Direct measurement of helical cell motion of the spirochete leptospira.

Leptospira are spirochete bacteria distinguished by a short-pitch coiled body and intracellular flagella. Leptospira cells swim in liquid with an asymmetric morphology of the cell body; the anterior end has a long-pitch spiral shape (S-end) and the posterior end is hook-shaped (H-end). Although the S-end and the coiled cell body called the protoplasmic cylinder are thought to be responsible for propulsion together, most observations on the motion mechanism have remained qualitative. In this study, we analyzed the swimming speed and rotation rate of the S-end, protoplasmic cylinder, and H-end of individual Leptospira cells by one-sided dark-field microscopy. At various viscosities of media containing different concentrations of Ficoll, the rotation rate of the S-end and protoplasmic cylinder showed a clear correlation with the swimming speed, suggesting that these two helical parts play a central role in the motion of Leptospira. In contrast, the H-end rotation rate was unstable and showed much less correlation with the swimming speed. Forces produced by the rotation of the S-end and protoplasmic cylinder showed that these two helical parts contribute to propulsion at nearly equal magnitude. Torque generated by each part, also obtained from experimental motion parameters, indicated that the flagellar motor can generate torque >4000 pN nm, twice as large as that of Escherichia coli. Furthermore, the S-end torque was found to show a markedly larger fluctuation than the protoplasmic cylinder torque, suggesting that the unstable H-end rotation might be mechanically related to changes in the S-end rotation rate for torque balance of the entire cell. Variations in torque at the anterior and posterior ends of the Leptospira cell body could be transmitted from one end to the other through the cell body to coordinate the morphological transformations of the two ends for a rapid change in the swimming direction.

Simple and rapid method for selective enumeration of lactic acid bacteria in commercially prepared yogurt by image analysis and K-means clustering.

We developed a simple and rapid method based on the combination of image analysis and k-means clustering to selectively enumerate cocci and bacilli from among lactic acid bacteria (LAB) in commercially prepared yogurt. We used our previously reported method for recovering only LAB without non-microbial substances from commercial yogurt, and found that the shape and light intensity of LAB cell images taken by optical microscopy were factors that could distinguish cocci and bacilli, allowing the selective enumeration of LAB. Also, k-means clustering was executed on a dataset of the mean light intensity and aspect ratio of each LAB obtained by image analysis, and each LAB in the image could be automatically assigned to either the cocci or bacilli group. The results obtained by this automated method were in good agreement with those obtained by manually counting the LAB under a microscope, with an overall error within 10%. In addition, this method could provide results within a few hours, which is approximately 1/32 of the time required for the conventional colony-counting method.

Volatile biomarkers for early-stage detection of insect-infested brown rice: Isopentenols and polysulfides.

To reduce food loss from stored products by insect attack, monitoring and early detection of insects are essential. Presently, monitoring with pheromone traps is the primary method for detection; however, traps are effective only after the insects propagate. Detection and identification of the early volatile biomarkers arising from insect-infested brown rice was performed in this study to develop an alternative detection strategy. Brown rice was infested with eggs of seven insect species, including Sitophilus zeamais and Plodia interpunctella. Infested rice emitted at least one of the volatile compounds prenol, isoprenol, dimethyl disulfide, and dimethyl trisulfide (DMTS). In particular, isopentenols were generated by moths within one week of infestation, whereas they were not released from non-infested rice. DMTS was detected from all insect-infested brown rice, especially S. zeamais and P. interpunctella. These volatiles are potential early biomarkers for the presence of insects in brown rice.

Direct Imaging of Carboxymethyl Cellulose-mediated Aggregation of Lactic Acid Bacteria Using Dark-field Microscopy.

The ecological functions of lactic acid bacteria (LAB) have been utilized in human life for food processing and probiotic therapy. Understanding the interaction mechanisms between LAB and food ingredients may help to clarify the fermentation process and physiological functions of LAB in the production of fermented foods made from plant materials and dairy products. However, the interaction mechanisms have yet to be fully clarified. Although laser diffraction was used for measuring the size changes of aggregates caused by the interaction between LAB and food ingredients, aggregate sizes could not be determined because of the precipitation of aggregates and its disruption from stirring. Therefore, a microscopy-based method for directly visualizing their interactions is required. We directly observed aggregation processes of LAB cells mediated by water-soluble polysaccharides, carboxymethyl cellulose (CMC), by dark-filed microscopy (DFM). DFM could visualize CMC-mediated cell aggregation with high contrast in real time, and revealed that the aggregates were formed by repeated collisions of LAB cells in a suspension. This suggests that our method can be used as a useful assay to directly visualize grain formation caused by interactions between LAB cells and various polysaccharides in food ingredients.

Forced rotation of Na+-driven flagellar motor in a coupling ion-free environment.

Rotational characteristics of Na+-driven flagellar motor in the presence and absence of coupling ion were analyzed by electrorotation method. The motor rotated spontaneously in the presence of Na+, and the rotation accelerated or decelerated following the direction of the applied external torque. The spontaneous motor rotation was inhibited by removal of external Na+, however, the motor could be forcibly rotated by relatively small external torque applied by the electrorotation apparatus. The observed characteristic of the motor was completely different from that of ATP-driven motor systems, which form rigor bond when their energy source, ATP, is absent. The internal resistance of the flagellar motor increased significantly when the coupling ion could not access the inside of the motor, suggesting that the interaction between the rotor and the stator is changed by the binding of the coupling ion to the internal sites of the motor.

Asymmetric swimming pattern of Vibrio alginolyticus cells with single polar flagella.

The swimming pattern of bacteria with single polar flagella has usually been described as "run and reverse". We observed the swimming traces of monotrichously flagellated Vibrio alginolyticus cells and examined the relationship between the swimming pattern and the sense of progress. Swimming in regions other than a solid surface was confirmed to be linear run and reverse. Near a solid surface, the traces consisted of "run and arc"; the cells were found to curve sharply during backward swimming, while they progressed linearly during forward swimming. The "run and arc" swimming pattern may play an important role in the chemotaxis strategy of marine bacteria at solid surfaces.

Effect of risk information exposure on consumers' responses to foods with insect contamination.

This study explores the impact that scientific information about insect contamination of food has on consumer perceptions. Participants (n = 320, Japanese consumers) were randomly assigned to 1 of 8 information-type conditions: (1) information about insect type, (2) information about contamination processes, (3) information about the safety of contaminated food, (4, 5, 6) combinations of 2 of (1), (2), and (3) above, (7) all information, and (8) no-information, and asked to rate their valuation, behavioral intention, and attitude toward food with insect contamination. Results demonstrated that some combinations of scientific information that include the safety of the contaminated food are effective to reduce consumers' compulsive rejection of insect contamination in food, whereas the single presentation of information about insect type increases consumers' explicit rejection of both the contaminated product and the manufacturer. These findings have implications for the coordination of risk communication strategies.

On-chip cell immobilization and monitoring system using thermosensitive gel controlled by suspended polymeric microbridge.

In this paper, we describe the fabrication of a temperature-controlled microfluidic chip for cell immobilization using a thermosensitive hydrogel of poly (N-isopropylacrylamide) (PNIPAAm). A mixture of PNIPAAm solution, yeast cells, and Calcein-AM fluorescent dye is flowed in the microchannel, and the indium-tin-oxide (ITO) microheaters that were fabricated by micromachining technology heat a PNIPAAm gel. However, if the gel is very thick, it blocks the observation of the culturing cells and reduces the SNR. To address this problem, we fabricate a suspended microbridge above the microheater that limits the height of the gel, ensuring that it forms a thin and transparent layer above the heater. Microheaters and suspended biocompatible microbridge are integrated on a chip in which yeast-cell immobilization can be performed by gelation of a PNIPAAm solution.

A function of polar flagellum and anisotropic growth in Vibrio alginolyticus early-phase colonies.

We continuously observed growth of Vibrio alginolyticus early-phase colonies on agar plates by phase-contrast microscopy. Two mutants defective in motility on solid surfaces were used in this study: one (YM4) can swim in liquid environments using its polar flagellum, and the other (NMB198) cannot swim because it lacks any flagella. We found that isolated colonies of YM4 were generally more circular than those of NMB198. This observation suggests that YM4 cells moved slightly within a colony by the function of their polar flagella. For clustered colonies, where the distance between the colonies was short (<50 microm), the colonies of YM4 grew rapidly along the line between them, but they grew slowly in the lateral directions. Some colonies of NMB198 grew toward neighboring colonies. These observations indicate colony-to-colony interaction.

Difference in bacterial motion between forward and backward swimming caused by the wall effect.

A bacterial cell that has a single polar flagellum alternately repeats forward swimming, in which the flagellum pushes the cell body, and backward swimming, in which the flagellum pulls the cell body. We have reported that the backward swimming speeds of Vibrio alginolyticus are on average greater than the forward swimming speeds. In this study, we quantitatively measured the shape of the trajectory as well as the swimming speed. The trajectory shape in the forward mode was almost straight, whereas that in the backward mode was curved. The same parameters were measured at different distances from a surface. The difference in the motion characteristics between swimming modes was significant when a cell swam near a surface. In contrast, the difference was indistinguishable when a cell swam >60 microm away from any surfaces. In addition, a cell in backward mode tended to stay near the surface longer than a cell in forward mode. This wall effect on the bacterial motion was independent of chemical modification of the glass surface. The macroscopic behavior is numerically simulated on the basis of experimental results and the significance of the phenomenon reported here is discussed.

A fluid-dynamic interpretation of the asymmetric motion of singly flagellated bacteria swimming close to a boundary.

The singly flagellated bacterium, Vibrio alginolyticus, moves forward and backward by alternating the rotational direction of its flagellum. The bacterium has been observed retracing a previous path almost exactly and swimming in a zigzag pattern. In the presence of a boundary, however, the motion changes significantly, to something closer to a circular trajectory. Additionally, when the cell swims close to a wall, the forward and backward speeds differ noticeably. This study details a boundary element model for the motion of a bacterium swimming near a rigid boundary and the results of numerical analyses conducted using this model. The results reveal that bacterium motion is apparently influenced by pitch angle, i.e., the angle between the boundary and the swimming direction, and that forward motion is more stable than backward motion with respect to pitching of the bacterium. From these results, a set of diagrammatic representations have been created that explain the observed asymmetry in trajectory and speed between the forward and backward motions. For forward motion, a cell moving parallel to the boundary will maintain this trajectory. However, for backward motion, the resulting trajectory depends upon whether the bacterium is approaching or departing the boundary. Fluid-dynamic interactions between the flagellum and the boundary vary with cell orientation and cause peculiarities in the resulting trajectories.

A mathematical explanation of an increase in bacterial swimming speed with viscosity in linear-polymer solutions.

Bacterial swimming speed is sometimes known to increase with viscosity. This phenomenon is peculiar to bacterial motion. Berg and Turner (Nature. 278:349-351, 1979) indicated that the phenomenon was caused by a loose, quasi-rigid network formed by polymer molecules that were added to increase viscosity. We mathematically developed their concept by introducing two apparent viscosities and obtained results similar to the experimental data reported before. Addition of polymer improved the propulsion efficiency, which surpasses the decline in flagellar rotation rate, and the swimming speed increased with viscosity.

Reevaluation and reduction of a PCR bias caused by reannealing of templates.

We reevaluated the bias toward a 1:1 ratio of products in multitemplate PCR used in ecological studies and showed that the template reannealing at the annealing step would not cause the bias; however, the preferential homoduplex formation during temperature decrease from denaturation to annealing step would cause the bias.