Starch analysis

Staining solutions are one way to make comparative analysis of starch granules more accessible, depending on what type of stain is specifically used. For example, as stated by Kovárník and Benes, Lugol's solution stains the grains a dark blue color, in order to distinguish the starch grain from other common structures that can appear similar in size and shape.[1] In addition to this technique, the use of CongoRed dye marks the damage, making it easier to study, compare, and to anlayze differences in the damage of particular taxa. Specifically, the intensity of the red color depends on how damaged the grain is.[1] Lastly, Trypan blue is another way to stain grain damage within starch grains, only staining the damaged grains, not the undamaged.[1] Specifically, it can be important to incorporate the staining process into an analysis due to the fact that there are several other structures found naturally that have similar characteristics to starch when compared under a microscope.[1]

There are several ways to go about extracting the starch granules from stone tools. One technique as explained by Kovárnik and Benes described as the most common, is through pipetting the surface of the objects on the most common areas in which starch grains are found, for example fissures, or others divetts on the artifact.[1] The water droplets from the pipetting technique are then collected onto slides to be utilized in comparative analysis. This is a common technique for the use of understanding specifically how stone tools were used, and what types of plants were being exploited during the time being studied.[1]

Another common approach is to dislodge the starch grains through sonication, which is a laboratory technique that uses sound waves to "agitate particles" in order to convert an electrical signal into a vibration which in turn breaks down a substance.[5] In terms of starch grain analysis, the starch grains are released by use of an ultrasonic bath and the distilled water containing the sample particles is then centrifuged. This is the process in which the specimens are spun and separated by density as a result of centripetal force, or due to the fictitious centrifugal force that is felt by the specimens due to their reference frame.[6] Utilizing this force allows for the separation of solutions of different densities.[6] Thus the least dense component, consisting of the starch grain particles is separated and a microscopic slide can be prepared.[1]

Starch grain analysis through dental calculus can provide a plethora of information when it come to diet reconstruction of past societies.[2] Specifically, dental calculus is a layering found on the teeth that is formed from plaque, "after mineralization."[1]

In a study conducted by Tao et al., the research group followed the methods laid out by Piperno and Dillehay in 2008 and Li et al. in 2010.[2] In these specific examples, they ground the dental calculus by use of mortar and pestle, and centrifuged the mixture containing the dental calculus as well as small amounts of Calgon in order to break the particles into a dispersed state.[2] Once the extracts were dissolved in hydrochloride and rinsed with acetone, the residues were extracted and comparative analysis could begin.[2] This is just one way in which to go about extracting starch grain residues from dental calculus, with another example being the use of sonification as described above.[1]

There are several structures that are observed when doing comparative analysis to determine the plant taxa in which the starch grain belongs. First, starting on a slightly larger scale, the granule types such as simple, compound, or semi-compound, sizes, and shapes are observed.[7] Next there is the hilum, which is the area in which protein layers are deposited.[8] Hilums differ in their positioning on the starch granule and this positioning can differ between taxa.[1] In addition to the hilum, "lamellas" are observed which are the different growth layers in which are only sometimes visible microscopically.[1] The fissures can also be observed, however these are only common in some starch grains, not all.[7] In total, starch grain analysis through the use of a computer database is the primary way in which starch grains are differentiated.[1] This is helpful in terms of a supplemental approach to understanding plant exploitation of past societies.

Archaeological research focused on residue adhering to artifacts start at lower magnifications, commonly using a stereoscope. Most data obtained at this stage is qualitative, an important first stage to fuller analysis. Magnifications of between x10 and x50 are sufficient to locate target residues, describe features and confirm internal structures of the identified residues.[14]

Modern light, high powered microscopes have an internal light source, allowing illumination with both transmitted and reflected light. These microscopes can provide a magnification of up to x1000: good enough to provide clear images of starch granules as small as a few micrometres in diameter.[15]

Starch granules show different sizes. For example;

• Tapioca starch: 5-35 µm
• Potato starch: 15-100 µm
• Maize starch: 5-25 µm
• Rice starch: 3-8 µm

but all are generally under 100 micrometres in size, and are, therefore, best observed under compound microscopes equipped with various lighting conditions and magnifications from x200 to x800.[16]

The starch grains are also compared to standard reference collections for comparison. Archaeologists and researchers can consider four issues in classification of the plant(s) and its use(s):

• Determination of whether evidence for the utilization of plants is present
• Study of the assemblage variation
• Determination of the presence of particular plant species
• Assign percentages of starch granules within a sample to a particular taxon, and present quantitative data regarding relative abundance within the sample. Identification of ancient starch is fairly easily for the first three levels of classification, whilst the fourth level requires continued improvement in the description, classification, and identification of individual starch granules.[17]

Sampling a sediment core or stratigraphic profile to gather information about an environment requires a detailed understanding of the way the sediments were formed.[19]

Most extraction techniques follow a general methodology of:

• sample preparation (sieving, drying, or soaking)
• disaggregation and deflocculation to break up the elements of the sample into single particles
• removal of undesired particles (sands, silts, minerals, organics).
• chemicals preservation of the starch granules.[20]

Starch granules are mounted onto a slide, using a variety of mounting medias including, but not limited, to water, glycerol, and glycerine jelly. It is important that the material is dried thoroughly before being mounted to ensure that no further degradation of the sample occurs. The slide is then viewed, as appropriate, for identification and counting.[21]

After the starch granules have been examined, the findings are then recorded and interpreted with respect to the research questions that are being investigated.