Bone preparation at the KCCAMS laboratory

doi:10.1016/j.nimb.2009.10.061

Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms

Volume 268, Issues 7–8, April 2010, Pages 906-909

https://doi.org/10.1016/j.nimb.2009.10.061 Get rights and content

Abstract

Collagen purification by ultrafiltration [1] is becoming widely accepted as a superior ¹⁴C sample preparation method for removing environmental contamination from archaeological bone. We present an overview of bone sample preparation methods used at the KCCAMS laboratory, UC Irvine. Methods development data are also discussed, including results of tests of gelatinization temperature, evaluation of 10 kDa vs. 30 kDa molecular weight (MW) ultrafiltration, improvements to ultrafilter cleaning methods, tests of ultrafilter memory, and ultrafilter glycerin contamination. These tests were not intended to be exhaustive, but rather to show how standard procedures can be validated and improved with relatively little effort.

Introduction

Radiocarbon dating of bone is particularly difficult because the porous and easily alterable apatite matrix of bone allows potential contamination of bone collagen by environmentally mobile soil carbon. Brown et al. [1] developed a modified Longin method using ultrafiltration to select large protein fragments (hereafter referred to operationally as “collagen”) most likely to be uncontaminated. This technique has been used in North America for over 20 years (all bone samples prepared by the RIDDL group [1], and subsequently at the CAMS/LLNL and UC Irvine AMS labs, have been prepared with this technique) and is now also being used extensively by researchers at Oxford [2]. Here we describe the technique as used at the UCI-KCCAMS lab and report on tests to maximize collagen yields and to characterize process blanks.

Section snippets

Methodology

Collagen production at the UCI-KCCAMS lab follows a modified Longin method [1]. Approximately 100 mg of bone is manually cleaned and crushed to particles roughly 2 mm in size. The sample is then decalcified in 0.5 N HCl for 24–36 h. After rinsing with Milli-Q water to neutral pH, the sample is hydrolyzed with 0.01 N HCl at 60 °C for 8–12 h. The high MW fraction of the gelatin solution is selected using precleaned 30 kDa Centriprep ultrafilters (Fisher Scientific). The sample is ultrafiltered twice,

Decalcification

Acid strength and decalcification time should be optimized in order to maximize collagen yield. Collagen yield decreases strongly with increasing acid strength and HCl strengths above 1 N should not be used (Fig. 1). Decalcification time is highly dependant on bone preservation state and the size of bone pieces chosen (Fig. 2). We use a 24–36 h decalcification period, with visual inspection to check for completion. Decalcification temperature appears to have little effect on the yield (Fig. 2).

Gelatinization

Conclusions

These and similar samples tests can help to validate bone preparation procedures and can point the way to further improvements such as the rapid gelatinization developed by Semal and Orban [4]. A significant obstacle in such tests has been the lack of a well characterized “dirty bone” standard that is known to be seriously contaminated. We urge all laboratories undertaking bone preparation to be aware of the need for suitably large samples of such material and to report them to the community if

References (7)

P. Semal et al.
J. Arch. Sci.
(1995)
T.A. Brown et al.
Radiocarbon
(1988)
C.B. Ramsey et al.
Radiocarbon
(2004)

There are more references available in the full text version of this article.

Cited by (82)

Climate change without extinction: Tasmania's small-mammal communities persisted through the Last Glacial Maximum–Holocene transition
2022, Quaternary Science Reviews
Global ecosystems underwent major changes through the Quaternary, as climates cycled from cool and dry glacial conditions to relatively warm and humid interglacial conditions. How these changes affected the diversity and composition of small-mammal communities is mostly unknown, especially for the southern-temperate regions of Australia. We used fossil assemblages from owl regurgitates to investigate changes in small mammals from Tasmania's forest environments over the last 20,000 years, encompassing the transition from cold glacial conditions of the Last Glacial′ Maximum (LGM) into the warmer Holocene. During the early part of this period, Tasmania was connected to mainland Australia via a low-elevation land bridge, but was transformed into a large island by rising sea levels during the Late Pleistocene – Holocene transition. Despite these changes, the regional small-mammal fauna appears to have been persistent over this entire period, with no loss of species. However, the relative abundance of most species changed dramatically, apparently in response to habitat changes, which also provoked substantial shifts in community composition and diversity. A strong indicator of the magnitude of change is Mastacomys fuscus, a graminivorus specialist murid, which dominated the assemblage prior to and during the LGM, but declined as temperature and sea level rose during the Late Pleistocene – Holocene. In contrast, the generalist rodent Pseudomys higginsi, along with the small forest-dependent marsupials Cercartetus spp. and Antechinus spp., became more common during the Holocene. The continued survival of these species to the present, despite divergent environmental needs, implies that Tasmania's complex geomorphology provided multiple refugia that conferred resilience in the face of environmental change.
Variable kinship patterns in Neolithic Anatolia revealed by ancient genomes
2021, Current Biology
The social organization of the first fully sedentary societies that emerged during the Neolithic period in Southwest Asia remains enigmatic,¹ mainly because material culture studies provide limited insight into this issue. However, because Neolithic Anatolian communities often buried their dead beneath domestic buildings,² household composition and social structure can be studied through these human remains. Here, we describe genetic relatedness among co-burials associated with domestic buildings in Neolithic Anatolia using 59 ancient genomes, including 22 new genomes from Aşıklı Höyük and Çatalhöyük. We infer pedigree relationships by simultaneously analyzing multiple types of information, including autosomal and X chromosome kinship coefficients, maternal markers, and radiocarbon dating. In two early Neolithic villages dating to the 9th and 8th millennia BCE, Aşıklı Höyük and Boncuklu, we discover that siblings and parent-offspring pairings were frequent within domestic structures, which provides the first direct indication of close genetic relationships among co-burials. In contrast, in the 7th millennium BCE sites of Çatalhöyük and Barcın, where we study subadults interred within and around houses, we find close genetic relatives to be rare. Hence, genetic relatedness may not have played a major role in the choice of burial location at these latter two sites, at least for subadults. This supports the hypothesis that in Çatalhöyük,3, 4, 5 and possibly in some other Neolithic communities, domestic structures may have served as burial location for social units incorporating biologically unrelated individuals. Our results underscore the diversity of kin structures in Neolithic communities during this important phase of sociocultural development.
Impact of varying HCl concentrations on the δ15N values of human rib bones used for dietary reconstruction
2021, Journal of Archaeological Science: Reports
Citation Excerpt :
Concerning the HCl concentration, in 2010, Pestle observed no difference in %C, %N, atomic ratio of carbon to nitrogen (C:N), δ13C and δ15N values obtained from four protocols with varying HCl concentration and duration (n = 2): 0.2 M for 24 h, 0.1 M for 48 and 72 h, and 0.05 M for 120 h, concluding that stronger HCl concentrations should be used to reduce acidification time and increase productivity. Yet, stronger acids than 1 M should not be used as demonstrated by Beaumont and colleagues (2010, Fig. 1). The complete Sealy et al. (1986) method recommends soaking samples in 0.125 M NaOH for 20 h in order to remove humic acid after bone demineralization.
While the effect of HCl on bone collagen extraction and IRMS measurements have been addressed previously, the effects of the variation of HCl concentration on the measurement of δ¹⁵N analysis have never been reported. To this effect, “chunks” of four right rib shafts from one individual of the skeletal collection of Sainte-Marie-de-Beauce Cemetery (Canada, 1748–1878) were separated in 15 subsamples: three unacidified aliquots of different grain sizes (0–0.125 mm, 0.125 to 1 mm and >1 mm) and four aliquots that underwent three different HCl treatments (1%, 2% or 5%). Each aliquot was analyzed in duplicate or triplicate for δ¹⁵N depending on material availability. The unacidified samples show the largest δ¹⁵N variation (7.5 to 9.2‰) and the mean δ¹⁵N increase with HCl acid concentration, from 9.0‰ vs. AIR (1%) to 9.4‰ (5%). Also, we observe variations in δ¹⁵N from one analytical sequence to another while controls remain invariant. Therefore, we conclude that powdered samples (0–1 mm grain size) have to be thoroughly washed prior to analysis to remove soil contamination in trabecular bone, and that increases in δ¹⁵N in correlation with HCl concentration is likely the result of protein hydrolysis extent from 2 to 5% HCl solutions. Thus, unacidified >1 mm and samples acidified with 1% HCl provide the most reliable %N, C:N and δ¹⁵N values. The analytical sequences of the same sample producing different δ¹⁵N values, while control standards are invariant, remain unexplained. Replication of this study on cortical bones, while monitoring the impact on amino acids, would help to better understand acid pre-treatment effects on δ¹⁵N analysis for diet reconstruction.
Coast-proximal inland archaeology and the Vancouver Island marmot (Marmota vancouverensis)
2021, Journal of Archaeological Science: Reports
Recent archaeological research in coastal areas of British Columbia has focused on investigations of coastline sites and marine-based subsistence. This study differs in its investigation of the High Burrow Site (DhSh-2), a late Holocene coast-proximal inland camp at which terrestrial taxa comprised the majority of the faunal assemblage and Vancouver Island marmot (Marmota vancouverensis) was the most numerous taxon represented. Analysis of the marmot assemblage indicated that bone density mediated attrition influenced the relative abundance of skeletal elements more so than did the transport of marmot portions away from the site. The distribution of marmot bones at coastline sites supports this conclusion and may link changes in regional settlement patterning across south Vancouver Island with the decline of marmot hunting at High Burrow in the late prehistoric. This study thus draws our focus from coastlines upland to interior landscapes as vital components in the interpretation of prehistoric subsistence and settlement along the coast.
Minimally invasive bone biopsies of fully wrapped mummies guided by computed tomography and fibre-optic endoscopy: Methods and suggested guidelines
2020, Journal of Archaeological Science: Reports
Citation Excerpt :
This amount is especially recommended in the case of ancient Egyptian material as Egyptian samples commonly underwent an additional cleaning step with benzene to remove contamination with bitumen based embalming substances (Aufderheide et al., 2004). For soft tissue, a minimum amount of about 100 mg could be enough (Beaumont et al., 2010). As mentioned above, the combination of CT imaging and fibre-optic endoscopy for minimal invasive sampling of fully wrapped mummies has the major advantage that it allows the detection of ways of access to the inner mummy parts.
Recent advances in the recovery and analysis of ancient DNA (aDNA) and application of isotopic analysis of tissue obtained from mummified human remains has been accompanied by continued advances in non-invasive imaging using X-ray computed tomography (CT) and use of minimally invasive surgical techniques employing small fibre-optic endoscopes. We used these state-of-the-art techniques to examine ancient Egyptian mummies in the Egyptian Museum and Papyrus Collection of Berlin, obtaining bone samples for aDNA, stable isotope analyses and radiocarbon dating. CT and endoscopic guidance were applied to locate and biopsy bones using pre-existing access points in order to avoid any further damage to the precious specimens. Here we provide guidelines to obtain bone samples of fully wrapped mummies in a minimally invasive way, by using a combination of computed tomography and endoscopic guided biopsy.
Fish δ13C and δ15N results from two Bronze/Iron Age sites (Tell Tweini & Sidon) along the Levantine coast
2020, Journal of Archaeological Science: Reports
Stable isotope ratio measurements of fish remains from archaeological sites are relatively rare so here we report results of 201 fish remains from two Bronze and Iron Age sites along the Syrian (Tell Tweini) and Lebanese (Sidon) coast to document the inter- and intra-specific variation of the δ¹³C and δ¹⁵N isotope values. Due to poor preservation, successful isotopic results were only obtained from 42 specimens (21%). Our results were combined with previously published fish isotopic results from Sidon (n = 16) so that a total 58 specimens representing 16 different fish taxa are presented. A wide variation was observed between species that appears to be related to the ecology of the fish, in particular their salinity tolerance and feeding behaviour. The largest intra-specific variation was observed in mullets (Mugilidae) and seabreams (Sparidae) in both δ¹³C and δ¹⁵N values, and it appears, after comparison with published data from the Northern Aegean, some clustering occurs with location. However, the data clustering is not discrete enough to allow provenancing of fish remains from archaeological sites in this region. This large series of fish isotope values can also serve as an isotopic baseline for other studies, including the dietary reconstruction of Eastern Mediterranean human remains.

View all citing articles on Scopus

¹: Address: Cotsen Institute of Archaeology, University of California, Los Angeles, CA 90021, USA.

View full text

Article preview

Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms

Abstract

Introduction

Section snippets

Methodology

Decalcification

Gelatinization

Conclusions

References (7)

J. Arch. Sci.

Radiocarbon

Radiocarbon

Cited by (82)

Climate change without extinction: Tasmania's small-mammal communities persisted through the Last Glacial Maximum–Holocene transition

Variable kinship patterns in Neolithic Anatolia revealed by ancient genomes

Impact of varying HCl concentrations on the δ<sup>15</sup>N values of human rib bones used for dietary reconstruction

Coast-proximal inland archaeology and the Vancouver Island marmot (Marmota vancouverensis)

Minimally invasive bone biopsies of fully wrapped mummies guided by computed tomography and fibre-optic endoscopy: Methods and suggested guidelines

Fish δ<sup>13</sup>C and δ<sup>15</sup>N results from two Bronze/Iron Age sites (Tell Tweini & Sidon) along the Levantine coast