Atomic force microscopy of Bacillus spore surface morphology

Bacillus spore surface morphology was imaged with atomic force microscopy (AFM) to determine if characteristic surface features could be used to distinguish between four closely related species; Bacillus anthracis Sterne strain, Bacillus thuringiensis var. kurstaki, Bacillus cereus strain 569, and Bacillus globigii var. niger. AFM surface height images showed an irregular topography across the curved upper surface of the spores. Phase images showed a superficial grain structure with different levels of phase contrast and significant differences in average surface morphologies among the four species. Although spores of the same species showed similarities, there was significant variability within each species. Overall, AFM revealed that spore surface morphology is rich with information, which can be used to distinguish a sample of about 20 spores from a similar number of spores of closely related species. Statistical analysis of spore morphology from a combination of amplitude and phase images for a small sample allows differentiation between, B. anthracis and its close relatives.     

Using capillary forces to estimate the adhesion strength of Magnaporthe grisea spores on glass

The Magnaporthe grisea is a fungus whose spores strongly adhere to plant leaves, and to solid surfaces in general. In this note, we give an estimate of the adhesion force, F_det, of a spore on a glass surface, in water. F_det is defined as the force to be applied to a spore to pull it out from the surface. In our experiments, spores are detached from the glass in a few milliseconds by means of a capillary force. The latter is provided by a water/air or a water/oil interface, in contact with the spore body. We thus find F_det of the order of 10⁻⁶ N.     

Strong tolerance of blue-green alga Microcystis flos-aquae to very high pressure

It was shown in our previous reports that a few spores of moss Venturiella could tolerate the very high pressure of 20 GPa for 30 min and germinated a protonema to the length of 30 μm. However, these spores did not grow any further, and disappeared at around 30 days of incubation after seeded. On the other hand, colonies of blue-green alga Microcystis flos-aquae came to appear about 76 days after the moss spores were seeded. Many of these colonies appeared at the places where the moss spores had disappeared. These colonies were formed by the algae that had adhered to the spore cases of the moss and survived after exposure to the very high pressure of 20 GPa. Though the appearance of the colonies of high pressure exposed algae was delayed by about 50 days compared with that of the control group which was not exposed to high pressure, there seems no difference in their shape and color from those of the control group. The pressure tolerance of blue-green alga is found to be enormously strong, and it can survive after exposure to the high pressure which corresponds to the depth of about 550–600 km from the surface of the Earth, just above the lower mantle.     

拉曼光谱测定单个细菌芽孢吡啶二羧酸浓度及机理研究

应用激光镊子拉曼光谱技术(LTRS)测定19株芽孢杆菌的单个细菌芽孢吡啶二羧酸(DPA)浓度,并验证了系统的重现性。一束30 mW,785 nm的近红外激光导入倒置显微镜,形成光镊,随机俘获水溶液中的单个芽孢,同时收集其拉曼光谱信号。细菌芽孢的DPA是以与钙离子形成的鳌合物(Ca-DPA)形式存在,收集到的芽孢拉曼光谱信号反映的是Ca-DPA信息,选用信号最强、与Ca-DPA浓度成线性关系的1 017 cm-1作为其定量的谱峰。通过1 017 cm-1的峰强,推算出Ca-DPA的浓度。结果显示,单个芽孢的拉曼光谱能够很好地反映出单个芽孢的特征信息,不同种、同种不同菌株之间的芽孢的DPA浓度有所不同,同一菌株的不同芽孢个体之间DPA浓度也存在差异。该方法不需要复杂的分离、纯化过程,可在水溶液中直接俘获单个芽孢并收集其拉曼光谱信号,便可得到单个芽孢的信息,读出DPA的浓度水平。     

Probing the mechanism and Ca-DPA concentration of individual Bacillus spores using trapping and Raman spectroscopy

Measuring the levels of 2,6-pyridine dicarboxylic acid (DPA) in bacteria spores could provide the information about the DPA function, resistance mechanism and the mechanism of spore germination. The authors have measured levels of Ca-DPA of individual spores of different 19 kinds of Bacillus which from different sources, species, and strains by using laser tweezers Raman spectroscopy (LTRS). Also we have verified the reproducibility of the system simultaneously. To investigate the biochemical components and structure in single spore, a Raman tweezers setup was used to record the Raman spectrum of single spore. A NIR laser beam (30 mW, 785 nm) was introduced into an inverted microscope to form a tweezers for trapping the spore suspended in water, and the Raman scatter was excited by the same beam. Raman spectra of 30 spores of 19 bacillus strains which collected from different area in China were recorded, and 100 spores of B. subtilis ACCC10243 were measured. A spore of the same strain was probed 100 times for verifying the reproducibility of the LTRS system. A Matlab 7.0 edited program and Origin 8.0 were used to process the spectral data. Because Ca-DPA is the chelate of DPA and the calcium ion, and the strongest Raman bands at 1 017 cm(-1) was from Ca-DPA component of the spore, its intensity was linearly with the Ca-DPA concentration. Therefore, the 1017 cm(-1) bands of Ca-DPA could be used as the quantitative standard peak, and then calculated the concentration of Ca-DPA could be calculated according the intensity of 1017 cm(-1) peak. The results showed that Raman spectra of single spore can reflect the characteristics information of it. The diversity of Ca-DPA levels not only happened between different species and strains of bacillus, but also happened between different individual spores in the same strains of bacillus. Conclusion from these measurements is that there is heterogeneity in different individual spores. It is convenient to trapping and collecting its Raman spectrum in water directly, and then get the information of the level of DPA, without the complex preparation of separating, purifying spores and abstracting DPA, so we predict LTRS as a high sensitivity, high accuracy, rapid and effective method in the research of individual spores.     

Preserving life of moss Ptychomitrium under very high pressure

The experiment on the search for life under very high pressure done on small animal, tardigrade, has been extended to the moss Ptychomitrium. Several spore placentas of the moss Ptychomitrium were sealed in a small Teflon capsule together with fluorinate as liquid pressure medium. The capsule was put in the center of a pyrophillite cube. This cube was compressed by six tungsten-carbide second-stage anvils with a front edge length of 4.0 mm. These anvils were compressed by a first stage, 250 ton press. It was proven that 80-90% of the spores were alive and germinated after being exposed to the maximum pressure of 7.5 GPa for up to 48 h. Furthermore, a relatively high germination rate of about 35% was retained even after exposure to 7.5 GPa for 6 days. The pressure tolerance of moss is found to be much stronger than tardigrades.     

光镊捕获金福菇孢子的拉曼光谱分析

使用光镊拉曼光谱系统俘获悬浮在生理盐水中的单个金福菇孢子,激发并收集其拉曼信号,结果显示单个金福菇孢子的拉曼光谱基本能呈现其内含物的组成和结构信息,脂类物质是其主要成分。同种栽培方式的3个不同菌株单个金福菇孢子的平均拉曼光谱信号基本一致,分别对其进行多元统计分析(PCA),无法区分其拉曼光谱信号;同一菌株不同栽培方式的单个孢子平均拉曼光谱的差异光谱表明其主要成分相同,都以脂类物质为主;而源自于脂类1743、1655、1442、1125、1080、1070、876、728cm-1等峰的信号强度基本一致,说明其脂类的含量基本相同。从单细胞拉曼光谱角度初步分析金福菇孢子,为光镊拉曼光谱技术研究食用菌孢子萌发机理提供有意义的参考。     

Development of Aptamer Beacons for Rapid Presumptive Detection of <Emphasis Type="Italic">Bacillus</Emphasis> Spores

A library of 92 DNA aptamer sequences was developed against Bacillus anthracis (nonpathogenic Sterne strain) spores and anthrose sugar immobilized on magnetic beads. The selected DNA sequences were studied for similarities and potential binding pockets between the B. anthracis spore and anthrose aptamers. Several recurring loop structures were identified and tested for their potential to act as aptamer beacons when labeled with TYE 665 dye on their 5′ ends and Iowa Black quencher on their 3′ ends. Of these candidate sequences, two beacons designated BAS-6F and BAS-6R emerged which gave strong fluorescence responses at high spore concentrations (greater than 30,000 spores/ml). These aptamer beacons also detect B. cereus and B. thuringiensis spores with greater fluorescence intensity, but do not strongly detect vegetative cells from an array of other bacterial species. BAS-6F and 6R are also not capable of detecting pure anthrose, thereby probably ruling that epitope out as a spore surface target for these particular beacons. While not extremely sensitive, the BAS-6F and 6R aptamer beacons are potentially valuable for rapid presumptive detection of anthrax or Bacillus spores in suspect powders or bioterrorist activity where spore concentrations are anticipated to be high. The sequence similarities of these beacons to other published Bacillus spore aptamers are also discussed.     

Nanomechanical characteristics of SnO2:F thin films deposited by chemical vapor deposition

The nanoindentation characterizations and mechanical properties of fluorine-doped tin oxide (SnO₂:F) films deposited on glass substrates, using chemical vapor deposition (CVD) method, were studied, which included the effects of the indentation loads, the loading time and the hold time on the thin film. The surface roughness, fractal dimension and frictional coefficient were also studied by varying the freon flow rates. X-ray diffraction (XRD), atomic force microscopy (AFM) and frictional force microscopy (FFM) were used to analyze the morphology of the microstructure. The results showed that crystalline structure of the film had a high intensity (1 1 0) peak orientation, especially at a low freon flow rate. According to the nanoindentation records, the Young's modulus ranged from 62.4 to 75.1 GPa and the hardness ranged from 5.1 to 9.9 GPa at a freon flow rate of 8000 sccm. The changes in measured properties were due to changing loading rate.     

Direct whole-mount imaging of fungal spores by energy-filtering transmission electron microscopy

Whole-mount fungal spores were examined by energy-filtering transmission electron microscopy. Conidia of Penicillium species and Ustilaginoidea virens were suspended in distilled water and directly placed on a glow-discharged formvar-coated copper grid. Energy-filtered images were taken from 0 to 100eV loss regions. Due to their considerable inherent thickness, their globose morphology was evident. In zero-loss images, the fungal spores appeared to have higher contrast in general, showing darker periphery than unfiltered images. Most spores in zero-loss images exhibited almost homogeneous electron density across the spores. The contrast was partially inversed in low-loss images where more details of the outer cell wall ornamentations of spores could be discerned than zero-loss images. As obvious advantages of whole-mount spore imaging, it allows for ensuring two-dimensional images with higher spatial resolution than light microscopy and conventional scanning electron microscopy. If a higher resolution is needed to observe fungal surface structures such as fimbriae and rodlet layers, or discriminate an outer sheath enveloping spores, whole-mount spore imaging can be employed to unravel structural details.     

Distortion of spores of moss Venturiella under ultra high pressure

In our previous studies on the tolerance of living organisms such as planktons and spores of mosses to the high hydrostatic pressure of 7.5 GPa, we showed that all the samples could be borne at this high pressure. These studies have been extended to the extreme high pressure of 20 GPa by using a Kawai-type octahedral anvil press. It was found that the average diameter of the spores of Venturiella exposed to 20 GPa for 30 min was 25.5 μm, which is 16.5% smaller (40.0% smaller in volume) than that of the control group which was not exposed to high pressure. The inner organisms showed a further extent of plastic deformation. As a result, a gap appeared between the outer cover and the cytoplasm. A relationship has been obtained between the survival ratio and plastic deformation of spores of moss Venturiella caused by the application of ultra high pressure.     

热压协同处理对接种于鸡腿菇中的细菌芽孢灭活效果的影响

以鸡腿菇为原料,以凝结芽孢杆菌芽孢和嗜热脂肪芽孢杆菌芽孢为对象菌,研究中温与超高静压协同处理条件下两种对象菌的灭活效果及其相应的动力学变化趋势。研究结果表明:在相同条件下,鸡腿菇中凝结芽孢杆菌芽孢的致死效果优于嗜热脂肪芽孢杆菌;接种于鸡腿菇中的两种对象菌芽孢致死效果均低于磷酸缓冲液中的效果,说明鸡腿菇对这两种细菌芽孢有一定的保护作用。利用线性模型、Weibull模型和Log-logistic模型,对鸡腿菇中两种对象菌芽孢的灭活效果进行拟合,以均方差和回归系数为评价指标,分析了3种模型的拟合效果。结果表明:在所研究的条件下,Log-logistic模型的拟合效果最好,Weibull模型次之,线性模型最差。因此可以通过Log-logistic模型来预测热压协同处理下细菌芽孢的灭活效果。     

Laser‐Raman and atomic force microscopy assessment of the chlorococcalean affinity of problematic microfossils

Organic‐walled microfossils of uncertain origin, classified to an informal group named acritarchs, are most commonly interpreted as the resting cysts of marine eukaryotic phytoplankton. Some acritarchs have recently been interpreted as vegetative cells of chlorococcalean green algae, based on internal bodies that have been interpreted as their asexual reproductive structures (spores). To verify this interpretation, we applied confocal Raman spectroscopy and atomic force microscopy (AFM) to study the ultrastructure and nanostructure of exceptionally preserved acritarchs with internal bodies from the early Silurian cherts (ca 430 Ma‐old) of Frankenwald (Germany). Three‐dimensional Raman mapping showed the spatial distribution of carbonaceous material and other minerals in the walls of the analysed internal bodies and confirmed that these structures are comparable with spores of chlorococcalean microalgae. Our findings document therefore the oldest thus far known vegetative cells of sporulating green algae. The combination of confocal Raman and AFM techniques yielded detailed information about the nanostructure and fossilisation mode of the mineralised organic walls of both the central vesicles and the enclosed spore‐like bodies. Copyright © 2011 John Wiley & Sons, Ltd.     

Inactivation of Bucillus Stearothermophilus by pulsed electricfield

B. Stearothermophilus remains alive even under intense electric field, since spores formed are highly resistant to damage from electric field effects. This paper describes the effects of pulsed electric field on B. Stearothermophilus (ATCC no, 12980). An aqueous suspension of B. Stearothermophilus spores was placed between parallel planar electrodes and subjected to pulsed electric fields of up to 189 kV/cm, which are obtained from a high-voltage pulsed voltage generator. Either a Blumlein generator or a magnetic compression circuit with a semiconductor opening switch was used as the pulsed voltage generator. The spores that survived the treatment were detected by a colony counting method. The growth activities were measured using a microbial calorimetry technique. The temperature and pH of the spore suspension remained unchanged during the treatment. It was found that the growth activity of B. Stearothermophilus was affected by the pulsed electric field. while no remarkable lethal effect was observed in the range of up to 40 kV/cm     

Moderate hydrostatic pressure–temperature combinations for inactivation of Bacillus subtilis spores

We report the effect of using moderate hydrostatic pressure, 40–140?MPa, at moderate temperature (38–58°C) to inactivate Bacillus subtilis spores in McIlvaine's citric phosphate buffer at pH 6. We have investigated several parameters: pressure applied, holding time, pressure cycling, and temperature. The kinetics of spore inactivation is reported. The results show that spore inactivation is exponentially proportional to the time the sample is exposed to pressure. Spore germination and inactivation occur at the hydrostatic pressures/temperature combinations we explored. Cycling the pressure while keeping the total time at high pressure constant does not significantly increase spore inactivation. We show that temperature increases spore inactivation at two different rates; a slow rate below 33°C, and at a more rapid rate at higher temperatures. Increasing pressure leads to an increase in spore inactivation below 95?MPa; however, further increases in pressure give a similar rate kill. The time dependence of the effect of pressure is consistent with the first-order model (R²?>?0.9). The thermal resistance values (Z_T) of B. subtilis spores are 30°C, 37°C, and 40°C at 60, 80, 100?MPa. The increase in Z_T value at higher pressures indicates lower temperature sensitivity. The pressure resistance values (Z_P) are 125, 125 and 143?MPa at 38°C, 48°C, and 58°C. These Z_P values are lower than those reported for B. subtilis spores in the literature, which indicates higher sensitivity at pressures less than about 140?MPa. We show that at temperatures <60°C, B. subtilis spores are inactivated at pressures below 100?MPa. This finding could have implications for the design of the sterilization equipment.     

Inactivation of food-borne enteropathogenic bacteria and spoilagefungi using pulsed-light

The lethality of high-intensity pulsed-light emissions from low and high ultraviolet (UV) light sources on predetermined microbial populations has been investigated. Prior to treatment, the bacterial enteropathogens Bacillus cereus, Escherichia coli, and Salmonella enteritidis and the food-spoilage fungi Aspergillus niger and Fusarium culmorum were seeded separately onto the surface of either tryptone soya yeast extract or malt extract agar plates. Prescribed microbial population densities were applied to the test media and these samples were exposed to one of two light sources. These were low-pressure, xenon filled, flash lamps that produced either high or low UV intensities. They were operated in pulsed mode, being driven by a stacked Blurnlein table generator. Microbial samples were treated by exposure to different numbers of light pulses. The treated bacterial populations were reduced by ~8 log orders after 1000 light-pulses of the higher UV intensity light and the fungal counts had a corresponding reduction of 4.5 log orders. The fungus, Aspergillus niger, was shown to be significantly more resistant in spore form to the intense UV light compared with Fusarium culmorum. This resistance has been attributed to the high level of UV absorbance associated with the dark pigment present in A. niger. The pulsed light source of lower UV intensity was shown to be significantly less effective in reducing microbial populations     

Surface Modified Long Period Fiber Grating Sensor for Rapid Detection of Aspergillus Niger Fungal Spores

We present development of a compact and label-free sensor based on the surface modification of copper vapor laser fabricated long period fiber gratings for detection of airborne Aspergillus niger (A. niger) fungal spores. Surface of sensors were functionalized with monoclonal glucose oxidases IgG1 for target-specific covalent binding. In process of functionalization and binding of 10³ cfu/ml of pathogenic A. niger fungal spores, notable shorter wave transition in resonance wavelength from 1562.93 nm to 1555.97 nm, and significant reduction in peak loss from 61.72 dB to 57.48 dB were recorded. The implementation was cost effective and yielded instantaneous results.     

High precision localization methods for HZE-particles

For experimental investigations in the field of microdosimetry of heavy ions a high precision localization method for biological objects relative to the path of the penetrating ion is mandatory. New methods are described for corn seeds of Arabidopsis thaliana and spores of Bacillus subtilis. In the latter case the actual penetration point of the particle in the spore layer is determined by an individual microetching technique with an accuracy of about 0.2 μm. This is done after removing the spores from the hit area by “break-through” etching. With the help of reference spores the impact parameter is determined for correlating the biological effects with the physical event. Besides the aspect of water during the track development is described.     

Angularly resolved light scattering from aerosolized spores: observations and calculations

Angularly resolved elastic light scattering patterns from individual aerosolized Bacillus subtilis spores were qualitatively compared with simulations. Two-dimensional angular optical scattering patterns of the spores were collected for polar scattering angles varying from approximately 77 degrees to 130 degrees and azimuthal angles varying from 0 degrees to 360 degrees . Computations were performed with single T-matrix formalism by simulating a spore with three different particle shapes: (1) a finite-length cylinder with spherical end caps, (2) a spheroid, and (3) two spheres in contact. Excellent agreement between computation and measurement was found for the finite-length cylinder with spherical end caps, poorer agreement was found for the spheroids, and the poorest agreement was for the two spheres in contact.