Phylogenetic identification and in situ detection of individual microbial cells without cultivation.

The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.     

基于盐酸硫胺保护的荧光铜纳米簇的制备及其对微生物样品中铁离子的特异性检测

【目的】采用盐酸硫胺(aneurine hydrochloride,VB₁)作为保护配体和还原剂,制备荧光稳定的VB₁保护的铜纳米簇(aneurine hydrochloride protected copper nanoclusters, VB₁-Cu NCs),并用于痕量Fe³⁺的检测。【方法】使用VB₁作为保护配体和还原剂,合成VB₁-Cu NCs。通过紫外-可见吸收光谱、荧光光谱和粒径进行表征,并探究了VB₁-Cu NCs的pH响应性、对Fe³⁺的选择性以及线性范围。【结果】制备的VB₁-CuNCs具有良好的水溶性,优异的稳定性和超细的尺寸。VB₁-Cu NCs作为荧光探针检测Fe³⁺,在0–5μmol/L和5–500μmol/L范围内均呈良好的线性,检测限为0.085μmol/L。利用该方法检测实际微生物样品毛癣菌(Trichophyto...     

Accumulation of fluorescent dyes in tumor and normal cells of man

The comparative accumulation of fluorescein Na2-salt (FINa) by the established cell lines of human tumors (cancer of the uterine body, urinary bladder, Wilms tumor, chorionepithelioma, melanoma, rhabdomyosarcoma, osteosarcoma) and human normal fibroblast cultures was obtained. The tumor cells of the different genesis is characterized by its own parameters of FINa accumulation. The most pronounced accumulation of dye has been noted for cells of cancer of the uterine body, the urinary bladder and chorionepithelioma. The normal cells accumulated FINa less considerably. Mechanism of the selective accumulation of dye by the tumor cells was discussed.     

Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy

The green fluorescent protein (GFP) has proven to be an excellent fluorescent marker for protein expression and localisation in living cells [1] [2] [3] [4] [5]. Several mutant GFPs with distinct fluorescence excitation and emission spectra have been engineered for intended use in multi-labelling experiments [6] [7] [8] [9]. Discrimination of these co-expressed GFP variants by wavelength is hampered, however, by a high degree of spectral overlap, low quantum efficiencies and extinction coefficients [10], or rapid photobleaching [6]. Using fluorescence lifetime imaging microscopy (FLIM) [11] [12] [13] [14] [15] [16], four GFP variants were shown to have distinguishable fluorescence lifetimes. Among these was a new variant (YFP5) with spectral characteristics reminiscent of yellow fluorescent protein [8] and a comparatively long fluorescence lifetime. The fluorescence intensities of co-expressed spectrally similar GFP variants (either alone or as fusion proteins) were separated using lifetime images obtained with FLIM at a single excitation wavelength and using a single broad band emission filter. Fluorescence lifetime imaging opens up an additional spectroscopic dimension to wavelength through which novel GFP variants can be selected to extend the number of protein processes that can be imaged simultaneously in cells.     

Localization of endoplasmic reticulum in living and glutaraldehyde-fixed cells with fluorescent dyes

Certain fluorescent dyes, previously reported to localize mitochondria, when used at higher concentrations also localize a continuous net-like structure in both living and glutaraldehyde-fixed cells. A similar reticular structure can be detected by phase-contrast microscopy and whole-mount electron microscopy in potassium permanganate-fixed cells as well. This structure is mostly tubular, with some patch-like areas, and is likely to be the endoplasmic reticulum (ER). The organization of the reticular structure is sensitive to colchicine and rotenone but not to cytochalasin B, taxol, monensin, the calcium ionophore A23187, 12-O-tetradecanoylphorbol 13-acetate, or hydrocortisone.     

Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples

Potent PCR inhibitors in blood and soil samples can cause false negative results from PCR-based clinical and forensic tests. We show that the effect of these inhibitors is primarily upon Taq DNA polymerase, since mutational alteration of the polymerase can overcome the inhibition to the extent that no DNA purification is now required. An N-terminal deletion (Klentaq1) is some 10–100-fold inhibition resistant to whole blood compared to full-length, wild-type (w.t.) Taq, which is strongly inhibited by 0.1–1% blood. Further mutations at codon 708, both in Klentaq 1 and Taq, confer enhanced resistance to various inhibitors of PCR reactions, including whole blood, plasma, hemoglobin, lactoferrin, serum IgG, soil extracts and humic acid, as well as high concentrations of intercalating dyes. Blood PCR inhibitors can predominantly reduce the DNA extension speed of the w.t. Taq polymerase as compared to the mutant enzymes. Single-copy human genomic targets are readily amplified from whole blood or crude soil extract, without pretreatment to purify the template DNA, and the allowed increase in dye concentration overcomes fluorescence background and quenching in real-time PCR of blood.     

The use of NASBA for the detection of microbial pathogens in food and environmental samples

The isothermal amplification method nucleic acid sequence-based amplification (NASBA), which amplifies RNA, has been reported as useful for the detection of microbial pathogens in food and environmental samples. Methods have been published for Campylobacter spp., Listeria monocytogenes and Salmonella enterica ser. Enteritidis in various foods and for Cryptosporidium parvum in water. Both 16S rRNA and various mRNAs have been used as target molecules for detection; the latter may have advantages in allowing specific detection of viable cells. Most of the methods to detect pathogens in foods have employed enrichment in nutrient medium prior to NASBA, as this can ensure sensitivity of detection and encourage the detection of only viable target cells. Although a relatively recent method, NASBA has the potential for adoption as a diagnostic tool for environmental pathogens.     

Methyl tert-butyl ether biodegradation by microbial consortia obtained from soil samples of gasoline-polluted sites in Mexico

Microbial consortia obtained from soil samples of gasoline-polluted sites were individually enriched with pentane, hexane, isooctane and toluene. Cometabolism with methyl tert-butyl ether, (MTBE), gave maximum degradation rates of 49, 12, 32 and 0 mg g(-1)protein h(-1), respectively. MTBE was fully degraded even when pentane was completely depleted with a cometabolic coefficient of 1 mgMTBE mg(-1)pentane. The analysis of 16S rDNA from isolated microorganisms in the pentane-adapted consortia showed that microorganisms could be assigned to Pseudomonas. This is the first work reporting the cometabolic mineralization of MTBE by consortium of this genus.     

Oxidative chemistry of fluorescent dyes: implications in the detection of reactive oxygen and nitrogen species

HE (hydroethidine), a widely used fluorescent dye for detecting intracellular superoxide, undergoes specific oxidation and hydroxylation reactions. The reaction between HE and O2?- (superoxide radical) yields a diagnostic marker product, 2-hydroxyethidium. This is contrary to the popular notion that O2?- oxidizes HE to form ethidium. HE, however, undergoes a non-specific oxidation to form ethidium in the presence of other oxidants (hydroxyl radical, peroxynitrite and perferryl iron) and other dimeric products. The mitochondria-targeted HE analogue Mito-SOX? undergoes the same type of oxidative chemistry to form products similar to those formed from HE. On the basis of the oxidative chemical mechanism of HE and Mito-SOX?, we conclude that flurorescence microscopy or related techniques are not sufficient to measure the superoxide-specific hydroxylated products. HPLC methodologies are required to separate and identify these products. Peroxynitrite reacts rapidly and stoichiometrically with boronates to form specific products. Assays using fluorescent-based boronate probes will be more reliable for peroxynitrite determination than those using either dichlorodihydrofluorescein or dihydrorhodamine.     

Factors underlying membrane potential-dependent and -independent fluorescence responses of potentiometric dyes in stressed cells: diS-C(3)(3) in yeast

The redistribution fluorescent dye diS-C(3)(3) responds to yeast plasma membrane depolarisation or hyperpolarisation by Delta psi-dependent outflow from or uptake into the cells, reflected in changes in the fluorescence maximum lambda(max) and fluorescence intensity. Upon membrane permeabilisation the dye redistributes between the cell and the medium in a purely concentration-dependent manner, which gives rise to Delta psi-independent fluorescence responses that may mimic Delta psi-dependent blue or red shift in lambda(max). These lambda(max) shifts after cell permeabilisation depend on probe and ion concentrations inside and outside the cells at the moment of permeabilisation and reflect (a) permeabilisation-induced Delta psi collapse, (b) changing probe binding capacity of cell constituents (inverse to the ambient ionic strength) and (c) hampering of probe equilibration by the poorly permeable cell wall. At low external ion concentrations, cell permeabilisation causes ion outflow and probe influx (hyperpolarisation-like red shift in lambda(max)) caused by an increase in the probe-binding capacity of the cell interior and, in the case of heat shock, protein denaturation unmasking additional probe-binding sites. At high external ion levels minimising net ion efflux and at high intracellular probe concentrations at the moment of permeabilisation, the Delta psi collapse causes a blue lambda(max) shift mimicking an apparent depolarisation.     

Optimization of microbial DNA extraction and purification from raw wastewater samples for downstream pathogen detection by microarrays

Numerous waterborne pathogens are difficult to detect and enumerate with accuracy due to methodological limitations and high costs of direct culturing. The purity of DNA extracted from wastewater samples is an important issue in the sensitivity and the usefulness of molecular methods such as polymerase chain reaction (PCR) and hybridizations on DNA microarrays. Ten different DNA extraction procedures, including physical and chemical extraction and purification steps, were examined to ascertain their relative effectiveness for extracting bacterial DNA from wastewater samples. The quality of the differentially extracted DNAs was subsequently assessed by PCR amplification and microarray hybridization. Our results showed that great differences existed among the ten procedures and only a few of the methods gave satisfactory results when applied to bacterial pathogens. This observation suggested that the extraction method needed to be carefully selected to produce significant and confident results in the detection of pathogens from environmental samples.     

Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR

Quantitative analysis of anthrax spores from environmental samples is essential for accurate detection and risk assessment since Bacillus anthracis spores have been shown to be one of the most effective biological weapons. Using TaqMan real-time PCR, specific primers and probes were designed for the identification of pathogenic B. anthracis strains from pag gene and cap gene on two plasmids, pXO1 and pXO2, as well as a sap gene encoded on the S-layer. To select the appropriate lysis method of anthrax spore from environmental samples, several heat treatments and germination methods were evaluated with multiplex-PCR. Among them, heat treatment of samples suspended with sucrose plus non-ionic detergent was considered an effective spore disruption method because it detected up to 10(5) spores/g soil by multiplex-PCR. Serial dilutions of B. anthracis DNA and spore were detected up to a level of 0.1 ng/ microliters and 10 spores/ml, respectively, at the correlation coefficient of 0.99 by real-time PCR. Quantitative analysis of anthrax spore could be obtained from the comparison between C(T) value and serial dilutions of soil sample at the correlation coefficient of 0.99. Additionally, spores added to soil samples were detected up to 10(4) spores/g soil within 3 hr by real-time PCR. As a consequence, we established a rapid and accurate detection system for environmental anthrax spores using real-time PCR, avoiding time and labor-consuming preparation steps such as enrichment culturing and DNA preparation.     

A red fluorescent carbon dots with good water solubility for rapid detection of Al3+ in actual samples

We developed a facile strategy for the fabrication of red fluorescent carbon nanodots (R-CDs) and demonstrated their applications for Al³⁺ sensing. Red-emission carbon dots (CDs) were synthesized using a simple hydrothermal treatment with citric acid and urea as precursors, manifesting intriguing red-emission behaviour at 610 nm. With increasing Al³⁺ concentration, the fluorescence band at 610 nm decreased gradually. Monitoring the intrinsic fluorescence variation (I_610nm), as-prepared CDs were developed as an effective platform for fluorescent Al³⁺ sensing, with a linear range of 0.5–60.0 μM and a detection limit of 3.0 nM. More importantly, R-CDs have been applied successfully to the analysis of Al³⁺ in actual samples with satisfactory recoveries in the range 97.12–102.05%, which indicated that obtained CDs could be implemented as an effective tool for the identification and detection of Al³⁺ in actual samples.     

The extremely rapid oligonucleotide hybridization and high throughput detection of microbial gene sequences using fluorescence polarization

The hybridization of oligonucleotide sequences complementary to the genes of Shiga toxins (verotoxins) types 1 and 2 of enterohaemorrhagic Escherichia coli (EHEC) and human hepatitis C virus (HCV) was monitored using fluorescence polarization under the reaction condition of high salt concentration (0.8 M NaCl), which was optimized to obtain a higher rate of hybridization. The time courses of hybridization of fluorescently labeled oligomers (probe DNAs) with the amplified DNA or RNA of the genes were recorded. Two methods, the asymmetric PCR and NASBA, were used to amplify the genetic DNA of Shiga toxins and that of RNA in HCV, respectively. Probe DNA sequences were designed which hybridized extremely rapidly with amplicons of the genes of Shiga toxins types 1 and 2 and that of HCV. In the cases using the three different DNA probes, the hybridization was 90% complete in about 1 min, considerably faster than that of the 3 min reported previously. The rapidity of this hybridization could not be explained by the melting temperature or the G+C content of the probe sequences but its relationship with high order structure of the single stranded DNA or RNA of the amplicons in the solution was strongly suggested.     

Biodegradation of BTE in soil by indigenous microbial populations with and without biogenic substrates

Degradation of benzene, toluene, and ethylbenzene (BTE) by microbial populations indigenous to the soil and populations proliferated from the indigenous using biogenic substrates were compared. The reaction system consisted of aerobic microcosms representing an unsaturated soil. Microcosms supplemented with glucose and citrate, when compared to the unsupplemented microcosms, showed increases in bacterial counts, but the overall degradation rates for B, T, or E were reduced in spite of shorter lag times. Both biogenic substrate supplements were non-beneficial for BTE degradation due largely to the preferential and healthy growth of the indigenous populations on the biogenic substrates, and thus the urgency of developing a favorable amount of BTE degraders was reduced.     

The application of pH-sensitive fluorescent dyes in lactic acid bacteria reveals distinct extrusion systems for unmodified and conjugated dyes

Membrane translocation is a crucial issue when addressing the activity of both cell-penetrating and antimicrobial peptides. Translocation is responsible for the therapeutic potential of cell-penetrating peptides as drug carriers and can dictate the killing mechanisms, selectivity and efficiency of antimicrobial peptides. It is essential to evaluate if the internalization of cell-penetrating peptides is mediated by endocytosis and if it is able to internalize attached cargoes. The mode of action of an antimicrobial peptide cannot be fully understood if it is not known whether the peptide acts exclusively at the membrane level or also at the cytoplasm. Therefore, experimental methods to evaluate and quantify translocation processes are of first importance. In this work, over 20 methods described in the literature for the assessment of peptide translocation in vivo and in vitro, with and without attached macromolecular cargoes, are discussed and their applicability, advantages and disadvantages reviewed. In addition, a classification of these methods is proposed, based on common approaches to detect translocation.     

Spectral imaging detection and counting of microbial cells in marine sediment

Semiautomated detection and counting techniques for microbial cells in soil and marine sediment using microscopic-spectral-imaging analysis were developed. Microbial cells in microscopic fields were selectively detected from other fluorescent particles by their fluorescent spectrum, based on the spectral shift between the conjunction and nonconjunction of DNA fluorochrome (SYBR Green II) with nucleic acids. Using this technique, microbial cells could be easily detected in soil and 30-cm deep sediment samples from Tokyo Bay, both of which contain particles other than microbial cells. Total cell density was semiautomatically estimated at 1-6 x 10(9) cells cm(-3) of sediment sampled at different depths in Tokyo Bay, which corresponded to 65-106% (mean 88%) of visual direct counting. This technique may be useful for detecting microbial cells in soil and sediment samples from the deeper subsurface environment.