A Color Image Analysis Method for Assessment of Germination Based on Differential Fluorescence Staining of Bacterial Spores and Vegetative Cells Using Acridine Orange

Color fluorescence image analysis of acridine orange (AO) stained germinating Bacillus subtilis var. niger bacteria revealed a cell population initially dominated by small green spores followed by the emergence of at least three additional discernible subpopulations in response to stimulation with D-glucose. These subpopulations were small, round or oblong red cells; intermediate to large metachromatic cells; and large red rods. Large green rods were rarely observed. An increase in red emissions (i.e., putative RNA synthesis) was sometimes seen as early as 90 min after exposure to D-glucose and uptake of AO at room temperature. This may represent either metabolic recovery from quiescence or RNA synthesis associated with germination. In the absence of D-glucose, or using autoclaved bacteria in the presence of glucose, no relative increase in the red signal was observed despite hours of observation. Digital image analysis was used for relative measurement of red, green and blue signals and to correlate the size of various subpopulations with their fluorescence color emissions over time. Image analysis demonstrated a trend toward increasing size and red emission in the presence of glucose. The average red emission was found to be a good discriminator of the various subpopulations, while the average green emission was approximately equal among the subpopulations making it a poor discriminator. These data suggest that AO staining might be used for rapid computer-assisted discrimination of spores vs. vegetative cells.     

NADH荧光法快速检测细菌总数

基于细菌胞内NADH的荧光特性及其在胞内含量稳定的特性, 建立一种快速检测细菌总数的新方法。该荧光法的NADH检测限为1 nmol/L, NADH含量在10 nmol/L~0.2 mmol/L间与荧光强度呈良好线性关系(R2 =0.9905)。经离心获得菌体细胞, 热Tris-HCl法提取胞内NADH, 以 342 nm为激发波长, 461 nm为发射波长测定提取液荧光强度, 1 h内可检测到样品1×104 CFU/mL菌数。结果表明该方法快速、灵敏、简便、重复性好, 可适用于食品卫生与安全、环境检测等领域活细菌数量的定量检测。     

Protection of Bacterial Spores in Space, a Contribution to the Discussion on Panspermia

Spores of Bacillus subtilis were exposed to space in theBIOPAN facility of the European Space Agency onboard of the Russian Earth-orbiting FOTON satellite. The spores were exposed either in dry layers without any protecting agent, or mixed withclay, red sandstone, Martian analogue soil or meteorite powder,in dry layers as well as in so-called `artificial meteorites', i.e. cubes filled with clay and spores in naturally occurring concentrations. After about 2 weeks in space, their survival was tested from the number of colony formers. Unprotected spores in layers open to space or behind a quartz window were completely or nearly completely inactivated (survival rates in most cases10^-6). The same low survival was obtained behind a thin layer of clay acting as an optical filter. The survival rate was increased by 5 orders of magnitude and more, if the spores in the dry layer were directly mixed with powder of clay,rock or meteorites, and up to 100% survival was reached in soilmixtures with spores comparable to the natural soil to spore ratio. These data confirm the deleterious effects of extraterrestrial solar UV radiation. Thin layers of clay, rock or meteorite are only successful in UV-shielding, if they are indirect contact with the spores. The data suggest that in a scenario of interplanetary transfer of life, small rock ejecta ofa few cm in diameter could be sufficiently large to protectbacterial spores against the intense insolation; however, micron-sized grains, as originally requested by Panspermia, may notprovide sufficient protection for spores to survive. The data arealso pertinent to search for life on Mars and planetaryprotection considerations for future missions to Mars.     

Rapid Identification of ESKAPE Bacterial Strains Using an Autonomous Microfluidic Device

This article describes Bacteria ID Chips ('BacChips'): an inexpensive, portable, and autonomous microfluidic platform for identifying pathogenic strains of bacteria. BacChips consist of a set of microchambers and channels molded in the elastomeric polymer, poly(dimethylsiloxane) (PDMS). Each microchamber is preloaded with mono-, di-, or trisaccharides and dried. Pressing the layer of PDMS into contact with a glass coverslip forms the device; the footprint of the device in this article is ～6 cm(2). After assembly, BacChips are degased under large negative pressure and are stored in vacuum-sealed plastic bags. To use the device, the bag is opened, a sample containing bacteria is introduced at the inlet of the device, and the degased PDMS draws the sample into the central channel and chambers. After the liquid at the inlet is consumed, air is drawn into the BacChip via the inlet and provides a physical barrier that separates the liquid samples in adjacent microchambers. A pH indicator is admixed with the samples prior to their loading, enabling the metabolism of the dissolved saccharides in the microchambers to be visualized. Importantly, BacChips operate without external equipment or instruments. By visually detecting the growth of bacteria using ambient light after ～4 h, we demonstrate that BacChips with ten microchambers containing different saccharides can reproducibly detect the ESKAPE panel of pathogens, including strains of: Enterococcus faecalis, Enteroccocus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter aerogenes, and Enterobacter cloacae. This article describes a BacChip for point-of-care detection of ESKAPE pathogens and a starting point for designing multiplexed assays that identify bacterial strains from clinical samples and simultaneously determine their susceptibility to antibiotics.     

Development of a New, Combined Rapid Method Using Phage and PCR for Detection and Identification of Viable Mycobacterium paratuberculosis Bacteria within 48 Hours

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h.     

赤芝孢子粉中一个葡聚糖的分离纯化与结构鉴定

从破壁的赤芝 (Ganodermalucidum (Fr.)Karst)孢子粉中分离纯化到一个水溶性的、以 1,4连接的D_葡萄糖为主链、在 6位接 1,6连接葡萄糖的α_D 葡聚糖。高效分子排阻色谱测定 ,重均分子量为 9.3× 10 3 ,[α]2 1D = 174.2° (c 0 .87,H2 O)。通过糖组成分析 ,甲基化反应 ,乙酰解及一维和二维核磁等光谱解析 ,确定其结构如下 :　　　　　　　　　　　　　　　　α_D_Glcp_(1→ 6 )_α_D_Glcp_(1→ 6 )_α_D_Glcp　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　 1　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　↓　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　 6　　　　　　　〔→ 4)_α_D_Glcp_(1→ 4)_α_D_Glcp_(1→ 4)_α_D_Glcp_(1→ 4)_α_D_Glcp_(1→ 4_)_α_D_Glcp_(1→〕n     

Structural Characterization and Immunomodulating Activity of a Complex Glucan from Spores of Ganoderma lucidum

A polysaccharide with a molecular weight of 1.26×10⁵, obtained from the sporoderm-broken spores of Ganoderma lucidum, was purified by anion-exchange and gel-permeation chromatography. This polysaccharide had a strong effect on suppressing the antibody production and the Con A or LPS induced lymphocyte proliferation in mice. Chemically, the structure of the polysaccharide was identified by methylation analysis, 1D, 2D NMR and ESI-MS spectroscopic studies of the native one and of the oligosaccharide fragments generated by partial acid hydrolysis, Smith degradation, and acetolysis. It was concluded that the intact polysaccharide was a complex β-D-glucan consisting of a (1→6)-linked backbone chain, in which every other glucosyl residue was substituted at C-3 or C-4 with mono-, di- and trisaccharide branches.     

Spores of a New Microsporidan Species Parasitizing Molluscan Neurons

Synopsis.
A new species of Microsporida was observed in neurons of the marine mollusc Aplysia californica and the ultrastructure of its spores was investigated. The spores were ˜ 1.3 m long and had a thick 3-layered coat. An anchoring disc and polar aperture were present. The polaroplast occupied most of the anterior half of the spore. The vesicular portion included lengths of loosely packed lamellar structures as well as the usual vesicular profiles embedded in fibrillar material; it was entirely surrounded by tightly packed, electron-dense lamellae. The polar tube had 5 or 6 coils. In this stage of the life cycle (mature spores), the parasites did not appear to be disturbing the host cells. The organism was named Microsporidium aplysiae sp. n.     

Rapid and Simple Quantification of Bacterial Cells by Using a Microfluidic Device

This study investigated a microfluidic chip-based system (on-chip flow cytometry) for quantification of bacteria both in culture and in environmental samples. Bacterial numbers determined by this technique were similar to those obtained by direct microscopic count. The time required for this on-chip flow cytometry was only 30 min per 6 samples.     

2-Heptanone Production by Spores of Penicillium roquefortii in a Water-Organic Solvent Two-Phase System

The production of 2-heptanone from octanoic acid may be performed by free and entrapped spores of Penicillium roquefortii in a water-organic solvent two phase system.

An industrial, isoparafflnic solvent, i.e. Hydrosol IP 230 O.S., which may be considered as tetradecane, is well suited for the process. Activities nearly double those achieved with aqueous systems are observed using an initial fatty acid content in the organic layer close to 100 mM and a ratio of the volume of the organic phase to the total volume of the medium of 0.88. The presence of the solvent allows a better recovery of the metabolite by lowering its activity coefficient.

Fed-batch experiments performed in an aerated, stirred reactor show that the bioconversion may proceed in the two-phase system for at least 300 h. These conditions allow conversion of 750 mM (108 g · 1^-1) fatty acid, and production of 600 mM (68.5 g · 1^-1) 2-heptanone.     

银耳碱提孢子多糖A-BTF的分离与结构研究

银耳孢子发酵粉(Tremella fuciformisBerk),用热水煮提后,除去水溶性多糖。其沉淀用1M NaOH提取,Sevage法除去蛋白,用乙醇沉淀得到粗多糖。粗多糖经DEAE-32-cellulose和sephadex G-200反复分离后,纯化得到分布均一的多糖A-BTF。HPGPC测定A-BTF的分子量为67000,糖组成分析显示主要由葡萄糖组成。多糖A-BTF的甲基化产物,经水解、还原、乙酰化,通过GC-MS分析表明,主要含有1,6连接的葡萄糖和1,3,6连接的甘露糖,另外还有1,4连接的葡萄糖少量的半乳糖和1-NH2-来苏糖,末端为端基连接的葡萄糖。     

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type¹. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines². Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism^3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation⁴. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines^5,6,7. A link between IL-1β release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes⁸. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.     

Nocturnal Fungi: Airborne Spores in the Canopy and Understory of a Tropical Rain Forest1

Pathogens and other symbiotic fungi that infect above‐ground plant parts commonly disperse as airborne spores. Here we present diel patterns of the density of airborne fungal spores in the canopy and understory of a tropical rain forest. Spores were 52‐fold more abundant in the understory than in the canopy. Additionally, spores were 5‐ to 35‐fold more abundant at night than during the day, associated with environmental conditions conducive to germination and plant infection.     

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.     

基于特异性PCR和荧光检测技术快速鉴定艾纳香

为建立快速准确的艾纳香分子鉴定方法。采取筛选艾纳香及其混伪品基因组DNA的提取方法,针对艾纳香特异性位点设计引物,优化PCR扩增条件,荧光检测扩增产物。结果表明碱裂解法更适于艾纳香基因组DNA的提取;叶绿体基因（tDNA）特异引物能特异性扩增艾纳香DNA,其扩增产物荧光检测呈绿色,混伪品无反应发生。试验结果显示该法简化了分子鉴定过程,省时节力,且结果准确可靠,可作为艾纳香植物和药材的鉴定方法。     

Rapid Identification of Candidate Genes for Seed Weight Using the SLAF-Seq Method in Brassica napus

Seed weight is a critical and direct trait for oilseed crop seed yield. Understanding its genetic mechanism is of great importance for yield improvement in Brassica napus breeding. Two hundred and fifty doubled haploid lines derived by microspore culture were developed from a cross between a large-seed line G-42 and a small-seed line 7–9. According to the 1000-seed weight (TSW) data, the individual DNA of the heaviest 46 lines and the lightest 47 lines were respectively selected to establish two bulked DNA pools. A new high-throughput sequencing technology, Specific Locus Amplified Fragment Sequencing (SLAF-seq), was used to identify candidate genes of TSW in association analysis combined with bulked segregant analysis (BSA). A total of 1,933 high quality polymorphic SLAF markers were developed and 4 associated markers of TSW were procured. A hot region of ~0.58 Mb at nucleotides 25,401,885–25,985,931 on ChrA09 containing 91 candidate genes was identified as tightly associated with the TSW trait. From annotation information, four genes (GSBRNA2T00037136001, GSBRNA2T00037157001, GSBRNA2T00037129001 and GSBRNA2T00069389001) might be interesting candidate genes that are highly related to seed weight.     

Fluorescence In Situ Hybridization Method Using a Peptide Nucleic Acid Probe for Identification of Salmonella spp. in a Broad Spectrum of Samples

A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. The probe theoretical specificity and sensitivity were both 100%. The PNA-FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species confirmed the predicted theoretical values of specificity and sensitivity. The PNA-FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water, and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. After the use of an enrichment step, the PNA-FISH method was able to detect 1 CFU per 10 ml of blood (5 × 10⁹ ± 5 × 10⁸ CFU/ml after an overnight enrichment step) and also 1 CFU per 10 g of PIF (2 × 10⁷ ± 5 × 10⁶ CFU/ml after an 8-h enrichment step). The feces and water samples were also enriched according to the corresponding International Organization for Standardization methods, and results showed that the PNA-FISH method was able to detect Salmonella immediately after the first enrichment step was conducted. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4′,6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples and takes less than 20 h to obtain a diagnosis, except for PIF samples, where the analysis takes less than 12 h. This procedure may be used for food processing and municipal water control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity.Salmonella spp. are enteropathogenic bacteria that cause diseases that range from a mild gastroenteritis to systemic infections (5, 18) The disease severity is determined by the virulence characteristics of the Salmonella strain, host species, and host health condition. Phylogenetic analysis has demonstrated that the genus Salmonella includes two species: Salmonella bongori and Salmonella enterica. Salmonella strains are conventionally identified and classified according to the Kauffmann-White serotyping scheme, which is based on antigenic variation in the outer membrane (23). To date, more than 2,500 Salmonella serovars have been identified, and most of them are capable of infecting a wide variety of animal species and humans (33). Salmonella can be transmitted directly by person to person via the fecal-oral route or by contact with external reservoirs if fecal contamination of soil, water, and foods occurs. It is therefore necessary to develop robust detection methods for all of these sample types.The diagnostic method currently used for Salmonella detection is bacterial culture (International Organization for Standardization [ISO] method 6579:2002), a time-consuming and laborious process (40). A rapid and reliable tool to assist disease control management should aim to reduce salmonellosis in both people and animals. For this purpose a number of assays, such as the enzyme-linked immunosorbent assay (ELISA), PCR, and fluorescence in situ hybridization (FISH), have been developed to decrease the time required to identify Salmonella in food, feces, water, and other clinical samples (8, 10, 14, 15, 25, 26, 31, 41).Several authors have compared some of these approaches, especially culture-based, ELISA, and PCR methods, for Salmonella detection. Some authors found that PCR and ELISA-based methods failed to detect some samples that were positive by culture method (12, 13, 36, 39, 40). Even so, PCR-based methods have proved to be more accurate. Other work showed that when a selective enrichment step was performed before PCR, all Salmonella samples recovered by the culture method were detected. Moreover, the presence of Salmonella that was not recovered by the culture method could be detected by PCR (13, 35). These studies revealed that the enrichment step could increase the molecular assay sensitivity by eliminating problems such as the low numbers of bacteria and the presence of inhibitory substances in certain types of samples, such as food and fecal matter (11, 28, 36). However, PCR-based methods usually require a DNA extraction step, and none of the methods referred to above allows a direct, in situ visualization of the bacterium within the sample.FISH is a molecular assay widely applied for bacterial identification and localization within samples (2, 3). The method is usually based on the specific binding of nucleic acid probes to particular RNAs, due to their higher numbers of copies in the cells. There are already some studies reporting Salmonella detection by FISH using DNA probes (21, 29). A recently developed synthetic DNA analogue, named peptide nucleic acid (PNA), capable of hybridizing to complementary nucleic acid targets, has made FISH procedures easier and more efficient (38, 42). PNA-FISH methods have been successfully applied to the detection of several pathogenic microorganisms (6, 16, 17, 19, 22, 30, 34, 37, 42). For Salmonella, a PNA probe, designated Sal23S10, that targets the 23S rRNA of both Salmonella species has been already developed (31). However, the probe is also complementary to Actinobacillus actinomycetemcomitans, Buchnera aphidicola, and Haemophilus influenzae 23S rRNAs.In this paper, we identify and describe the design of a new fluorescently labeled PNA probe for the specific identification of the Salmonella genus. A novel, rapid, and reliable PNA-FISH method that can be easily applied to a great variety of sample types, either clinical or environmental, has consequently been developed and optimized.     

Rapid Identification of Rice Samples Using an Electronic Nose

Four rice samples of long grain type were tested using an electronic nose (Cyranose-320).Samples of 5 g of each variety ofrice were placed individually in vials and were analyzed with the electronic nose unit consisting of 32 polymer sensors.TheCyranose-320 was able to differentiate between varieties of rice.The chemical composition of the rice odors for differentiatingrice samples needs to be investigated.The optimum parameter settings should be considered during the Cyranose-320 trainingprocess especially for multiple samples,which are helpful for obtaining an accurate training model to improve identificationcapability.Further,it is necessary to investigate the E-nose sensor selection for obtaining better classification accuracy.A re-duced number of sensors could potentially shorten the data processing time,and could be used to establish an application pro-cedure and reduce the cost for a specific electronic nose.Further research is needed for developing analytical procedures thatadapt the Cyranose-320 as a tool for testing rice quality.     

Rapid Identification of Major-Effect Genes Using the Collaborative Cross

The Collaborative Cross (CC) was designed to facilitate rapid gene mapping and consists of hundreds of recombinant inbred lines descended from eight diverse inbred founder strains. A decade in production, it can now be applied to mapping projects. Here, we provide a proof of principle for rapid identification of major-effect genes using the CC. To do so, we chose coat color traits since the location and identity of many relevant genes are known. We ascertained in 110 CC lines six different coat phenotypes: albino, agouti, black, cinnamon, and chocolate coat colors and the white-belly trait. We developed a pipeline employing modifications of existing mapping tools suitable for analyzing the complex genetic architecture of the CC. Together with analysis of the founders’ genome sequences, mapping was successfully achieved with sufficient resolution to identify the causative genes for five traits. Anticipating the application of the CC to complex traits, we also developed strategies to detect interacting genes, testing joint effects of three loci. Our results illustrate the power of the CC and provide confidence that this resource can be applied to complex traits for detection of both qualitative and quantitative trait loci.