The following protocol is the one that I currently use after more than a decade of work with starch residues. I am constantly trying to improve the methods, so various steps may change slightly from time to time.

Please read the entire protocol before beginning your work. This step will ensure that you have all the materials that you need before you begin, and you will also gain an understanding of what types of sampling are possible.

Starch Extraction Protocol

In the field, ideally, all artifacts and sediment samples should be collected and bagged separately without washing. While washing is a traditional step in the collection and curation of artifacts, it will remove some and possibly all of the residues from the artifacts. If you did not have the ability to direct activities in the field, or if you are working with older museum collections, you will probably still be able to extract some starch residues from the artifacts.

Before beginning starch extractions from artifacts, work surfaces and all tools in the laboratory should be cleaned thoroughly to rid them of any potential materials that could contaminate your samples. To maintain your laboratory in a clean state, prohibit food from entering the area and designate separate areas for the processing of modern and archaeological samples. Be sure to use powder-free gloves, and wash your hands thoroughly, gloves on, between each sample.

1. Sampling of artifacts. Decide if you want to do stepwise sampling, or if you wish to do a single extraction from the artifact.

For stepwise sampling:

A. Use a clean brush to remove any clinging sediment from the artifact. Collect all sediment in a new, clean, 15 ml test tube and proceed to step 5.

B. Use a clean brush and some deionized, distilled, or reverse-osmosis-filtered water to clean the same artifact. Rinsing with a wash bottle while brushing works well. Collect all the effluent into a clean container and proceed to Step 2.

For single step sampling, and for the final step in stepwise sampling, place the artifact into a clean container, cover it with deionized, distilled, or reverse-osmosis filtered water, and place the container in a sonic bath.

i. Allow the artifact to sit undisturbed for a period of five minutes to “soak.” While I have not experimented to see if there is a difference in extraction, this step may help loosen any adhering starches.

ii. Turn on the sonicator and let it run for a period of five minutes. I use ten minutes if the artifacts have not been cleaned previously to sonication. You may see material “puff” out from the artifacts.

iii. Remove the container from the bath. Using clean, sterile tongs or your immaculately clean gloves, remove the artifact from the container, rinse it with a wash bottle, and set it aside. Proceed to Step 2.

2. Settling. Containers with wash water from artifacts should be set aside so that the residues can settle to the bottom of the container. If you wish to do so, you can measure the depth of the water in the container and calculate how long the residues will take to settle using Stoke’s Law. I use a particle measurement constant of 1 micron. Otherwise, you can let the material settle overnight and continue with the procedure the following day.

3. Decanting. Once the residues have settled in the container, very gently pour off the water on top of the container. If you are uncomfortable pouring, you can use a large pipet to remove this material. When there is a very small amount of water remaining in the container with the residue, swirl it gently to dislodge anything that is clinging to the bottom of the container, and decant or pipet it into a new test tube. You may need to rinse stubborn residues with a wash bottle.

4. Centrifugation. The tubes that hold the water and residue can now be centrifuged at 1000 RPM for ten minutes. Pour off the effluent on top of the pelleted material in the bottom of the tube. If you use a higher speed, you risk pelleting the materials in the cone of the tube so they can’t be easily combined with the heavy liquid in the next step.

5. Heavy liquid flotation. Now you must decide if a heavy liquid flotation will be necessary to separate the residues from the sediment. If you see a bit of sediment in the tube, it is best to do a flotation. If you try to put sediment on the slide, you will find that it is not a time saver at all. It takes much more time to scan while looking around all the sediment particles, and, more importantly, these particles could obscure the residues from view. If the tube residue is clear, or nearly so, you can proceed directly to step 6.

I use Cesium chloride for a heavy liquid because it is readily available here and makes for a very clean float, even when the sediments are highly organic. I have used Sodium polytungstate successfully with sandy sediments, but it does not work well with organic sediments. There are other heavy liquid materials out there, and many can be used for starch extractions. Experiment on modern samples first and be sure to rinse all chemicals from the samples as soon as possible after flotation.

A. Using a new pipet, add a small amount of heavy liquid of density1.8 g/cm3 to the tube. It is not necessary to fill the tube to the top - I find that a small amount of heavy liquid works just as well as a large quantity. You can eyeball the amount as about two times the amount of sediment when you have small quantities of residue in the tube. I use a minimum of one ml of heavy liquid for a flotation.

B. Agitate the tube to mix the liquid with the residue, and place the tube in the centrifuge.

C. Centrifuge at a speed of 1000 RPM for 2 minutes to 5 minutes, dependent upon the height of the heavy liquid in the test tube. Smaller volumes take less time to separate cleanly.

D. Pour the effluent that is on top of the sediment into a new test tube.

E. Repeat the flotation. Repeating the flotation will capture more starches, and the second flotation will often extract a few phytoliths. The first flotation should displace any water in the sediment thus making the second more effective.

6. Rinsing. This step ensures that the heavy liquid is removed from the sample, and it should be completed immediately following the flotation.

A. Fill the tube with the heavy liquid in it to the top with deionized, distilled, or reverse-osmosis filtered water.

B. Centrifuge at 1500 RPM for ten minutes.

C. Pour off the effluent on top of the residue. Use a bottle to store this hazardous waste and dispose of it in compliance with your local regulations.

D. Repeat two times for a total of three rinses.

7. Observation. At this point, you should have a small quantity of either clear or whitish residue in the bottom of a test tube. You can now transfer the residue to a microscope slide and observe what you have extracted. Always use new slides and clean them in ethanol to remove the thin film of oil that is applied in the factory. I store mine in a mix of 50/50 ethanol/water in a slide staining jar so they are always ready. Clean the cover glasses with a lint-free wipe and ethanol. I mount the residue suspension with a drop of water/white liquid glycerin mixture that is a 50/50 mix. This medium prevents the rapid evaporation of the water to give you a bit more time on the scope. Slides can be sealed for storage with either a euparol mounting medium or nail polish around the edges of the cover slip.

What if the artifact is too large for a sonication? You can put very large artifacts into clean basins and wash them using a sonic toothbrush. Use wash bottles to keep the water flowing over the artifact, and be sure to use a new or boiled brush head for each sample. The same protocol can be used for artifacts that cannot be removed from the field. Let the sediment settle in the basin then proceed from Step 3.

Sampling Sediments:

1. Place 25 ml of sediment in a test tube with about 5 ml of baking soda, a deflocculant.

2. Fill the tube with water, agitate, and allow to sit for a period of about three days (72 hours), agitating the tubes when you happen to walk past them. This step will disperse the sediments so that any starches that might be bound within the soil particles will become freed.

**Note that very acidic sediments will foam up when water and baking soda are added to the tube. To prevent loss of your sample, add the water drop by drop when working with acidic sediments.**

3. Centrifuge the sediment at 1000 RPM for ten minutes and pour off the effluent.

4. Proceed to the heavy liquid flotation as described in step 5 above and continue through the steps. Use as much heavy liquid as you can: usually about 20 ml will fit in a 50 ml tube with a sediment sample.

Spot Sampling:

From time to time, you may wish to extract residues from a specific area of an artifact.

1. Locate the area of the artifact you wish to sample.

2. Using a new, plastic pipet, place a drop or two of water on the spot in question.

3. Allow it to sit for a minute or so, agitate the water with the pipet tip, and siphon it off into the pipet.

4. You can now place this material directly from the pipet onto a slide with your mounting medium and observe it.

References used in developing this protocol.

While many protocols have been published since, these four references mark the beginnings of the field and document initial experiments by researchers. For my dissertation research, I used Loy’s article as a starting point. Piperno and Holst’s article describes stepwise sampling. Therin’s thesis documents damage to starches when they are left soaking overnight in chloride salts.

Loy, T.H. (1994) Methods in the Analysis of Starch Residues on Prehistoric Stone Tools. Pp. 86-113 in: J. G. Hather (Ed.), Tropical Archaeobotany: Applications and New Developments. Routledge.

Perry, L. (2001) Prehispanic subsistence in the middle Orinoco basin: starch analyses yield new evidence. Doctoral dissertation, Southern Illinois University Carbondale, Illinois.

Piperno, D.R. and I. Holst. (1998) The presence of starch grains on prehistoric stone tools from the humid Neotropics: indications of early tuber use and agriculture in Panama.
Journal of Archaeological Science 25: 765–776.

Therin, M. (1994) Subsistence through starch: The examination of subsistence changes on Garua Island, West New Britain, Papua New Guinea, through the extraction and identification of starch from sediments. BA (Hon), University of Sydney.

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