2.2. Scanning electron microscopy
To study the external morphology of radular teeth in the scanning electron microscope radulae were cleaned in one of the following three ways: (1) by ultrasonic treatment, followed by drying in air; (2) by incubation in 1 N NaOH during 1 to 4 h at 100°C, followed by drying in air; (3) by incubation in a sodium tetraborate-buffered solution (pH 9) of trypsin at 25°C, after which the radulae were fixed in 2% OsO4 and dried at the critical point of carbon dioxide. After cleaning, the radulae were mounted on aluminium stubs with a carbon-containing adhesive. The internal morphology of the radular teeth was studied in SEM either in deliberately broken teeth of mounted specimens, or in etched sections of teeth embedded in a polymethylmetacrylate-containing substance called Technovit (Kulzer) or in Epon (10:0). Embedment in Technovit occurred after air-drying the radulae; embedment in Epon was done after dehydration of the radulae in a graded series of ethanol and subsequent infiltration with propylene oxide. The blocks containing the specimens were ground down until suitable sections through teeth were obtained and were then polished. Etching of the polished sections was done by incubating the blocks in an aqueous solution of 4 N HCl during a period of up to 24 h. The etched specimens were then cleaned in distilled water, dried at the critical point of carbon dioxide and mounted on aluminium stubs with a carbon-containing adhesive. All mounted specimens were finally coated with a 10-nm thin layer of gold and viewed in either an ISI SEM type DS 130 at 5–12 kV or a Jeol 35C at 25 kV.