Sterilization by electrohydraulic discharges

High voltage electric discharges between two electrodes immersed in a liquid (“electrohydraulic discharges”) inactivate microorganisms suspended in the liquid. The intense pulse of UV radiation emitted from the plasma formed between the electrodes causes most of the bactericidal effects, rather than shock waves, or free radicals or other chemical species formed in the liquid medium. A method of sterilizing materials without contamination from electrode debris is described. Possible applications and limitations of the technique are outlined.     

液电效应处理污水的实验研究

本文研究了液电效应对污水的杀菌净化作用，介绍了实验方法，给出了实验结果。液电效应所产生的强压力冲击波、强紫外光具有很强的杀菌作用。杀菌率与投入到单位体积中的能量有关，投入的能量越多，灭菌效果越好。     

Attachment and Detachment of Living Microorganisms Using a Potential-Controlled Electrode

We developed an electrical modulation method for attachment and detachment of microorganisms. Living microorganisms suspended in non-nutritive media such as PBS(?) and artificial seawater were attracted by and selectively attached to indium tin oxide (ITO)/glass electrode regions to which a negative potential was applied. The microorganisms suspended in LB medium and glucose solution were not attracted to the ITO electrode. Dead microorganisms were not attracted to the ITO electrode. The living microorganisms were retrieved after detachment from the ITO electrode by application of a high-frequency triangular wave potential. When we applied this method to separate microorganisms from deep-sea sediment, bacteria belonging to 19 phyla and 23 classes were collected without undesirable high molecular weight contaminants such as humic acids. At the phylum and class level, respectively, 95 and 87 % of the phylotypes among electrically retrieved bacteria were common to the gene clones from the direct sediment DNA extraction. This technique is a novel useful method to prepare bacterial cells in a single population or a community for metagenomic analyses.     

Hydrodynamic interaction of two unsteady model microorganisms

The study of pair-wise interactions between swimming microorganisms is fundamental to the understanding of the rheological and transport properties of semi-dilute suspensions. In this paper, the hydrodynamic interaction of two ciliated microorganisms is investigated numerically using a boundary-element method, and the microorganisms are modeled as spherical squirmers that swim by time-dependent surface deformations. The results show that the inclusion of the unsteady terms in the ciliary propulsion model has a large impact on the trajectories of the interacting cells, and causes a significant change in scattering angles with potential important consequences on the diffusion properties of semi-dilute suspensions. Furthermore, the analysis of the shear stress acting on the surface of the microorganisms revealed that the duration and the intensity of the near-field interaction are significantly modified by the presence of unsteadiness. This observation may account for the hydrodynamic nature of randomness in some biological reactions, and supersedes the distinction between intrinsic randomness and hydrodynamic interactions, adding a further element to the understanding and modeling of interacting microorganisms.     

Dye-coupled electrode system for the rapid determination of cell populations in polluted water.

We determined cell populations in polluted waters by using a fuel cell-type electrode. The electrode was constructed from a platinum anode, a silver peroxide cathode, and a membrane filter for retaining microorganisms. The principle of cell number determination is based on sensing a redox dye reduced by the microorganisms with the electrode. Sample solutions containing microorganisms were membrane filtered, and the resulting filter containing microbial cells was attached to the surface of a platinum anode. The electrode was immersed in phosphate buffer solution (0.05 M, pH 7) containing a redox dye (2,4-dichlorophenol-indophenol), and the current generated was measured. The response time of the electrode system was 10 to 20 min, and the current generated was proportional to cell populations above 10(4) cells/ml.     

坛紫菜叶状体的无菌化培养及其应用

对坛紫菜叶状体的无菌处理方法进行了优化,并利用紫菜外生菌对无菌处理后的紫菜叶片进行了人工感染。经0.7%KI和0.1%(w/v)氨苄青霉素对紫菜叶片进行预处理后,利用氨苄青霉素、硫酸链霉素、新霉素、庆大霉素及卡那霉素等5种抗生素及其不同组合对紫菜叶片进行处理,筛选出最佳抗生素组合、浓度和培养条件。结果表明:氨苄青霉素(终浓度300μg/mL)、卡那霉素(终浓度100μg/mL)与庆大霉素(终浓度100μg/mL)3种抗生素组合对坛紫菜叶状体进行无菌处理18 h,对紫菜细胞的毒害较小,并且3种抗生素合用对89.5%紫菜外生细菌的抑菌率达80%以上;外生菌感染结果研究表明:用105/mL真菌孢子液和细菌菌液回染紫菜,培养22 d后,实验组紫菜生长状况较对照组好,对照组紫菜出现褪色。用108/mL真菌孢子液分别回染健康紫菜、穿刺紫菜(用灭菌刀片在紫菜表面划1～2 mm的伤口),分别在18℃、28℃和35℃条件下培养。实验结果表明28℃和35℃高温和穿刺均会影响无菌紫菜健康生长,且加菌紫菜在高温和穿刺条件下,则会出现明显病斑,而18℃培养的加菌紫菜则生长良好。     

Quantitation of the Tetramethyl-p-Phenylenediamine Oxidase Reaction in Neisseria Species

The tetramethyl-p-phenylenediamine oxidase reaction commonly used in the Kovacs oxidase test was quantitatively estimated for various Neisseria species employing standardized resting cell suspensions. This genus of microorganisms exhibited very high tetramethyl-p-phenylenediamine oxidase rates comparable to that of Azotobacter and Pseudomonas, and this reaction was found to be a valid measurement for the respiratory capability possessed by this group of organisms.     

Use of a lysoenzyme preparation from Streptomyces recifensis subsp. lyticus 2435 for isolating DNA from staphylococcal cells

It was shown that the preparation 2435 from Streptomyces recifensis subsp. lyticus, including a complex of bacteriolytic and concomitant enzymes provided lysis of thick staphylococcal suspensions within 15 to 20 minutes under optimal conditions after preliminary treatment of the cells with 0.1 M cystein-HCl. A procedure was developed for isolating DNA from the cells of staphylococci and other microorganisms based on enzymatic lysis. In terms of major physicochemical properties, the preparations of DNA were not inferior to the preparations of DNA isolated by the classical Marmur technique with Kirbi's deproteinization and had transforming activity. The developed procedure for isolation of DNA with using the lysoenzyme preparation widened the possibilities of investigating the genetics of staphylococci and other microorganisms.     

Rapid enumeration of microorganisms in foods by the direct epifluorescent filter technique.

Filtration of "stomachered" food suspensions through nylon filters (pore size, 5 microns) removed most of the food debris without affecting the recovery of microorganisms. Two to ten milliliters of these prefiltered suspensions could be filtered in the direct epifluorescent filter technique (DEFT). The technique takes less than 30 min to complete and has a lower sensitivity of less than 60,000 microorganisms per g for all products examined. Vegetative bacterial cells, spores, fungal hyphae, and yeasts could be distinguished with the technique. For fresh meat and fish, the DEFT count of prefiltered suspensions agreed well with the plate count of unfiltered suspensions over the range of 10(4) to 10(10)/g (correlation coefficient of 0.91). For frozen meat and fish and frozen vegetables, the two counting methods had correlation coefficients of 0.87 and 0.66, respectively. The poor correlation for frozen vegetables was due to the inclusion in the DEFT count of nonviable bacteria killed by the blanching process used to inactivate enzymes. Good agreement was obtained between the prefiltered DEFT count and unfiltered plate count for cooked meats, cream doughnut, and whole peppers. Possible reasons for the poor agreement between the DEFT count and plate count for certain products are discussed.     

A rapid electrochemical method for assimilation test of microorganisms

Summary A microbial electrode consisting of the immobilized microorganisms to be tested and an oxygen electrode was used to study the assimilation characteristics of microorganisms. When a sample solution containing a substrate was injected into the microbial sensor system, the current of the sensor markedly decreased with time if the microorganisms assimilated the substrate. On the other hand, no current decrease was observed if the microorganisms could not assimilate the substrate. Assimilation characteristics of various microorganisms such as molds, yeasts, bacteria, actinomycetes and activated sludges were tested with various substrates. The time required for a test was 30 min per substrate by the pulse method (sample injection period, 5 min). Good correlations were obtained between this electrochemical method and the conventional growth test. The fundamental differences between the two methods and the application of the electrochemical method are discussed.     

微生物流式分选的单细胞样品制备及存活率提高的方法优化

【背景】以流式细胞技术为代表的高通量筛选技术能够高效筛选具有目标性状的微生物工程菌株。在流式分选中微生物的粘连会造成分析数据不准确,分选纯度降低,因此快速简便的单细胞样品制备是流式检测的关键。优势菌大多是通过筛选偶联荧光蛋白的随机突变库获得,阳性率低,杂质和死细胞的自发荧光较强,容易混入分选门内造成存活率降低,亟须提高分选存活率的方法。【目的】建立一种简便的微生物流式分选的单细胞样品制备方法,并通过碘化丙啶(propidium iodide, PI)染色提高分选样品存活率。【方法】分别在大肠杆菌、枯草芽孢杆菌、谷氨酸棒状杆菌和酵母菌4种底盘细胞中探索超声波、消化酶、表面活性剂及超声-表面活性剂联合作用4种方式对单细胞制备效率的影响。提高微生物流式分选存活率,用常压室温等离子诱变(atmospheric and room temperature plasma, ARTP)技术处理含有绿色荧光蛋白(green fluorescent protein, GFP)的酿酒酵母HZ848 (简称HZ848-GFP),形成不同强度GFP文库后,按照GFP强度分选全细胞和PI染色阴性细胞的前0.5%,统计单细胞存活率。【结果】酵母细胞分散条件为：0.01% Tween-80联合超声1 min,单细胞率达到88%以上,PI染色细胞破损率＜1.4%。谷氨酸棒状杆菌单细胞分散条件为：0.01% Tween-80联合超声5 min,单细胞率达到97%以上,PI染色细胞破损率＜1%。分选存活率结果表明,未用PI染色的酿酒酵母分选后单细胞存活率是4.3%,用PI染色去除死细胞后再分选单细胞存活率是18.3%,后者是前者的4.3倍,且具有显著性差异。【结论】本研究为微生物流式分选建立了一套简单快捷的单细胞样品制备方法,证实了PI染色法能够显著提高分选样品存活率。     

Extracellular iron reductases: identification of a new class of enzymes by siderophore-producing microorganisms

This study identifies extracellular iron reductases in culture supernatant fluids of the siderophore-producing microorganisms Escherichia coli and Pseudomonas aeruginosa. These enzymes were constitutively produced and reduced and released iron from a variety of ferric chelators. Dialyzable cofactors, necessary for the transfer of electrons in the enzymatic reduction of iron, were identified. The reductases were sensitive to treatment with proteinase K and guanidine-HCl, were not associated with siderophore activity, and were apparently released from the cell as extracellular enzymes. The acquisition of 59Fe2+ by cell suspensions of E. coli and P. aeruginosa was saturable, suggesting that the ferrous iron generated by these reductases can be bound and transported. Salmonella typhimurium mutants feoB, tonB, entB, and entBfeoB, deficient in numerous known iron uptake pathways, were found to exhibit substantial extracellular iron-reducing activities over that of the parent. A hypothesis is proposed in which the extracellular iron reductases excreted by siderophore-producing microorganisms may be responsible for the mobilization of iron during conditions of iron repletion when siderophores are repressed and may also function in concert with siderophores during periods of iron starvation.     

Physical properties of microbial suspensions

Physical properties of suspensions of Saccharomyces cerevisiae, Candida utilis and Escherichia coli (density, viscosity and surface tension) were measured in synthetic suspensions formed of centrifuged biomass and supernatants from various stages of batch cultivation in the range from 0 to 10% w/v for yeasts and from 0 to 0.25% w/v for bacteria. Surface tension was also measured in native suspensions in the range of 0 less than or equal to Cm less than or equal to 2.0% w/v. All single cell suspensions were found to be Newtonian in behaviour. Densities strictly obey the mixing law, viscosities of Saccharomyces cerevisiae suspensions were correlated by an empirical relation in dependence of Cm and t, surface tensions were correlated graphically for suspensions of Saccharomyces cerevisiae and Escherichia coli, since experiments with both microorganisms have shown that the previously published approximate correlation can safely be used.     

Electrode system for determination of microbial cell populations in polluted water

Microbial cell populations in polluted water were determined by using a fuel cell-type electrode. The electrode was composed of a Pt anode, a Pt-K3Fe(CN)6-K4Fe(CN)6 cathode, and a cation-exchange membrane for separating two electrode compartments. The principle of microbial cell number determination is based on sensing a redox dye reduced by microorganisms with the electrode. Sample solutions containing microorganisms, a redox dye (thionine), and peptone were purged with oxygen-free nitrogen during the determination. A linear relationship was obtained between the increasing rate of current and the number of microbial cells measured by the colony count method above 10(4) cells per ml. The determination time varied with the number of microbial cells determined from 20 to 60 min for 3.6 x 10(6) and 3.6 x 10(4) cells per ml, respectively.     

Electrode system for determination of microbial cell populations in polluted water.

Microbial cell populations in polluted water were determined by using a fuel cell-type electrode. The electrode was composed of a Pt anode, a Pt-K3Fe(CN)6-K4Fe(CN)6 cathode, and a cation-exchange membrane for separating two electrode compartments. The principle of microbial cell number determination is based on sensing a redox dye reduced by microorganisms with the electrode. Sample solutions containing microorganisms, a redox dye (thionine), and peptone were purged with oxygen-free nitrogen during the determination. A linear relationship was obtained between the increasing rate of current and the number of microbial cells measured by the colony count method above 10(4) cells per ml. The determination time varied with the number of microbial cells determined from 20 to 60 min for 3.6 x 10(6) and 3.6 x 10(4) cells per ml, respectively.     

16S rDNA analysis reveals low microbial diversity in community level physiological profile assays

The metabolic diversity of microbial communities is fundamental for the multiple soil functions mediated by microorganisms. Community level physiological profiles (CLPPs) based on sole C source oxidation have been used as a fast and reproducible tool to study soil microbial functional diversity because the utilisation of available carbon is the key factor governing microbial growth in soil. Our aim was to assess the phylogenetic affiliation of the microorganisms responsible for C consumption after inoculating Biolog plates. For this purpose, two semi-arid Mediterranean forest soils with significantly different patterns of C consumption and microbial community structure were used. Following the inoculation of the Biolog plates, suspensions from seven wells were sampled after 1, 2 and 7 d of incubation. DNA was extracted and the microbial communities analysed by polymerase chain reaction followed by denaturing gradient gel electrophoresis (PCR-DGGE) and sequencing of excised bands. Despite major differences in the microbial communities of the soils studied, their DGGE banding patterns after incubation were similar for all the analysed C source suspensions. Microorganisms belonging to beta-Proteobacteria (Ralstonia sp. and Burkholderia sp.) and alpha-Proteobacteria (Rhizobium sp.) were dominant. These opportunists had a competitive advantage under the conditions at which the CLPPs were analysed. This study reveals that significantly different CLPP patterns can be generated on the basis of only 3-4 genera, as reflected by PCR-DGGE analysis. Also for this reason, CLPPs based on incubations of soil suspensions should just be used as a screening method and always be accompanied by other techniques for community analysis.     

In Vitro Catabolism of Histidine by Mixed Rumen Bacteriaand Protozoa

An in vitro study was conducted to examine the metabolism of histidine (His) by mixed rumen bacteria (B), mixed rumen protozoa (P), and a combination of the two (BP). Rumen microorganisms were collected from fistulated goats fed with lucerne cubes (Medicago sativa) and a concentrate mixture twice a day. Microbial suspensions were anaerobically incubated with or without 2 mm each of His, or histamine (HTM), or 1 mm urocanic acid (URA) at 39°C for 12 h. His and other related compounds in both supernatant and microbial hydrolysates were analyzed by HPLC. After 6- and 12-h incubations, the net degradation of His was 26.1% and 51.7% in B, 13.5% and 20.9% in P, and 21.7% and 46.0% in BP, respectively. The rate of the net degradation of His in B (98.0 μmol/g microbial nitrogen/h) was about 2.6 times higher than that of P during a 12-h incubation period. His was found to be degraded into urocanic acid (URA), imidazolelactic acid (ImLA), imidazoleacetic acid (ImAA), and histamine (HTM). Of these degraded His was mainly converted into URA in all microbial suspensions. The production of ImLA and ImAA was higher in B than in P suspensions, whereas the production of HTM was higher in P than in B suspensions. From these results, the existence of diverse catabolic routes of His in rumen microorganisms was indicated. Received: 23 May 2000 / Accepted: 31 July 2000     

Aeration without air: oxygen supply by hydrogen peroxide.

Oxygen has been supplied to suspensions of microorganisms kept under nitrogen by the addition of hydrogen peroxide. If catalase was present in the suspension and the flow was adjusted to the rate of oxygen consumption, the cells grew at rates identical to the controls incubated under air. The applicability of oxygen supply by hydrogen peroxide and its limits are discussed.     

Estimating the Size and Concentration of Unicellular Microorganisms by Light Scattering

The optical density of suspensions of microorganisms was measured at two different positions in a spectrophotometer cuvette compartment. From these two measurements, the particle size and the particle concentration could be estimated. Estimates of particle numbers were not significantly different from those obtained by direct microscopic counting.     

Possibilities of enhancing the sensitivity of the coagglutination reaction

The influence of heating Shigella suspension at 60 degrees C (for 3 minutes) and 100 degrees C (for 30 minutes), as well as adding extraneous microorganisms (Escherichia coli, Proteus) and homologous antibodies to these suspensions, on the sensitivity of the coagglutination test has been studied. The possibility of enhancing the sensitivity of this test 10 to 100 times by heating Shigella suspensions at 100 degrees C for 30 minutes has been shown.