Quantitative Aspects of Growth of the Methane Oxidizing Bacterium Methylococcus capsulatus on Methane in Shake Flask and Continuous Chemostat Culture

S ummary : Experiments were directed towards the production of high biomass concentrations in cultures of Methylococcus capsulatus. In shake flasks the effects of ammonium ion, phosphate ion and various trace metals on growth were studied. In the chemostat the effects on growth of methane limitation, oxygen limitation, aeration, dilution rate, pH value and temperature were studied and carbon balances were made in steady state conditions. Growth in the fermenter was stimulated by the use of Amberlite CG–120 ion exchange resin in the medium. The probability that Amberlite removed a growth, inhibitor is discussed.     

Novel Technique for Isolating Microstructures Present in Shake Cultures of the Fungus Ceratocystis ulmi

Microstructures found in shake cultures of Ceratocystis ulmi, the fungus causing Dutch elm disease, have been isolated by a novel technique using the effect of bubbling gas through the culture filtrate.     

Rapid Method for the Radioisotopic Analysis of Gaseous End Products of Anaerobic Metabolism

A gas chromatographic procedure for the simultaneous analysis of (14)C-labeled and unlabeled metabolic gases from microbial methanogenic systems is described. H(2), CH(4), and CO(2) were separated within 2.5 min on a Carbosieve B column and were detected by thermal conductivity. Detector effluents were channeled into a gas proportional counter for measurement of radioactivity. This method was more rapid, sensitive, and convenient than gas chromatography-liquid scintillation techniques. The gas chromatography-gas proportional counting procedure was used to characterize the microbial decomposition of organic matter in anaerobic lake sediments and to monitor (14)CH(4) formation from H(2) and (14)CO(2) by Methanosarcina barkeri.     

SERS标记方法在生化分析中的应用

表面增强拉曼散射(SERS)标记方法结合现代生物标记方法与SERS光谱技术,使吸附到金或银等贵金属表面的标记分子的拉曼信号显著增强,并将其作为标记示踪信号,具有生物兼容性好、灵敏度高、分子特征性强和快速简便等优点,已成为新颖的标记示踪技术的研究热点之一。本文综述近年来SERS标记技术应用于基因分析、蛋白质检测、微生物检测、肿瘤靶向和小分子物质的最新进展,着重介绍蛋白质和小分子物质的检测,并展望了今后的发展方向。     

A Novel Chromatographic Method for the Preparation of High Density Lipoproteins

High density lipoproteins (HDL) were isolated by a procedure employing polyanion precipitation and column chromatography. The product was free of low density lipoproteins (LDL) but serum albumin (HSA) was still present. The remaining HSA was removed by an immuno-adsorbent column. The HDL isolated by our method was compared to another HDL preparation isolated from the same plasma sample by the combination of ultra centrifugation and gel chromatography.¹ It was found to have approximately the same lipid and protein composition as the HDL isolated by conventional techniques.¹ Minor differences included a higher phospholipid and apoprotein E content and lower triglyceride and ApoC II content of the HDL isolated by column chromatography. The method described here is considerably less tedious than earlier techniques, can be scaled up without substantial increase in labor and results in an approximately 30% higher yield than the method described by Rudel et al.¹     

Micro Radiometric Method for Study of Acid-insoluble Materials in Monolayer Cell Cultures

A simple radiometric procedure for study of acid-insoluble products synthesized in monolayer cell cultures is described. Cell cultures were produced directly on the bottom surface of scintillation vials or on glass cover slips (8 X 30 mm). The cells were labeled and extracted; the radioactivity was determined while the cells remained affixed to the glass surface upon which they were grown. This procedure enabled rapid investigations of certain biosynthetic processes to be carried out by using many individual cell cultures. The method was applied to an investigation of (3)H-thymidine incorporation induced by vaccinia virus in a 5-bromodeoxyuridine-resistant cell line. (14)C-labeling was evaluated as an alternate procedure for cell quantitation.     

Quantitative Method for the Gas Chromatographic Analysis of Short-Chain Monocarboxylic and Dicarboxylic Acids in Fermentation Media

A method for the preparation and gas chromatographic analysis of the butyl esters of volatile (C-1-C-7) and nonvolatile (lactic, succinic, and fumaric) acids in microbial fermentation media is presented. Butyl esters were prepared from the dry salts of the acids. The esters were separated by temperature programming on a column of Chromosorb W coated with Dexsil 300 GC liquid phase and analyzed with a flame ionization detector. Apparent recoveries with butanol-HCl or butanol-H2SO4 as butylating agents were 80 to 90% for most acids. Chromatographic profiles of the butyl esters demonstrated that both volatile and nonvolatile acids can be detected and separated in 24 min on a single column. Standard calibration curves (peak area versus concentration) of the butyl esters were linear in the range of 5 to 40 mumol of acid per ml. The advantages of using an internal standard (heptanoic acid) for quantitating fatty acids in a mixture are given. Chromatograms of butylated fermentation media in which rumen anaerobic bacteria were grown illustrated that this method is useful for determining short-chain volatile and nonvolatile acids of toxonomic significance.     

A Validated Method for Rapid Analysis of Ethane in Breath and its Application in Kinetic Studies in Human Volunteers

Oxidative stress may initiate lipid peroxidation that generates ethane. Ethane, at low concentrations, is eliminated by pulmonary exhalation. Previous methods have not allowed frequent sampling, thus ethane kinetics has not been studied in man. A validated method over the range 3.8-100,000 ppb with a limit of quantitation of 3.8 ppb (CV 9.3%) based on cryofocusing technique of a 60 ml breath sample allowed frequent sampling. Due to a rapid analytical procedure batches of more than 100 samples may be analyzed. In human volunteers (24-55 years) uptake was studied for up to 23 min <formula>(<italic>n</italic>=9)</formula>, elimination was studied for 210 min <formula>(<italic>n</italic>=9).</formula> Ethane was inhaled (concentrations varied from 16 to 29 ppm (parts per million)) through a non-rebreathing system; sampling was performed with short intervals from the expiratory limb. Samples were also drawn from the inhalatory limb. Ninety-five percent of steady state (inspired) concentration was reached within 1.75 min. Five percent of the initially inhaled concentrations was found in exhaled air 1.5 min after termination of inhalation. A terminal mean half life of 31 min for ethane was also observed. The data indicate that frequent sampling will be necessary to capture relevant changes in breath ethane.     

Development of a Bacterial Cell Enrichment Method and its Application to the Community Analysis in Soybean Stems

A method was developed for enriching bacterial cells from soybean stems which was recalcitrant for a culture-independent analysis of bacterial community due to the interference with plant DNA. Stem homogenates were fractionated by a series of differential centrifugations followed by a Nycodenz density gradient centrifugation. The efficiency of bacterial cell enrichment was assessed by ribosomal intergenic spacer analysis (RISA). The intensity and the number of bacterial amplicons of RISA were markedly increased in the DNA extracted from the enriched bacterial cells compared to that in the DNA directly extracted from soybean stems. The phylogenetic diversity of the enriched bacterial cells was evaluated by analyzing a clone library of 16S rRNA gene in comparison with those of the culturable fractions of the enriched and non-enriched stem-associated bacteria, endophytic bacteria, and epiphytic bacteria. The results indicated that the method was able to enrich both endophytic and epiphytic bacteria from soybean stems, and was useful to assess the bacterial diversity based on a 16S rRNA gene clone library. When the sequence data from all clones (1,332 sequences) were combined, 72 operational taxonomic units were affiliated with Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteroidetes, which also provided the most comprehensive set of data on the bacterial diversity in the aerial parts of soybeans.     

A Cultural Method for the Detection of Pullulan—Degrading Enzymes in Bacteria and its Application to the Genus Bacillus

Pullulan in a solid growth medium can be precipitated selectively with ethanol. Clear zones due to pullulan hydrolysis surround microbial colonies which produce the appropriate enzyme. When this procedure was applied to 297 strains representing 26 species of the genus Bacillus, 138 (46%) of the organisms were shown to hydrolyze pullulan.     

A Simple Method for Density Gradient Zonal Electrophoresis with Imidazole-Glycine Buffer and its Application to a Study of Cyclic Amp-Binding Protein

A system of sucrose density gradient electrophoresis with an imidazole-glycine buffer is described which allows the use of a simple apparatus without separate buffer reservoirs, electrode chambers and bridges. Changes in pH during electrophoresis are held to a minimum. The procedure is easy and capable of high resolution. An example of the application of this method to a preparation of a cyclic AMP binding protein is shown.     

A Modified Cooling Method and its Application in Drosophila Experiments

Chilling is a cost-effective and safe method of immobilising flies in Drosophila experiments. However, should condensation form on the plate, it would be fatal to the flies. Here we describe a modified cooling method using reusable commercial ice pack(s) (ca. 400 ml, 2–3 cm tall) rather than crushed ice. The ice pack is covered with a piece of filter paper which absorbs the condensed water formed on the cold surface, and the moistened filter paper does not wet the wings of flies inverted onto it. An ice pack (ca. 400 ml) takes effect in at least 1 h, and its height is shorter than the maximum specimen height of a dissecting microscope, so that flies can be checked while they remain on the cold surface. This method overcomes the disadvantages of the indigenous cooling anaesthetising approach and makes it practically feasible. We also describe an easy-to-make fly-transferring toolkit which will greatly facilitate fly transferral when it is combined with our chilling method. In our design, the flies were directed out of a vial by a blue pipette tip, which is inserted in a foam stopper and channelled down into another vial by a glass funnel inserted in another foam stopper which is escape-proof.     

Microtiter Method for the Evaluation of Viable Cells in Bacterial Cultures

A microtiter technique was investigated as a means of evaluating viable cells in bacterial cultures. Parallel experiments were performed employing the conventional agar plate method along with the microtiter procedure. Statistical analyses showed that the correlation between the two methods was highly significant. With this new method, many samples were analyzed simultaneously, and readable results were obtained in 12 to 15 hr. Other advantages of this method were substantial savings of time, space, and materials. Also, the applicability of this method to estimates of mixed bacterial populations was demonstrated by studying the associative growth of two bacterial cultures.     

The Relationship between Methane Production and Concentrations of Hydrogen in the Aqueous and Gaseous Phases during Rumen Fermentation in vitro

S ummary : When rumen micro-organisms were incubated in vitro with a wide range of concentrations of hydrogen (H) in the gas phase, the amount of methane produced was linearly related to the amount of H taken up. The rate of utilization of dissolved H for methane synthesis was close to the rate of entry of H into solution from the gas phase. Hydrogen produced from formate accumulated in the aqueous phase, but methane production was not related to its concentration.     

Applicability of Peak-Scaled Nonstationary Fluctuation Analysis to the Study of Inhibitory Synaptic Transmission in Hippocampal Cultures

At present, there are no direct methods to determine the number of synaptic receptor-related channels activated in the course of synaptic transmission (N) or a value of the single-channel conductance (γ). Peak-scaled nonstationary fluctuation analysis (PS NSFA) should be considered the most well-developed indirect approach used for estimating these parameters. Despite the relatively wide using of this approach for the analysis of various synaptic currents, some aspects of possible errors that can occur in the course of data acquisition or their subsequent processing have not been studied. We examined in detail the problem of applicability of PS NSFA in the study of spontaneous and evoked GABA-ergic inhibitory postsynaptic currents (IPSCs). IPSCs were recorded using a dual patch-clamp technique from hippocampal neurons growing in low-density cultures. Parameters of the recorded IPSCs and values for different components of GABA-ergic synaptic transmission reported earlier were used for simulations and PS-NSFA analysis. In Monte Carlo computer simulations of evoked IPSCs, the influence of series resistance, background noise, asynchronicity of transmitter release, GABA_A channel properties, dendritic attenuation, and instrumental filtering on γ estimates obtained by PS NSFA was examined. We concluded that the γ and, consequently, N values may be satisfactorily estimated by the suggested approach using spontaneous and evoked IPSCs recorded in inhibitory synaptic connections in hippocampal cultures within a wide range of experimental conditions. We also estimated the mean of the single-channel conductance of synaptic GABA_A receptors in neurons from primary hippocampal cultures and found that this value (29 ± 5 pS) agrees well with the high conductance of single synaptic GABA_A receptors observed in acute hippocampal slices. This indicates that dissociated cultures are an adequate model for studying the properties of synaptic GABA_A receptors. Neirofiziologiya/Neurophysiology, Vol. 37, No. 4, pp. 379–388, July–August, 2004.