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Preservative qualities of recentand fossil resins: Electronmicrograph studies on tissuepreserved in Baltic amberGeorge O. Poinar Jr. a & Roberta Hess aa Department of Entomological Sciences , Universityof California , BerkeleyPublished online: 28 Feb 2007.

To cite this article: George O. Poinar Jr. & Roberta Hess (1985) Preservative qualitiesof recent and fossil resins: Electron micrograph studies on tissue preserved in Balticamber, Journal of Baltic Studies, 16:3, 222-230

To link to this article: http://dx.doi.org/10.1080/01629778500000141

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P R E S E R V A T I V E Q U A L I T I E S O F R E C E N T A N D F O S S I L

R E S I N S : E L E C T R O N M I C R O G R A P H S T U D I E S O N T I S S U E

P R E S E R V E D I N B A L T I C A M B E R

George O. Poinar, Jr. and Roberta Hess, University of California, Berkeley

The origin of the use of resins as p rese rva t ives is a lmost as elusive as are those components in resin which are responsible for their conservat ive qualities.~ There are two maj or a t t r ibu tes of resin which contr ibute to i ts p reserva t ive nature: the first consists of antibiotic qualit ies which re tard or des t roy bacter ia and fungi; the second includes the abil i ty of resin itself to preserve the t issue of embalmed organisms. There are adequate records showing tha t the Egyp t i ans recognized the usefulness of resin as an embalming agent, a pract ice described by some of the classical wri ters who observed the operat ion and spoke to the emba lmers abou t their methods. This use of resin was apparent ly reserved for the nobility or wealthy citizens who could afford the luxury. Cheaper methods of embalming included drying and soaking the corpse in sal t baths . Herodo tus gives an account of E g y p t i a n embalming with myrrh, a resin f rom the Commiphora plant , in this case p robab ly C. molmok

They take first a crooked piece of iron and with it draw out the brain through the nostrils, thus getting rid of a portion, while the skull is cleared of the rest by rinsing with drugs. Next they make a cut along the flank with a sharp Ethiopian stone and take out the whole contents of the abdomen, which they then cleanse, washing it thoroughly with palm wine, and again frequently with an infusion of pounded aromatics. After this they fill the cavity with myrrh, with cassia and with other sort of spicery except frankincense and sew up the opening. Then the body is placed in natrum (saltpeter or soda) for seventy days and covered entirely over. Then the body is washed and wrapped round, from head to foot, with bandages of fine linen cloth, smeared over with gum, which is used generally by the Egyptians in the place of glue, and in this state is given back to the relatives. 2

Myrrh is an oleoresinous mater ial containing a mix tu re of resin, gum, and essential oils. I t possesses antiseptic, as t r ingent , stomachic, and carmina-

JBS, Vol. XVI, No. 3 (Fall 1985) 222

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Preservative Qualities of Amber 223

tive properties. Tincture of myrrh has been used to treat sores in the mouth. 3

Researchers who have investigated corpses that were embalmed or other- wise treated with resin have made some interesting observations. Barraco and others examined an Egyptian mummy that had been embalmed in 170 B.C. ± 70 years. 4 Resin had been poured into the cranial cavity, thorax, abdomen, and pelvis, as well as throughout the wrappings. The authors could find no sign of bacteria or fungi in the samples they examined, Lewin found cells with intact nuclei and cytoplasmic organelles in the hand of an Egyptiam mummy of about 400 B.C. ~ The body had been wrapped with numerous layers of hnen bandages soaked in resin. ;Fhe practice of soaking the wrappings with resin was widespread and may have aided in preserving the skin and underlying tissues. Other materials were sometimes also added. Thus, in analyzing the composition of wrappings of an Egyptian mummy of about 20 B.C., Benson and his colleagues noted the presence of beeswax and bitumen as well as galbanum, a gum resin obtained from the plant Ferula galbaniflua. 6

Resins were also used on wounds to prevent secondary infection and hasten healing. Yet another antimicrobial use of resin was to prevent spoilage of wines. The now famous Greek Retsina wines had their origin in ancient times when wine spoilage was a serious problem. The addition of pine resin to wines helped preserve them and the Greeks then became accustomed to the taste and have continued the tradition to the present. 7 A similar practice of resinating wines was also followed by the Romans and possibly other cultures. The actual components of the resin responsible for these preservative and antimicrobial qualities are not known, although the various sugars, terpinoids, and acids found in resin are likely possibilities.

After a certain degree of polymerization and cross-bonding, resin becomes harder and less soluble. When this slow process continues for millions of years, the resin becomes aged or fossilized and when the hard- ness reaches 2-2.5, the refractive index 1.5-1.6, the specific gravity between 1.06 and 1.10, and the melting point 250-300°C, we consider the material a fossilized resin or type of amber. Amber occurs in various locations throughout the world. The most famous amber comes from the Baltic and was produced by now-extinct forests some 40 million years ago. The next largest readily available source of amber comes from the Dominican Republic, with other additional significant sites in the State of Chiapas in Mexico; in Cedar Lake, Manitoba, Canada; near Point Barrows, Alaska; in Siberia; and in Lebanon.

One of the fascinating aspects of amber that has intrigued man for cen- turies is the presence of enclosed organisms entrapped when the resin was soft and then preserved for millions of years. The fine detail on the external surfaces of organisms, e.g., the cuticular hairs and other ornamentation on insects, has fascinated scientists and allowed them to conduct detailed com- parisons with current forms. It was generally considered, and often was

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224 Journal of Baltic Studies

true, that insects entombed in amber were represented only by their exter- nal surface structure which had been maintained in more or less normal position by the resin. Since the resin normally could not penetrate through the thick cuticle of these anthropods, internal soft tissues had degraded either by autolysis or through decompostion by microbial agents which then perished inside the organism's body cavity. However, in some hard-bodied insects injured in their attempts to free themselves from the resin or in soft bodied insects, the resin was able to partially penetrate into the body cavity and make physical contact with the soft tissues, resulting in varying degrees of preservation.

While examining insect fossils in Baltic amber, we came across a well- preserved female fungus gnat [Mycetophilidae: Diptera} that had preserved internal soft tissues. The piece of yellow transparent Baltic amber was broken through the abdomen of the fly; the body cavity had already been partly filled with the original plant sap which had since become amber (Fig. 1). The remainder of the fly's body cavity was filled with Araldite 6005 and polymerized for eight hours at 60°C. The blocks were trimmed and sectioned for electron microscopy on a Porter Blum MT2 ultramicrotome using glass knives. Sections were stained with saturated aqueous uranyl acetate for twenty minutes followed by lead citrate for five minutes. The sections mounted on slot grids were observed in a Philips EM 300 electron microscope.

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Fig. 1 Low Magnification Scan of a Fly Abdomen in Amber The scan illustrates the preserved hypodermal tissue, including the epidermis and under- lying muscle layer (H). The cuticle {C) is intact at this level in the fly; however, some amber (A) is present in the abdominal cavity.

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Preservative Qualities of Amber 225

Confirmation of the piece containing the fly as Baltic amber was performed through infrared spectroscopy by Professor Curt W. Beck of Vassar College, Poughkeepsie, New York. Beck and others have shown that Baltic amber is distinguishable from related ambers by an absorption band of medium intensity at 1,160 to 1,150 cm "1 {8.6 to 8.7 microns} preceded by a more or less flat shoulder of nearly 0.5 micron width. Using a 1.5 mg sample of the amber piece containing the fly specimen, a spectrum was obtained which matched that of exceptionally well-preserved Baltic amber. 8

Within the fly's abdomen was a strip of hypodermal tissue adjacent to the cuticle which was exceptionally well preserved {Fig. 1). Other tissue more centrally located lacked substructure and was composed of empty cell outlines. The hypodermal tissue was approximately 12 microns wide and contained epidermal cells and adjacent muscle tissue in various degrees of preservation. The electron microscope revealed distinct epidermal cells con- talning nuclei (Figs. 2, 3, 4, 5}, which were identified on the basis of size, distribution, and fine structure similar to that shown by nuclei in standard electron microscope studies fixed by gluteraldehyde alone or as processed with inert dehydration. Two distinct nuclear ultrastructures were observed. In a few cells the nuclei were elongate, irregular in outline, and contained areas of electron dense nucleoplasm that possibly corresponded to chromatin (Fig. 2). More frequently the nuclei were elongate and principally composed of electron dense nucleoplasm (Figs. 3, 4, 5}. A structure cor- responding to the nuclear envelope could be easily discerned surrounding the denser nuclei. The ground substance of the cytoplasm contained many membranous profiles forming globular units with electron dense centers or oriented in curvilinear arrays (Figs. 2, 3, 6). In general, the spacing be ~ tween the membranous arrays fell into two general categories: those measuring between 100 and 150/k and resembling myelin swirls, and those between 200 and 400 A resembling smooth endoplasmic reticulum (Fig. 6). In the majority of the epidermal cells examined, individual organelles other than nuclei were not easily distinguished, although in some cells organelles were preserved. Electron dense granules and dense granules containing membranous profiles ranging in size from 0.25 to 1 microns were observed (Fig. 4); these granules may correspond to autophagic vacuoles or lysosomes. Also present were elongate structures with membranous transverse septa which resembled mitochondria (Fig. 4). Forms similar to lipid droplets were observed along with electron dense structures of an undetermined nature (Fig. 5). Also observed were spherical to ellipsoidal electron dense bodies 175 to 250 /~ in diameter which appeared to correspond to ribosomes (Fig. 7).

The muscle fibers lying beneath the epidermal cells showed the greatest degree of preservation, and in longitudinal sections the bundles of muscle fibrils were clearly recognizable (Fig. 8). Interspersed between the myofibrils were extremely long mitochondria with prominent cristae. Associated with the muscle bundles were trachea and tracheoles closely applied to the muscle

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226 Journal of Baltic Studies

, ,~: F '¸

Fig. 2 Inc lus ions in an A b d o m i n a l Cell of a F ly in A m b e r I This well-preserved nucleus (N) shows electron-dense areas (G) believed to represent chromatin interspersed with less dense areas of nucleoplasm. The cytoplasm has a substruc- ture of membranous profiles and contains many vacuoles (V).

Fig. 3 Inc lu s ions in an A b d o m i n a l Cell of a F ly in A m b e r I I This nucleus (N) is electron-dense almost throughout. A weU-defined membrane (E), probably the nuclear envelope, is seen at the periphery. The cytoplasm contains many membranous profiles, some linearly aligned (P) and others in random swirls (S). Vacuoles (V~ are also present.

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Prese rva t ive Quali t ies of A m b e r 227

Fig. 4 Inc lus ions in an Abdomina l Cell of a F ly in A m b e r I I I Cell organelles are present here. Electron-dense bodies, probably lysosomes {L), are ob- served along with elongate structures with membranous transverse septa corresponding to mitochoudria (M). The nucleus (N} is of the electron-dense type with a prominent envelope (E). The cytoplasm is vacuolated {V}.

Fig. 5 Inclusions in an Abdominal Cell of a Fly in Amber IV Lipid droplets (D) predominate in this cell along with some irregular electron-dense patches (I). The nucleus {N) is of the electron-dense type.

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228 Journal of Baltic Studies

Fig. 6 Inc lus ions in an Abdomina l Cell of a F ly in A m b e r V This high-magnification micrograph demonstrates the principal forms of membranous profiles in the cytoplasm: curvilinear arrays spaced 100-150/~ apart which resemble myelin swirls (S}, and those spaced 200-400 ~ apart which may be smooth endoplasmie reticulum ~ERI.

Fig. 7 Inc lus ions in an Abdomina l Cell of a F ly in A m b e r VI Electron-dense particles (R} 175-250 ~ in diameter occur dispersed throughout this cell and probably correspond to ribosomes.

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Preserva t ive Qualities of Amber 229

surface or within the cell surface (Fig. 8). In the inset of Fig. 8 the p l a sma membrane was easily discerned on the exterior of the trachea, and the inner lipoprotein cuticulin layer was present adjacent to the small inner tubercles.

The pho tog raphs presented here of the hypodermal t issue in a fossil- ized fly represent the oldest known cells containing recognizable inclusions. In our effor ts to determine why these cells, millions of years old, are so well preserved, two possibilities seemed mos t likely. The first was a type of physical preservat ion known as inert dehydrat ion, potential ly an impor-

Fig. 8 Muscle Tissue of Preserved Fly in Amber Muscle tissue represents the most well-preserved tissue observed in the fly. Muscle fibrils IF} in longitudinal section are interspersed with elongate mitochondria IM}. Trachea IT} are closely opposed to the tissue surface or interdigitated within the cell. Inset: Higher magnification of trachea and associated tissue shows the plasma membrane ~P) on the surface of the trachea, the lipoprotein cuticulin layer (X} on the inner surface, the inner tubercules {l), muscle fibrils {F}, and the cristae {B} of the mitochondria.

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230 Journal of Baltic Studies

ran t factor in the preserva t ion process. Sugars and terpines presen t in the original t ree sap could have replaced the water f rom the adjacent t i ssues causing t hem to dehydrate . This type of physical p reserva t ion can be obtained when fresh t issues are t rea ted with ethylene glycol. 9 However , this process is not known to preserve lipids, and in the present examina- tion of insect cells in Baltic amber there were m a n y lipid s t ruc tu res still recognizable. For this reason we feel t h a t o ther components p resen t in the original resin were responsible for the remarkable preservat ive s ta te of these cells. These specific components are not known, but this process has resulted in an ex t reme case of mummificat ion, involving the preserva t ion of cells by na tura l embalming.

Tha t Balt ic amber i tself is considered to have medicinal proper t ies is a t t e s ted to b y m a n y people living in the Balt ic s ta tes , where t radi t ions of taking amber internally or rubbing it externally as a cream on the skin have persisted up to the present, w Other than the personal sat isfaction reported f rom these practices, there has been no scientific evidence of the healing or preservat ive qualities of Baltic amber for the human organism. However, pract ices which have endured often have some plausible foundation, and it m a y well be tha t amber has a t least some of the cura t ive proper t ies t ha t have been a t t r ibu ted to it. H

NOTES

1 In the following discussion, resin is meant to include the water insoluble components of tree sap that persist after most of the essential oils and other components have been removed.

2 Herodotus, The Persian Wars, trans. George Rawlinson {New York: Modern Library, 1942), 156.

3 John van Beek, "Frankincense and Myrrh," The Biblical Archaeologist 23 {1960), 70-95; Larousse des plantes qui gu~rissent (Paris: Librairie Larousse, 1974), 171.

4 R. A. Barraco, T. A. Reyman, T. A. Cockburn, "Paleobiochemical Analysis of an Egyptian Mummy," Journal of Human Evolutior~ 6 (1977), 533-46.

5 P. Lewin, "Palaeo-electron Microscopy of Mummified Tissue," Nature, 213 (January 1967), 416-17.

6 G. G. Benson, S. R. Hemingway, F. N. Leach, "Composition of the Wrappings of an Ancient Egyptian Mummy," Proceedings of the British Pharmcological Conference in Journal of Pharmacy and Pharmacology, 30 (1978, Supplement), 78.

7 Edward Hyams, Dionysus: A Social His tory of the Wine Vine (New York: Macmillan, 1965), 165.

8 Curt W. Beck, Personal Correspondence, May 25, 1981. 9 Daniel C. Pease, "The Preservation of Unfixed Cytological Detail by Dehydration

with 'Inert' Agents," Journal of Ultrastructural Research, 14 (1966), 356-76. 10 Patricia Rice, Amber. The Golden Gem of the Ages {New York: Van Nostrand Reinhold

Co., 1980), 127. 11 The authors would like to thank the following persons for assistance in locating

pertinent literature dealing with resinated wines: Mr. John W. McConnell, Univer- sity Library, Davis, California and Dr. Maynard Amarin, The Wine Institute, San Francisco.

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