Comparative Investigation of Alternative Negative Staining Reagents in Bacterial Morphological Study

Negative staining is an effective method that can be used for electron microscopic study to observe fine structural morphology without destruction of bacterial structure. Although uranium acetate is used worldwide as a general dyeing solution, it is extremely difficult to use it by a new purchase at a research institution because it falls under the nuclear regulation substance in Japan. Therefore, we examined alternative reagents for negative staining that could replace uranium acetate through bacterial observation with an electron microscope. Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pyogenes were examined by four stain reagents (phosphotungstic acid (PTA), EMstainer, TI blue, and uranium acetate). Pre cultured bacteria were stained with each stain reagents on a copper grid, washed with PBS, and observed with a transmission electron microscope. In the comparison between bacterial structures, the cell wall structure and bacterial flagella could be observed well in the order of PTA, EMstainer, and uranium acetate. With TI blue staining, flagella could be observed very poorly. In comparison between bacteria, gram negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, could be observed well as compared with gram positive cocci such as Staphylococcus aureus and Streptococcus pyogenes. The uranium acetate looked very coarse in background particles. Since crystals tend to precipitate, TI blue also required filtering, and electron beams were absorbed by the agglomerated crystals, and the frequency of electronic burning occurred high frequency. In this study, there was clear difference in the observation conditions depending on the type of bacteria and the kind of the staining reagents. Especially, it was confirmed that good negative staining features of Pseudomonas aeruginosa by electron microscope were obtained by PTA and EMstainer staining. These alternative reagents are considered to be a candidate for a negative staining.

LicT Modulates Biofilm Formation of Streptococcus pyogenes

Streptococcus pyogenes frequently causes purulent infections in humans. Biofilm formation is an important virulence property of S. pyogenes because of decreased susceptibility of bacteria to antibiotic treatment. Biofilm is composed of various types of matrix including glycocalyx which is an important exocellular matrix material related to bacterial sugar metabolism. A putative antiterminator protein, LicT (Spy0571), is one of the components of the glucose-independent β-gluco-side-specific phosphotransferase system (PTS). Although the PTS, a carbohydrate metabolic system, may play a role in biofilm formation, the relationship between LicT and biofilm formation has not yet been elucidated. Here, we evaluated whether LicT affected biofilm formation in modified chemically defined medium (CDMM) supplemented with glucose or β-glucoside:salicin. We created licT- and licT-complemented mutant strains from S. pyogenes 1529. Although the licT mutant strain tended to have higher growth rate than wild-typestrain in CDMM with glucose, it had a significant lower growth rate than the wild-type strain in CDMM with salicin. In addition, the licT mutant exhibited lower biofilm formation in CDMM containing salicin than the wild-type strain by 96 well plate analysis and confocal laser scanning microscopic analysis. Our results suggest that LicT plays an important role in biofilm formation of S. pyogenes.

Admission and Traffic Control Schemes Suitable for QoS Applications in WLAN Systems

This paper proposes the use of admission and traffic control schemes for real-time applications. The admission control scheme determines the admission of high-priority real-time applications such as voice and video streams in terms of their bandwidth utilization time (medium time), whereas the traffic control scheme maintains the communication quality of applications permitted admission by restricting other traffic. Owing to the use of contention-based access, a conventional scheme without admission control will degrade the communication quality when the number of terminals using high-priority applications increases. Moreover, only the capabilities (i.e., frame and sequence procedures) of admission control are defined in the IEEE 802.11e standard;the detailed usage in terms of the application characteristics is not specified, and it may be difficult to achieve a sufficient level of quality of service (QoS). The proposed schemes achieve the optimum QoS for actual services. The software used in the proposed schemes was implemented into hardware at the access point, and was evaluated experimentally. Based on the evaluation results, excellent performances with high QoS applications were obtained.