A convenient sample preparation protocol for scanning electron microscope examination of xylem-occluding bacterial biofilm on cut flowers and foliage

Microbes and their exopolysaccharides (EPS) can block xylem vessels, thereby increasing the hydraulic resistance and decreasing the vase life of cut flowers and foliage. Scanning electron microscopy (SEM) provides a powerful tool for investigation of bacteria-induced xylem occlusion. However, conventional preparation protocols for SEM involving chemicals can cause loss of hydrated EPS material, and thereby damage the bacterial biofilms during dehydration. A modified chemical fixation protocol involving pre-fixation with 75 mM lysine plus 2.5% glutaraldehyde followed by the normal fixation in 3% glutaraldehyde was, therefore, tested for improved preservation of bacterial biofilm at the stem-ends of cut Acacia holosericea foliage stems. Stem-end segments with different stages of bacterial growth were obtained from stems stood into water. The lysine-based protocol was compared with four other processing protocols of critical point drying (CPD) without fixation (control), freeze-drying (FD), conventional chemical fixation followed by drying with hexamethyldisilazane (HMDS), and conventional chemical fixation with CPD. The non-fixed control. FD and the glutaraldehyde fixation with HMDS drying gave poor preservation of hydrated material, including bacterial EPS. Conventional glutaraldehyde fixation followed by CPD was superior to these three methods in terms of better preserving the EPS. However, this fourth method gave condensation of biofilms during dehydration. In contrast, the modified lysine-based protocol resulted in superior preservation of EPS and biofilm structure. Thus, this fifth method was the most appropriate for examination of bacterial stem-end blockage in cut ornamentals. (C) 2012 Elsevier B.V. All rights reserved.

Three-dimensional reconstruction of black tiger prawn (Penaeus monodon) spermatozoa using serial block-face scanning electron microscopy

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) was used in this study to examine the ultrastructural morphology of Penaeus monodon spermatozoa. SBF-SEM provided a large dataset of sequential electron-microscopic-level images that facilitated comprehensive ultrastructural observations and three-dimensional reconstructions of the sperm cell. Reconstruction divulged a nuclear region of the spermatophoral spermatozoon filled with decondensed chromatin but with two apparent levels of packaging density. In addition, the nuclear region contained, not only numerous filamentous chromatin elements with dense microregions, but also large centrally gathered granular masses. Analysis of the sperm cytoplasm revealed the presence of degenerated mitochondria and membrane-less dense granules. A large electron-lucent vesicle and "arch-like" structures were apparent in the subacrosomal area, and an acrosomal core was found in the acrosomal vesicle. The spermatozoal spike arose from the inner membrane of the acrosomal vesicle, which was slightly bulbous in the middle region of the acrosomal vesicle, but then extended distally into a broad dense plate and to a sharp point proximally. This study has demonstrated that SBF-SEM is a powerful technique for the 3D ultrastructural reconstruction of prawn spermatozoa, that will no doubt be informative for further studies of sperm assessment, reproductive pathology and the spermiocladistics of penaeid prawns, and other decapod crustaceans. J. Morphol., 2016. (c) 2016 Wiley Periodicals, Inc.