Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis

Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.

Compartments of the foot: topographic anatomy

Recent publications have renewed the debate regarding the number of foot compartments. There is also no consensus regarding allocation of individual muscles and communication between compartments. The current study examines the anatomic topography of the foot compartments anew using 32 injections of epoxy-resin and subsequent sheet plastination in 12 cadaveric foot specimens. Six compartments were identified: dorsal, medial, lateral, superficial central, deep forefoot, and deep hindfoot compartments. Communication was evident between the deep hindfoot compartment and the superficial central and deep central forefoot compartments. In the hindfoot, the neurovascular bundles were located in separate tissue sheaths between the central hindfoot compartment and the medial compartment. In the forefoot, the medial and lateral bundles entered the deep central forefoot compartment. The deep central hindfoot compartment housed the quadratus plantae muscle, and after calcaneus fracture could develop an isolated compartment syndrome.

Resin composites: Modulus of elasticity and marginal quality

OBJECTIVE To investigate how the modulus of elasticity of resin composites influences marginal quality in restorations submitted to thermocyclic and mechanical loading. METHODS Charisma, Filtek Supreme XTE and Grandio were selected as they were found to possess different moduli of elasticity but quite similar polymerization contraction. MOD cavities (n=30) were prepared in extracted premolars, restored and then subjected to thermocyclic and mechanical loading. Marginal quality of the restorations before and after loading was analyzed on epoxy replicas under a scanning electron microscope. The percentage of gap-free margins and occurrence of paramarginal fractures were registered. Modulus of elasticity and polymerization contraction were analyzed with parametric and margins with nonparametric ANOVA and post hoc Tukey HSD or Wilcoxon rank-sum tests, respectively. The number of paramarginal fractures was analyzed with exact Fisher tests (α=0.05). RESULTS Grandio demonstrated significantly more gap-free enamel margins than Charisma and Filtek Supreme XTE, before and after loading (p<0.01), whereas there was no difference between Charisma and Filtek Supreme XTE (p>0.05). No significant effect of resin composite (p=0.81) on the quality of dentine margins was observed, before or after loading. Deterioration of all margins was evident after loading (p<0.0001). More paramarginal enamel fractures were observed after loading in teeth restored with Grandio when compared to Charisma (p=0.008). CONCLUSIONS The resin composite with the highest modulus of elasticity resulted in the highest number of gap-free enamel margins but with an increased incidence of paramarginal enamel fractures. CLINICAL SIGNIFICANCE The results from this study suggest that the marginal quality of restorations can be improved by the selection of a resin composite with modulus of elasticity close to that of dentine, although an increase in paramarginal enamel fractures can result as a consequence.