10 - Mounting Media.pdf

Mounting media

1. Cellular Pathology
Lecture #10
Mounting Media
Ephraim Imhotep Zulu, BScBMS, MScPath
University of Zambia
School of Medicine
Department of Biomedical Sciences

2. Sunday, January 3, 2016 2

3. Mounting Media
•Mountingofsectionsunderacoverslipisessential
togetthebestandclearestviewofthespecimen.
•Mountingmediaareneededformakingpermanent
slides.
•Themountingmediumholdsthespecimensinplace
betweenthecoverslipandtheslide.
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4. •Themountingmediumshouldhaveahighrefractive
index(RI).
•MosttissueshaveanRIofbetween1.5and1.55,soa
mountingmediumwithanRIinthisrangewillgive
maximumclarity.
•Therearetwomajortypesofmountingmediausedand
thedifferenceisinthesolvent.
•1.Aqueousmedia-Usedformaterialwhichisunstained,
stainedforfat,ormetachromaticallystained.
•2.Resinousmedia-Forroutinestaining.
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5. CriteriaOfAcceptableMountingMedia
•Refractiveindex(RI)
•Inabilitytocausestaintodiffuseorfade
•Appearanceonsettingiecrackorgranularappearance
•Abilitytodrytoanonstickyconsistencyandharden
relativelyquickly
•Inabilitytoshrinkbackfromedgeofcover-glass
•Itshouldbefreeflowingandfreeofbubbles
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6. Criteria Of Acceptable Mounting Media..,
1.RefractiveIndex
•Shouldbeascloseaspossibletothatofglassi.e.1.5
•IftheRIismuchlowerthan1.5,thentissueswillnotbe
completelytransparentanddiffractionwilloccur.
•LowerRIreducesclarityandgivesomecontrasttoeven
unstainedtissues.
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7. Criteria Of Acceptable Mounting Media..,
2.EffectsOnTheStainItself
•Somemediawillcausefadingegacidicmountingmaterials,
especiallyinthelight.
•Somemediamayactassolventsforthedyesandasa
consequencethedyediffusesorleachesoutintothe
mountant.
•Thiswillgraduallyobscurethetissues.
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8. Criteria Of Acceptable Mounting Media..,
3.Clarity
•Somemediacanbecomeopaqueastheydryoutandarenot
suitableforlong-termpreservation.
4.Fluorescence
•Criticalforfluorescencemicroscopy
•iteliminatestheneedtouseaspecialmountantwhen
fluorescenceisbeingused.
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9. Criteria Of Acceptable Mounting Media..,
5.Setting
•Theabilityofamountanttodryorsetquicklyandholdthe
coverslipinplaceisveryuseful.
•Manyaqueous-basedmediafailtohardensufficientlyand
thecoverslipwillneed‘ringing’topreservethesection.
•Ringingisthetermusedforsealingtheedgesofacoverslip
whenthemountingmediumdoesnotset.
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10. Resinous Mounting Media
•Commonesttypes
•Arebasedonhydrophobicorganicsolvents,usually
xylene
•Needthesectiontobedehydratedandcleared
beforemounting.eg
-Canadabalsam-Naturalresin(R.I.-1.52)
-D.P.X.(R.I.1.52)
-Eukitt
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11. Canada Balsam
•Naturalresin
•Itisusedas60%resinbyweightinxylene.
•Slidestakefewmonthstodry.
•Theyellowcolourofthemountanthardlyseemstomatter
whenviewedthroughthemicroscope
•Willcausefading,especiallyofbasicdyessinceitsacidic
•Notverycheaptoobtain.
•itsopticalpropertiesdonotdeterioratewithagehenceit
hasthelongstorageabilityoftheslides.
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12. DPX
•Asyntheticpolystyreneresinthatisdissolvedinxyleneand
hassomeplasticizeradded.
•Theinitialscomefromthecomponents:Distrene80,
plasticizerandxylene.
•Itisawater-whiteclearsolution.
•Ithasverylittletendencytofadedyesandhardensinabout
24hours.
•Thespecimensdonotneedringing.
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13. Eukitt
•Isaveryfastdryinggeneral-purposeresin-based
mountingmedium.
•Willsolidifywithinabout20minutes.
•Thespecimensmustbefreeofwaterandplaced
firstinalcoholandtheninxylenepriortomounting.
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14. Euparal
•Containssandarac,eucalyptol,paraldehyde,camphor,
andphenylsalicylate.
•Iscommonlyusedtomounthistologicalspecimensand
insects.
•Donotembedspecimenswhichcontainwater,thismay
resultinacloudingofthemountingmedium.
•Ithasarelativelylongdryingtimeofafewdays.
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15. Clear Nail Polish
•Canbeusedtosealthesidesofthecoverslipwhen
usingaqueousmountingmedia.
•Canalsobeuseddirectlyasamountingmedium.
•Thespecimensmustfirstbedehydratedinalcohol
andcanthenbedirectlymounted(withoutxylene)in
nailpolish
•Itisreadilyavailableanditavoidstheuseoftoxic
organicsolventstotreatthespecimens.
•Itseemstoshrinkalotwhenmakingverythick
mounts(suchaswholeinsects).
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16. Aqueous Mounting Media
•Usedformountingsectionsfromdistilledwater
whenthestainswouldbedecolorizedorremovedby
alcoholandxylene.
•Requireadditionofbacteriostaticagentstoprevent
thegrowthoffungi.
•Mostarebestconsideredastemporarymountsand
needringingtoholdthecoverslipinplaceand
preventdryingout.
•Tissuesdonotneedanytreatmentbeforemounting
andcanbemounteddirectlyfromwaterorbuffer.
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17. Glycerol
•AtrihydricalcoholwithahighRI.
•Isausefulmediumforfluorescentstaining,eg
immunofluorescentantibodytechniques.
•Itneitherhardensnordriesout.
•Theadditionofp-phenylenediamineissaidtoretard
thefadingoffluorescence.
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18. Glycerol Jelly
•Maycontainphenolwhichmakesithazardousfortheuse.
•Thehandlingofthismountingmedium,isalsonottooeasy.
•Bubblesareaproblemwiththismedium.
•Italsodoesnotshrink.
•TheusualformulationhasalowerRI(1.42)thanmost
mountingmedia.
•iscommonlyusedtopreservepollensamples.
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19. Apathy’s Medium
•ThisusesagumandsucrosetoraisetheRI.
•IthasanRIofaround1.5,soitcangivenicely
transparentpreparations.
•Ithasatendencytocrystallizeinstorageandcan
setbydryingbutthisisquiteslow.
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20. Polyvinyl Alcohol Media
•Aresyntheticandlessliabletobacterial
contaminationthantheorganic-basedmountants,
althoughtheadditionofphenolisstilladvisable.
•Theydissolveinwaterorbufferbutneedconstant
stirring.
•Theysolidifyslowlybyevaporationbutspecimens
canberingedtopreventthis.
•Thesearemorepermanentthantheotherwater-
basedmountingmedia,butarestillnotasgoodasa
resinousmedium.
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21. Temporary Mounts Need Ringing
•Ringingisthetermusedforsealingtheedges
ofacoverslipwhenthemountingmediumdoes
notset.
•Ringingwasoriginallysocalledbecausethe
coverslipswereroundandsotherewasaring
ofthesealantroundthecoverslip.
•Goodtemporaryringingcanbeachievedina
numberofwaysusingOrdinarynailvarnish,
Manystyrene-basedcements,Paraffinwax,
etc.
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22. Use Of Coverslips
•Mountingofsectionsunderacoverslipisessentialto
getthebestandclearestviewofthespecimen.
•Thecoverslipshouldhaveathicknessof0.17mmfor
thebestresults.
•Thickercoverslipswillinterferemarginallywiththe
clarityand
•verythickcoverslipsmayevenpreventtheoil-
immersionlensbeingusedastheycanhaveagreater
thicknessthanthenormalworkingdistanceofthe
oil-immersionlens.
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23. Storage of slides
•Mountedslidesshouldalwaysbecarefullylabelled
andstoredhorizontallyuntilfullydryandset
whentheycanbestoredontheiredgeorend.
•Stainedslidesshouldbestoredawayfromlight
asthedyeswillfadeeveninthebestmountant.
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24. References & Credits
•Cook,D.J(2006).BiomedicalSciencesExplained:
CellularPathology.Butterworth Heinemann,
Oxford.ISBN:10:194842305
•OliverKim.(2013).Anoverviewofmountingmedia
formicroscopy.MicrobeHunter.com
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25. End Of Lecture
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Ephraim Imhotep Zulu –2015/16