Scd 040

The GI tract from each P. monodon was dissected out and then fixed in 3% glutaraldehyde in 0.1M phosphate buffer pH 7.4. The samples were stored in the dark overnight at 4°C and then they were rinsed twice with 0.1 M phosphate buffer for 10 min followed by once with distilled water for 10 min. Thereafter they were dehydrated through a graded ethanol series, i.e. 10 min each in 30, 50, 70, 95% followed by 10 min each in three changes of absolute ethanol. The samples were subsequently critical-point dried using carbon dioxide as the transitional fluid and then mounted on stubs. During mounting, the samples of the foregut, midgut and hindgut were split longitudinally to expose the gut contents and the inner lining of the gut. After sputter coating the samples with gold using a Balzers model SCD 040, the specimens were examined in a JEOL model JSM-5410LV scanning electron microscope.

Some aspects of the analysis required experimentally charred reference material, in particular the preservability of starch granules in charred state and the formation of cavities during charring.
Triticum spelta ‘Bauländer Spelz’ was cultivated in autumn sowing by the Mechler family in Altheim-Walldürn, southwestern Germany, and harvested as Grünkern, i.e. in an unripe state [87 ] after nine months of growth. The grains were charred in a Nabertherm NA 15/65 muffle furnace under various charring regimes: at low temperature (230° C) for 7, 9, 12, and 24 hours [88 ], and at high temperature (300°C) for 6 hours.
Sprouted two-rowed hulled barley (Hordeum vulgare subsp. vulgare f. distichon) was acquired from the Durst Malz company, Bruchsal-Heidelsheim, Germany, in various states of germination (3 days and 5 days) and charred using the same muffle furnace under varying conditions (dry, soaked in water, and with water) at low temperature (230° C) for 24 hours [88 ].
SEM imagery for the Grünkern was produced using a Zeiss DSM 940 after sputter coating with gold/palladium in a Balzers SCD 040, for the barley malt with a JEOL JSM-6390LV after carbon coating using a JEOL-4X vacuum evaporator.