Dp72 digital

Different protocols were employed for scanning electron microscopy (SEM) analyses, depending on the strains studied and the institution where the study was conducted. The original material from La Gomera was cleaned of organic matter according to Hendey [26 ], except that cells were collected by centrifugation rather than sedimentation during repetitive washings (3–4 times) in distilled water. Cleaned cells were mounted on glass slides with Naphrax and observed in LM using an Olympus BX51 (Tokyo, Japan), with a DP72 digital camera, in differential interference contrast and phase contrast. For SEM examinations, cleaned frustules were air-dried onto circular coverslip mounted on an aluminum stub and sputter coated with either gold, platinum, or palladium-platinum alloy. Observations were done, depending on the facilities, using a JUC5000 or JEOL JFC2300 HR High Resolution Fine and JEOL 7600F or JEOL J 6335F or JEOL JSM-5600 (JEOL, Tokyo, Japan) or FEG Tescan MIRA3 microscope (Brno, Czech Republic). For TEM material was air-dried onto 200 mesh Formvar covered copper grid. The TEM observations were made using a JEOL JEM-1010 (Tokyo, Japan) operated at 80 kV, equipped with a GATAN Orius SC1000 digital camera (Pleasanton, CA, USA). Measurements of valve structural elements were made from SEM images using the ImageJ software (http://rsb/info/nih/gov/ij (accessed on 1 September 2010)).

Microscopic analysis of the samples was performed in order to compare the morphology of pollen grains and to show changes occurring during the fermentation process. The observations were performed using an epifluorescence microscope BX-51 (Olympus, Shinjuku, Japan) equipped with a DP-72 digital camera and Cell D software. A water drop smear of each homogenized sample was made on a glass slide and observed at 200× magnification.