Techniques in Cell Biology

Describes the cytological techniques

1. Lecture on
Cytological Techniques
Dr.Manikandan Kathirvel M.Sc., Ph.D., (NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
(Reaccredited with "A" Grade by NAAC)
Affiliated to Bengaluru North University,
K. Narayanapura, Kothanur(PO)
Bengaluru
Email: [email protected]
ORCID ID: 0000000270066334

2. 1. Microtome
2. Microscopy techniques
3. Staining techniques
4. Centrifugation

3. Cytological Techniques
-Cytologicaltechniquesaremethodsusedinthestudyormanipulationofcells.
-Theseincludemethodsusedincellbiologytoculture,track,phenotype,sortandscreen
cellsinpopulationsortissues,andmolecularmethodstounderstandcellularfunction.
-Mustbeabletoidentifynormalcellsfromabnormalcells,andinflammatoryfromnon-
inflammatorycells.
Theycanbeobservedeitherdirectlyorafterpreservationunderthemicroscope.
1.Fordirectobservation,thespecimenneedssufficientcontrast.Directobservationis
possiblebyusingvitalstains.
Vitalstains:-Vitaldyesorstainsaretakenupbylivingcellswithoutkillingthem.They
selectivelystainintracellularstructureswithoutaffectingcellularmetabolismandfunction.
Forexample,JanusgreenBselectivelystainsmitochondria,Golgiapparatus,nuclear
chromatininadividingcellcanbestainedbymethyleneblue;NeutralReddyeorCongo
Reddyecanbeusedtostainyeastcells.
2.Preservedandstainedtissues:-Fordetailedmicroscopicstudy,tissuescontainingcells
arepassedthroughvariousstages.Thestagesofcellpreparationonaglassslideinvolves
killing,fixation,dehydration,embedding,sectioning,stainingandmounting.

4. PREPARATION OF BIOLOGICAL
SPECIMENS FOR LIGHT MICROSCOPY

5. Sample collection

6. Sample collection

7. Sample collection

8. EXFOLIATIVE CYTOLOGY
Sample collection

11. Methods:Thereare5methodsbywhichthecytologicalspecimensarepreparedfor
microscopicalobservation.Theyare:
1. Teasing
2. Smear preparation
3. Squash preparation
4. Whole mounting
5. Microtomy
1. Teasing:
Themusculartissueisobservedbyteasing.Abitoftissueisteasedinasalinesolution.Itis
stainedbysafraninoreosin.Thestainedmaterialismountedonaslideandisobserved
underthemicroscope.
2.SmearPreparation
Fluidtissuessuchasbloodareobservedbysmearpreparation.Smearisathinfilmof
tissueonaslide.
a.Adropofbloodistakenonaslideandismadeintothinfilmwiththehelpofedgeof
anotherslide.
b.Itisdriedandstainedandcoveredwithacoverglassandobservedthemicroscope.

12. 2. Smear Preparation: METHODS OF SMEAR PREPARATION:
i.streaking
ii.spreading
iii.pull apart
iv.touch or impression smear
STREAKING
-Usedforpreparingmucoidsecretions,vaginal
secretions,sputumandgastriccontent
-useaspatula,dissectingneedleorapplicator
stickandstreakinazigzagfashion.
SPREADING
-usedforthickmucoidsecretions
-smearsoffreshsputumandbronchialaspirates
PULLAPART
-forserousfluids,concentratedsputum,and
enzymaticlavageformtheGIT,smearsof
urinarysediment,vaginalpoolandbreast
secretions.
TOUCHIMPRESSION
-ImpressioncytologybeingcollectedFroma
patient,usingasterileglassslidewith
polishededges.

13. 3. Squash preparation
i.Soft tissues such as testis, onion root tip, etc are observed by squash preparation.
ii.The soft tissue is placed on the slide, the cover glass is dropped over it and gentle
pressure is applied on the cover glass.
iii.The material is made into a thin layer.
-Fairly accurate,
-Simple and reliable tool for diagnosis of lesions.
Based on two essential factors:
• Availability of very small tissue fragments & good preservation of fine cellular details.
• Not effected by edema, hemorrhage, necrosis & calcification.
4. Whole mounting:
Certain objects are transparent and they are mounted entirely as such. This process is called
whole counting and the slide is called whole mount.
5. Microtomy: a technique to make a thin, transparent sections of tissues and cells of
Preserved tissues:-For detailed microscopic study, tissuescontaining cells are passed
through various stages. The stages of cell preparation on a glass slide involves killing,
fixation, dehydration, embedding, sectioning, staining and mounting.

14. PREPARATION OF BIOLOGICAL SPECIMENS
FOR MICROSCOPICAL OBSERVATION
Steps involved in Microtomy
Steps involved in Microtomy

15. Microtomy:isacytologicaltechniquebywhichextremelythintransparentsections
oftissuesandcellsarepreparedformicroscopicobservations.
Fordetailedmicroscopicstudy,tissuescontainingcellsarepassedthroughvariousstages.
Thestagesofcellpreparationonaglassslideinvolveskilling,fixation,dehydration,
embedding,sectioning,stainingandmounting.
1).Killingandfixation:-FIXATIONOFCYTOLOGYSPECIMENS
Fixationisthepreservationofthecellstructureofthematerialinalifelikecondition.
Fixationmeans:
-preventionofdegenerationofcellsandtissue
-preservationofcellsascloseaspossibletothelivingstatespecificperiodsoftime
changesthephysicalandchemicalstateofthecells.
-Fixationisdonebychemicalsorfreezing.ThechemicalusedforfixationiscalledFixative.
-Thisprocesscausessuddendeathofcellsortissuesandpreservesfreshlykilledtissuesin
aslifelikeaconditionaspossible.Agoodfixativepreventsbacterialdecayandautolysis.
-Itwillalsomakecellcomponentsmorevisibleandpreparethecellforstaining.
-ThecommonlyusedfixativesareAceticacid,formalin,picricacid,osmiumtetroxide,
Formaldehyde,Bouin'ssolutionandCarnoy'sfluid.

16. 1).FIXATIONOFCYTOLOGYSPECIMENS
Fixationmeans:
-preventionofdegenerationofcellsandtissue
-preservationofcellsascloseaspossibletothelivingstatespecificperiodsoftime
changesthephysicalandchemicalstateofthecells.
ANAPPROPRIATEFIXATIVEFORCYTODIAGNOSTICPURPOSESSHOULDPERFORMTHE
FOLLOWINGFUNCTIONS
a.Penetratecellsrapidly
b.Minimizecellshrinkage
c.Maintainmorphologicintegrity
d.Deactivateautolyticenzymes
e.Replacecellularwater
f.Facilitatediffusionofdyesacrosscellboundaries
g.Helpcellsadheretoaglasssurface
h.Provideconsistentresultsovertime

17. 2).Dehydration:-istheremovalofwatermoleculesfromfixedtissues.Inthisprocess
watervapourareremovedfromcellsortissuesusingchemicalagents.Itisdonebyusing
ethanolandbenzene.
Thefixedtissueispassedthroughtheseriesofincreasingconcentrationsofalcoholsuch
as30% 50% 70% 90% Absolutealcohol(95%).
Aftertreatedwith100%alcohol,thematerialisclearedwithbenzene.Forelectron
microscopepropyleneoxideisused.
Chemicalremovalofwaterandfixativefromthespecimen
•Replacethemwithdehydratingfluid-dehydrant
•Manydehydrantsarealcohols.Severalarehydrophilicsoattractwaterfromtissue.
•Practicedingradedseries
•Progressivelydecreasingconcentrationofwater
•Progressivelyincreasingconcentrationofdehydrant.
•Commondehydrantsareethylalcohol,acetone,normalbutylalcohol,tertiary
•butylalcoholGlycerine,Dioxanetc.
•Progressivelyincreasingconcentrations–10%,20%,30%,40%……100%
•Timerequired–softtissues~30minutes–Hard/largetissue-~6-12hrs.
Clearing:(Dealcoholization)-Removalofalcoholfromthetissues.
Clearingagents-Xylene,Toluene,Chloroform,Benzene,Petroletc

18. 3).Embedding:-isBlockmaking.Thetissuesareembeddedwithsupportingmediumsuch
asmoltenparaffinwaxforsufficienthardness.
Ithardensuponcoolingandprovidesenoughsupporttoallowthinsections.
Verythinsectionsneedtobetakenforelectronmicroscopy.Henceplasticsareusedfor
embedding.

19. 4).Sectioning:-Theembeddingmaterialiscutintothinsectionsofneededthickness.Itis
donebyusinganinstrumentcalledmicrotome.Microtomeisaninstrumentusedtocut
sectionsofdesiredthickness.
Therearethreetypesofmicrotome.
Theyare:
a.Ordinarymicrotome
b.Freezingmicrotome
c.Ultramicrotome
Forlightmicroscope,thethicknessofthesectionshouldbe6-8microns.
Forelectronmicroscope,thethicknessofthesectionshouldbe50-200millimicrons.

20. 5).Staining:-isaprocessbywhichthesectionsarecoloredwithsuitablestain.
-Useofdyestoprovidecolortovarioustissueconstituents
-Thesectionsareimmersedindyesthatstainstructuresofthecellcomponent.
Forexample,cytoplasmstainspinkwitheosin.
Nucleusstainsbluewithhaematoxylinorredwithsafranin.
6).Dehydration:-Stainedsectionsareimmersedinethanoltoremovewater.Thetissue
becomesmoretransparent.Dehydrationisdonegraduallybyusingaseriesofincreasing
concentrationsofethanolinwater.Finallythesectionisplacedin'absolute'alcohol.
30% 50% 70% 90% Absolutealcohol(95%).
7).Mounting:-Cleanedsectionsaremountedonaslideusingasuitablemediumlikecanada
balsam.Adropofcanadabalsamisplacedonthesectionandacoverslipisplacedoverit
andthemediumisallowedtodry.
8).Labelling:Assoonastheslideisprepared,itismarkedandlabelled.
canadabalsam: A natural resin used as a mounting medium. Canada balsam is a commonly used mounting medium to
prepare permanent slides for microscopy. It is produced from the resin of the balsam fir tree and can be combined
with xylene-containing specimens.

21. Slides showing embedded
thin slice of the tissue
samples after microtome
Observation under light
microscope
Microtome

22. Tissue sectioning

23. Slides showing embedded thin slice of the tissue samples after microtome
Under microscope

24. 1. Microtome
2. Microscopy techniques
3. Staining techniques
4. Centrifugation

25. Centrifugation

26. Centrifugationisatechniqueofseparatingofsubstanceswhichinvolvestheapplicationof
centrifugalforce.
Theparticlesareseparatedfromasolutionaccordingtotheirsize,shape,density,the
viscosityofthemediumandrotorspeed.Thecentrifugeiscommonlyusedinlaboratoriesfor
theseparationofbiologicalmoleculesfromacrudeextract.Centrifugationisthetechnique
ofseparatingcomponentswherethecentrifugalforce/accelerationcausesthedenser
moleculestomovetowardstheperipherywhilethelessdenseparticlesmovetothecenter.
Principle:
1.Thecentrifugeconsistsofamotorandrotor.
2.Thesampletobeseparatedisplacedontherotor.Themotormakestherotortospin.
3.Thespinningproducesaforcecalledcentrifugalforce.
4.Theforcewhichcausesasubstancespinningroundanaxis,tomoveawayfromthe
centreiscalledcentrifugalforce.
5.Themovementofparticlesinacentrifugalfieldiscalledsedimentation.
6.Therateofmovementofparticlesiscalledsedimentationrate.
7.Thesedimentationratedependsonthesizeanddensityoftheparticles.
8.Inasolution,particleswhosedensityishigherthanthatofthesolvent,sink(sediment),
andparticlesthatarelighterthanitfloatstothetop.
9.Thegreaterthedifferenceindensity,thefastertheymove(sediment).Thisisfollowedby
smallerandlessdenserparticles.

28. The sedimentedparticle is called pellet. The solution above the pellet is called
supernatant.
The speed of centrifuge is expressed as rpm (revolution per minute).
The greater the rpm, the greater will be the centrifugal force.
The rate of sedimentation depends on –
•The density of the particles
•The size of the particles
•The viscosity of the medium
•The gravitational pull
Revolutions Per Minute (RPM) in regards tocentrifugationis simply a measurement of how fast thecentrifugerotor does a full rotation in one minute.

29. TypesofCentrifuge
LOW-SPEEDCENTRIFUGE
1)Low-speedcentrifugeusedforsedimentationofheavyparticles
2)Thelow-speedcentrifugehasamaximumspeedof4000-5000rpm
3)Theseinstrumentsusuallyoperateatroomtemperature.
4)Twotypesofrotorsareusedinit:
•Fixedangle-Inangletype,theholdersandsampletubesarekeptatanangleof30°Cfrom
thecentralaxis.
•Swingingbucket-Theholdersandsampletubesswingupandrunhorizontallywhile
spinning.
5)Itisusedforsedimentationofredbloodcells,theparticlesaretightlypackedintoapellet
andsupernatantisseparatedbydecantation.

30. HIGH-SPEED CENTRIFUGES
1.High-speed centrifuges are used in more sophisticated biochemical applications,
higher speeds and temperature control of the rotor chamber are essential.
2.The high-speed centrifuge has a maximum speed of 15,000 –20,000 RPM
3.The operator of this instrument can carefully control speed and temperature which is
required for sensitive biological samples.
4.Three types of rotors are available for high-speed centrifugation-
•Fixed angle
•Swinging bucket
•Vertical rotors
ULTRACENTRIFUGES
1.It is the most sophisticated instrument.
2.Ultracentrifuge has a maximum speed of 65,000 RPM (100,000’s x g).
3.Intense heat is generated due to high speed thus the spinning chambers must be
refrigerated and kept at a high vacuum.
4.It is used for both preparative work and analytical work.

31. Types of Centrifugation
I).DifferentialPelleting(differentialcentrifugation)-theparticlesareseparatedat
differentspeedsatdifferenttimes.Itisusedintheseparationofcellularcomponents.
1.Itisthemostcommontypeofcentrifugationemployed.
2.Tissuesuchastheliverishomogenizedat32degreesinasucrosesolutionthat
containsbuffer.
3.Thehomogenateisthenplacedinacentrifugeandspunatconstantcentrifugal
force(700rpmfor10min)ataconstanttemperature.
4.AftersometimeasedimentformsatthebottomofacentrifugecalledpelletI
(Nuclearfraction)andanoverlyingsolutioncalledsupernatantI.
5.Theoverlyingsolutionisthenplacedinanothercentrifugetubewhichisthen
rotatedathigherspeeds(10,000rpmfor20min)inprogressingsteps.
6.AftersometimeasedimentformsatthebottomofacentrifugecalledpelletII
(Mitochondrialfractionsuchasmitochondria,lysosomes,peroxisomes)andan
overlyingsolutioncalledsupernatantII.
7.Theoverlyingsolutionisthenplacedinanothercentrifugetubewhichisthen
rotatedathigherspeeds(100,000rpmfor1hr).
8.AftersometimeasedimentformsatthebottomofacentrifugecalledpelletIII
(Microsomefractionsuchasribosomes,golgiapparatus,endoplasmicreticulum)
andanoverlyingsolutioncalledsupernatantIII(containscytosol,Proteins,lipids,
carbohydrates).

32. Differential Pelleting
(differential centrifugation)

33. II).DensityGradientCentrifugation
1.Thistypeofcentrifugationismainlyusedtopurifyviruses,ribosomes,membranes,etc.
2.Asucrosedensitygradientiscreatedbygentlyoverlayinglowerconcentrationsofsucrose
onhigherconcentrationsincentrifugetubes
3.Theparticlesofinterestareplacedontopofthegradientandcentrifugein
ultracentrifuges.
4.Theparticlestravelthroughthegradientuntiltheyreachapointatwhichtheirdensity
matchesthedensityofsurroundingsucrose.
5.Thefractionisremovedandanalyzedbymassspectrometry.
DensityGradientCentrifugation

34. DensityGradientCentrifugation