A study of naturally mummified human tissue from Historic Cave, Limpopo, South Africa S. Karodia a , J.I. Phillips b,c , A.B. Esterhuysen a, ⁎ a Department of Archaeology, School of Geography, Archaeology and Environmental Studies, University of the Witwatersrand, South Africa b Department of Pathology, National Institute for Occupational Health, National Health Laboratory Service, South Africa c Department of Biomedical Technology, Faculty of Health Sciences, University of Johannesburg, South Africa abstract article info Article history: Received 31 July 2015 Received in revised form 11 December 2015 Accepted 20 January 2016 Available online 29 January 2016 This study examines desiccated tissue samples from mummified human remains removed and excavated from Historic Cave in the Makapan Valley, Limpopo Province using scanning electron microscopy (SEM), energy- dispersive spectroscopy (EDS) and light microscopy. A range of methods are employed to clean, rehydrate and stain the desiccated tissue, and the outcomes are compared. Through this comparative analysis it is possible to determine the most suitable method for examining desiccated tissue from Historic Cave. In the SEM analysis, epidermal keratinocytes and vellus hairs were observed on the surface of the skin tissue. Histological analyses demonstrated the exclusive preservation of collagen fibres in the muscle tissue, the connective tissue and the skin tissue. This suggests that the collagen fibres play an integral part in preserving the structure of desiccated tissue that is devoid of cellular elements. © 2016 Elsevier Ltd. All rights reserved. Keywords: Historic Cave Makapan Valley South Africa Mummified tissue 1. Introduction Makapan Valley is situated in Limpopo Province, South Africa, about 15 km from Mokopane; previously known as Potgietersrus (24° 08′.27.32″ S, 29° 11′ 55.55″ E) (Fig. 1). The Makapan Valley comprises an extensive pre-Cambrian dolomite cave system and is famous for the palaeoanthropological, archaeological and historical material it has produced. Some of the better known caves within this cave system include Cold Air Cave, Limeworks Cave, Cave of Hearths, and Historic Cave (Fig. 1). In 2004 this site was recognised by UNESCO, and the valley was added to the list of World Heritage sites in South Africa. Historic Cave is famous for a siege that took place there in 1854. Briefly, members of the Kekana Ndebele chiefdom took refuge in the cave and were placed under siege by early European settlers. The Kekana remained trapped in the cave for approximately a month (25th October–21st November) after which a severely depleted group surrendered. According to contemporary accounts the Kekana had insufficient water in store and many succumbed to dehydration. 1 Subsequently, during the 20th century, two near-complete mummies were removed without authorization from Historic Cave, but were handed over to and housed in the Raymond Dart Collection at the University of the Witwatersrand. One of these, a young child was described by Cresswell (1993, also presented in Esterhuysen et al., 2009) and the other, an adolescent female, by Esterhuysen et al. (2009: 1044 - 1047) (Fig. 2). Archaeological excavations carried out in Historic Cave between 2001 and 2007 produced additional human skeletal remains some of which had mummified tissue attached. Natural mummification occurs when dehydration or desiccation precedes decomposition so that tissue, like skin, survives the decaying process. To date researchers have identified a number of factors, both extrinsic and intrinsic to the corpse, which may influence the rate and nature of post-mortem change. External environmental factors include temperature, humidity, anaerobic vs aerobic conditions, the chemical composition and pH of soil, the presence or absence of clothing, body emollients, and micro-organisms, insects and carnivores activity (Murphy et al., 2003: 625; Aufderheide, 2003, Campobasso et al., 2001). Intrinsic determinants include body fat, body position, and the degree of dehydration at the time of death (Amendt et al., 2004: 53; Aufderheide, 2003; Guillen, 2004). The finding of mummified remains affords the opportunity to study the soft tissues of individuals from historic sites. Examination of the soft tissues can provide information about microanatomy and may provide evidence relating to disease processes and even shed light on the cause of death. In addition to studies using light microscopy, electron microscopic techniques can be applied to the soft tissues. In order to conduct such studies the tissue has to be processed to make it amenable to microscopic investigation. This involves rehydration techniques, and in the case of tissue contaminated with dust or soil, cleaning techniques. This study explores techniques that render human, mummified, soft tissues from Historic Cave amenable to microscopic investigation. There are numerous studies using SEM as a method of analysis for mummified tissue. For example, the preserved skin structure from the Journal of Archaeological Science: Reports 6 (2016) 24–34 ⁎ Corresponding author. E-mail address: Amanda.Esterhuysen@wits.ac.za (A.B. Esterhuysen). 1 SS 7 R733/54 Verslag (Report) van M.W. Pretorius 6 December 1854. http://dx.doi.org/10.1016/j.jasrep.2016.01.024 2352-409X/© 2016 Elsevier Ltd. All rights reserved. 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