4.2. Thin Sections

In studying dental annulations, 'the best methodology involves microtome sectioned, decalcified, haematoxylin stained preparations, but these are generally unsuccessful for archaeological material. Alternatives need to be found' (Hillson, 1996: 209). In an effort to resolve these problems, different methodological procedures have been tested by our research group, including: 1) manual cutting with a handsaw and grinding (polishing) with different abrasive papers; 2) frozen cutting with a cryostat microtome after decalcification of the tooth, using alternatively HNO3, HCOOH, or EDTA; and 3) embedding in resin and cutting with a diamond blade microtome. Charles et al. (1989), Buikstra and Ubelaker (1994), and Kvaal et al. (1996) consider decalcified sectioning techniques more accurate for counting annulations. However, in archaeological samples that are exposed to a variety of taphonomic changes, the best results have been obtained by means of direct cutting of the root with the microtome (Geusa, 1995; Geusa et al., 1996a, b, 1997a, c). A test of staining techniques (alizarin red, toluidine blue) did not produce improvements in the contrast among the bands.

In order to cut undecalcified teeth with a rotating blade microtome, each tooth had to be cleaned in an ultrasonic bath for 10 minutes, dried for 24 hours at room temperature or in an oven (70°C) for 6 hours, embedded in epossidic resin, and placed in a vacuum chamber for 10-15 minutes to eliminate air bubbles. With the Epo-Kwick Buehler resin the specimen was ready to be cut after two hours; while using the Epo-Thin Buehler resin it was necessary to wait 24 hours.

Alternatively, some specimens were embedded in Biodur E12 resin and placed in a vacuum chamber for 24 hours at 0.7 bar. This resin polymerised in three days.

We preferred to use transverse instead of longitudinal cuts in order to obtain more histological sections, because transverse cuts can yield more than 20 sections whereas longitudinal cuts produce only 2-3 sections per tooth. Using a Leica 1600 rotating blade microtome, each specimen was crosscut in the central third of the root in several sections with a thickness of 60-120 µm.

Each histological section was dehydrated in increasing concentrations of ethyl alcohol and in xylene, and finally mounted on a slide using Eukitt (Kindler GmbH & Co). In total, 613 sections have been prepared.