STARCH : RECOVERY

The next stage of analysis involves extracting starch granules from the sample so that they can be analysed microscopically. This process can be as simple as removing a small amount of sample from the specimen and applying it directly to a slide. Often, however, more complex procedures are required to break down the sample matrix and then separate and concentrate the starch.

Starch extraction methods are still being developed and often need to be adapted to suit the type of material under analysis and individual research goals. The main issue to be aware of is that starch granules are much more fragile than other plant microremains such as pollen or phytoliths, therefore aggressive extraction techniques should be avoided. For example, starch granules can be damaged by heat above about 35°C (particularly in the presence of moisture), strong acids and oxidizers, and excessive mechanical processing such as grinding. It is therefore useful to test extraction techniques first on reference materials (e.g. Torrence and Therin 2006). If a number of microremains (e.g. phytoliths, pollen) are to be recovered from the same sample, then a sequential protocol that accommodates each residue type should be designed. Starch is usually extracted first in these cases because it is most easily damaged (see Coil et al. 2003 and Korstanje 2003). Below are some examples of starch recovery methods used for different types of archaeological materials.

Sediments

The most common method for extracting starch from sediments and other bulk samples such as coprolites involves heavy-liquid flotation. First, the sediment is disaggregated and dispersed, and unwanted sediment components such as clays, carbonates and organics are removed, either by chemical dissolution or physical separation. Starch granules are then separated from the sediment matrix by floating them in a heavy-density liquid prepared at a specific gravity (s.g.) higher than that of starch (~1.5) but lower than other inorganic particles (generally > 2.0). Sodium polytungstate is the most commonly used heavy-liquid, as it is non-toxic, does not damage starch and can be recycled (Torrence and Therin 2006). Extractions generally involve the following steps and an example of a full protocol can be seen below.

Step 1: Weigh out a suitable sub-sample of sediment (usually 1-5 g) with an analytical balance. Samples should be dry so that weights and starch quantifications are accurate.
Step 2: Break up aggregates by gently crushing the sediment in a mortar and pestle.
Step 3: Digest organics with a weak oxidising agent such as 5% hydrogen peroxide, keeping treatment times short (30 mins max.) to avoid damaging starch. The reaction can be vigorous, so keep an eye on the sample and stir regularly.
Step 4: Remove large particles by wet sieving the sediment through a 250-300 µm mesh whilst agitating with a glass stirring rod. In this example, a dispoable nylon mesh held in a plastic frame is used and the sediment and water are collected in a glass beaker.
Step 5: Disperse clays by washing the sediment with a chemical deflocculant (e.g. sodium hexametaphosphate), which breaks electrostatic bonds between charged clay particles. These are then left in suspension and poured off after centrifugation.
Step 6: Remove light organics by floating in heavy liquid prepared at a specific gravity of 1.3. Next, float the starch fraction using heavy liquid at a specific gravity between 1.79 and 2.0. Carefully remove the supernatant with a pipette or aspirator. Rinse, dry and weigh the extract, which is now ready for microscopy.

Heavy-liquid separation protocol for starch

The following protocol for the extraction of starch from sediments is from Therin and Lentfer (2006). It was designed to extract starch granules from very clayey soils containing only a small organic component.

I. Disaggregation and removal of large particles and organics

  1. Dry sediment in oven at 35°C. Sieve through a 2 mm mesh. Weigh out 5 g of sieved sediment.
  2. Gently crush the sediment with a mortar and pestle. Transfer sediment to a 200 ml beaker.
  3. Add 20 ml of 5% hydrogen peroxide (H2O2) to the beaker in increments of 5 ml. Stir and leave for 30 minutes.
  4. Sieve sediment suspension though a 250 µm sieve with distilled water (d.H2O) and transfer into 50 ml falcon tube. Discard the > 250 µm fraction or store for subsequent examination.
  5. Centrifuge suspension in tubes at 2500 rpm for 3 minutes. Decant supernatant and discard.

II. Deflocculation and removal of clay

  1. Fill tube with 5% sodium hexametaphosphate warmed to 35°C, shake or vortex, and centrifuge at 2500 rpm for 1 minute. Decant supernatant containing suspended clay and discard. Repeat until supernatant is clear.
  2. Rinse by filling tube to 50 ml with d.H2O and centrifuge at 2500 rpm for 2 minutes. Decant supernatant and discard. Repeat twice.
  3. Dry sediment at 35°C in oven.

III. Extraction of starch grains using heavy-liquid flotation

  1. Add 8 ml of sodium polytungstate (SPT) heavy-liquid solution at density 1.3g/cm3 to 50 ml centrifuge tube containing dried sediment. Centrifuge at 2500 rpm for 12 minutes. Decant and discard supernatant.
  2. Add 5 ml of SPT liquid solution at density 2.0g/cm3 to tube. Centrifuge at 2000 rpm for 10 minutes. Decant supernatant into new 50 ml tube. Repeat.
  3. Fill new tube containing the starch residue to 50 ml with d.H2O. Centrifuge at 3000 rpm for 10 minutes. Decant and discard approx. two-thirds of the supernatant. Repeat this step three times. Decant all of the supernatant in the final repetition.
  4. Fill tube to 50 ml with d.H2O and centrifuge at 2500 rpm for 2 minutes. Decant and discard supernatant.
  5. Transfer residue to pre-weighed vial for storage.
  6. Dry residue at 35°C in oven. Weigh extract and prepare slide for microscopy.

Artefacts

Samples for starch analysis are removed from artefacts in a step-wise procedure designed to remove granules attached directly to the artefact surface and those present in adhering sediment in separate stages. Qualitative and quantitative comparisons between these samples can be used to assess whether starches derive directly from tool use or from the surrounding sediment. The general process is as follows (see Barton et al. 1998; Pearsall et al. 2004; Perry 2004; Perry et al. 2006; Piperno 2006:98; Zarillo and Kooyman 2006):

  1. Dry brushing: Gently brush the artefact with a clean brush to remove loosely adhered sediment, which can then be combined with any sediment that has detached in the sample bag. This sample represents the depositional matrix immediately surrounding the artefact, which is the most likely source of artefact contamination (Barton et al. 1998). Starch can be separated from this sample using the heavy-liquid flotation method described above. Sediment collected from around the artefact during excavation serves as an additional control and should be processed in the same manner.
  2. Spot sampling: Next, remove residue samples from various locations on the used and non-used areas of the tool with a small volume of water (10-20 µl) applied with a pipette. The aqueous extract can be mounted directly on a clean microscope slide. Particular attention should be paid to pits and cracks where granules could have been embedded during use. Alternatively, granules trapped in these voids can be removed using a needle probe (Piperno et al. 2004). It can be useful to examine the artefact under a dissecting microscope during this process to locate possible starchy deposits. Don’t forget to record the sample locations on a drawing or photograph of the artefact. Use-wear and residue spatial patterns should also be recorded as these can reveal important functional and taphonomic clues (see Fullagar 2006a, 2006b for more information).
  3. Wet brushing: Remove all remaining sediment from the used and non-unused areas of the artefact separately by scrubbing with a clean brush and cool, distilled water. Collect the extracts and concentrate by centrifugation. Separate starch further using heavy-liquid flotation if necessary.
  4. Sonication: Finally, remove well-adhered starches (particularly those trapped in micro-pits or -crevices) by sonicating the artefact for 5-10 minutes. Concentrate the extract and separate the starch with heavy liquid if necessary.
Removing starch residues from a grindstone with distilled water and a toothbrush (Step 3, above). The aqueous extract is pooled and collected with a pipette.

Charred samples

Extracting starch from charred residues on pottery or other carbonised food remains usually requires chemical treatment to break down the charred matrix and liberate the entrapped starch granules. Hydrogen peroxide (H2O2), sodium hydroxide (NaOH), sodium hypochlorite (bleach, NaOCl) and heated nitric acid (HNO3) have all been used in the past (Boyd et al. 2006, 2008; Cortella and Pochettino 1994; Crowther 2009; Scott-Cummings 2006; Zarillo et al. 2008), but the effects of some of these reagents on starch have not yet been thoroughly tested and some may be damaging (e.g. Cortella et al. 2001; see also Crowther 2009). The extraction procedure illustrated below is recommended (Crowther 2009).

It can also be useful to examine microstructural features of charred or desiccated food remains with scanning electron microscopy or in thin section to establish whether characteristic alterations induced by different processing methods (e.g. wet or dry cooking, fermentation) are visible. Methods for these types of analyses are described by Samuel (1996; 2006) and Valamoti et al. (in press).

Step 1: First, scrape off the surface soil layer with a clean scalpel blade. Retain for analysis as a control sample. Then, scrape a sample of charred residue from the freshly exposed surface onto a clean piece of aluminium foil with a clean blade.
Leave some sample for future analyses. In this case, less than half was removed.
Step 2: Fold the foil around the residue and gently crush with a pestle to disaggregate the charred matrix.
Step 3: Transfer the residue to a vial and treat with a weak oxidising agent (e.g. 5% hydrogen peroxide, 0.125% sodium hydroxide) for 30 minutes. Agitate regularly.
Step 4: Dilute the sample with distilled water and sonicate for 5 minutes to disaggregate the residue. Rinse and dry. If necessary, larger residue samples can be processed with a heavy-liquid flotation step to further separate the starch (Zarillo et al. 2008).

Dental calculus

  • First, wash the tooth and calculus to remove any adherent sediment, either by gently brushing and rinsing with water or by sonicating in water for 5-10 minutes.
  • Thin calculus deposits can be carefully scraped from the tooth with a dental pick and mounted directly on slide (Piperno and Dillehay 2008) or removed chemically by submersing the tooth in weak (4%) hydrochloric acid (Boyadjian et al. 2007). It should be noted, however, that the latter method may damage the tooth enamel, particularly on fragile specimens, and is therefore not recommended if other analyses such as microwear are to be undertaken. Likewise, take care not to damage the tooth when removing the calculus mechanically with a dental pick.
  • Thicker accumulations, such as those shown in the example below, may require chemical digestion to release the entrapped starch. Recent studies (Hardy et al. 2009; Henry and Piperno 2008) recommend the following steps:
  1. After washing, carefully detach the calculus from the tooth with a dental pick (below right).
  2. If there is a lot of adherent sediment, perform a second washing step by either sonicating the calculus in water or briefly immersing it in weak hydrochloric acid (HCl) to remove the outer calculus layer. Rinse the calculus with distilled water.
  3. Deflocculate the sample by washing in 5% sodium hexametaphosphate. Rinse.
  4. Dissolve the calcified matrix by immersing the calculus in weak (10% or 4.6M) HCl for up to 12 hours. Agitate regularly.
  5. Rinse and dry the extract, and prepare slides for microscopy.
  • Target different areas of the same specimen with visible calculus deposits or where starch residues may be trapped, such as in the crown of molars, near the gum line, and in caries.
Starch grains derived from consumed plant foods can be recovered from dental calculi (tooth tartar), which are calcified deposits of dental plaque that accumulate on teeth. This archaeological example was excavated from an Anglo-Saxon burial, northern England.
Carefully detach the calculus with a dental pick onto a clean sheet of tin foil (which is non-static and can be folded to transfer the calculus into a vial for chemical processing).

Avoiding contamination in the laboratory

It is important that archaeological samples don't become contaminated with modern (and ancient) starch during the extraction process. Here are some simple controls that should be adopted:

  • Monitor and control airborne contaminants as much as possible. Place contamination slides around the laboratory and check them regularly for background starch. Filter the laboratory air supply if possible.
  • Wash hands thoroughly before beginning work and don't consume food or drink in the laboratory, which contravenes most safety protocols and is also a potential source of starch contamination.
  • Wear non-powdered gloves. The powder in gloves is manufactured from starch, which poses a major contamination risk.
  • Use distilled water at all times and prepare fresh reagents as often as possible. Work with small aliquots of reagents so that if contamination does occur, it will not be widespread. If reagents such as heavy liquids are recycled, they should be filtered first at < 1 µm.
  • Use sterile, disposable equipment where possible (e.g. pipette tips, weigh trays, centrifuge tubes).
  • Clean re-useable laboratory materials as soon as possible after use by scrubbing with detergent. In addition to mechanical cleaning, it is good practice also to wash materials with a starch-destroying chemical (e.g. 5% potassium- or sodium hydroxide, 10% bleach), by submerged in boiling water, or by sonication. Rinse thoroughly with distilled water after cleaning to remove any remaining residue.
  • Prepare reference materials in a separate location to archaeological samples and use separate sets of reagents and processing equipment (e.g. mortars and pestles) to reduce the potential for cross-contamination.