The Enzymatic Extraction

of Papyri from Cartonnage:

Å Control Study by Light- and Scanning

Electron Microscopy

ly PER R. FLOOD & ØYSTEIN SøENDELBO

IN'rnoouc'rtotr

As mentioned in previous publications (\Øendelbo i975, a, b, c), there shouldbe no theoretical objections to the use of proteolytic enzymes for the freeingof papyri from cartonnage. The papyrus sheets stem from the pithy ma-terial of Cyperus papyrus and consist almost entirely of structural materiale. g. cellulose, hemicelluloses, lignin etc. which are inert to the protelyticenzymes used. Only 2-3olo D. \Ø. of the pithy material may consist ofproteins (Grant 1970), which might possibly be attacked by the enzymes.Although it is unlikely that an eventual "digestion" of these proteins shouldcause a.ny detrimental effects to the structure of papyrus, a light- andscanning electron microscopic study was undertaken, to see if any signifi-cant structural differences could be detected between "untreated" and"treated" papyrus.

Me:Tnnrel. al.io Mt.rrrons

Papyrus from the same collection as thar used in the previous publishedworks (\Øendelbo 1,975, a,b) was used (gesso cartonnage, abour 2100 yearsold). By treated papyrus is meant papyrus exposed to the same enzy-matic preparation (Pancreas Trypsin NOVO, 6 Anson u/ gr.), for thesame time (30 minutes) and under the same working conditions (pH,buffer solution, temp.) as we use for the extraction of papyri from carron-nage during restoration; i. e. pH 8, Sørensen's phosphate buffer, temp. *40" C, concentration of enzymes in the solution, 1 0/0.

By untreated papyrus is meant papyrus that has been exposed to thesame procedures with the exception of the use of enzymes. For scanningelectron microscopy, small fragments of the two types of papyrus speci-

R.statr.ator L 1075 :53 59 t3

Tnxl ,ro rne Frcrrnrs

FIGS. 1a and b.

Scånning electron micrographs of the same region of the naturål surface of a påpyrusfragment al before and b) after enzymåtic ireatment. Three grånules present in bothpictures are indicåted by various symbols for ease of orientation. Note that most of thecontamination layer in a) has been removed by the treatment b). Maqntfication X 300,

FIGS.2a and b.

Scanning electron mjcrographs of the internal structure of a fragment of papyrus a) beforeand bl after enzymatic treatment. Details trom 3 cells are marked by various symbols forease of orientation. Note that almost every detail is preserved after the treatment. Thediffuse light areås in al are artifacts due to electrjcal charging of the specimen. Magni-fication x 300.

FIGS.3a and b.

Light micrographs of toluidine lblue stained sections throrlgh a) untreated and b) enzyme-treated papyrus. The light roundish holes represent the plant cells and the dark meshwork.the walls of two adjoining cells. Note that the jnternal structure and the total djameterof the walls have not changed after the treatment. Magnification X 1520.

Tbe Enzymøtic Extract;on of Pøpyri from Cartonnøge

mens were mounted on aluminium studs with the aid of Scotch double

rJtoi.r".uo" (Minnesota Mining and Manufacturing C')' The studs with

4;;;eateJj p)py..r, *"r" first soaked in distilled water' air-dried' and

then e"arnit å in a JEOL JSM U-3 scanning electron microscope' operated

at 25 KY. Photographs *"." -"d" of easily identifiable details- Later the

,f".i-"n raod, *-"r"^."-o-red and then exposed to the enzymatic solution'

Åf*, o bri"f rinse in distilled water, air drying and evaporation of gold-

prtt*t- from a heated tungsten filament at 1O-a Torr, these "treated"

å"^p1", *"r" examined again in the scanning microscope and photographs

-rå of th" ,o-" easily identifiable details previously photographed'

Fo, light microscopy, small fragments of "treated" and "untreated"

prpy.u, "*.."

fixed in ilutaraldehyde, rinsed in distilled water' dehydrated

i,l'in"r""rit g concentration of ethanol, exposed to moderate -Yacuum

to

."lnor," ai. iro- the interior of the sample, and finally infiltrated by

propylene oxide and embedded in Epon (Luft 1961) ?olymerization was

r.hi"""d by incubation at i 60o C for 3 days' Sections were cut 1 4m

thick and stained by toluidine blue (Grimley et aI' 1'965) or a modification

of the Periodic acid-schiff reaction (Pearse 1961)'

Rpsur'rs

The scanning electron microscope is particularly suited for the exemination

of small details in the surface topography of specimens'

The external swrlaces of the papyrus revealed abrupt changes from the

untreated state to the treated state (Figs' 1a and 1b)' The untreated surface

(Fig. 1a) appeared to contain numerous granules and fibres partly em-

å"ai.a l" Jtt "-orpholr,

material. After the enzyr'atic treatment (Fig 1b)

most of these ,.tbrturr.", had been removed, revealing a much smoother

surface where the orientation of plant fibres could be seen'

Vhen internal sørJaces were.tn.o-,","d by stripping off the superficial

layer of plant fibres from fragments of the papyrus prior to the enzymatic

a.å"4-.*, no obvious changes from the untreated to treated state were

,".."ol"d iFigr. 2a and b) o,tly quite rarely did the details seem to be

changed or lost in the latter sample.

fhe tight microscope was used to examine thin sections through the

pupyrur.ih" outlines of the individual cells of the plant tissue were easily

identifiable. No direct comparison of the structure before and after treat-

ment could be made as these sections had to be made from two separate

pieces of the papyrus sheet, one untreated and one treated' Although the

56

PBn R, Flooo & Ø:rsruN lVrNnrrso

size and form of the individual plant cells showed great variability, thestructures in their limiting walls were quite similar in the untreated (Fig.3a) and treated (Iig.3b) specimens. No tendency to cell dissociation wasnoted in any of the specimens examined.

DlscusstoN

It may be worthwile to mention a few iimitations of the applied techniques.High quality scanning electron micrographs can only be obtaired from

specimens coated with heavy metals to enhance secondary electron emissionand to prevent electrical charging up. Such metal coating could not beperforn.red on the untreated samples as the coating might prevent theaccess of the enzyme preparation to the specimen during the subsequenttr€atment. Accordingly the micrographs of the untreated samples revealsome artifacts not present in thc micrographs of the treated sarnples. There soaking and re-drying of the specimens from untreated to treated statemay also give slight differences in the surface pattern due to the randomcollapse of delicate structures. Vith these two limitations in mind it isconcluded that the scanning electron microscopic study revealed no signifi-cant differences in the internal structure of treated and untreated papyrus,whereas pronounced differences were seen on the natural surface of thepapyrus; or in orher terms, even thick layers of contamination could beremoyed without any visible harmful effects to the plant tissue.

Regarding the light microscopical technique used, it n.ray be said thatsmall quantitative- or patchy differences are difficult to verify, especiallywhen the tissue is as heterogeneous as in the present måterial. A greatnun.rber of rather large samples, both treated and untreated, and exactmeasurerlents of various structures through the microscope would be need-ed to achieve this. Unfortunately, our limited supply of papyrus did notallow us to perform such analyses. \Øe can therefore only conclude thatno obvious differences between the untreated and treated samples couldbe detected.

From the reported observations it seems justified to stare that rheproteolytic enzymes have no light-microscopically visible detrimental ef-lects on the structure of papyrus rissue.

Tbe Enzytnatic Extrøction ot' Papyri lrorn Cartonnøge

Suulrenrrs

The structure of papyrus extracted from cartonnage by means of pro-teolytic enzymes (trypsin and chymotrypsin), were compared with those

of untreated papyrus from the same collection by the use of light- andscanning microscopy. No significant differences were observed, and it is

concluded that the enzyme extraction technique causes no microscopicallyvisible detrimental effects to papyrus.

Die Struktur von Papyrus, freigemacht von Kartonage durch die Anven-dung proteolytischer Enzymen (Trypsin und Chymoterypsin) wurde mitder Struktur von unbehandeltem Papyrus aus derselben Kollektion durchLicht- und scanning Mikroskopie vergleichen. Keine signifikante Differen-zen wurden beobachtet, und es ist konkludiert, dass die Enzymekstrak-tionstechnik keine mikroskopische sichtbare schådliche lX/irkungen aufPapyrus verursacht.

La structure de papyrus extraits de cartonnages au moyen d'enzymesprot6olytiques (trypsine et chymotrypsine) a dtd compar6e par microscopielumineuse et par scanning avec celle de papyrus non traitds de la rnåme

collection. Aucune diff6rence significative r''ayant 6t6 constatde, on en a

conclu que la technique de I'extraction enzymatique n'a aucun effetnuisible, visible au microscope, sur les papyrus.

AcrNovtrncrltBNTs

The authors v/ant to express their sincere gratitude to Professor RichardF{olton Pierce, Ph. D., Departments of Classics, University of Bergen, forhis continued support of this work. The permission of Professor, Dr. philos.A. P. Nygaard to use the laboratories and equipment of the Departmentof Biochemistry, University of Bergen is highly appreciated.

This study has been financially supported by the Norwegian Research

Council lor Science and the Humanities.

RlrgnrNces

Crimley. P. M.. Albrechr. J. M. and Michel;tch. H. l.: Prepararion of large epoxy se,ttons

Jor l;ght nicroscopy as dn adjønct to fine-structure st dies, St^1D TecLnol. 40, 352-366(1965).

Vcndelbo, Ø.: The remopal of papyus lrom gesso cartonage roitb some rcmarhs on gLaed

?aPi,ri. Syn$olae Osloenses. 1, 1975^, 155-62.

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PBn R. Frooo &.ØYsrrrN IFnNou,lo

Vendelbo, Ø.: Tbe fueeing ol papyri lrom cartonnage, Restaurator. 2 (1975).: 41-52.enzyrnatic dllroacb. XIV. International Congress of Papyrologists, Oxford, 24,31 July1975b. Proceedings. In press,

'\Tendelbo, Ø.: Tbe free;ng ol papyri t'rom cdrtonndge, Restaurator. 2 (1975\: 41-52. Inpress.

Luft, J. H.: Improøements in epoxy resin enbedding methods. J. Biophys. Biochem.Cytol. 9,409414, 1961.

Pearse, A. G. E.: Histochernishr. Theoretical and applied. 2. ed. 998 pp. Churchill, London,1961.

Grant, cited in Thompson, K. & J. J. Gaudet: The ecology ol Cyperøs gagltrøs - a repiezøol pøpyrøs and its role in trop;aal sruamps. Arch. hydrobiol. 1975. In press.

Per R. Flood and Øystein Wendelbo .

Department of Anatomy and T'he University LibraryUniversity of BergcnÅrstadveien 19

5000 BergenNorway

Correspondence to:Deputy librarian, Dr. Ø. Iflendelbo, M. D.University Library of Bergen:De prehliniske institutterÅrstadveien 19

5000 Berg€nNorway

Editort noter This article was submitted for publication J^nuary 17th,7975.

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