Microtomy - Preparation of Histological Slides

Microtomy, or the preparation of tissue slides, is the foremost technique used in histological studies. This presentation is a brief overview of the technique and the steps involved.

1. MICROTOMY
PREPARATION OF HISTOLOGICAL SLIDES
SYED MUHAMMAD KHAN
BS HONS. ZOOLOGY

2. HISTOLOGY
Histologyisthestudyoftissuesandhowthesetissuesare
arrangedintoorgans(“Histo”inGreekmeanstissueorweb).
Tissuesconsistofcellsandextracellularmatrix.
Thefunctionofthetissuedependsontheinteraction
betweenthecellsandtheextracellularmatrix.
Thestudyoftissuesisdependentonmicroscopyandother
advancesinbiologicaltechniques.

3. MICROTOMY
Microtomy/SectionCuttingisthetechniqueofmaking
verythinslicesoftissuespecimensforthemicroscopic
examination.
Itisusedtostudyvariouscomponentsofthecellsor
tissueslikelipids,enzymes,antigensorantibodies
(Immunohistochemistry),organelles,etc.
Itcanalsobeusedtoidentifyabnormalitiesoratypical
appearanceinthetissue(ifpresent).

4. MICROTOMY
The main stages involved in the preparation of histology
slides via microtomyare:
1.Fixation:to prevent cell decay and to preserve it in a
life-like appearance.
2.Processing:dehydration, clearing and embedding.
3.Sectioning:cutting very thin sections of the wax
embedded block.
4.Staining:to create visible contrast.
5.Mounting:to create a permanent slide.

5. FIXATION
Fixationisthepreservationofbiologicaltissuesfrom
decayduetoautolysisorputrefaction.
Samplesofbiologicaltissuearefixedtopreservethe
cells/tissuesinasnaturalastateaspossible.
Chemical fixativesareverycarefullyselected
substanceswhosepropertiesmustmeetmanycriteria.
Eventhemostcarefulfixationaltersthesampletoa
certainextentandmaypotentiallyintroduceartifacts.

6. FIXATION
Artifactsarestructuresorfeaturesintissuethatinterfere
withnormalhistologicalexamination,i.e.pigments
formedbyfixatives.
Thechoiceoffixationmethodandspecificfixativemay
dependonthesubsequentprocessingsteps.
Fixationisareactionbetweenthefixativeandproteins
inthespecimenwhichformagel,keepingeverythingas
theirinvivorelationtoeachother.

7. FIXATION
The aims of fixation are as follows:
1.To prevent autolysis and bacterial attack.
2.To fix the tissues so they will not change their volume
and shape during processing.
3.To prepare tissues and leave them in a condition which
will allow clear staining of sections.
4.To leave the tissues in a life-like state.

8. FIXATION
ChemicalFixation:biologicalstructuresarepreservedina
stateasclosetothatofthelivingtissueaspossible.
Thisrequiresachemicalfixativethatcanstabilizeproteins,
nucleicacidsandmuco-substancesoftissuesbymaking
theminsoluble.
Somechemicalfixativesinclude:(1)Aceticacid,(2)
Formaldehyde–10%,(3)Ethanol,(4)Glutaraldehyde,(5)
Methanol,(6)Picricacidand(7)Osmicacid(Osmium
tetroxide).
Thetissuesamplesmustbecutintosmallcubes,around1cm
x1cmx1cminsize,andbeputinthefixative(i.e.10%
formaldehyde/formalin)for24–28hours.

9. FIXATION
Frozen Sections
1.Small pieces of tissue (typically 5 mm x 5 mm x 3 mm) are placed in
a cryoprotectiveembedding medium.
2.Then they are snap frozen(rapid cooling for preservation) in
isopentane(an alkane) –cooled by liquid nitrogen.
3.The tissue is then sectioned in a freezing microtome
(cryomicrotome–discussed later).
4.Sections are then fixed by immersion in a specific fixative or series
of fixatives for a carefully controlled period of time.

10. PROCESSING
Aim: to embed tissues in a solid medium firm enough to support
them and give them sufficient rigidity to enable thin sections to be
cut, and yet soft enough not to damage the knife or the tissues.
The stages of processing are:
1.Dehydration (removal of water)
2.Clearing (removal of alcohol)
3.Infiltration & Embedding (preparation of a paraffin wax block)

11. STEPS INVOLVED IN TISSUE PROCESSING

12. PROCESSING
Dehydration
1.Dehydrationremovesthefixativeandwaterfromthetissues
andtoreplacethemwithadehydratingfluid.
2.Specimensaredehydratedinanascendingethanolseries:10%,
20%,50%,70%,95%and100%–absolute(around30minutesin
each).
3.Somecommon dehydratingagentsinclude:(1)Ethanol,(2)
Methanoland(3)Acetone.
4.Tissuesmaybeheldandstoredindefinitelyin70%ethanol
withoutharm.

13. PROCESSING
Clearing
1.In this process, an organic solvent such as xylene is used
to remove the alcohol and allow infiltration with paraffin
wax.
2.Some clearing agents include: (1)Xylene, (2)Toluene,
(3)Chloroform, (4)Benzene and (5)Propylene oxide.

14. PROCESSING
Infiltration&Embedding
1.Thetissueisfirstputin58
o
Chot
paraffinwaxforonehour.
2.Thewaxinfiltratesitand
replacesxylene.
3.Tissuesaresurroundedbya
mediumsuchasparaffinwax
(tomakeablock).
4.Whenthewaxsolidifies,itwill
providesupportandfirmnessto
thetissueduringsectioning.
Wax embedded tissue

15. PROCESSING
The overall aims of embedding are:
1.To improve ribboning
2.To increase hardness
3.To decrease melting point
4.To improve adhesion between specimen and wax

16. PROCESSING
Embeddingisdoneinmolds/cases,forexample:(1)paperboat
mold,(2)metallicboatmold,(3)peel-awaydisposablemold,and
(4)basemold.
Peel-away
Disposable Mold
Embedding Rings
Metallic
Base Mold
Cassette Bases

17. SECTIONING
Sectioningistheproductionof
thinslicesofwax-embedded
tissuesviaamicrotome.
Sectionsare5μmthickforlight
microscopyand80-100nmfor
electronmicroscopy.
Microtome isamechanical
instrumentusedtocutbiological
specimensintoverythinsections
formicroscopicexamination.
Thinsectionsofparaffinembedded
tissuebeingcutbyamicrotome

18. SECTIONING
Mostmicrotomesuseasteelblade(ultramicrotomesusea
diamondknife).
Theyareusedtopreparesectionsofanimalorplanttissuesfor
histology.
Oncethesectionshavebeenextracted,theyareputon
warmwater(toflattenthem).
Thentheyarepickedfromunderneathbyaglassslide.
Theslidewiththesectiononit,isallowedtodryat37
o
C,so
thatthesectionadherestoit.

19. TYPES OF MICROTOMES
RotaryMicrotome
•Itisthemostcommonlyused
microtome.
•Itisusedforsectioningofparaffin
embeddedblocks.
•Itcanalsobeusedforfrozen
sectionsincryostatandalsofor
resinembeddedcases.
•Sectioningoccursbyrotational
movement ofthemicrotome
headcontainingtheblockacross
theblade.
Rotary Microtome

20. TYPES OF MICROTOMES
RockingMicrotome
•Itisasmallmicrotomethathas
tworockingarms.
•Onecutsthesections.
•Theotherfeedsthroughthe
tissueblock.
•Itislimitedtosectioningsmall
softblocksasitusesspring
actiontocut.
Rocking Microtome

21. TYPES OF MICROTOMES
BaseSledgeMicrotome
•Inthismicrotome,thesampleis
placedintoafixedholder
(shuttle),whichthenmoves
backwardsandforwardsacrossa
knife.
•Thepressureappliedtothe
sampleduringthecutcanbe
reduced.
•Typicalcutthicknessachievable
onasledgemicrotome is
between1and60µm.
Base Sledge Microtome

22. TYPES OF MICROTOMES
SlidingMicrotome
•Itisanunusuallydesigned
microtome withablade
movingovertheblock,rather
thantheblockmoving.
•Itisgood forcelloidin
sectioning,althoughitcan
produce good paraffin
sectionstoo.
Sliding Microtome

23. TYPES OF MICROTOMES
Ultramicrotome
•Itisamicrotomeusedmainlyfor
electronmicroscopy.
•Itallowsthepreparationofextremely
thinsections.
•Diamondknives(preferably)andglass
knivesareusedinthismicrotome.
•Tocollectthesections,theyare
floatedontopofaliquidastheyare
cutandarecarefullypickeduponto
gridssuitableforTEMspecimen
viewing. Ultramicrotome

24. TYPES OF MICROTOMES
Cryomicrotome
•Itisusedforcuttingfrozen
samples.
•Thereducedtemperatureallows
thehardnessofthesampletobe
increasedwhichallowsthe
preparationofsemi-thinsamples.
•Howeverthesampletemperature
andtheknifetemperaturemust
becontrolledinordertooptimize
theresultantsamplethickness.
Cryomicrotome

25. TISSUE SECTIONS
Transverse Section (T.S)
Horizontalsectioncutmadeinaplane
atrightangletothelongitudinalaxisof
thebodyofasubject.
T.Sgoesbetweenlateralends.
Itisusuallycomparativelyshorter.
Thenumber ofpossibletransverse
sectionsthroughaspecimen is
comparativelymore.
Longitudinal Section (L.S)
Verticalsectionthatiscutalongthe
longestaxisofasubject.
L.Srunsthroughtheanteriorposterior
axis.
Itisusuallycomparativelylonger.
Thenumberofpossiblelongitudinal
sectionsthroughaspecimen is
comparativelylesser.

26. STAINING
Stainingreferstotheuseofstainstomakecellsand/orcellular
structuresvisibleandtoenhancethecontrastofamicroscopic
image.
Themountedsectionsaretreatedwithanappropriatehistology
stain.
Biologicaltissueshaveverylittlevariationincolors/shadeswhen
viewedusingeitherunderamicroscope.
Stainingbiologicaltissuesisdonetobothincreasethecontrastof
thetissuesandalsotohighlightsomespecificfeaturesofinterest.

27. STAINING
Thestainingofparaffinembeddedsectionsisdoneinthefollowing
way:
Deparaffinization:Thewaxofthesectionisremovedbyapplying
xylene.
Re-hydration:Thespecimenisrehydratedviaadescendingethanol
series:90%,70%,50%,20%,10%anddistilledwater(around30
minutesineach).

28. STAINING
Hematoxylin&EosinStaining
1.Thetissueisfirststainedwithhematoxylinfor10minutes(thisstaingives
abluecolortothenuclei).
2.Thenthetissueisrinsedindistilledwaterandthendippedinacid
alcoholuntilonlythenucleiarestainedblue.
3.Theslideisagainwashedwithdistilledwaterandtheneutralizedwith
alkalinetapwater(Scott’stapwater).
4.Itiswashedagainindistilledwaterandthenstainedwitheosinforjust
10secondstostainthecytoplasm,extracellularmatrix,collagenand
erythrocytesred.

29. STAINING
Dehydration:Specimenaredehydratedonceagain,inan
ascendingethanolseries:10%,20%,50%,70%,95%and100%
ethanol(around30minutesineach).
Clearing:Thespecimenisclearedinaclearingagentsuchas
xylenefor15minutestoremovealltracesofalcoholandraise
therefractiveindexofthespecimentomakeitmore
transparent.

30. MOUNTING
Mountingisdonetopreserveand
supportastainedsectionfor
microscopicexamination.
Tomountaslide:
A.ApplyasingledropofDPX
Mountantupontissuesection.
B.Holdcoverslipat45
o
allowingthe
droptospreadalongtheedgeof
theslip.
C.Letgoofslipandallowmedium
tospreadslowly
Mounting Procedure

31. HISTOLOGICAL SLIDES
H&E stained sections (top left to bottom right) –lung tissue (of an emphysema patient),
retina, cartilage, muscle tissue, kidney and liver.