A New Fluorescence Staining Assay for Visualizing Living Microorganisms in Soil

5- (and 6-)Sulfofluorescein diacetate (SFDA), which is converted to a fluorescent product by intracellular esterase activity, was used to stain living microorganisms, including bacteria, Saccharomyces cerevisiae, and fungi, in soil. SFDA (1 mM) dissolved in ethyl alcohol was added to an intact soil sample, and the preparation was examined with an epifluorescence microscope. Bright single cells and colonies of live bacteria were observed without interference from the autofluorescence of soil minerals and detritus. Cultured Escherichia coli was killed through heat treatment; thus, SFDA was concluded to stain only living cells. Microbial colonies obtained from natural soils and various cultured strains were tested. It was found that 151 of 154 colonies from natural soils were stained and that hyphae and spores from 1 of 28 cultured microbial strains were not stained. The SFDA method was successfully used to visualize and count bacteria in soil samples from Mount Shiga in Japan.     

Use of the Scanning Electron Microscope for Viewing Bacteria in Soil

Scanning electron microscopy was used for viewing Bacillus cereus and Staphylococcus aureus in three different soils. Both organisms were detected in the test soils at an approximate concentration of 10⁷ cells per gram of soil; theoretically, the minimal number of microorganisms required for detection with the scanning electron microscope technique was between 10⁷ and 10¹⁰ cells per gram of soil. Because the concentration of cells was critical, the use of scanning electron microscopy as an extraterrestrial life detection instrument would be limited with soils containing more than 10⁷ bacteria per gram of soil.     

Kinetic Study on Conformational Change in a Single Molecular Species, β3, of β-Conglycinin in an Acidic Ethanol Solution

The conformational change in a single molecular species, beta3, of beta-conglycinin in an acidic ethanol solution was kinetically studied by the stopped-flow technique, utilizing the intrinsic fluorescence of proteins and the fluorescence of 1-anilinonaphthalene-8-sulfonic acid (ANS) bound to the proteins. The time-course of the intrinsic fluorescence changes clearly showed the rate of conformational change below and above 25% ethanol to be quite different from each other. ANS could bind well to the protein in an ethanol concentration range of 15-25%. However, the rate of conformational change of the protein corresponding to that for ANS binding could not be obtained at less than 25% ethanol, while the rate of conformational change agreed well with that for ANS binding at more than 25% ethanol. In addition, the process showing the greatest and slowest ANS binding was not apparent in the denaturation of beta-conglycinin under the conditions employed. These results lead to the conclusions that the beta-conglycinin structure could be maintained in the mild molten globule-like denaturation state, and that various tertiary structural changes could take place without any significant effect on the high sensitivity of intrinsic fluorescence after the secondary structural changes.     

Vitality measurement using spectrum shift in Hoechst 33342 stained cells.

A bivariate flow cytometric technique has been developed in which Hoechst 33342 stained vital cells are discriminated from early damaged cells by differences in their fluorescence emission spectra. A discrete population of cells with intact cell membranes passing from vital to dead could be identified by using propidium iodide exclusion in combination with a concentration-independent spectrum shift of Hoechst fluorescence to longer wavelengths. The appearance after injury of two subsets of cells with intact membranes representing vital and early damaged cells, respectively, indicates two types of binding of Hoechst 33342 to these cells, presumably resulting from differences in chromatin structure. The method was tested in murine and human hemopoietic cell lines by cytotoxic treatment with cytosine arabinoside and interleukin 3 deprivation in factor-dependent lines. The power of the method was demonstrated by a cell cycle analysis of the early damaged cells after both cytotoxic treatment and growth factor deprivation.     

影响引人微生物根部定殖的因素

从外界引入的各类有益微生物如生防菌(BCA)和根际促生菌或增产菌(PGPR,YIB)到种子表面随其生根发芽而蔓延或直接到根表沿根分布定殖．外来微生物在根际定殖的过程为与根尖接触,沿根分布,最后在根际建立自己的种群．定殖的位点以PGPR为例,是表皮细胞间隙,或侧根、根毛基部．外来微生物在根际定殖动态变化的原因,由于根际生物的和非生物的因素引起的．生物因子除去外来微生物本身的生理特性,还有根际土著微生物与外来微生物的相互作用,更重要的是植物基因型对微生物定殖的影响．非生物因子包括土壤环境、土壤结构和含水量,土壤温度和土壤pH值均能影响外来微生物在根部的定殖．     

The effect of surface and membrane potential on 1-anilino-8-naphthalenesulfonate binding with E. coli membrane

Dependence of ANS fluorescence on the surface potential of E. coli under lowered resistance of the bacterial membrane and after application of the positive diffusion potential inside the cell was investigated. It was shown that in the absence of the latter ANS binding in de-energised bacteria occurs mainly at the outside surface. It may be due to the high negative charge of the inner side of the cytoplasmic membrane. According to produced evaluation the potential of this surface is 120 +/- 25 mV. The data obtained suggest that low ANS fluorescence in intact cells is due to the membrane modification on energisation.     

土壤微生物生物量碳氮磷与土壤酶化学计量对气候变化的响应机制

微生物和土壤酶是陆地生态系统中生物地球化学循环的重要驱动力,深入理解微生物在生态系统中的调节作用以及气候变化过程中微生物量和土壤酶的响应机制是生态学领域关注的重要科学问题.本研究从气候因素角度出发,基于生态化学计量学理论,综述了微生物和土壤酶在陆地生态系统碳氮磷循环中的作用,以及土壤微生物生物量碳氮磷和土壤酶化学计量对气候变化的响应机制,即: 改变微生物代谢速率和酶活性;调整微生物群落结构;调整微生物生物量碳氮磷与土壤酶化学计量特征;改变碳氮磷养分元素利用效率.最后分析当前研究的不足,并提出了该领域亟待解决的科学问题: 综合阐明土壤微生物和土壤酶对气候变化的响应机制;探究土壤微生物和胞外酶养分耦合机理;深入探究土壤微生物量和土壤酶化学计量特征对气候变化的适应对策.     

Survival of Microorganisms in a Rock Bed Under Conditions Simulating Solar Heat Storage

A laboratory-scale unit containing about 360 kg of washed river gravel was designed to [ill] the use of rocks for heat storage. The unit was operated under varying conditions of temperature, relative humidity, and the addition of volatile nutrients over a 4-month period. Effluent air and rock surfaces were monitored for the presence of microorganisms. After 2 weeks, virtually no microorganisms were detected in the effluent air except when dry soil or compost was added as the inoculum. A small number of heat-resistant bacteria, but no fungi, were found to survive on the rock surfaces. Microorganisms isolated were either sporeforming bacteria or actinomycetes closely resembling Thermoactinomyces vulgaris. Microbial colonization of rock beds used for solar heat storage does not appear likely under routine operation.     

A fluorescent probe response to the interaction of pyocin R1 with sensitive cells.

Additon of pyocin R1, a bacteriocin of Pseudomonas aeruginosa, to sensitive cells caused a fluorescence increase of 8-anilino-1-naphthalenesulfonate (ANS) in the cell suspension. The reaction was rapid, starting with a short time lag after adsorption of pyocin onto the cells and finishing within several minutes. The fluorescence response was attributed to the interaction of the cell body and ANS, not to that of the medium outside the cells and ANS. The maximal amplitude of fluorescence after pyocin addition was dependent on temperature, and the relation appeared to be biphasic. Similarly, Arrhenius plots of the initial rate of fluorescence change were biphasic. The transition of slopes in both cases occurred in the temperature range between 18 and 19 degrees. These results suggest that ANS interacts with lipids in the cell envelope and that pyocin causes a structural change of the cell envelope leading to increased fluorescence of ANS.     

Trifluoroethanol and acetonitrile induced formation of the molten globule states and aggregates of cellulase

A systematic investigation on the effects of trifluoroethanol and acetonitrile at various concentrations on cellulase (EC 3.2.1.4) was studied by enzyme assay, intrinsic fluorescence, ANS binding, circular dichroism and ATR-Fourier transform infra red spectroscopy. The results show the presence of molten globule states at 3% (v/v) TFE and 80% (v/v) ACN. Cellulase aggregates at 25% (v/v) TFE and 90% (v/v) ACN, as detected by decrease in intrinsic and ANS fluorescence, loss in tertiary structure and enzyme activity, increase non-native β-sheet structure as evident from far-UV CD and FTIR spectra, increase in Thioflavin T fluorescence and shift in Congo red assay.     

Flow cytometry applications in the food industry

Flow cytometry has become a valuable tool in food microbiology. By analysing large numbers of cells individually using light-scattering and fluorescence measurements, this technique reveals both cellular characteristics and the levels of cellular components. Flow cytometry has been developed to rapidly enumerate microorganisms; to distinguish between viable, metabolically active and dead cells, which is of great importance in food development and food spoilage; and to detect specific pathogenic microorganisms by conjugating antibodies with fluorochromes, which is of great use in the food industry. In addition, high-speed multiparametric data acquisition, analysis and cell sorting, which allow other characteristics of individual cells to be studied, have increased the interest of food microbiologists in this technique. This mini-review gives an overview of the principles of flow cytometry and examples of the application of this technique in the food industry.     

Microbial associations of soil types

Microorganisms are characterized by wide ranges of distribution. Some groups, however, are known to have zones of active proliferation, and the development of specific populations in discrete zones results in rather specific microbial associations in some soil types. The soils formed in warm climates are richer in microorganisms and contain more bacilli and actinomycetes. The spectrum of dominant microbial groups varies in different soil types.     

Microflora of the honeybee gastrointestinal tract

Microorganisms in the midgut and rectum of the honeybee were enumerated and characterized. Counts of aerobic microorganisms were distinctly lower than counts of anaerobes (10(5)-10(6) viable cells per g of intestinal content vs. 10(8)-10(9) per g). Total numbers of anaerobic microorganisms were almost identical with the count of anaerobic Gram-positive acid resistant rods. A higher number of coliform bacteria and Bacillus spp. was detected in the rectum (10(5) per g). Anaerobic and aerobic microorganisms, coliforms, enterococci, Bacillus spp., Pseudomonas spp. and yeasts were found in all bees; lactobacilli, staphylococci and moulds were not found.     

Interaction of a hydrophobic fluorescent probe with mouse embryo fibroblasts

Fluorescence photomicrographs show that the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) binds to hydrophobic components of intact 3T3 cells. Cells exposed to ANS exhibit fluorescence in the cytoplasm, intense nuclear membrane fluorescence, and well-defined fluorescent nucleoli. Fluorescence titrations of 3T3 cells with ANS show a decrease in fluorescence intensity, a blue shift of native cell emission with increasing ANS concentration and the appearance of a new peak due to ANS fluorescence. These fluorescence effects are ascribed to energy transfer processes involving bound ANS and the tryptophan and tyrosine residues of cellular proteins. ANS bound to 3T3 cells appears to quench the long wavelength component of the cellular tryptophan fluorescence, resulting in an unmasking of tryptophan and tyrosine emission at shorter wavelengths.     

Distribution of phytoplasmas in infected plants as revealed by real-time PCR and bioimaging

Phytoplasmas are cell wall-less bacteria inhabiting the phloem and utilizing it for their spread. Infected plants often show changes in growth pattern and a reduced crop yield. A quantitative real-time polymerase chain reaction (Q-PCR) assay and a bioimaging method were developed to quantify and localize phytoplasmas in situ. According to the Q-PCR assay, phytoplasmas accumulated disproportionately in source leaves of Euphorbia pulcherrima and, to a lesser extent, in petioles of source leaves and in stems. However, phytoplasma accumulation was small or nondetectable in sink organs (roots and sink leaves). For bioimaging, infected plant tissue was stained with vital fluorescence dyes and examined using confocal laser scanning microscopy. With a DNA-sensitive dye, the pathogens were detected exclusively in the phloem, where they formed dense masses in sieve tubes of Catharanthus roseus. Sieve tubes were identified by counterstaining with aniline blue for callose and multiphoton excitation. With a potentiometric dye, not all DNA-positive material was stained, suggesting that the dye stained metabolically active phytoplasmas only. Some highly infected sieve tubes contained phytoplasmas that were either inactive or dead upon staining.     

Microscopic quantification of bacterial invasion by a novel antibody-independent staining method

Microscopic discrimination between extracellular and invasive, intracellular bacteria is a valuable technique in microbiology and immunology. We describe a novel fluorescence staining protocol, called FITC-biotin-avidin (FBA) staining, which allows the differentiation between extracellular and intracellular bacteria and is independent of specific antibodies directed against the microorganisms. FBA staining of eukaryotic cells infected with Gram-negative bacteria of the genus Neisseria or the Gram-positive pathogen Staphylococcus aureus are employed to validate the novel technique. The quantitative evaluation of intracellular pathogens by the FBA staining protocol yields identical results compared to parallel samples stained with conventional, antibody-dependent methods. FBA staining eliminates the need for cell permeabilization resulting in robust and rapid detection of invasive microbes. Taken together, FBA staining provides a reliable and convenient alternative for the differential detection of intracellular and extracellular bacteria and should be a valuable technical tool for the quantitative analysis of the invasive properties of pathogenic bacteria and other microorganisms.     

Effects of short-time drying on biofilm-associated bacteria

Microorganisms tend to form biofilms consisting of cells embedded in a highly hydrated extracellular polymeric matrix. The biofilm protects its inhabitants from antimicrobial agents, pH alterations, and confers protection against drying. It is known that biofilm-associated bacteria can survive for a while in the absence of water. When rehydrated, metabolic processes are quickly restored and microorganisms resume life. The aim of this study is to evaluate the survival of heterotrophic bacteria, sulphate-reducing bacteria and amoeba against short-time drying. Biofilms were allowed to grow for 30 and 60 days on stainless steel (316, 2B) coupons in annular biofilm reactor, which was fed with drinking water network under constant, non-turbulent shear stress and temperature. The results presented in this study indicate a role for biofilm layer in protecting biofilm-associated microorganisms from drying. The current study has provided that short-time (24 h) absence of water could not affect biofilm-associated heterotrophic microorganisms significantly, in terms of cell viability.     

Evidence for detachment of indigenous bacteria from aquifer sediment in response to arrival of injected bacteria

Two bacterial strains isolated from the aquifer underlying Oyster, Va., were recently injected into the aquifer and monitored using ferrographic capture, a high-resolution immunomagnetic technique. Injected cells were enumerated on the basis of a vital fluorescence stain, whereas total cell numbers (stained target cells plus unstained target and antigenically similar indigenous bacteria) were identified by cell outlines emanating from fluorophore-conjugated antibodies to the two target strains. The arrival of injected bacteria at the majority of monitored sampling ports was accompanied by simultaneous temporary increases in unstained cell counts that outnumbered the injected bacteria by 2- to 100-fold. The origin and mechanism of appearance of the unstained cells are considered.     

Evidence for Detachment of Indigenous Bacteria from Aquifer Sediment in Response to Arrival of Injected Bacteria

Two bacterial strains isolated from the aquifer underlying Oyster, Va., were recently injected into the aquifer and monitored using ferrographic capture, a high-resolution immunomagnetic technique. Injected cells were enumerated on the basis of a vital fluorescence stain, whereas total cell numbers (stained target cells plus unstained target and antigenically similar indigenous bacteria) were identified by cell outlines emanating from fluorophore-conjugated antibodies to the two target strains. The arrival of injected bacteria at the majority of monitored sampling ports was accompanied by simultaneous temporary increases in unstained cell counts that outnumbered the injected bacteria by 2- to 100-fold. The origin and mechanism of appearance of the unstained cells are considered.     

Isolation of hydrocarbon-oxidizing psychrophilic bacteria from oil-polluted soils

Microorganisms growing on a mineral medium with crude oil and its light fractions as only carbon and energy sources have been isolated from samples of oil-polluted soils collected in the Usa District (Komi Republic, Russia). For the first time, hydrocarbon-oxidizing psychrophilic bacteria of the genus Cytophaga have been found that are clearly capable of consuming crude oil hydrocarbons. A method for cultivating microorganisms on porous plastic is proposed. The data from the literature on the response of soil microbiota to oil pollution indicate that the pollution can activate or suppress the growth of various physiological groups of microorganisms [1]. Different soil and climatic conditions and pollution levels can give rise to different microbial cenoses, which include different associations and predominant microbial species.