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.

Lack of release of bound anthocyanins and phenolic acids from carrot plant cell walls and model composites during simulated gastric and small intestinal digestion

Separately, polyphenols and plant cell walls (PCW) are important contributors to the health benefits associated with fruits and vegetables. However, interactions with PCW which occur either during food preparation or mastication may affect bioaccessibility and hence bioavailability of polyphenols. Binding interactions between anthocyanins, phenolic acids (PAs) and PCW components, were evaluated using both a bacterial cellulose-pectin model system and a black carrot puree system. The majority of available polyphenols bound to PCW material with 60-70% of available anthocyanins and PAs respectively binding to black carrot puree PCW matter. Once bound, release of polyphenols using acidified methanol is low with only similar to 20% of total anthocyanins to similar to 30% of PAs being released. Less than 2% of bound polyphenol was released after in vitro gastric and small intestinal (S.I.) digestion for both the model system and the black carrot puree PCW matter. Confocal laser scanning microscopy shows localised binding of anthocyanins to PCW. Very similar patterns of binding for anthocyanins and PAs suggest that PAs form complexes with anthocyanins and polysaccharides. Time dependent changes in extractability with acidified methanol but not the total bound fraction suggests that initial nonspecific deposition on cellulose surfaces is followed by rearrangement of the bound molecules. Minimal release of anthocyanins and PAs after simulated gastric and S.I. digestion indicates that polyphenols in fruits and vegetables which bind to the PCW will be transported to the colon where they would be expected to be released by the action of cell wall degrading bacteria.

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.

Nano-silver treatments reduced bacterial colonization and biofilm formation at the stem-ends of cut gladiolus ‘Eerde’ spikes

Postharvest treatments with nano-silver (NS) alleviate bacteria-related stem blockage of some cut flowers to extend their longevity. Gladiolus (Gladiolus hybridus) is a commercially important cut flower species. For the first time, the effects of NS pulses on cut gladiolus ‘Eerde’ spikes were investigated towards reducing bacterial colonization of and biofilm formation on their stems. As compared with a deionized water (DIW) control, pulse treatments with NS at 10, 25 and 50 mg L−1 for 24 h significantly (P ≤ 0.05) prolonged the vase life of cut gladiolus spikes moved into vases containing DIW. The NS treatments enhanced floret ‘opening rate’ and ‘daily ornamental value’. Although there were no significant differences among NS treatments, a 25 mg L−1 NS pulse treatment tended to give the longest vase life and the best ‘display quality’. All NS pulse treatments significantly improved water uptake by and reduced water loss from flowering spikes, thereby delaying the loss of water balance and maintaining relative fresh weight. Fifty (50) mg L−1 NS pulse-treated cut gladiolus spikes tended to exhibit the most water uptake and highest water balance over the vase period. However, there was no significant difference between 25 and 50 mg L−1 NS pulse treatments. Observations of stem-end bacterial proliferation during the vase period on cut gladiolus spikes either with or without NS pulse treatments were performed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). As compared to the control treatment, they revealed that the 25 mg L−1 NS pulse treatment effectively inhibited bacterial colonization and biofilm formation on the stem-end cut surface and in the xylem vessels, respectively. In vitro culture of the bacterial microflora and analysis of biofilm architecture using CLSM revealed that NS treatment restricted bacterial biofilm formation. After static culture for 24 h at 35 °C with 25 mg L−1 NS in the medium, no biofilm form or structure was evident. Rather, only limited bacterial cell number and scanty extracellular polysaccharide (EPS) material were observed. In contrast, mature bacterial biofilm architecture comprised of abundant bacteria interwoven with EPS formed in the absence of NS.