

a transition solvent such as methylsalicylate17 or toluene should precede infiltration with wax. &$ shrinks tissues slightly

less than &$.+" hemical dehydration is suitable for rapid manual processing or machine processing, and is comparable to conventional dehydration for tissue morphology and

staining reactions.+" 'cidified &$4&$ can be reused several times, though dehydration

times need to be extended. The reagent is stored at 1: in a spark%proofed refrigerator.

$(enol, beechwood creosote and aniline facilitate dehydration when mixed withtransition solvents, as in Weigert=s carbol%xylol !xylene 9+ ml and phenol (+ ml#.*8 The

coupling action permits tissues and celloidin sections to be cleared from lower strength

alcohols. reosote and aniline are used less commonly though in a similar manner to

phenol. $henol consists of clear hygroscopic acicular crystals and is also available as503 w4w liquefied phenol. 2t is soluble in water, alcohol and most organic solvents

except petroleum ethers. oncentrated solutions coagulate nitrocellulose. ;n exposure toair and light, phenol and its solutions develop a pink to reddish discolouration. ontainers

must be protected from light and tightly sealed. $henol crystals and 503 concentrate

react violently with formaldehyde.

Clearinglearing is the transition step between dehydration and infiltration with the embedding

medium. any dehydrants are immiscible with paraffin wax, and a solvent !transitionsolvent, 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 transitionsolvent, and from transition solvent to wax"9,"5 !>ig 1#. 2n the final stage shrinkage may

result from the extraction of fat by the transition solvent."5

The term clearing arises because some solvents have high refractive indices !approaching

that of dehydrated fixed tissue protein# and, on immersion, anhydrous tissues arerendered transparent or clear.90 This property is used to ascertain the endpoint and





duration of the clearing step. The presence of opaque areas indicates incomplete

dehydration.

-owever, other solvents, notably chlorinated hydrocarbons, do not render tissuestransparent and the clearing endpoint !generally when the specimen sinks in the solvent#

must then be determined empirically. Transition solvents extract certain tissue substancessuch as lipids, but otherwise do not alter tissue reactivity nor behave as secondary

fixatives during processing.

hoice 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 sol"ents00/DROCAR!ONS

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 )'lene clear rapidly and tissues are rendered transparent, facilitatingclearing endpoint determination. oncerns over the exposure of personnel to xylene88

relate mainly to the use of the solvent in coverslipping rather than in processing and

xylene substitutes can be used in these circumstances. Eylene hardens tissues fixed innon%protein coagulant fixatives and prolonged clearing in the solvent should be avoided.

Tissues stabilised in protein coagulant fixatives !6ouin=s or S<S'# are less affected."9,(*

6enzene is more gentle and rapid than xylene and toluene and is probably the best

transition solvent, though toxicity and possible carcinogenicity81 preclude its use inhistology. 2ndustrial grade xylene may contain nearly (+3 of other solvents such as ethyl

benzene, with traces of benzene, odorous mercaptans and hydrogen sulphide. ;nly the

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

$etroleum sol"ents have a gentle, non%hardening action on tissues, clear more slowly

than xylene and toluene, and do not render tissues transparent. 6lends of aromatic,

naphthenic and aliphatic solvents !each with varying toxicity, flammability and solvent

action# can be used as xylene substitutes.88 any of these solvents have a particularly

strong petroleum odour which some people find ob@ectionable. Toxic effects of petroleumsolvents are broadly similar to those of pure hydrocarbons % skin degreasing, acute

intoxication and narcosis in high concentrations. 6lends with high aromatic andnaphthene but reduced paraffin content such as Shell E*69", 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 clearingaction. Ferosene, some xylene substitutes and Shellsol "8(8 have properties intermediate

between these two groups.



C(lorinated ('drocar%ons 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. embers of this group clearmore slowly but harden far less than xylene. 'lthough 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. hlorinated hydrocarbons are ozonedestroying chemicals, and from Ganuary "778 ","," trichloroethane and carbon

tetrachloride are banned from use under the ontreal $rotocol.9(

C(loroform is an expensive, heavy, highly volatile, slowly penetrating transition solvent.

2t causes less brittleness than xylene and is often used on dense tissues such as uterus andmuscle*( 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.

Car%on tetrac(loride has similar properties, but because of its high toxicity is now

rarely used in histology.

Tric(loroet(ane and other members of this group are commonly used as xylene9*,91 and

chloroform substitutes. They include ","," trichloroethane !","," T#, present in

2nhibisol) ","," T and perchloroethylene components of A$*0 and -istosol) andtrichloroethylene. 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.+7 'lthough mildly toxic !except at high concentrations#81 thedecision to substitute them for more toxic solvents must be soundly based !Table 9.*#9+.

6ecause 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.

9+

ESTERS

These are colourless flammable solvents miscible with most organic solvents and with

paraffin wax.

n1!ut'l acetate is used as a xylene substitute98 and nitrocellulose solvent.

Am'l acetate, met('l %en3oate and met('l salic'late 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. Theseesters 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. ethyl benzoate and methyl salicylate render tissues completelytransparent and are used for clearing helminth parasites for examination and whole

mounting. ethyl salicylate clears tissues from 783 ethanol, hardens less and has a more

pleasant odour than methyl benzoate. 2t causes minimal tissue shrinkage and hardening"5



and tissues can remain in it indefinitely without harm. This ester is one of the best though

expensive transition solvents.

TER$ENES

Terpenes are isoprene polymers found in essential oils originally derived from plants,99

though some are now synthesised. They are the earliest transition solvents to be used inhistology95 and include turpentine and oils of bergamot, cedarwood, clove, lemon,

origanum and sandalwood.8",97 2n general the natural oils are not highly pure compounds but contain several substances.

any terpenes clear tissues and celloidin sections from 503%7+3 alcohol, render tissues

transparent and have a slow gentle non%hardening action. ost are generally regarded assafe 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, withterpene/dehydrant or wax ratios of */", "/", "/* 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 *0 minute changes of toluene or similar solvent to remove theterpene before infiltration with wax. 6rief immersion in toluene does not negate the

effectiveness of the terpene. 'lternatively, tissues are given three, four or more changes

of wax until the terpene has been eliminated. 'lthough biodegradable, terpenes are notwater miscible and should not be flushed away with water, but disposed of by recycling

or incineration.

Cedar4ood oil& largely composed of cedrene, rapidly clears tissues from 7+3 alcohol,

hardens tissues the least of all the transition solvents, but is difficult to eliminate fromtissues during wax infiltration. 2t 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. ?ow viscosity refined oil should be used forclearing. >ormation of crystalline cedrol in cedarwood oil can be overcome by the

addition of " ml xylene or toluene to 50 ml cedarwood oil8+. edarwood oil is expensive,

but exhausted oil can be restored by filtering, then heating to 80: under vacuum for *0%80 minutes50.

-imonene !dH limonene# is derived from citrus fruit and is a component of various

proprietary blends of transition solvents such as learene, -emo%&e and -isto%lear

marketed as xylene substitutes. 2t is less viscous than cedarwood oil and is similar to theesters in clearing action and in elimination from wax. ?imonene may cause allergic skin

reactions.5",5(



Terpineol is a clear almost colourless mixture of isomers with a faint pleasant odour and

very low evaporation rate. 2t clears tissues from 503%703 alcohol with minimal

hardening. ?ike the other terpenes it is difficult to eliminate from paraffin wax. 2t 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 summarisedin Table *.

Infiltration and em%edding media and met(ods2deally an infiltrating and embedding medium should be/"7

• soluble in processing fluids

• suitable for sectioning and ribboning

• molten between *0: and 80:

• translucent or transparent) colourless• stable

• homogeneous

• capable of flattening after ribboning

• non%toxic

• odourless

• easy to handle

• inexpensive

2n addition the properties of the medium should approach those of the tissues to besectioned with regard to density, elasticity, plasticity, viscosity and adhesion and should

be harmless to the embedded material.

Barious substances have been used to infiltrate and embed tissues for microtomy. Aone

completely fulfil the foregoing criteria, and media are selected according to the nature ofthe task for which they are required.

Em%edding 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 areinfiltrated by immersion in a substance such as a wax, which is fluid when hot and solid

when cold. 'lternatively, tissues can be infiltrated with a solution of a substancedissolved in a solvent, for example nitrocellulose in alcohol%ether, which solidifies on

evaporation of the solvent to provide a firm mass suitable for sectioning.





Dou%le em%edding 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.

In"estment generally refers to the practice of embedding wax infiltrated tissues in

another wax, such as $iccolyte%paraffin wax, modified to provide improved tissue supportand sectioning qualities.

.acuum 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 sub@ected to high temperatures thus minimising heat%induced

tissue hardening, facilitates complete removal of transition solvents, and prolongs the lifeof wax by reducing solvent contamination. Bacuum infiltration requires a vacuum

infiltrator or embedding oven, consisting of wax baths, fluid trap and vacuum gauge, to

which a vacuum of up to 980 mm -g is applied using a water or mechanical pump.

odern tissue processors are equipped to deliver vacuum, or vacuum and pressure, to all

or some reagent stations during the processing cycle.

$arffin 4a)$RO$ERTIES

$araffin wax is a polycrystalline mixture of solid hydrocarbons produced during the

refining of coal and mineral oils. 2t is about two thirds the density and slightly moreelastic than dried protein.**

Wax hardness !viscosity# depends upon the molecular weight of the components and the

ambient temperature. -igh molecular weight mixtures melt at higher temperatures than

waxes comprised of lower molecular weight fractions. $araffin wax is traditionally

marketed by its melting points which range from *7: to 85:.

Tissue%wax adhesion depends upon crystal morphology of the embedding medium.

Small, uniform sized crystals provide better physical support for specimens through close

packing. rystalline morphology of paraffin wax can be altered by incorporatingadditives 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.51%58

MODI*IED $ARA**IN 5A,ES

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.+3 ceresin, 0."%+3 beeswax, rubber, asphalt, bayberry wax, or phenanthrene. 59



arly histological wax formulations*8,55%57 have largely been replaced by uniform, high

quality proprietary blends of histological paraffin waxes. 'dditives recently incorporated

in proprietary waxes include the following/

$iccol'te 667& a thermoplastic terpene resin added at the rate of +3%"03 to the

infiltrating wax,70 or to the final investing paraffin wax to improve tissue support for thin

sectioning and facilitate flattening and expansion of sections on the waterbath.7"%7(

$iccolyte mixtures cannot be used in certain models of fluid%transfer type tissue

processors.

$lastic pol'mers such as polyethylene wax, added to improve adhesion, hardness and plasticity.7* $olymer waxes are incorporated in the ma@ority of proprietary histological

paraffin wax blends presently available.

Dimet('l sulp(o)ide !&S;# added to proprietary blends of plastic polymer paraffin

waxes reduces infiltration times and facilitates thin sectioning.71 &S; scavengesresidual transition solvent and probably alters tissue permeability by substituting for or

removing bound water thus improving infiltration. Some individuals who handle &S;%

paraffin wax may experience an unpleasant and annoying oyster or garlic taste probably

caused by &S; metabolites.

7+

$ossible health risks associated with the use of &S;% paraffin wax88 are minimal if correct laboratory hygiene is practised.

Em%edding tissues in paraffin 4a)Tissues are embedded by placing them in a mould filled with molten embedding medium

which is then allowed to solidify. mbedding requirements and procedures are essentiallythe same for all waxes, and only the technique for paraffin wax is provided here in detail.

't the completion of processing, tissues are held in clean paraffin wax which is free of

solvent and particulate matter.

Cequirements for embedding are as follows/

• a supply of clean, filtered paraffin wax held at (%1: above its melting point.

• a cold plate to rapidly cool the wax.

• a supply of moulds in which to embed the tissues.

These elements are conveniently combined in commercially available embedding stations

!>ig +#. ;therwise a wax dispenser, embedding oven and ice will suffice. There are fourmain mould systems and associated embedding protocols presently in use78 !>ig 8#/

traditional methods using paper boats) ?euckart or &immock embedding irons or metal

containers) the $eel%a%way system using disposable plastic moulds and) systems usingembedding rings or cassette%bases which become an integral part of the block and serve

as the block holder in the microtome.

eneral Em%edding $rocedureMET0OD

" ;pen the tissue cassette, check against worksheet entry to ensure the correct number of

tissue pieces are present.

( Select the mould, there should be sufficient room for the tissue with allowance for at

least a ( mm surrounding margin of wax.







* >ill the mould with paraffin wax.

1 <sing warm forceps select the tissue, taking care that it does not cool in the air) at the

same time.+ hill the mould on the cold plate, orienting the tissue and firming it into the wax with

warmed forceps. This ensures that the correct orientation is maintained and the tissue

surface to be sectioned is kept flat.8 2nsert the identifying label or place the labelled embedding ring or cassette base onto

the mould.

9 ool the block on the cold plate, or carefully submerge it under water when a thin skinhas formed over the wax surface.

5 Cemove the block from the mould.

7 ross check block, label and worksheet.

ORIENTATION O* TISSUE IN T0E !-OCK

orrect orientation of tissue in a mould is the most important step in embedding.

2ncorrect placement of tissues may result in diagnostically important tissue elements

being missed or damaged during microtomy. 2n circumstances where precise orientationis essential tissue should be marked or agar double embedded. <sually tissues are

embedded with the surface to be cut facing down in the mould. Some general

considerations !>ig.9# are as follows/

• elongate tissues are placed diagonally across the block

• tubular and walled specimens such as vas deferens, cysts and gastrointestinal

tissues are embedded so as to provide transverse sections showing all tissue layers

• tissues with an epithelial surface such as skin, are embedded to provide sections in

a plane at right angles to the surface !hairy or keratinised epithelia are oriented toface the knife diagonally#

•

multiple tissue pieces are aligned across the long axis of the mould, and not placed at random.

&uring cooling, paraffin wax shrinks up to "+3, causing compression in tissues."9 Thiscompression is almost fully recovered when sections are floated on a warm waterbath8)

compression resulting from microtomy is only partially recovered.

$rocessing met(ods and routine sc(edulesTissues are usually more rapidly processed by machine than by manual methods,

although the latter can be accelerated by using microwave or ultrasonic stimulation. >or

routine purposes tissues are most conveniently processed through dehydration, clearing

and infiltration stages automatically by machine. There are two broad types of automatictissue processors % tissue%transfer and fluid%transfer types.

Automated tissue processingTISSUE1TRANS*ER $ROCESSORS

These processors are characterised by the transfer of tissues, contained within a basket,

through a series of stationary reagents arranged in%line or in a circular carousel plan. Therotary or carousel is the most common model of automatic tissue processor, and was





invented by 'rendt in "707.97 2t is provided with 7%"0 reagent and (%* wax positions, with

a capacity of *0%""0 cassettes depending upon the model. >luid agitation is achieved by

vertical oscillation or rotary motion of the tissue basket. $rocessing schedules !Table 1#are card%notched, pin or touch pad programmed.

Tissue%transfer processors allow maximum flexibility in the choice of reagents andschedules 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 day4night processing. 2n more recent

models !>ig.5# the tissue basket is enclosed within an integrated fume hood during

agitation and transfer cycles thus overcoming the disadvantages of earlier styles.

*-UID1TRANS*ER $ROCESSORS

2n fluid%transfer units !>ig.7# processing fluids are pumped to and from a retort in which

the tissues remain stationary. There are "0%"( reagent stations with temperatures

ad@ustable between *0%1+:, *%1 paraffin wax stations with variable temperature settings

between 15%85:, and vacuum%pressure options for each station. &epending upon themodel these machines can process "00%*00 cassettes at any one time. 'gitation is

achieved by tidal action. Schedules are microprocessor programmed and controlled.Bacuum%pressure cycles coupled with heated reagents allow effective reductions in

processing times and improved infiltration of dense tissues.

>luid%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 ventedthrough filters or retained in a closed%loop system. $rocessors 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 $iccolyte. Cepresentative schedules for rapid andovernight processing are provided in Table +.

TISSUE RECO.ER/ $ROCEDURES

$rocedures for recovery of tissues that have air dried because of mechanical or electricalfailure 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 8.

ENERA- CONSIDERATIONS

6askets and metal cassettes should be clean and wax%free.

Tissues should not be packed too tightly in baskets so as to impede fluid exchange.

$rocessors must be free of spilt fluids and wax accumulations to reduce hazards and toensure mechanical reliability.

>luid levels must be higher than the specimen containers.

Timing and delay mechanism must be correctly set and checked against the appropriate processing schedule.

' 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 processinganual 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/

o rapid processing of an urgent specimen

o delicate material

o very large or thick tissue blocks

o hard, dense tissues !nitrocellulose methods#

o special diagnostic, teaching or research applications

• small scale processing requirements

• resin embedding.

The main advantage of manual processing over automated methods lies in the flexibilityof reagent selection, conditions and schedule design to provide optimum processing for

small batches of tissues. xposure of tissues to the deleterious effects of some reagentscan 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 processingrather than risk the problems of under processed tissues. anual processing is accelerated

using microwave ovens or ultrasonics.

Schedules for rapid manual processing using chemical dehydration !Table 9#, one and

two day routine processing !Table 5#, and extended manual processing for large, thick, or

hard tissues !Table 7# are provided.

<niversal 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 "0# and dioxane

!Table ""#.

Aonetheless manual processing can be time consuming and inconvenient. are must be

exercised so that tissues are left overnight in reagents that will cause minimal harm. ' permanent series of solutions in wash bottles simplifies processing small single

specimens. Tissues are processed in tubes and agitated on a rotor. Ceagents are pipetted,or decanted through a fine sieve. ultiple specimens or large blocks are economically processed in large lidded @ars of processing fluids. The specimen to reagent volume ratio

should be at least "/+0. 'gitation is provided by a magnetic%stirrer.

&ehydrated tissues float on the surface when transferred to higher density transition

solvents such as chloroform or cedarwood oil. -owever, if placed in lower densitymixtures of dehydrant%transition solvent before finally transferring to pure transition













solvent, tissues will remain submerged throughout the clearing stage. 'n 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 thetransition solvent. Ceagents are carefully decanted and the specimen placed in a fresh

change of transition solvent.

Micro4a"e1stimulated processingCapid manual microwave%stimulated paraffin wax processing of small batches of tissues

gives excellent results which are comparable to tissues processed by longer automatednon%microwave methods.*1,79%"0"

$rocessing is undertaken in a dedicated microwave oven !>ig. "0# which is fitted with

precise temperature control and timer, and an interlocked fume extraction system to

preclude accidental solvent vapour ignition. 'gitation is provided by an air%nitrogensystem.

&omestic microwave ovens with a temperature probe and timer accurate to seconds aresuitable for tissue processing. ' turntable or in%built radiation disperser facilitates even

reagent heating. Toxic and flammable solvent vapours generated during processingcannot always be adequately vented from these ovens and present an ignition hazard if

the electrical system is unprotected. ;vens should therefore be used within a fume

cupboard to minimise this problem. alibration of domestic ovens is essential foroptimum 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.*1

E8UI$MENT

Tissues are processed in conventional plastic cassettes, including those with !provided themetal lids lie below the fluid#.7 Transparent glass or solvent%resistant plastic containers of

about (00 ml capacity are ideal for processing batches of up to "1 cassettes per container.

*I,ATION

>or rapid processing, tissues are fixed by microwave irradiation,7 or in 7+3 ethanol !800

ml#%polyethylene glycol $D 100 !1+ ml#"0( from which specimens can be transferred

directly to dehydrant. >ormaldehyde%fixed tissues must be rinsed in running tap water for+ minutes before microwave processing and an extra dehydration change incorporated in

the schedule. $rocessing times for formaldehyde%fixed tissues need to be increased above

those provided for coagulant%fixed tissues.*1 $icric acid fixed tissues should not be

microwave processed as there is an explosion risk even in well washed tissues.

*1

$rocessing schedules are provided in Table "(.

0INTS *OR MICRO5A.E $ROCESSIN

% Tissue blocks should be as thin as possible. ?ength and width are not as important.*1

• $rocess blocks of similar thickness together.

• Ceagent volumes should be at least +0 times that of specimen volume.







• The temperature probe should be placed centrally in processing baths.

• <se a dummy load to check heat generation should reagents boil on minimum

settings % an equal volume of reagent irradiated together with the primary loadeffectively halves the energy received by the primary load.

• $re%heat paraffin wax baths in a conventional oven.

•'n increase in the number of cassettes or fluid volumes will require aconcomitant increase in power and or time to achieve the correct processingtemperature.

Ultrasound1stimulated processing<ltrasonics are used in histopathology to accelerate fixation, tissue processing for

electron microscopy, the decalcification of bone, tissue softening in post%embedding

ad@uvants, improving the sensitivity of immunohistochemical reactions, conventional

staining and for accelerated tissue processing. <nfixed tissue blocks "%( mm thick can befixed and processed to paraffin wax using ultrasonic%stimulation in " hour 1+ minutes.10%1"

The most important effect of ultrasound at frequencies of "00 k-z%" -z is agitation. 'tlower frequencies cavitation phenomena and attendant heat, pressure and streaming

effects may damage tissues and care must be exercised.

$rocessing is performed in reagent containers suspended in a detergent solution within

the transducer tank of an ultrasonic cleaner operated at +0 watts. 'n immersion heater is

used to elevate bath temperatures for paraffin wax infiltration. Tissues are placed in metalor plastic cassettes for processing.

oagulant fixatives provide optimal stabilisation for ultrasonic%stimulated processing.10

Tissues are dehydrated in ethanol and cleared in toluene, or preferably methyl benzoate

or methyl salicylate !Table "*#. ells and organelles such as cilia, microvilli anddesmosomes are all well preserved. ;ld and friable specimens sometimes exhibit

marginal distortion and erosion.

Alternati"e em%edding media and processing met(ods'lternative embedding media may provide optimum support for tissues in applications

for which paraffin waxes are unsuited, for example when/

• tissue components are heat or reagent labile

• hard or dense tissues are inadequately supported

• adhesion between specimen and wax is poor

• very thick or very thin sections are required• sectioning whole organs such as lung or brain.

Cesin embedding methods are now used for many of these applications. Aon%resinousembedding media include those listed below.

A8UEOUS MEDIA

Agar has a high melting point and low gelling temperature of agar make it ideal for





double embedding multiple small tissue fragments. 'gar is generally unstained by

overnight stains, but will stain with alcian blue.

elatine is used for simple embedding in a similar manner to agar. -owever the lowmelting point of gelatine !*+%10:# makes it unsuitable for double embedding. 2t is used

in Dough and Wentworth=s whole%organ sectioning method and its variants, or simply tosupport large tissue blocks for " mm thick sectioning and subsequent three%dimensional

reconstruction."0* 2n phospholipid and enzyme studies tissues may be infiltrated andembedded in gelatine+",90 and the resulting blocks sectioned on a freezing microtome. This

technique has now largely been superseded by other media used for cryotomy.

Sodium car%o)' met('l cellulose !# used as an embedding medium for whole body sectioning techniques, was first developed for autoradiograph studies"01 and

subsequently refined for histological and histochemical applications."0+ >rozen tissues are

transferred from coolant directly into +3 , briefly placed under vacuum to remove

trapped air, then frozen to a solid block for sectioning.

$ol'"in'l alco(ol !$B'# is a highly polar, water soluble medium suited to a variety of

applications, in particular histochemical studies of lipids and enzymes."08%"09 Tissues are

infiltrated at elevated or room temperatures through an ascending series of aqueous $B'%

glycerol solutions and embedded in "+3 aqueous $B' which is slowly dried to produce afirm block. Sections are cut at "%"00 Im in the normal manner. -umidity control during

microtomy is important. $rocessing schedules take "%( weeks or longer, restricting $B'

to research applications. ' low molecular weight, low viscosity $B' is necessary for thismethod."09 Cenewed interest in $B' as an infiltrating and embedding medium for electron

microscopy"05 has resulted in refinements to the technique. ross%linking $B' with

glutaraldehyde provides a final hydrophobic block containing some "03 water, with

improved sectioning characteristics and good preservation of lipids, proteins andcarbohydrates. Tissues are infiltrated through aqueous $B' solutions at concentrations

from "3%(+3. $B' of molecular weight "1k&a, 773 hydrolysed gives the mostconsistent results"05, but must be fully hydrolysed with sodium hydroxide before use. The

application of $B' to immunohistochemical studies is worthy of attention.

5ATER1MISCI!-E MEDIA

$ol'et('lene gl'cols !$D# are water soluble media used for investigation of heat andsolvent%labile lipids and proteins"07%""0, and to overcome tissue shrinkage, damage and

distortion inherent in the paraffin wax technique. The polyethylene glycols, or

arbowaxes, are polymers of varying length !the numerical suffix denotes molecular

weight#. 't room temperature $D (00 and $D 800 are syrupy liquids, $D "000 issoft and slippery, $D "+00 is hard, and $D 1000 is hard and brittle. 2n general they are

less elastic, denser and somewhat harder than paraffin wax. rystal slip is a bigger

problem than in paraffin and sectioning deformation is mainly non%recoverable.** Tissuesare dehydrated by gradual infiltration through increasing concentrations of aqueous $D

solutions, to pure $D in which they are finally embedded. Sections are cut in a low

humidity environment, otherwise considerable difficulties arise. They are difficult toflatten without loss of tissue and adhere poorly to slides, leading to the development of



numerous flotation fluids.""0 ?ow viscosity nitrocellulose !?BA#89 or water insoluble

polyvinyl acetate resin""0 incorporated into $D dehydrating, infiltrating and embedding

solutions allow water flotation of sections. The $D dissolves, leaving the tissue in a thinfilm of ?BA or $B' which is mounted on albumenised slides in the usual manner. These

approaches !Table "1# surmount many of the previous problems inherent with $D. The

nitrocellulose is removed from sections by immersing in $D (00 for "+ minutes.

$ol'et('lene l'col1-.N Solutions89

REAENTS RE8UIRED

" $olyethylene glycol 7+ g

( ?BA -E *%+ or *0%+ +%"0 gThe ?BA must be finely divided and dried to facilitate dissolution in the $D media) +3

?BA is preferred as "03 ?BA is near saturation in $D.

MET0OD

The mixtures are heated to 80: and stirred to aid solution.

$roblems with this method include the high viscosity of infiltrating media necessitating

slow agitation and uneven distribution of ?BA in the final embedding mix which results

in crazed blocks. These can be overcome mostly by thorough blending of the ?BA and

$D. With current interest in immunohistochemistry, polyethylene glycols may warrantre%evaluation. -owever considerable time and patience are required when using these

waxes.

$ol'et('lene gl'col monostearate !Aonex 8*6#, a water soluble synthetic wax is usedin a similar manner to polyethylene glycol and polyester waxes with application in

histochemistry""" and botanical histology.""(

5ATER1TO-ERANT MEDIA

Diet('lene gl'col distearate is a hard, brittle, water tolerant ester !m.p. 19%+(:#. 2t hascertain deficiencies when used for routine embedding,""* unless combined with other

substances as in ester waxes. -owever it may be used unmodified for thin sectioning

!0.+%( Im# of freeze dried and osmium tetroxide fixed tissues for high resolution light

microscopy.""1%""+ Tissues are dehydrated and cleared as in the paraffin wax method.

Ester 4a)es, developed by Steedman,""8 and subsequently modified""9%""5 have low

melting points, are hard at room temperature and have good adhesive properties. They are

therefore ideal for supporting and serially sectioning refractory hard, chitinised material

such as arthropods, and tissues which heat%harden excessively. They are also used forsimple investment of paraffin blocks to be sectioned under hot conditions""7 and in double

embedding with agar. ster waxes are no longer commercially available and must be

prepared from the basic ingredients.

Ester 5a) 69:;""5 !m.p. 15:#

REAENTS RE8UIRED

" &iethylene glycol distearate 80 g





( Dlyceryl monostearate *0 g

* *00 polyethylene glycol distearate "0 g

MET0OD

" elt the diethylene glycol distearate and heat it until clear. 'dd the glyceryl

monostearate and when dissolved add the polyethylene glycol distearate.( >ilter through coarse filter paper supported in a ring rather than a filter funnel.

2n Tropical ster wax "780""7 !m.p. +0:# triethylene glycol monostearate "0 g issubstituted in the forgoing formula for the *00 polyethylene glycol distearate. This

modification permits sectioning and ribboning at room temperatures of *9.+:. Tissues

are infiltrated at +8%80:.

ster waxes are soluble in 7+3 ethanol, n%butanol, cellosolve and xylene. Tissues areusually dehydrated via (%ethoxyethanol in which the waxes are soluble, obviating the

need for a transition solvent. ster waxes do not charge with static electricity, and have

good ribboning, thin sectioning and glass adhesion properties.

$ol'ester 4a), developed by Steedman"(0 is a ribboning, low melting point wax whichreduces heat%induced artefacts. 2t is recommended for heat labile tissues,"(" to minimise

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 facilitateimmunohistochemical investigations as antigenic determinants are well preserved."(* The

main advantages of this medium are low melting point and infiltration directly from 783

ethanol permitting a near isothermic processing schedule for mammalian tissues !Table"+#. $olyester wax is no longer commercially available and must be prepared from the

basic ingredients.

$ol'ester 5a) 9;<6; !m.p. *5:#

REAENTS RE8UIRED

" 100 polyethylene glycol distearate 70 g

( "%hexadecanol !cetyl alcohol# "0 g

MET0OD

" elt the polyethylene glycol at 80° , then mix in the cetyl alcohol.

( ool 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. 6ecause of its low melting point, non%volatile transition solvents suchas cedarwood oil and other terpenes should not be used. $olyester wax adheres to metalembedding moulds and paper%boat or plastic peel%a%way moulds are recommended.

Aormally blocks are cut in cool room temperatures using a cooled knife. $olyester wax is

more conveniently sectioned on a cryotome at %+: to ((:. Sections are affixed togelatine subbed or aminoalkylsilane treated slides, or floated on amylopectin."(0 6locks

and sections are stored at 1:. $olyester wax is dissolved from sections with absolute

ethanol. Steedman"7 proposed simultaneous fixation and infiltration of tissues with picro%







formal%polyester wax and mercury%formal%polyester wax mixtures. These methods and

principles present possibilities for immunohistochemistry, but appear to have received

little attention.

0/DRO$0O!IC MEDIA

Nitrocelluloseelloidin !# and ?ow Biscosity Aitrocellulose !?BA#, mixtures of di% and tri%

nitrocellulose, are composed of yellowish%white matted filaments with the appearance ofraw cotton. Aitrocellulose is insoluble in water, but soluble in absolute ethanol%diethyl

ether, amyl acetate, methyl benzoate, methyl salicylate and (%ethoxyethanol and is set by

most hydrocarbon solvents. 2t is highly flammable, and must be kept alcohol%dampedwith n%butanol or as 53 solutions in ethanol%ether or "3 ?BA in methyl benzoate as it

is explosive if detonated when dry. elloidin solutions have a low tolerance of water and

dehydration must be thorough. ?BA tolerates up to 83 of water,"(1 has superior penetration and final block hardness and is supplied in various grades of viscosity and

nitrogen content.+*,89 Aitrocellulose tissue processing techniques are generally employed

for sectioning hard tissues such as bone,

"(+%"(8

for topographical studies of central nervoussystem tissues,"(9 or for delicate embryonic material. Tissues are processed at room

temperature producing minimal and shrinkage and hardening. 2mmunohistochemical

investigations such as immunophenotyping of lymphoid and non%lymphoid cells "(5 are

possible on nitrocellulose processed tissues.

Dou%le em%edding and dou%le infiltration met(ods&ouble embedding methods such as agar%paraffin embedding, are used when tissuesrequire external support or particular pre%embedment orientation. $araffin wax double

infiltration methods provide hard tissues with additional support provided by substances

such as agar or nitrocellulose, with the convenience and ease of wax microtomy.

AAR1$ARA**IN 5A, DOU!-E EM!EDDIN

&ouble embedding in agar%paraffin is a reliable and convenient method of handling

minute and friable tissue fragments such as curettings and endoscopic biopsies, which

can be lost during tissue processing."(7%"*+ 2t also overcomes the difficulty of manipulatingsmall tissue fragments during embedding and facilitates correct orientation and

identification of tissues for histochemical and immunohistochemical control tissues."*8

Agar Em%edding Medium

REAENTS RE8UIRED

" 'gar !technical or microbiological grade# ".+ % *.0 g

( &istilled water 70 ml* *93 formaldehyde solution "0 ml

MET0OD

" &issolve the agar in the distilled water using a boiling water bath, autoclave or

microwave oven.

( 'dd formaldehyde and mix well.* &istribute (0 ml aliquot=s into screw capped bottles. Store at 1:.



1 >or use melt the agar as previously indicated, cool to +0%80:, and hold in a 80:

oven. ?oosen container caps before remelting agar.

+ mbed tissues by pipetting agar solution over tissue fragments correctly oriented on aclean flat surface"(7 or membrane filter."0+

TEC0NICA- NOTES" Specimens can be embedded in silicon%rubber flat embedment moulds, moulds made

from cut%down plastic syringes,"*0 or in metal Tissue%Tek type moulds."*(,"*+

( <nstabilized agar embedments sometimes disintegrate during processing and must be

stabilised by the addition of (.+3%13 formaldehyde to the medium, or by immersing

blocks in 903 ethanol or fixative for "%* hours.

Agar1$araffin 5a) Dou%le Em%edding *or *ragments& !iopsies And *ria%le

Specimens"*+

REAENT RE8UIRED

'gar !prepared as above#

MET0OD

" >ill the appropriate sized Tissue%Tek base mould with agar medium cooled to

approximately 10:.

( Aumber embedding cassette, cross checked with request slip and specimen container.* $lace specimen in agar%filled mould and orientate.

1 'llow agar to solidify for + minutes. $lace embedding cassette on top of the mould to

identify the tissue.+ 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 *%+ mm width of

agar surrounding the tissue. $rocess normally.

Agar1$araffin 5a) Dou%le Em%edding *or !one Marro4 Aspirates And Cell

Suspensions Using T(e Collodion !ag Tec(ni+ue"*9

REAENT RE8UIRED

ollodion !add 13 nitrocellulose to (+ ml absolute ethanol / 9+ ml diethyl ether#

MET0OD

" $lace "%( ml of collodion into a "0 ml glass centrifuge tube. Cotate 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.( >ill the tube with 903 ethanol and stand for + minutes to harden the collodion. &rain

the ethanol, rinse with water.

* 'dd fixed aspirate or suspension to tube.1 entrifuge at (000 rpm for *0 seconds.

+ &ecant supernatant fluid.

8 $ipette "%( ml of agar, cooled to approximately 1+:, into the centrifuge tube.9 Capidly resuspend the specimen and centrifuge at (000 rpm for " minute.

5 'llow the mass to cool for + minutes.

7 <sing fine forceps, carefully detach the collodion bag and contents from the tube.



"0 Trim off unwanted collodion, and process as a normal specimen, taking care to orient

and embed the specimen correctly.

AAR 1 ESTER 5A, DOU!-E IN*I-TRATION

&ouble infiltration of tissues in agar and ester wax"*5%"*7 aids thin serial sectioning of

chitinised tissues at 0.+%".0 Im and is a possible alternative to pine resin%beeswax paraffin wax used to support plastic vascular prostheses for sectioning."10 The fine

crystalline nature and hardness of ester wax improves tissue%wax adhesion and providesadequate support for thin serial sectioning.

Agar1Ester 5a) Dou%le Infiltration"*5

REAENTS RE8UIRED

" +3 aqueous agar

ellosolve !(%ethoxyethanol#

MET0OD

" 2nfiltrate fixed tissues in +3 aqueous agar solution for " hour at +0%80:.( ;rientate tissues in an agar filled mould as indicated previously. 'llow the agar to set.

Trim the block.

* $ass the block through the following series, *0 minutes in each solution/ *03, +03,

903 ethanol) 703 ethanol plus cellosolve, (/") the same "/() pure cellosolve, * changes)cellosolve plus ester wax "/") pure ester wax, at least ( changes, the last overnight.

1 mbed as usual and cool rapidly.

TEC0NICA- NOTE

6uzzell"1" dehydrates in dioxane, with amyl acetate as a transition solvent. 6locks are

best cut using a retracting microtome.

NITROCE--U-OSE 1 $ARA**IN 5A, DOU!-E IN*I-TRATION

Aitrocellulose%paraffin wax double embedding is mainly used for brain, friable tissuesand decalcified bone and is particularly useful for whole body sections of small animals

and chitinous tissues. 2t 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 blocktrimmed, hardened in chloroform and infiltrated in paraffin wax, or !b#infiltrated with

thin low viscosity nitrocellulose !?BA# solutions which are integrated into a normal

processing schedule. $roprietary celloidin%ethanol%ether solutions provide a simple and

convenient double%embedding method !Table "8#. The principle drawbacks of thistechnique are prolonged exposure of tissues to absolute ethanol and the high flammability

and volatility of diethyl ether precluding machine processing. <se of methyl benzoate or

methyl salicylate as ?BA solvents overcome these deficiencies.

2n $eterfi=s classic technique,"1( tissues are dehydrated to absolute ethanol, infiltrated with

"3 celloidin in methyl benzoate with (%* changes over (1%9( hours !until clear#,

hardened in three changes of toluene for 5 hours, then infiltrated as usual in paraffin wax.





olnar=s variation"1* !Table "9# 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 *03 of theweight of the nitrocellulose"(9 and must be taken into consideration when preparing

solutions. Aitrocellulose dissolves slowly in methyl salicylate, and during preparationmixtures should be periodically shaken to assist dissolution. 2n recent times ?BA has

largely replaced celloidin.

Sections of double%embedded tissues may tend to wrinkle or curl on the waterbath.

>loating on 7+3 ethanol or Cuyter=s fluid"*7 softens the ?BA and facilitates section

flattening.

Met(ods for difficult tissues0ARD DENSE TISSUES

Tissues largely comprised of thickened keratin, dense collagen, closely packed smooth

muscle fibres, colloid, areas of haemorrhage, thrombi or yolk, can be hardenedexcessively when processed on routine schedules and consequently, sections may

crumble or shatter. 2deally the fixative, processing reagents, embedding medium andschedules should be selected to minimise hardening in these tissues. &espite careful

processing hard tissues frequently require treatment with post%embedding ad@uvants

before microtomy.

ammalian tissues such as uterus, scirrhous carcinoma, leiomyomas and keratinisedtissues are softened by fixing in 13 phenol in a mixture of absolute ethanol !9+ ml#,

water !"0 ml# and chloroform !"0 ml#,(7 or by treating fixed tissues using 13 aqueous

phenol for (1%9( hours.(7 Similar results are obtained by dehydrating tissues using phenol

in the first bath of absolute ethanol, or in all dehydrant baths !Table "5#.*(

Transitionsolvents such as chlorinated hydrocarbons and terpenes are recommended as they do not

exacerbate tissue hardness.

MUMMI*IED TISSUES

ummified archaeological specimens for histology are first rehydrated then processed on

a ?BA%paraffin wax double%infiltration schedule using phenol%ethanol to soften the

tissues and amyl acetate as the transition solvent."11%"1+

Met(ods *or T(e Reco"er' Of Dried And Mummified Tissues

REAENTS RE8UIRED

Solution !after Sandison"11

#'bsolute ethanol *0 ml>ormaldehyde, *93 0.+ ml

Sodium carbonate 0.( g

Water to "00 mlor

Ban leve J Coss= solution"1+







Trisodium phosphate 0.(+ g

Water "00 ml

MET0OD

" 2mmerse tissues in either solution for (1%9( hours or more, depending upon the nature

and thickness of the specimen !most tissues rehydrate and soften within 1%8 hours#.( $rocess re%hydrated tissues on a normal schedule beginning in 903 ethanol. Tissues

dried and hardened over years, and mummified archaeological specimens should berehydrated in Sandison=s solution, then double infiltrated in phenol%amyl acetate%?BA%

paraffin wax schedule.

/O-K/ TISSUES

Kolk%rich gonads, and muscle of marine and freshwater fish and invertebrates are

routinely fixed in >'' fixative !formaldehyde *93, "0 ml) glacial acetic acid, + ml)

calcium chloride dihydrate, ".* g#"18 and processed by the schedule given in Table "7.

This fixative has similar fixation image, processing and sectioning characteristics to

6ouin=s fluid, but without the hazard, cost and inconvenience of picric acid used in thisfixative. 6uffered or saline formaldehyde fixatives cause excessive hardening of these

tissues and are contra%indicated.

Steedman"7 recommends polyester wax for minimising heat%induced hardening ofdifficult tissues.

*ATT/ TISSUES

>atty tissues such as breast or lipoma may be inadequately processed in what is normallya successful schedule for other tissues. thanol is a poor fat solvent. To ensure complete

dehydration, a superior fat solvent such as acetone or isopropanol should be inserted

before the final absolute ethanol, and chloroform or ",",",%trichloroethane used as thetransition solvent.

Ackno4ledgments

The assistance of ?aurie Ceilly, Dary &oak and Savita >rancis in the preparation of this

chapter is gratefully acknowledged.

C>CASS'>TK &'T'


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Tissue Processing (1)

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