Microsampling protocol
Collect Samples
q Examine tooth to determine how sampling will be done, either by drilling off of the tooth, or by removing a flake.
q Use a flake of enamel if a flake, at least 1-2 mm2, looks ready to come off.
o Begin by cleaning the outer surface of the flake, while it is still attached to the tooth. Cleaning is done with carbide dental drill bits in a Foredom Drill, or similar device, at low RPM.
o Try to clean inside surface of the flake, if possible, while it is still attached to the tooth.
o Remove flake.
o Clean remaining dentine off of inside surface of flake.
o Grind flake in clean mortar and pestle.
o Place enamel powder in labeled microcentrifuge vial.
q If no flake available, select a part of the tooth, where enamel is thick, to commence drilling.
o Prepare surface of tooth for drilling, by cleaning off excess cementum or soil from surface using carbide dental drill bits
o Drill off about 15 mg of pristine enamel, being cautious to not drill into the dentine. Use low RPM drill, such as Foredom Drill. Collect powder on creased weighing paper.
o Place enamel powder into labeled microcentrifuge tube.
PREPARATION OF SAMPLES, DAY 1
Remove organic residues from enamel
30% H2O2 is dangerous
as it forms free radicals. While H2O2 burns are not
usually very severe, the scientist should wear latex gloves and eye protection
while using it. Use of a ventilated hood is recommended. Read warning label on
bottle.
q Add 1ml 30% H2O2 to each sample.
q Shake samples.
q Let sit overnight.
Sometimes gasses build up in vials after a
few hours of reacting, causing lids to pop off. Pressure may be relieved by
gently lifting the lid, without removing it entirely. Wear gloves while doing
this, to avoid H2O2 burns.
Other solvents may be used to remove organic
material from enamel samples. A 2% solution Sodium Hypochlorite (NaHClO3;
Lee-Thorpe and van der Merwe, 1991) or NaOCl (Vennemann et al., 2001) may also
be used.
PREPARATION OF SAMPLES, DAY 2
Rinsing samples and removal of carbonates
q One; remove H2O2.
Use latex gloves to avoid H2O2
burns.
Vent excess gas from vials (see above).
o Centrifuge samples for 5–10 minutes at 10,000 RPM in microcentrifuge.
o Pipette H2O2 off using pipetter and tips.
§ Replace pipetter tips after each sample.
o Add 1 ml deionized water.
o Shake up.
q Two; rinse.
o Centrifuge samples for 5–10 minutes at 10,000 RPM in microcentrifuge.
o Pipette water off using pipetter and tips.
§ Replace pipetter tips after each sample.
o Add 1 ml deionized water.
o Shake up.
q Three; rinse.
o Centrifuge samples for 5–10 minutes at 10,000 RPM in microcentrifuge.
o Pipette water off using pipetter and tips.
§ Replace pipetter tips after each sample.
o Add 1 ml deionized water.
o Shake up.
q Four; add acetic acid to remove carbonates.
o Centrifuge samples for 5–10 minutes at 10,000 RPM in microcentrifuge.
o Pipette water off using pipetter and tips.
§ Replace pipetter tips after each sample.
o Add 1 ml 0.1 N acetic acid.
o Shake up.
q Let sit overnight.
Be sure not to leave for more than one day,
as this may affect values for carbon isotopes (Lee-Thorpe et al., 1989;
Vennemann et al., 2001)
PREPARATION OF SAMPLES, DAY 3
Rinse acetic acid and dry samples
q One; remove acetic acid.
o Centrifuge samples for 5–10 minutes at 10,000 RPM in microcentrifuge.
o Pipette acetic acid off using pipetter and tips.
§ Replace pipetter tips after each sample.
o Add 1 ml deionized water.
o Shake up.
q Two; rinse.
o Centrifuge samples for 5–10 minutes at 10,000 RPM in microcentrifuge.
o Pipette water off using pipetter and tips.
§ Replace pipetter tips after each sample.
o Add 1 ml deionized water.
o Shake up.
A third water rinse may be added if the
scientist feels that it is necessary, for example, if samples are very large.
q Three; add EtOH to help dry samples.
Methanol may also be used instead of ethanol.
There is some question about whether Ethanol might affect isotopic analysis, as
it is similar in mass to CO2.
o Centrifuge samples for 5–10 minutes at 10,000 RPM in microcentrifuge.
o Pipette water off using pipetter and tips.
§ Replace pipetter tips after each sample.
o Add 1 ml 95% Ethanol.
o Shake up.
q Four; add EtOH to help dry samples.
o Centrifuge samples for 5–10 minutes at 10,000 RPM in microcentrifuge.
o Pipette EtOH off using pipetter and tips.
§ Replace pipetter tips after each sample.
It is possible to just pour off EtOH without
losing your sample down the drain
o Add 1 ml 95% Ethanol.
o Shake up.
PREPARATION OF SAMPLES, DAY 3, continued.
q Five; finish.
o Centrifuge samples for 5–10 minutes at 10,000 RPM in microcentrifuge.
o Pipette EtOH off using pipetter and tips.
§ Replace pipetter tips after each sample.
§ It is possible to just pour off EtOH without losing your sample down the drain
q Leave vials open, in a safe place, for samples to dry