Archaeobotany - Recognition

STARCH : RECOGNITION

Preparing slides for starch analysis

Most of the routine work in starch analysis is done with a polarising transmitted light microscope, in which starch granules are observed on glass microscope slides illuminated by a light source located underneath the microscope stage. The light passes through the transparent granule so that internal structural features (e.g. fissures, lamellae) as well as external morphology are visible. Scanning electron microscopy can also be used to examine three-dimensional granule morphology, but internal features are not visible using this technique and it is also much more time-consuming.

When preparing slides for starch analysis, a mounting medium with a refractive index higher or lower than that of starch (ri = 1.530) must be used (Field 2006). Temporary water mounts are useful if iodine potassium-iodide (IKI) staining (see below) is to be performed or if starch granules need to be recovered for other analyses such as SEM. Semi-permanent mounts (e.g. 50% glycerol) are often preferred as they a slightly more viscous and enable starch granules to be rotated more easily. Permanent media are generally not recommended, as starch granules cannot be rotated to view in three-dimensions and some media have been found to alter or degrade certain morphological features over time (Fullagar et al. 2006:605; Piperno 2006:96).

The typical procedure for preparing a slide involves the following steps (see also Field 2006):

Rehydrate dried extracts in vials with a standard volume of water (e.g. 500 µl). Agitate the extract so that the starch is evenly suspended and withdraw a small volume (e.g. 50 µl) with a pipette. Apply to a clean, glass microscope slide. Extracts removed directly from an artefact with a pipette or scraped from a calculus (without further chemical processing) can be applied direct to slide. Spread the extract out evenly across an area slightly smaller than the cover slip so that particles are not clumped or clustered.
For temporary water mounts, allow the extract to air-dry and cover with a glass slip. Tack the slip down with small drops of clear nail polish at each corner. When the slide is ready to be examined, rehydrate the sample by applying water along one edge of the cover slip with a pipette until the sample is fully immersed. Re-apply water as needed during microscopy if the mount begins to dry.
For semi-permanent mounts (e.g. 50% glycerol) apply the starch extract to the slide as above and allow to partially dry. Add a drop of the mounting medium and cover with a glass slip. Seal the edges with nail polish to prevent the slide from dehydrating.

The number of slides examined per sample will vary depending on the amount of extract removed and the amount of starch recovered in each extract. Multiple slides may need to be prepared if recovery rates are low.

Preparing a temporary water mount: 1. Start with a clean, glass microscope slide, the aqueous starch extract and a pipette. 2. Apply a small volume of starch extract to the slide using the pipette. 3. Cover with a glass slip and tack down with nail polish at each corner.

Is it starch? Scanning slides and techniques for detecting starch

Slides are usually scanned for starch systematically in transects at about x200-x400 magnifications, although higher magnifications may be needed to see smaller granules. It is best to scan slides in polarised light, in which starch granules can be readily spotted and differentiated from other components on the slide (e.g. organic and inorganic sediment particles, microcharcoal, biogenic silica such as phytoliths, diatoms and sponge spicules, plant tissues, pollen, etc.) by their bright appearance (birefringence) and distinctive interference figure in the shape of a Maltese cross. The cross is produced by the double refraction of light through the granule’s structure and its point of intersection corresponds with the position of the granule’s hilum.

Field of view at x400 magnifications in plane- (left) and cross-polarised (right) light. A small starch granule can be seen centre (yellow arrow) and three larger, out of focus granules (white arrows) are around the periphery. The slide also contains lots of micro-charcoal, sediment particles and other debris, making the starch difficult to spot.

Starch usually occurs as single, isolated granules but clusters, which are mostly likely to derive from a storage organ, can also be present. Another form to look out for are compound grains, which occur when more than one starch granule is produced in an amyloplast. These become fused together, forming aggregates comprised of multiple sub-granules that usually display their own extinction cross (Banks et al. 1973, 1974).

Compound grains can separate when mechanically disrupted, such as during grinding. In these cases, the sub-granules have pressure facets (flat faces) where they were once joined. Compound grains can have a variety of configurations depending on taxon of origin (see examples shown right). Single starch grains produced in an amyloplast are referred to as ‘simple’ granules.

Examples of different types of compound starch grains.

Some soil and plant components can appear superficially similar to starch to the untrained eye in terms of shape (particularly the more simple round/spherical granules) and because they display an extinction cross. Some common examples of starch-like residues that you should watch out for while analysing archaeological samples are shown below.

Bordered pits

Bordered pits are small openings in the cell walls of tracheary elements in plants. These small celluose-based structures display a cross-like figure in cross-polarised light, and, as for starch, the arms of the cross will move slightly upon rotation of the analyser (Loy 2006:124). Bordered pits are not solid spheres like starch, but are toroid shaped and can be easily recognised by the presence of a small hole or very thin central membrane, the border of which is often visible in plain transmitted light. The hole appears as a dark central region in cross-polarised light. Bordered pits are usually found in sheets of plant tissue and have poor relief owing to their essentially flat form. (400x magnification, scale bar = 10 µm)

Spherulites

Spherulites are microscopic bodies composed of radially- or concentrically-arranged crystals that produce birefringence and an extinction cross in cross-polarised light. The most common type noted in archaeological sediments is calcium carbonate ‘faecal spherulites’, which derive from the dung of various herbivores including sheep, goats and cows, particularly those that graze on calcareous pastures (e.g. Canti 1998). Sometimes spherulites crystallize in situ on slides, typically at the margin of a residue that has partially or completely dried prior to mounting. Spherulites can be differentiated from starch by their permanent (non-rotating) extinction cross (Loy 2006:123), or by using a lambda plate or quarter-wave plate in combination with cross-polarised light, under which conditions spherulites show specific colours in the white (birefringent) region between the dark extinction cross arms (Canti 1998:439). Calcium carbonate faecal spherulites will also readily dissolve in weak hydrochloric or acetic acid (starch should not be affected, at least over short time periods, e.g. <1 hour), and they will not stain with IKI. (500x magnification, scale bar = 10 µm)

Annular (plant rings)

These ring-like fragments of lignified cellulose display birefringence in cross-polarised light. Although close up these fragments clearly have a ring structure with distinct concentric internal and external borders, they can sometimes look like starch granules with weak internal birefringence when scanning slides at lower magnification. (400x magnification, scale bar = 10 µm)

Air bubbles under the coverslip

Air bubbles can sometimes get trapped in the mounting medium under the coverslip during slide preparation or rehydration. These form concentric rings with extinction cross-like figures in cross-polarised light. They can be readily distinguished from starch by their thick black borders and their lack of structure. Air bubbles can usually be removed by tapping gently on the coverslip, forcing the trapped air to the edge of the slide. (600x magnification, scale bar = 5 µm)

References

Canti, M. G. 1997. An investigation of microscopic calcareous spherulites from herbivore dungs. Journal of Archaeological Science, 24:219-31.

Loy, T. H. 2006. Optical properties of potential look-alikes. In R. Torrence & H. Barton (eds.) Ancient Starch Research, pp. 123-124. Walnut Creek, California: Left Coast Press.

Starch granules can usually be differentiated from these by considering a combination of characteristics, including birefringence, the formation of an extinction cross in cross-polarised light, morphology, and the presence of internal growth structures such vacuoles, fissures and lamellae (these features are illustrated in the Starch - Identification section). Another key feature of starch is that the polarisation cross rotates slightly when the analyser (the second polarising filter) is rotated. The film on the right demonstrates this feature.

video on chickpea polarisation | video on chickpea starch rotation

Watch these videos to see how chickpea (Cicer arietinum) starch granules become birefringent and form an extinction cross in cross-polarised light. Notice also how the extinction cross rotates, which is characteristic of starch.

Iodine potassium-iodide (IKI or Lugol’s stain) can also be used to help locate starch granules and may be essential for detecting damaged granules that have lost birefringence (Barton 2007; Loy 2006). Iodine stains starch granules various shades of blue, purple and red depending on the relative content of amylose (which gives a strong blue reaction) and amylopectin (which gives a more red-purple reaction), which vary between species. Care should be employed when using iodine staining, as strong solutions can produce a very dark reaction, in which case granules may not be detected (especially if there is lots of charcoal and other dark sediment components on the slide). A very weak solution (0.1-0.2%) is best, but experiment first with reference samples. Iodine staining can also be useful for differentiating starch from particles with similar morphological and optical properties (see examples here), as these usually will not give the blue-purple reaction characteristic of starch. Iodine can stain other plant polysaccharides, however, and should be used in conjunction with morphological features to identify starch positively.

Potato starch grain stained with iodine potassium-iodide (IKI), shown in plane- (left) and cross-polarised (right) light. Note how internal morphological features such as lamellae as well as the extinction cross are still visible because a very weak IKI solution was used. If the solution is too strong, granules will stain a very dark blue-black and will be difficult to recognise.

Another method for testing starch that has been developed recently is enzymatic digestion (Hardy et al. in press). This technique involves the use of enzymes called alpha-amylases, which specifically degrade starch, to hydrolyse suspected starch granules. Because this method is destructive, its routine use may not be desirable, but it could be useful in cases where starch granules have lost their native structure through damage and can not be identified confidently as starch, even with use of IKI.

Damaged starch

Damaged or modified starch granules may not display all the characteristics typical of native granules (particularly birefringence and an extinction cross) or their morphological and optical characteristics may be altered. As a result, damaged starch granules can be more difficult to recognise, but they can give important clues as to how plant foods were processed in the past (e.g. Babot 2003; Chandler-Ezell et al. 2006; Henry and Piperno 2009).

The main food processing methods that create recogniseable starch alteration signatures are mechanical processing (e.g. grinding, pounding), cooking, and fermentation.

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