Archaeobotany - Recovery

PHYTOLITHS : RECOVERY

Extraction procedure

The extraction procedure is an essential step to retrieve and concentrate phytoliths. It consists in the separation of the biogenic silica fraction (not only phytoliths but also quartz, diatoms, sponge spicules, etc.) from the soil matrix, so that the residue obtained can be mounted on a slide for observation under the microscope. There is no single method of phytolith extraction, and different researchers use different approaches. However, extensive research has been carried out comparing different extraction protocols (Powers and Gilbertson 1987; Buckler et al. 1994; Lentfer and Boyd 1998; Zhao and Pearsall 1998; Zucol and Osterrieth 2002; Piperno 2006) as well as approaches recovering multiple microfossil (e.g. phytoliths, pollen, starch, etc.) data sets (Coil et al 2003; Lentfer and Boyd 2000).

Some protocols have as a first step the fractionation of the sediment using a fine mesh to remove the bigger particles (Rosen & Weiner 1994) or to separate silt, clay and sand fractions (Piperno 2006). In some protocols this first step has been removed to reduce mechanical damage to the more delicate, ornamented morphotypes such as the fine spiny rods and dendritic forms, or the silica skeletons, as well as to speed up the processing (e.g. Madella et al. 1998).

Although there are differences between the various existing methods, all protocols consists of the following basic steps:

Removal of carbonates (to remove part of the mineral fraction and prevent chemical reactions with products used further down the extraction procedure);
Deflocculation (to loosen the electrostatic bounds between the different components of the sediment);
Removal of organic matter;
Removal of clays.

Also, most methods can be divided into two main groups: the ones that separate the phytoliths using a heavy liquid to float them and those that do not use this approach. The most commonly used heavy liquids are zinc bromide (ZnBr2) and sodium polytungstate [Na6(H2W12O40)H2O]. The latter is a non-toxic (Munsterman and Kerstholt 1996), high density (1·0–3·1 g/cm3) separating compound that generates an almost neutral solution (pH 6) of relatively low viscosity. Although sodium polytungstate is expensive, it can be recycled several times, using a membrane filter of 0.5 microns and the help of a vacuum pump. The filtered liquid is then evaporated at low temperature in a fume cupboard until it reaches the desired density.

Removal of carbonates.

Floating supernatant with phytoliths.

Also, most methods can be divided into two main groups: the ones that separate the phytoliths using a heavy liquid to float them and those that do not use this approach. The most commonly used heavy liquids are zinc bromide (ZnBr2) and sodium polytungstate [Na6(H2W12O40)H2O]. The latter is a non-toxic (Munsterman and Kerstholt 1996), high density (1·0–3·1 g/cm3) separating compound that generates an almost neutral solution (pH 6) of relatively low viscosity. Although sodium polytungstate is expensive, it can be recycled several times, using a membrane filter of 0.5 microns and the help of a vacuum pump. The filtered liquid is then evaporated at low temperature in a fume cupboard until it reaches the desired density.

A sodium polytungstate procedure (modified from Madella et al 1998)

Place 2–4 g of sediment in a clean 100 ml beaker and add 25 ml of a 7% solution of hydrochloric acid (HCl).
Place the beaker with the sample in a sand-bath at a temperature of 40ºC until the liquid is reduced to about 5 ml. Remove the beaker from the sand-bath and allow to cool.
Transfer the remaining liquid in a 15 ml laboratory tube with screw cap and add distilled water to 15 ml (if the volume of the sediment is quite substantial, it is possible to use 50 ml tubes - this is at discretion of the operator).
Shake the suspension gently with a tube-shaker, then centrifuge for 3 min at 1000 rpm.
After centrifuging carefully discard the supernatant, add distilled water to 15 ml, shake gently and centrifuge for 3 min at 1000 rpm (repeat this stage one more time if the second supernatant is still yellowish).
Discard the supernatant, add to 15 ml a 5% weight solution of sodium hexametaphosphate [(NaPO3)6] and leave overnight.
Shake gently and centrifuge the sample for 3 min at 1000 rpm and discard the supernatant. Repeat this step twice.
Add 33% volume hydrogen peroxide (H2O2), shake gently and leave at 50ºC until the reaction is over (normally few hours). Do not seal the tube to allow the release of gas.
Shake gently and centrifuge for 3 min at 1000 rpm and discard the supernatant. Repeat this step twice.
Add 10 ml of a solution of sodium polytungstate [Na6(H2W12O40)H2O] with a specific gravity of 2·35 g/cm3, shake gently and centrifuge for 3 min at 1000 rpm (if using the 50 ml tubes, add at least 25 ml of sodium polytungstate).
The floating fraction is recovered with a Pasteur or automatic pipette and transferred to a new tube. Shake gently and centrifuge the remaining suspension for 3 min at 1000 rpm and recover again.
Add distilled water to the recovered floating fraction to 15 ml and centrifuge for 3 min at 1500 rpm.
Pour out the supernatant leaving only the residue at the bottom.
Transfer the residue in the storage vial.
Let evaporate the water in the fume cupboard. Depending on the amount of water, this step could take from a few hours to some days.
The sample is ready to be mounted or stored.

To speed up the drying process it is possible to add the following steps after the supernatant has been poured out:

Add ethanol, gently shake and centrifuge for 3 min at 1500 rpm. Discard the supernatant. Repeat this step twice.
Transfer the residue in the storage vial.
Let the alcohol evaporate in the a fume cupboard.
The sample is ready to be mounted or stored.

Bibliography

Buckler, E. S. IV, Pearsall, D. M. and Holtsford, T. P. 1994. Zinc iodide and centrifugation allow rapid, inexpensive phytoliths separation. Phytolitharian Newsletter 8:2–3.
Coil J., Korstanje M. A. , Archer S., Hastorf C. A. 2003. Laboratory goals and considerations for multiple microfossil extraction in archaeology. Journal of Archaeological Science 30(8):991-1008.
Lentfer C.J. and Boyd W.E. 1998. A Comparison of Three Methods for the Extraction of Phytoliths from Sediments. Journal of Archaeological Science, 25(12):1159-1183.
Lentfer C.J. and Boyd W.E. 2000. Simultaneous Extraction of Phytoliths, Pollen and Spores from Sediments. Journal of Archaeological Science 27(5):363-372.
Madella M., Powers-Jones A.H. and Jones M.K. 1998. A Simple Method of Extraction of Opal Phytoliths from Sediments Using a Non-Toxic Heavy Liquid. Journal of Archaeological Science, 25(8):801-803.
Munsterman D. and Kerstholt S. 1996. Sodium polytungstate, a new non-toxic alternative to bromoform in heavy liquid separation. Review of Palaeobotany and Palynology 91:417-422.
Piperno D. R. 2006. Phytoliths: A Comprehensive Guide for Archaeologists and Paleoecologists. Altamira Press.
Powers A. H. and Gilbertson, D. D. 1987. A simple preparation technique for the study of opal phytoliths from archaeological and Quaternary sediments. Journal of Archaeological Science 14:529–535.
Rosen A.M. and Weiner S. 1994. Identifying ancient irrigation: A new method using opaline phytoliths from emmer wheat. Journal of Archaeological Science 21:132-135.
Zhao Z. and Pearsall D.M. 1998. Experiments for Improving Phytolith Extraction from Soils. Journal of Archaeological Science, 25(6):587-598.
Zucol A. and Osterrieth M. 2002. Técnicas de preparación de muestras sedimentarías para la extracción de fitolitos. Ameghiniana 39(3):379-382.