Seeingred:Theuseofabiological

staintoidentifycookedand

processed/damagedstarchgrains

inarchaeologicalresidues

JennaWeston AustralianMuseumBusinessServices

6CollegeSt

SydneyNSW2010Australia

Abstract

Starchyplantfoodsareamajorcomponentinthedietofalmostallpeoples,particularlyhunter-gathererssuch as Aboriginal Australians. However, the archaeological preservation of such plants is rare, as isother direct evidence of plant use by past peoples. While analysis of starchy residues preserved onartefactshasgainedacceptanceasaneffectivemethodforidentifyingstarchyplantuse,itisverydifficultusingthestandardmorphologicalmethodtoaccuratelyidentifystarchgrainsthathavebeendamagedbyprocessingactivitiessuchasmillingorcooking.Therefore,amethodispresentedfortheidentificationofsuchdamagedstarchgrainsusingthestainCongoRed,whichdyesdamaged(cookedorprocessed)butnot undamaged starch. This method has been applied to identify cooking or milling activities in thesubsistenceofhunter-gatherersfromsouth-eastQueensland,Australia.

Keywords: Congo Red; starch grains; cooking; residue analysis; bevel-edged artefacts; south-eastQueensland.

Introduction

ThefollowingpaperpresentstheresultsofafeasibilitystudythatwasinitiallyundertakenaspartofanHonoursthesisattheUniversityofQueensland(Lamb2003)underthesupervisionofDrTomLoy.Thiswork resulted in the establishment of a new method for determining cooking in archaeological plantresidues, which was published (Lamb and Loy 2005) and then presented at the 2005 AustralasianArchaeometry Conference. This paper provides a summary of the methods and archaeologicalapplicationspublishedbyLambandLoy(2005),andexpandsuponthe2005publicationwithnewdataon taphonomic controls and additional images. It is published in this forum subsequent to Dr Loy’sdeath,withacknowledgementforhisroleindevelopingthemethod.

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Background

Ashasbeenestablishedbyanumberofstudies(e.g.,BrandandCherikoff1985;Gould1969;Latz1995;Latz and Griffin 1978; Lee 1965, 1968; Meehan 1989; O’Connell et al. 1983; Peterson 1978), plant foods,particularlystarchyplants,areamajorpartofpeople’sdiets.Thisisespeciallytrueforhunter-gatherers,such as Aboriginal Australians (Beck et al. 1989:6; Kaberry 1935:6; Mountford 1960; Peterson 1973).However,theseplantsareoftennotpreservedinwholeforminthearchaeologicalrecord,particularlyinAustralia,anddirectevidenceofplantusebypastpeoplesisalsorare(Hather1991:661;Meehan1989:14;Pearsall 2000:153; Piperno and Holst 1998:765; Therin et al. 1999:439). Residue analysis, particularlyanalysisofstarchyresiduespreservedonartefacts,hasnowbecomeacceptedasaneffectivemethodforidentifying past plant use and starch grains, which seem to be fairly prolific in archaeological plantresidues,canalsoallowtheidentificationofcookinginresidueevidence(Babot2003;Bartonetal.1998;Fullagar1998;Loy1994;Loyetal.1992;PipernoandHolst1998;TorrenceandBarton2006).

StarchStarch grains have a very regular structure of amylose and amylopectin layers (Badenhuizen 1965:81;Banks and Greenwood 1975:242; Cortella and Pochettino 1994:172; Loy 1994:89; Reichert 1913:89). Thisstructureimpartsthedistinctive‘cross’thattheyexhibitwhenviewedmicroscopicallyincross-polarisedlight,whichwillrotateradiallywhenthepolariserplateisrotated.Thiscrosseffectisalsodisplayedbyfaecalspherulites,ooliths,somecoccoliths,someavianandreptileuricacidspheres,someplantcalciumoxalates and some other biologically precipitated compounds, but each of these has a much higherrefractive index than starch, so viewing residues through a low refractive index medium like watermakes thesecompoundsalmost invisibleowing toexcessivelyhighrelief (BanksandGreenwood1975;Canti1998;Loy1994;Reichert1913).

The regular structure of starch is altered by cooking, however, a feature which allows theidentification of cooking via residue analysis. This is important because it can provide additionalevidencethatanartefactwasusedinhumansubsistence-relatedactivities,namelythecookingofplantfoods,which is auniquelyhumanactivity.Other evidenceof thehumansubsistenceuseof anartefactmay include the presence of other food residues from plants, use wear, and ethnohistoric accounts ofcertainstonetoolsbeingusedtopreparefood.

Theknowneffectsofcookingonstarchincludemainlyswellingandlossoftheregularstructureof thegrains,withacorresponding lossof theextinctioncross (BanksandGreenwood1975;Halletal.1989;Loy1994).Thisisaprocessknownasgelatinisation,anditoccurswhenthegrainisinthepresenceof moisture and is heated at temperatures greater than 30°C. During this process, the hydrogen bonds

linkingtheamylosechainsareprogressivelybrokenandthegrainabsorbswater,which iswhatcauses

ittoswell.Thetemperatureandrateatwhichagraingelatinisesdependonfactorssuchasthepresenceofadditiveslikeglucoseormineralsalts,andthesizeandshapeofthestarchgrain.Thislossofthecrosseffect in cooked starch grains makes it very difficult to identify them accurately using the standardmorphological method, because their appearance depends on the stage of gelatinisation, and at somepoint in this process the cross becomes completely invisible (Banks and Greenwood 1975; Loy 1994;Reichert1913).

Onewaytoaddressthisproblemofhowtoidentifycookedstarchliesintheeffectsofcookingonstarchgrains.The lossof thestructurefromthebreakingof theirhydrogenbonds,andtheswellingcaused by absorption of water, should allow cooked grains to absorb certain stains where raw andundamagedstarchgrains,whicharehydrophobic,willnot.

CongoRedCongoRed(CI22120,CIname‘DirectRed28’;empiricalformulaC32H22N6O6S2Na2)isawater-solubledyeand a suspected carcinogen, which depends on linear hydrogen bonding for staining (Conn and

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Lillie 1969). This stain has been used in other studies as a general contrast stain for cellulose, amyloidfibrilsandagriculturalstarchproducts(Chouetal.2001;ConnandLillie1969;RameshandThranathan1999).However, itstainsproteinslikeamyloidfibrilsonlyinacidoralkalineconditions,soataneutralpH only starch and cellulose will stain (Badenhuizen 1965:86; Conn and Lillie 1969; Cortella andPochettino 1994; Khurana et al. 2001 [pH2–4]; Loy 1994:92; Mehta and Rajput 1998 [pH3.5]; Reichert1913). Congo Red appears to react with the amylose in starch, which is exposed when grains aredamagedbystructurelossandswollenwithwater,aswhentheyhavebeencooked.

The amylose in starch is based on the same monosaccharide molecule as cellulose (Figure 1).However,asFigure1shows,themoleculeisbondedinadifferentwayineach.Thereforebothcellulose,andstarchgrainsthathavebrokenbonds,willstainwithCongoRed,buttheirappearanceandstructurearevisiblydifferent,sotheycanbedifferentiatedunderthemicroscope.

Methodology

Inordertotesttheeffectsofthestain,itwasnecessarytouseitonbothexperimentalandarchaeologicalresidues.Fortheexperimentalphase,threeplantswerechosenwhicheachhadhighstarchcontentandwere present in south-east Queensland, and for all of which there are ethnohistoric accounts of theirhavingbeencookedand thenpoundedbetween stonesbyAboriginalpeople in this area.Theseplantswere Alocasia macrorrhiza (elephant ear; see Brown 1893, Roth 1901, Thozet 1866), Blechnum indicum(Bungwahlfern;seeThrelkeld1825,Watkins1891),andCastanospermum australe(MoretonBaychestnut;seeBanfield1908,Moore[citedinMaiden1900],Roth1901).Thestarchyrootorseedfromeachoftheseplantswastaken,andrawsamplesweresmearedontoslides.Theremainingroot/seedwasthencookedin an electric frypan filled with sand (to simulate roasting as if in a fire), and cooked samples weresmeared onto slides. Another specimen of the three plants was then taken, and part of each wasprocessedwhen rawbypoundingbetween twostones, thenanotherpartof eachwasprocessed in thesamewayafterhavingbeencookedinafire(ondifferentdaysanddifferentplacesinthefencedareaoftheUniversityofQueensland’sTARDISexcavationsite,soastoavoidcontamination).Aftertheresidueshaddried,sampleswereextractedfromtheexperimentalstonesandplacedontomicroscopeslidesusingtheprocedureoutlinedinLambandLoy(2005:1435[Table1]).

After microscopic examination of the characteristics of each of the raw, cooked, raw andprocessed,andcookedandprocessedstarchsamples,eachwasthenstainedwithCongoRed,usingtheprocedureoutlinedinLambandLoy(2005:1435[Table1]),beingverycarefultotakeprecautionsagainstthe possible carcinogenic properties of the stain. This was achieved mainly by wearing a lab coat andnon-starch-powdered gloves, and sealing the slide covers once the stain was applied. The Congo Redsolution used was originally made up by dissolving 50 mg of the powder into 50 ml of water; in this

Figure1.

Primarystructureofcelluloseandα-amylose(fromstarch).nmaybeseveralthousand(afterVoetandVoet2004:365-6).

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situation face masks and eye goggles were worn in addition to the lab coat and gloves. The solutionsapplied to theslidesallhadthe lowrefractive indexofwater, toavoidconfusionwith thecrosseffectsgivenbynon-starchresidues,aspreviouslymentioned.

Archaeologicalresidueswereinitiallytakenfromthreebevel-edgedartefactsfromtheSouthernCurtis Coast (Figure 2). These artefacts (Figures 3–5) were provided by Dr Sean Ulm, having beencollected by him during his PhD research (Ulm 2006).A further archaeological residue was recoveredfromafourthbevel-edgedartefactwhichwasexcavatedfromashellmiddenontheGoldCoast(Robinsetal.2005).

Figure2.

LocationofSouthernCurtisCoastfromwheretheinitialthreearchaeologicalartefactswererecovered(Ulm2006:15).

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Figure3.

Bevel-edgedartefact1,surface-collectedfromanerodingcreeksectionatEurimbulaSite1(ES1)onthewesternbankofRoundHillCreekaspartoftheGoorengGoorengCulturalHeritageProject(GGCHP),on9March1999.ES1isanextensiveopenmiddencomplexlocatedinEurimbulaNationalPark,withdepositsdatingtothelast3,200years(Ulm2006:177).

Figure4.

Bevel-edgedartefact2,surface-collectedfromanerodingcreeksectionatES1onthewesternbankofRoundHillCreekaspartoftheGGCHP,on3June2001.Scale=5cmunits.

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Figure5.

Bevel-edgedartefact3,found20mfromSquaresOandPattheIronbarkSiteComplex(ISC)aspartoftheGGCHP,

on13February1998.ISCisanextensivestonequarry/shellmiddensitecomplexlocatedonthelowersouthernbankofMiddleCreekestuary,withdepositsdatedtothelast500years(Ulm2006:131).

Residueswereextractedfrommultiplepositionsoneachartefact,mainlyalongtheedgesandonthe flat, bevelled surface. All archaeological residues were extracted using the procedure outlined inLambandLoy(2005:1436[Table2]).

Afterexaminingeachresidueslideandnotingthepresenceandcharacteristicsofthestarch(andotherplantresidues),eachslidewasstainedusingthesameprocedureasfortheexperimentalresidues(LambandLoy2005:1435[Table1]).

Results

Samplesofstarch fromtheprocessedrawplants tended tostain light red inregular lightandretainedtheir cross effect in cross-polarised light, often as four dark points in their outline (Figure 6a and b).Partially-cookedgrainsmayalsohavethisappearancewhenstained,butwouldhaveswollensomewhat,soifthespeciesoftheplantresidueisknown,thisdistinctioninactivitymaybedetermined.

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The raw, unprocessed starch samples did not stain, unless they had been unintentionallydamagedduringtheexperiments.However,thewell-cooked,gelatinisedgrainsstainedbrightred,oftenwith either an orange-red or green-gold glow. These grains were not seen in regular light withoutstaining,butapplicationofCongoRedrevealedthattheyhadswollenquitedramatically.IntheinstanceofMoretonBaychestnut(seeninFigures7–8),thegrainshadswollentoaroundtentimestheiroriginalsize. Some pitting, layering and cracking can also be seen in the swollen grains (Figure 8). Generally,cookedgrainsdidseemtoretainenoughoftheirstructureforthemtomaintaintheirprimarilyroundoroval shape, although extensive swelling associated with heating appears to be able to change themorphology of grains somewhat (e.g. from round to oval-shaped; see Figures 7–8) and for a modifiedcrosstoappear,asfourdarkerpointsintheiroutline,whenviewedincross-polarisedlight.

6b.6a.

Figure6.Damagedstarchgrainsfromraw,processed

sample,400xmagnification,stained

(a)

transmittedlight

(b)

cross-polarisedtransmittedlight.

Alocasiamacrorrhiza

Figure7.

Starchgrainsfromraw sample(cross-polarisedtransmittedlight,400xmagnification,stained).Scalebar20μm.

Castanospermumaustrale Figure8.

Starchgrainsfromcooked

sample(transmittedlight,400xmagnification,stained).Scalebar20μm.

Castanospermumaustrale

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Substantialquantitiesofbothstainedandunstainedstarchgrainswereseeninthearchaeologicalresidues.Theunstainedgrainswereinterpretedasbeingrawandunprocessed,ornotheavilyprocessed.Thestainedgrainswereconsideredtobelongtooneoftwocategories.Figures9(aandb)and10(aandb)showsomegrainsthatarethoughttohavebeenprocessedtosomeextentbutwereuncooked,becausetheystainedslightlyredbutretainedtheirextinctioncross.Incontrast,Figures11–15showstainedgrainsarguedtohavebeencooked,becausetheystainedbrightredinregulartransmittedlight,andhadonlyamodifiedcrossasfourdarkpointsintheoutlineofthegrainwhenviewedincross-polarisedlight.

11b.11a.

Figure11.

StarchgrainfromArtefact#2,400xmagnification,stained(a)transmittedlight(b)cross-polarisedtransmittedlight.

10b.10a.

Figure10.Starchgrains(arrowsinB)frombevelledface,Artefact#3,400xmagnification,stained(a)transmittedlight(b)cross-polarisedtransmittedlight.

9b.9a.

Figure9.

Damagedstarchgrainfrombevelledface,Artefact#2,400xmagnification,stained(a)transmittedlight(b)cross-polarisedtransmittedlight.

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12b.12a.

Figure12.

GelatinisedstarchgrainfromArtefact#2,400xmagnification,stained

(a)transmittedlight

(b)cross-polarisedtransmittedlight.

Figure13.

Groupofstarchgrainsfromslopedface,Artefact#1(transmittedlight,400xmagnification,stained).

Figure15.

Gelatinisedstarchgrainfrombevelledface,Artefact#2(transmittedlight,400xmagnification,stained).

Figure14.

Gelatinisedstarchgrainfrombevelledface,Artefact#1(cross-polarisedtransmittedlight,400xmagnification,stained).

The fourth archaeological artefactproduced plant-dominated residues, with pre-dominantly small starch grains (approximately10–15 microns in diameter), many of whichstained and hence were interpreted as havingbeen processed or cooked. The grain shownbelow in regular transmitted (Figure 16a) andcross-polarised light (Figure 16b) was assessedas having been cooked because it was stainedbrightredwithamodifiedcrossintheoutline.

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Figure16.

Damaged(12μm)starchgrain,400xmagnification,stained(a)transmittedlight(b)cross-polarisedtransmittedlight.

16b.16a.

Congo Red was observed to stain sclereids, tracheids, parenchymal tissue (Figure 17), wallthickenings, bordered pits (Figure 18) and cellulose. However, while these stained red in regular light,theyusuallyappearedgreenoryellowincross-polarisedlight,ratherthanredaswasthecasewiththestarchgrains.

DiscussionofResultsNothingsimilarinappearancetothegelatinisedstarchgrainswasobservedtostaininthesameway,andthestainedstarchgrainsthatwereconcludedtohavebeendamagedthroughprocessingwerealsoquitedistinctive in appearance. It was therefore concluded that each artefact had most likely been used toprocessastarchyplantorplants,atleastoneofwhichwascookedtosomeextentbeforebeingprocessed.The described studies have therefore ascertained that Congo Red appears to be a feasible method foridentifyingcookingandprocessinginpastsocieties.

Figure17.

Parenchymatissuefromstainedsection,Artefact#2(transmittedlight,200xmagnification,stained).

Figure18.

Borderedpits(arrows)fromArtefact#2(cross-polarisedtransmittedlight,400xmagnification,stained).

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TaphonomicStudiesSeveral brief taphonomic studies were also conducted to ascertain the extent of non-processing-relatedstarch contamination on the stone artefacts. The possibility of ground contamination was tested bytrampling a sterile (i.e. newly manufactured) tool into the ground where experimental processing wasundertaken.Soilsamplesexaminedbeforeprocessingrevealedalmostnostarchgrains;afterprocessingstarchwaspresentinthesoilandhencewasapotentialsourceofcontaminationiftoolsweretrampledintothesoil.Starchgrains,plantmaterialanddirtwererecordedonthetoolswhichweretrampledintosoiloftheprocessingarea,althoughonly23%ofthetotalrecordedontheprocessingtoolswaspresent(seeTable1,ToolType2).

Thepossibilityofcontaminationthroughtouchwastestedbyhandlingcleanexperimentaltoolsafter the user had processed each plant (Table 1, Tool Type 3). On these tools, starch grains and plantmaterialwerealso seenand thequantitywasabout13%of that seenonprocessing tools (seeTable1).

All tools were subjected to the contamination mechanism for the same amount of time (ten seconds),

butitislikelythatmoregrainswouldhavebeentransferredtothetoolsiftheyweresubjecttoalongerperiodofexposureorhandling.

Lastly,thepossibilityofaircontaminationwastestedbyplacingacleanmicroscopeslideintheopenairneartheplacewhereprocessingoccurred;onlythreestarchgrainswerefoundonthisslide.

Therefore,itisconcludedthatcontaminationoftoolscanoccurthroughhandlingandtrampling.Use-wearanalysisprovidesapotentialmethodforidentifyingwhenthisisthecasewitharchaeologicaltoolspreservingresidues.

It is possible that natural attacks on starch by enzymes in biologically active soils may causedamage that would stain with Congo Red. This possibility has not been tested as part of the currentresearch. However it seems unlikely that enzyme attack would swell the starch grains in the way thatcookingdoes.Althoughitmaybemoredifficulttodistinguishprocessingdamagefromenzymedamage,use-wearanalysisoftoolsshouldenableabetterunderstandingofwhetherthismaybethecase.Inbothcases further experimental studies may provide useful insights into the potentially confusing effect ofenzymedamage.

ArchaeologicalImplications

TheuseofCongoRedwhenworkingwithstarchyresiduesfromarchaeologicalartefactsmeansthat a cultural association may be more confidently made between the tool and its use in subsistenceactivity,particularlyplantfoodpreparation.ThisisbecauseCongoRedonlystainsstarchthathasbeendamaged,whether thedamagehasoccurredthroughprocessingor throughcooking,orboth,anddoesnotstainundamagedgrains.ThereforetheuseofCongoRedcanmakeasignificantcontributiontothepreponderanceofevidenceforhumansubsistenceuseofanartefact.

Tool type Number of recorded Percentage compared starch grains with processing tools

1.Experimentalprocessingtools 146 N/A2.Groundcontaminatedtools 19 13.013.Touchcontaminatedtools 34 23.294.Aircontaminationslide

3 2.06

Table1.

Quantitiesof

starchgrains(rawandcooked)recoveredfromprocessing,tramplingandtouch-testexperiments,andaircontamination.Valueswerecalculatedusingoneunstainedmicroscopeslideforeachactivity.

Alocasiamacrorrhiza

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PossibleApplicationsCongoRedmaybeusefulintestingtheaccuracyofethnohistoricaccountsofstarchyplantpreparation,particularlythoseplantsthatneedtobecookedorprocessedpriortoconsumptionbecausetheyaretoxicor unpalatable raw (for example, inAustralia, cycads as well as Alocasia and Castanospermum australe).

Itmayalsobeusedtocontributetothedebateontheoriginsofcontrolledfireuse,bybeingappliedtoartefactsassociatedwithearlyhumanssuchasHomo erectus,anddetectingwhethertheseartefactsretainthe residuesof cookedplants.The fact thatCongoRedstainscellulose isalsouseful,becausecellulosecanoftenbedifficulttodistinguishmicroscopicallyfromresiduessuchasbonecollagenfibres.

FurtherWorkThereareanumberofstudiesthatshouldpreferablybedonetosupporttheuseofCongoRedstainingofarchaeologicalresidues.Firstly, furthertaphonomicstudiesshouldbecompleted, lookingspecificallyatwhethercookedstarchgrainsarepreservedandunderwhatcircumstances,andwhethertheirdepositionfor archaeological time spans changes their structure or appearance. Secondly, a reference collectionshould be compiled with samples of archaeological and experimental cooked starches. Experimentalsamples should be obtained from as many different starch species as possible, and controlledexperimentsshouldbeconductedonspeciesofdifferentsizes,usingavarietyofcooking temperaturesandcookingtimes,anddifferentmethodsofcookingandprocessing.Thirdly,examiningtheuse-wearontoolswithresiduesstainedwithCongoRedshouldbeundertakentodeterminewhethertheevidenceforprocessing of either cooked or raw starchy plants is in accordance with the conclusions drawn fromresidueanalysisandstaining.

Acknowledgements

Thanks to: the lateDrTomLoy, formerlyof theSchoolofSocialScience,UniversityofQueensland forsupervision and collaboration in developing the method; Dr Sean Ulm, Aboriginal and Torres StraitIslander Studies Unit, University of Queensland; Dr Richard Robins, Everick Heritage Consulting;Eastern Yugambeh Limited; Nathan Woolford, Aboriginal and Torres Strait Islander Studies Unit,UniversityofQueensland;SueNugent,AlisonCrowther,MichaelHaslam,MegHeaslop,LukeKirkwoodandDrGailRobinson,SchoolofSocialScience,UniversityofQueensland;andEnvironmentalResourcesManagement(Australia)PtyLtd.

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