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the presence of glycogen in rat liver following in vitro processing in decalcifying agents

the presence of glycogen in rat liver following in vitro processing in decalcifying agents

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Published by Kush Pathak

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Published by: Kush Pathak on Apr 01, 2012
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699
THEPRESENCEOFGLYCOGENINTHERATLIVERFOLLOWING
I.VVITRO
IROCESSINGINDECALCIFVINGAGENTSJ.R.TROTT
FacultyofDentistry,UniversityofManitoba,Winnipeg,Canada
ReceivedforpublicationMay31,1961Inearlierattemptstopreserveghycogenimithemucoperiosteumofjawsitwasfoundthat
themethodsusedwereinadequate(8).Ifthe
mucoperiosteumwasremovedfrom
thepalateofarat,fixedinaceticalcoholfortnahin,and
stainedby-theperiodic-acidSchiffreaction
(
PAS),largeamountsofglycogenwerefoundintheoralepithelium.However,ifthejaws
decalcifiedthenthisgly-cogenaslost.ThereseemedtobethreePossibilitiestoconsider.First,themethodsusedforthefixationandstainingofglycogenmaybeinadequate,second,
iftheglycogenwasadeqcmatelvfixed,thenthe
acids
usedfordecalcificationmay-
beremoving
it,
orthird,itmay-benecessaryforaminimumamountofglycogentobepresentinthetissuesbeforeitcanhedcmotistratedhistochemically(1).Thefirstoftheseh)ossil)ilitieshasbeen
comisideredelsewhere(9).This
communicationdealswiththeactionofdecalcifyimugagentsonglycogen
in
livers.Previouspertinentpublicationsincludethefollowing:SchajowiczandCabrini(6)havediscussedtheeffectoftuitricacid,hy-drochloricacidandformicacid:20sodiumcitrateequalparts,on
thehistochemicalbehaviourof
glycogeninboneandcartilage.Theypoimutoutthath)eriodhic-acidSchiiffpositivesul)stanc-tSumudergolittlemodification,exceptforglycogen,
whichisquicklylostexceptincartilagecclls.
SchiajowiczandCabrinialsotestedtheeffectofdisodiumethylenediaminetetraacetate(EDTA),at
pH
7.0onhiistochemicalreactiotis
in
bone,cartilageandkidney.Concerninggly-cogen,theresultswerethesameasbefore.Loe(3),inhisstudyonbone,fixedtissue
in
l0formalinfor48hoursI)efOreclecalcifyingin
EDT;
itt
1)H7.2..\lthoughperiodicacidSchiiffpositivema-terialwas
seemiinthecytoplasmofosteoblasts,it
istiot
t-learwhetherthmcauthorconsidlersthistobeglycogemiornot.
METHODsANDMATERIALs
Twoexperitiientswereperformiie(ltotesttimeeffectofdecalcifyingsolutionsontheglycogencontentofratlivers(Sprague-Dawleystrain).
1.A
liverwastakemifroma1miiommtholdmimIcrat,dividedinto4piecesandl)lacedlitmmmnedi-ately’
ill
aceticacidalcoholformiialinandfixedfor24hours.ThepHofthefixativewas4.0.Afterfixationonepieceoftissue
was
processedinitiiediatelyasdescribede-viously(9)andsectionscutat6
They’
werestainetlbytheperiodicacidSchiffreaction.Oftheremilainingthreepiecesofliver,one
was
placedintotrichboraceticacidatpH0.6,anotherinfortnicacidandsodiumcitrateaccordingtotheformmmlaeofMorse(5),at.pH2.5,anotherinEDTAadjustedwithNaOHtopH7.5.2.Asixweekoldnmalerate
killedandthelivercarefullyslicedintopiecesaproxi-mately3mmthickbeforeheitmgfixedinaceticacidalcoholformahinfor6hours,24
hours,
48hoursamidsevendays.
Thespecimiietmsweredividedintothreegroups.Thefirstgroup
was
usedascontrolsandfollowingfixationwereprocessedasabove.Atiothergroupwa.sl)laeedinEI)TAat1)H7.5for7
days.
Thelastgroupwasplaced
in
formiiicacidandsochitmmcitrate,asinthepreviousexperiment,for7days.cotitrols,aknownperiodicacidSchiffposi-tiveslidewasalwayspassedthrough(lie
solutions
at.
thesametitneastheexperimentalslides,and
aslidefromeachexperimentalgroupwas
digestedin
chia.stasebeforestaining(4).
Glycogenwas
estimatedinthesections
as
0,
+,++,+++,
and+++
+.
Notiote
was
mmiake
ofthedensit
,
distribution,ordegreeofpolariza-tion,asthishadbeenconsideredpreviously(9).
REsULTsANDDI5CUSsmON
Thefirstrequirementfordcmonstratimmgglycogemi
intissuesis
thatitmustbeadledluatelvfixed
in
thecells.Itwas
found
previouslythat
for
rouititierocessitigaccticacid
alcoholformahiti
 
TABLEII
PreservationofLiverGlycogenafter7DaysinDifferentDecalcifyin.gSolutions
AllsectionsshownstainedbyperiodicacidSchiffmethod,photographedat250X.
FIG.1.Control
ratliver,fixedsevenday-sinaceticalcoholformalimi
FiG.
2.Ratliver,fixedsixhoursinaceticalcoholformalin,leftsevemiday-sinEI)TAsolution.Fto.3.Ratliver,fixedsevemidaysinaceticalcoholformalin,left.sevenclaysinE1.)TAsoluition.Ftc;.
4.
Ratliver,fixedsevendays
iii
aceticalcoholformalin,leftinformicacidandsodiumcitrateseverdirtys.
Ftu.
5.Ratliver,fixed
iii
aceticalcoholformalimifortwenty--foumrhoumrs,thetuplaced
in
5(trichlor-aceticacidforsevemiclays.
FIG.
6.Asectionof
rat
liver,fixedinaceticalcoholformalinfortwenty-fourhours,thenplacedin
El)TAsolutionforsevendays.
Thesizeofthespecimenwa.snotcontrolled.Noticetheperipheraldensityofglycogen.FmG.7.AnotherillumstrationofthesamesectionasFigure
6,takendeeperinthesection,showing
scatteredglycogen.
700
J.R.TROTT
TABLEI
Pre8ervationofLiverGlycogenAfterDifferentTimesofFixationandMethodsofDecalcification(7Days)
Fixation(Acetic
AlcoholFormalin)
DecaicifyingAgent(7days)Control
EDTAFormicacid
6hours++++++024hours+++++048hours+++++++0
7days++++++++0
Solution
pH
Glycogen
Control
5%
TrichloraceticAcidFormicAcid&SodiumCitrate
(Itiorse,
1945)EDTA.0.62.57.5
++++
00+++gavethemostsatisfactoryresultsinratliver.However,longerfixationmightbedesirablefor
tissuesthataretobe(lecalcified.FromTableI,wherethesizeofspecinienwas
controlled,thelengthoffixationdidnotappear
tohaveanygreatbearingon
theamountof
glycogemulostduringthesevendaysinthe
EDTA
solution(Figs.1,
2,3).It
willheseen
that
formicacid-sodiumcitrateremovesall
theglycogenfromthetissueaseffectivelyas
adiastasecontrol(Fig.4).Similarresultswereoi)tainedfromthe
first
experiment(TableII),
where
thesizeofthespecimtnwastiotcontrolled.However,therewasatendencyfortheglycogeninthe
sectiontohe
unevenlydistributed(Figs.
5,6,
7).Deeper
areasshowlessgly-cogemithatuthosenearthetissuesurface;the
3+figuredoesnot
reflectthis.Thusitappearsdesirablenottohave
specimensthickerthanabout
3mmifcomplete
andadequatefixationofglycogenistooccur.Thelengthoffixationdoesnotappeartomake
muchdifference,ascanbeseenfrom
TableI.Itisinterestingtoobservethateventhoughthe
1)H
of
thefixativeis4.0,glycogenisstillretained.Schajowicz&Cabrini(7)suggestthatthere
isacapsulearoundthecartilagecellswhich
allowsglycogentoberetainedinthecellduring
subsequentdecalcificationprocedures.
Thisappearsunlikely,becauseifthecapsuleallowsfreeaccessoffixativetothecell,decalcifyingagentsshouldalsohaveaccess.Fromthetwo
experimentsthatare
reported,andfrom
pre-viouswork,itwouldappear
thatadequate
fixation,followedbytheuseofchelatingagents
forthedecalcificationofboneandcartilage,are
thenecessaryprerequisites
if
glycogenistobedemonstratedat
all.ItisdoubtfulifLoe(3)
used
thebestfixativeforthedemonstrationofglycogen,eventhoughheusedEDTAatapHof
7.2
fordecalcification.
SUMMARY
Ithasbeenshownthatglycogencanbeade-quatelyretainedinratliversafterfixationwith
aceticalcoholformalinfor6hours,24hours,48hoursor7
clays,followedby
treatmentwith
clisocliutnethyhenecliaminctetraacetateadjusted
to
pH
7.5.Formicacid-sodiumcitrateor5%
trichloraceticacidsolutionsremovetheglycogenfollowingfixation.Acknowledgments:ThisworkwasmadepossiblebyagrantfromtheNationalResearchCouncilofCatuaclaand!theauthorisgrateful
fortheirsupport.Heisalso
muchindebtedtothetechnicaladviceofMissM.MelvilleoftheDepartmentofPathology,Faculty
d)f
Medicine,Universityof
Manitoba.Thetechnicalworkwas
doneby-Mrs.D.
Hart,N.R.C.laboratory
technician,an(ltypingbyMiss
V.
Rees.

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