TITLE: Dental Calculus Field-Sampling Protocol (Sabin version)
AUTHORS: Susanna Sabin, James Fellows Yates

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to sample dental calculus from skeletal remains for biomolecular analysis. This protocol is particularly recommended for sampling calculus from teeth attached to a jaw bone, skull, or skeleton. The primary use-case is for DNA and proteomic analysis. </div></div>

[STEPS]
?. [Workstation Preparation]
Put on two pairs of gloves.
Replacement The outer layer will be replaced after each sampling.
?. [Workstation Preparation]
Cover work surface with two layers of aluminum foil.
Replacement The top layer of foil must be replaced after coming into contact with sampling debris. Alternatively, the top layer of foil can be replaced after each sampling.
?. [Workstation Preparation]
Sterilise all utensiles with by wiping with alcohol wipes OR paper towels with some bleach (1:10 dilution with HPLC water).
Bleach If you use bleach, ensure that you wipe down the metal utensils with (preferably HPLC) water to prevent corrosion.
?. [Workstation Preparation]
Replace top layer of your gloves.
?. [Sampling Preparation]
Label tube ready for sampling.
Tubes Tubes should be closed at all times following removal from packaging before and after depositing the calculus sample. This will limit contamination.
?. [Sampling Preparation]
Fill in your metadata sheet with corresponding tubes IDs.
?. [Sampling Preparation]
Label all bags for placing corresponding tubes in.
?. [Sampling Preparation]
Lay out fresh sheet of weighing paper. Create a gentle crease in the weigh paper to ease specimen collection and handling.
Recommendation Place the weighing paper inside a weighing boat for better sample containment during the sampling process, or create a foil barrier surrounding the weighing paper for the same purpose.
?. [Sampling Preparation]
If possible, remove the tooth from the jaw. Locate the dental calculus.
Identification Dental calculus deposits can vary a lot in size and morphology. Large deposits should be relatively clear to identify. Smaller deposits will often have a rough surface, and form a ridge that would follow the gum line. Very small deposits may also be patch round shapes.
Avoid touching the calculus as much as possible, to reduce the risk of contamination.
Check that there are no fractures or other indications of a weakened structural integrity of the tooth. If this is identified, please consult with the collection curator or manager whether they are comfortable in proceeding with sampling.
?. [Sampling Preparation]
Write on a piece of paper the name of the sample and/or label on the tube. Place the label next to the weighing paper.
?. [Sampling Preparation]
Place the tooth on the weighing paper, and take photographs of the tooth as a whole. If the tooth is still attached to a larger specimen, take photographs of the tooth from as many angles as possible. Include close ups of the calculus at different angles. Ensure the sample label is clearly visible.
Measurements If possible, include a scale for measurement.
?. [Sampling Preparation]
Record the location of the selected calculus deposit on your metadata sheet (e.g. lingual, buccal surface etc). Also add other information such as whether there is evidence of disease or consolidants.
?. [Sampling Procedure]
Position the specimen such that you can scrape the calculus from the tooth with a downward motion towards the fold in the weighing paper.
?. [Sampling Procedure]
Take the sterile dental scalar. Scrape the broad flat edge against the calculus deposit only, without touching the enamel, along the surface of the tooth in a downward motion towards the fold in the weigh paper.
Powdery If the calculus deposit is coming off in a powdery form, try to go deeper into the bowl and scrape directly into the crease of the weighing paper. This will minimise loss from static and airflow, as well as reduce contamination of your workstation.
Force Each deposit of calculus have different adhesion to the tooth. Try gentle pressure at first and increase if necessary. Don't be surprised if it suddenly pops off. The foil or weighing boat should catch this.
Do not use the pick of the scalar, as you may scratch or damage the enamel surface. This will confound microwear and other scientific analysis.
?. [Sampling Procedure]
Gently tap the collected calculus from the weighing paper into the labelled tube, using the crease as a guide.
?. [Sampling Procedure]
If some calculus has not fallen directly onto the weighing paper, use sterile forceps/tweezers to place the calculus deposit(s) directly in the tube
?. [Sampling Procedure]
Close the cap of the tube securely. Place tube in pre-labelled sample bag.
?. [Post-Sampling Procedure]
Sterilise all utensils used by wiping with alcohol wipe or bleach on a paper towel.
Bleach If you use bleach, ensure that you wipe down the metal utensils with (preferably HPLC) water to prevent corrosion.
?. [Post-Sampling Procedure]
Replace outer layer of gloves.
?. [Post-Sampling Procedure]
Photograph the tooth again, focusing on the sampling site.  Again ensure the sample ID is visible in the picture.
?. [Post-Sampling Procedure]
Record any other observations from sampling, such as an approximate weight of the calculus sample.
?. [Post-Sampling Procedure]
Return tooth to appropriate storage. Discard the weighing paper and weighing boat (if used). If necessary or desired, discard the top layer of foil from the work surface and lay out a fresh sheet of foil for continued sampling.
?. [Sampling Preparation]
Replace gloves and repeat sampling preparation