Laboratory processing of samples

In document Agriculture in Tongan Prehistory: An Archaeobotanical Perspective (Page 77-80)

Each of the samples collected in the field was first removed from their original containers and put into a new sterilised vial with 70% Ethanol and 30% De-ionised water for storage until further processing began. Each sample was then divided into four sub-samples to allow for starch extraction, histological thin sectioning and experimental charring for parenchyma from dried and fresh states.

Figure 5.1 Flowchart showing methodology for the imaging and recording of starch and parenchyma within the reference collection

Starch processing

The starch samples were processed using a methodology established by Ussher (2009, 2012) in creating a comparative collection for the Marquesan archipelago, and including methods suggested in Field (2006). Two methods were used to extract starches from the parenchymatous tissues of the vegetative organs sampled. The first of these were to simply cut a fresh section from the organ and press that section onto the slide (Gott in Field 2006). The second was to gently crush the sample in a mortar and pestle with some distilled water, and then put this

60 extracted residue onto a slide using a pipette (Lentfer, pers.com). The slide was then covered with a Petri dish until the residue had dried. Glycerol was used as a permanent mountant, due to the reflective and viscous properties of this compound which allows starch granules to be rolled and viewed during light microscopy (Field 2006:112). Finally, each sample was covered with a cover-slip and sealed with nail polish.

Histology

A histological thin section was made from each plant species and organ collected, to demonstrate parenchymatous cell arrangement, structure and contents. The samples collected were removed from the 70% Ethanol, and a small 1cm cubed fragment was taken from each sample and placed into a mixture of 95% (70% dilution) alcohol, 1% glacial acetic acid, and 4% formaldehyde. This fixative formula was recommended by Hather (2000:78) and is known as FAA (Formalin-Acetic-Alcohol) (Miksche and Berlyn 1976:30). This fluid is stable, has a good hardening action and material can be stored in it for years. The samples were left in the FAA mix fixative for approximately two weeks, by which time the organs had soaked up the mixture and partially solidified/preserved as dead plant tissue. This solution enabled greater precision during microtomy to cut thin sections.

Thin sections were made by Anne Prins in the Histology Laboratory at the John Curtin Medical Research Centre. Several methods for processing samples prior to microtomy were tested. A white potato (Solanum tuberosum) was also preserved in the fixative formula, and then used to experiment with microtomy and staining. The Standard Bouins Cycle technique was applied to prepare the samples after they were placed into small histological baskets. This technique involves several stages of soaking samples in varying percentages of ethanol warmed to 40˚C, starting with 70% and finishing in 100%, then baths of chloroform at 40˚C and paraffin wax at 60˚C. These were then placed in the microtome and cut into 5, 8, 12, 15 and 20µm thin sections. Experimentation with the white potato indicated that the most appropriate section size during microtomy was 15µm, as thinner sections tended to rip and thicker sections were difficult to mount onto the slides. The paraffin embedded thin sections were then mounted on heated slides and placed in xylolto dissolve the wax, and finally washed with 100%, 90% and 70% ethanol, and tap water prior to staining.

As part of the staining process the sections were treated with 3% glacial acetic acid for three minutes, and then washed well with running tap water. The slides were then immersed in Alcian Blue (1%) stain with a pH of 2.5 for 30 minutes, washed in running tap water again, and finally immersed in a Safranin (0.02%) counter-stain for five minutes and then dehydrated. After staining, the sections were blotted, allowed to air dry and cover-slipped with Leica Micromount used as a permanent mountant.

61 The experimental thin sections were observed using light microscopy at x100 and x200 magnification. The cell walls were in good condition, and many starch granules were still visible and displayed birefringence in some samples. The fact that starch was preserved in these thin sections was surprising, as starch generally gelatinises at between 30-60˚C (Barton and Torrence 2006). Here the starch was not only still in native condition, but also abundant within the cell structures. It was therefore decided that the Paraffin embedded preparation was the most appropriate for the remainder of the samples within the reference collection. The samples were prepared in the same way as the experimental white potato and then analysed using light microscopy in the Microscopy Laboratory in the Department of Archaeology and Natural History at the ANU.

Experimental charring

To understand the morphological changes that occur during charring, plant materials within the reference collection were experimentally charred in a muffle furnace. These samples were then compared with the parenchyma cell organisation and structures observed in the thin sections. Recommended temperatures and length of time for charring vary according to the type of plant tissue, i.e. woody or non-woody tissue. Orvis and others (2005) suggest charring woody stem material, soft-leaf tissue, needle-leaf tissue and monocot tissue at 550˚C for only eight to nine minutes. Boardman and Jones (1990) tested a range of temperatures, times and atmospheric conditions (oxidised or reduced). It was discovered that most plant components (granules, glumes, rachis and straw) will carbonise after 1-2 hours at 300-250˚C, with greater distortion at higher temperatures even within shorter time periods (1990:5-9). Similarly, Wright (2003) experimented with charring achenes, kernels, seeds and rind segments of a variety of species. Variables tested included thermal atmosphere, temperature, duration and moisture content. Overall, some generalities could be made regarding the carbonisation process including that the higher the temperature, and longer the exposure, the more likely the specimen will ash rather than carbonise, and that moist specimens generally survived oxidation better than dry samples. Finally, a reducing atmosphere was more conducive to preservation by carbonisation than oxidation (2003:582). The results of these previous studies were used to determine the techniques used within the current research.

Two small 1cm cubed samples were cut from the remaining plant material for each sample, and wrapped in tin foil. One sub-sample of each sample was placed into large tin trays, and then put into an incubator at 60˚C for 48 hours to dry. The second sample was placed into a pre-heated muffle furnace at 300˚C for 2 hours (Hather 2000:85), or until the samples had turned to charcoal (Boardman and Jones 1990:3). They were then removed from the furnace and cooled. Once dried, the remaining samples were also charred in the furnace. All samples were then fractured to reveal a flat plane that could be viewed using Reflected light microscopy and Scanning Electron Microscopy (SEM) (Hather 2000:76).

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Recording

In document Agriculture in Tongan Prehistory: An Archaeobotanical Perspective (Page 77-80)