Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

This method can answer key questions in archeology, paleontology, and ecology, such as domesticated animal diets, animal management strategies, and animal mobility. The main advantage of this technique is that it is relatively nondestructive and that it is focused on a material that is reasonably resistant to chemical changes in the burial environment. Individuals new to this method will struggle because there are no available visual aids for the sampling of paleontological and archeological remains of humans and animals.

Visual demonstration of this method is critical because it is difficult to learn and practice on archeological materials is limited. To begin bulk sampling, set up a handheld drill as outlined in the text protocol, with a clean diamond-tipped drill of even shape, attached via a chuck to the drill setup. Using the drill, clean the outer surface of the tooth by gentle abrasion.

Next, tare a 1.5-milliliter microcentrifuge tube on an appropriately accurate balance. Then, place clean aluminum foil or weighing paper under the drill to collect the enamel powder. Use the drill to deliver gentle and even abrasion, creating a groove parallel to the entire growth axis.

Be aware that the amount of sample powder drilled will vary depending on the equipment used for analysis, the pretreatment protocol, and the size of the tooth. Collect the resulting enamel powder, and transfer it into a 1.5-milliliter microcentrifuge tube. Record both the sample number and enamel weight in a laboratory notebook, as well as an electronic database.

After each tooth is drilled, clean any used drill bits using 0.5-molar hydrochloric acid on a robust tissue. Wash the drill bits again with an organic solvent on a new robust tissue. Clean the drill itself using either a vacuum or compressed air.

Once a drilling session is complete, thoroughly clean the workspace using either a dedicated vacuum cleaner or a dustpan and brush. Wipe down the sampling space with methanol. Clean the drill using either the vacuum or compressed air.

For sequential sampling, first clean the surface of the tooth by removing a very thin layer of outer tooth enamel. Choose an appropriate drill bit for the sample, as the width of the sampling line is determined by the diameter of the drill bit. To begin drilling, press the tooth enamel against the drill bit and apply pressure.

Drill along the buccal surface perpendicular to the growth axis of the tooth, running from the enamel-root junction to the top of the crown. Make sure to drill from the apex to the base of the crown at the location of the enamel-root junction, resulting in a groove across the enamel layer. After the drilling is complete, weigh the sample to ensure there is enough enamel powder, and record the weight.

Repeat the drilling process several times per tooth, with each consecutive line parallel to the previous sample line and about one to two millimeters apart. After this, use calipers to measure the distance of each sampling line from the enamel-root junction. Record each distance for comparison.

To begin, use a pipette to add 0.1-molar acetic acid to each sample at a concentration of 0.1 milliliters per one milligram of enamel. Agitate the samples either by shaking or by using an agitator. Let the agitated samples sit for 10 minutes.

Centrifuge the samples at 13, 700 g for two minutes. Using a clean pipette, replace the acetic acid with one milliliter of ultrapure water, and agitate either by shaking or by using an agitator. Centrifuge again at 13, 700 g for two minutes.

Repeat the process of replacing the ultrapure water and centrifuging two additional times. Test the samples for neutrality. If the sample has not reached neutrality, repeat the washing process until it does.

Then, use a pipette to remove the remaining fluid without disturbing the supernatant. Place a precut one centimeter by one centimeter square of Parafilm over each microcentrifuge tube. Using a sharp object, make a small hole in the Parafilm to ensure that the sample dries appropriately, and place the tubes in a freezer overnight.

The next day, transfer the samples to the freeze dryer to remove any remaining fluid. Once the samples are dry, remove the Parafilm, and close the microcentrifuge lids. Check the tube labeling, and remark the tubes as necessary.

In this study, incremental enamel bioapatite samples are prepared. Each incremental sample location is numbered, and the distance between each location and the enamel-root junction is measured. Diverse carbon and oxygen stable isotope results from two representative sheep confirm that these animals lived in different environments.

Sheep A is from a tropical grassland and shows incremental oxygen 18 in a narrow range, between 3.3 and 5.1 per mil. This suggests ingestion of water from sources with similar isotopic values and a lack of strong seasonal shifts in precipitation. In contrast, sheep B, which is from a temperate dry steppe grassland, has oxygen 18 values with a high amplitude of variation, ranging from negative 5.2 to negative 13.1 per mil.

This indicates strong seasonal variation in precipitation. Stable carbon isotope values suggest that sheep A's diet consists mainly of C4 plants, while sheep B's diet consists mainly of C3 vegetation. Incremental oxygen 18 and carbon 13 values for a human tooth from a rainforest environment are highly constrained.

These values, which fall within a two per mil range, suggest a lack of variation in foraging strategies over the period of enamel mineralization. While attempting this procedure, it is important to remember to practice sequential sampling on modern teeth before drilling ancient samples that are not replaceable. Sequential sampling may take several hours of practice before proficiency is reached.

For ancient samples, it is important to choose teeth that can withstand the sampling process. Teeth that are poorly preserved or are liable to crumble are unlikely to prove useful for detailed sequential sampling. Following this procedure, other methods such as strontium isotope analysis can be attempted in order to understand human and animal mobility.

After its development, this technique paved the way for researchers in the fields of archeology and paleontology to explore animal herding and migration strategies across diverse regions and time periods. Don't forget that working with drills and acetic acid can be extremely hazardous, and precautions such as googles, gloves, and face masks should always be taken while performing this procedure.