Rapid and Sensitive Detection of Bacteria by Gas Chromatography

A gas chromatograph fitted with electron capture and flame ionization detectors was employed for the rapid detection of bacteria by analysis for their metabolic products. The presence of Proteus vulgaris, Streptococcus faecalis, S. liquefaciens, Escherichia coli B, Bacillus cereus, and B. popilliae was detected in 2 to 4 hr in media inoculated with less than 10(4) cells per ml, whereas a 7- to 12-hr growth period was required for the detection of products formed in cultures of Serratia marcescens, Aerobacter aerogenes, E. coli K-12, Staphylococcus aureus, and Salmonella typhimurium. Metabolites elaborated by the equivalent of less than a single cell of B. cereus, S. faecalis, P. vulgaris, or E. coli B were sensed by the electron capture detector. The flame ionization detector was generally not as sensitive. Volatile metabolites were identified, and their concentrations were determined.     

Thin Layer Chromatography of Radiolabeled Oligonucleotide Analogues: A Rapid and Sensitive Purity Assay

Abstract

Oligonucleotide analogues are being developed for use in cell culture, animals and for therapy. Prior to use it is important to have an indication of the oligomers' purity. Thin layer chromatography (TLC) has been applied to analyze hosphoromonothioate and phosphorodithioate oligonucleotides radiolabeled with either ³²P Or ¹⁴C. TLC coupled with radioactivity has been compared to Polyacrylamide Gel Electrophoresis (PAGE) and High Pressure Liquid Chromatography (HPLC). TLC is a rapid and sensitive alternative to these methods and is particularly suited for chemically modified oligonucleotides.     

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients'' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.     

Rapid, Sensitive Assay for Staphylococcal Enterotoxin A by Reversed Immuno-osmophoresis

Reversed immuno-osmophoresis using phenylsulfonated immune gamma(2)-globulin is a rapid sensitive method of assaying for staphylococcal enterotoxin A. The technique is compared with other serological methods.     

A New Method for Rapid Determination of Biocatalyst Process Stability

In developing enzyme-catalysed processes, a highly active, selective and especially stable biocatalyst forms the basis for an economic industrial technology. Enzymatic stability is one of the most important properties, but there are no good methods for determining it quickly and efficiently. In order to solve this problem, a new method for the “accelerated measurement of activity and stability of enzymes (AMASE)” has been developed. Compared to conventional methods, the new approach differs in two main points. First, the process of ageing is accelerated by a permanent increase in temperature during a continuous experiment, which generates a non-stable response of the enzyme-reactor system. Second, the evaluation procedure is strongly based on a mathematical-mechanistic approach consisting of four steps: assume a deactivation mechanism, formulate the mathematical model, determine the model parameters and finally calculate the biocatalyst characteristics. In this paper, the application of the method for the Lipozyme IM-catalysed hydrolysis of triglycerides in different media is presented.     

A Method of Determination of Penta-, Tetra-, Tri-, and Disaccharides from Xylan by Ion-exchange Chromatography

Bacillus subtilis B7, a tmrA mutant, shows both tunicamycin resistance and a-amylase hyperproductivity. The tmrA characters can be transferred simultaneously to recipient cells by DNA-mediated transformation. We found a typical gene amplification phenomenon in the tmrA transformants and B7 strain. The amplified unit, 16.3kb in size, covers a-amylase structural gene amyE to another tunicamycin resistance gene tmrB, which is located 9kb downstream of the amyE gene. About 10 repeating units are supposed to be tandemly repeated in the transformants. Amplification of the wild amyE and tmrB genes could be the cause of the α-amylase hyperproductivity and tunicamycin resistance of the tmrA transformants and B7 strain.     

A Method for Fractional Determination of Soybean Sterols in Four Classes by Florisil Column Chromatography

A procedure for fractional determination of soybean sterols is presented. Sterols in lipid extracts were fractionated into four classes, fatty acid esters, the free form, acylated glucosides and non-acylated glucosides, by Florisil column chromatography. Sterol contents in the four classes were determined colorimetrically with ferric chloride-perchloric acid reagent. Before the colorimetry, the fatty acid ester fraction was hydrolyzed with ethanolic KOH, and the sterol was isolated as tomatinide. The free sterol fraction was directly treated with tomatine solution. The tomatinides were dissociated with dimethyl sulfoxide. To avoid the contamination of pigments from the acylated glucoside fraction, the second Florisil column was rinsed with diethyl ether between the elution with the first solvent (0 to 50% diethyl ether in n-heхane) and that with the second solvent (0 to 30% methanol in diethyl ether).     

Chelex-100快速提取放线菌DNA作为PCR扩增模板

旨在建立有效扩增16S rRNA基因序列的放线菌DNA快速提取的方法。采用Chelex-100法提取放线菌DNA,使用PCR扩增16S rRNA基因序列评价提取核酸的质量。结果显示,Chelex-100法能够在10 min之内从放线菌中快速提取DNA,所提取的DNA可以直接用于PCR扩增反应,PCR扩增产物电泳条带清晰,符合理论预期结果。因此,Chelex-100法提取放线菌DNA可以作为16S rRNA基因序列PCR扩增的模板,该方法具有经济、简便、快速的特点,适合于放线菌菌株大规模地筛选和分类鉴定。     

A Simple and Sensitive Method for the Quantitative Analysis of Chloroplast Lipids by Use of Thin Layer Chromatography and Flame Ionization Detector

The CD spectrum of lutein drastically changed with added amounts of sodium dodecyl sulfate (SDS) in buffers (pH 7.0) at phosphate concentrations below 70 mm. The CD pattern was inverted at particular binding ratios of SDS to the lutein, depending on the lutein concentration. The ratios were 30, 15 and 10 for lutein concentrations below 3 µm, between 3 and 8 µm, and above 8 µm, respectively. The sedimentation analysis showed strong dependence of the aggregate size of lutein on both the SDS and phosphate concentrations. The size became larger with a decrease of SDS concentration and with an increase of the salt. The sedimentation constants of these aggregates were small in comparison with those expected on the basis of parallel filtration measurements, suggesting that the aggregate was considerably porous. These results indicate that the size of the lutein aggregate is larger than 4500 Å as far as the ordinary CD pattern remains and it becomes smaller as the ordinary pattern changes to the inverted one. A card-pack structure with a chiral nature is discussed as a model of the present aggregate. In the progress of binding the SDS to the lutein, the packed lutein molecules must be twisted from the native form to the subsequent one.     

A Method for the Rapid Enumeration of Mycoplasma Species growing in Broth Culture

S ummary : A method is outlined whereby Mycoplasma species growing in broth culture can be rapidly counted, using the relationship between the number of organisms and the mean lactate dehydrogenase (LDH) content of each organism. The uses of this technique are discussed.     

一种根系分泌物中有机酸的前处理和高效液相色谱检测方法

改进了根系分泌有机酸的前处理和高效液相色谱检测方法.将DEAE纤维素层析法直接用于根系分泌物中有机酸的富集浓缩,并与旋转蒸发浓缩的回收率进行了比较.结果表明,DEAE纤维素柱层析有较高的回收率,可用于根系分泌物中有机酸的富集浓缩,特别适用于大批量样品的前处理.确定了反向高效液相色谱同时检测草酸、酒石酸、马来酸、苹果酸、乙酸、柠檬酸、琥珀酸等7种有机酸的条件.玉米根系分泌物样品中可检出苹果酸等5种有机酸.     

Sensitive and Rapid Detection of Edwardsiellosis in Fish by a Loop-Mediated Isothermal Amplification Method

Here we report a rapid and sensitive method (using loop-mediated isothermal amplification [LAMP]) for the diagnosis of edwardsiellosis, a fish disease caused by Edwardsiella tarda, in Japanese flounder. A set of four primers was designed, and conditions for the detection were optimized for the detection of E. tarda in 45 min at 65°C. No amplification of the target hemolysin gene was detected in other related bacteria. When the LAMP primers were used, detection of edwardsiellosis in infected Japanese flounder kidney, and spleen and seawater cultures was possible. We have developed a rapid and sensitive diagnostic protocol for edwardsiellosis detection in fish. This is the first report of the application of LAMP for the diagnosis of a fish pathogen.     

A Rapid and Simple Method for the Determination of Picogram Levels of 3-Methoxytyramine in Brain Tissue Using Liquid Chromatography with Electrochemical Detection

Abstract: A rapid and simple technique using solvent extraction, ion-pairing extraction, and high pressure liquid chromatography with electrochemical detection has been developed for the determination of 3-methoxytyramine in striata of rats killed by microwave irradiation. The method is specific and reproducible (coefficient of variation among replications, ±4%); recovery of authentic 3-methoxytyramine added to the samples is 45–50%. 3-Methoxytyramine levels found with this technique in rat striata were 15 ± 1.7 ng/g. The method has a sensitivity of about 0.2 pmol per brain sample. Monoamine oxidase inhibition with pargyline increased 3-methoxytyramine levels in rat striata, while catechol- O -methyltransferase inhibition with 3',4'-dihydroxy-2 methylpropiophenone completely depleted 3-methoxytyramine. The effects of nomifensine, quipazine, caroxazone, piribedil, and D-amphetamine were also examined. The 3-methoxytyramine concentrations in the brains of animals killed by decapitation or by microwave irradiation were compared.     

A Rapid,Simple Method for the Determination of 6-Benzyladenine by Pyrolysis Mass Spectrometry

Pyrolysis mass spectrometry (PyMS) is a rapid, simple, high-resolution analytical method based on thermal degradation of complex material in a vacuum. PyMS was used for the quantitative determination of 6-benzyladenine (BA) supplemented to agar-solidified culture media (ASM) in this study. When subjected to PyMS, pure BA generated prominent fingerprint peaks. The peaks at m/z 68 and 123 were chosen for the quantitative measurement of BA because of the highest signal among those generated from pure BA and because of one of the highest masses among those with a prominent signal, respectively. To establish a standard curve for BA concentration in ASM, the combined peak intensity at m/z 68 and 123 was plotted against BA concentration ranged from as low as 0.44 μM after logarithmic transformation of both parameters. A linear regression line was yielded, which indicates BA concentration in ASM is directly proportional to the peak intensity, with R ² = 0.9052, significant at the 99% level. These results suggest that PyMS enables the quantitative determination of growth regulators and other related compounds in plant materials in a rapid, simple, sensitive, accurate manner.     

A Sensitive and Simple Method for the Determination of Fatty Acid Hydroperoxides with Horseradish Peroxidase

Glycine, glycylglycine, glycine methyl ester and glycine ethyl ester were found to be effective for the production and release of γ-galactosidase by Escherichia coli. Addition of an appropriate concentration of glycine and glycylglycine to the culture increased total enzyme production 6 to 7-fold and extracellular enzyme production over 240-fold at 24 hr cultivation. The enzyme synthesis was stimulated even at the exponential period of growth, and 93% of enzyme was found in the culture fluid at 24 hr cultivation on addition of 1.2% glycine. A large amount of protein was also accumulated in the culture fluid. A micrograph showed glycine gave swollen and irregular cells, indicating that the cell surface was altered. Various amino acid analogues and some antibiotics had a small or no effect on the production and release of the enzyme as compared with glycine. Polypeptone or brain heart infusion was needed as a nitrogen source for efficient production of the enzyme.     

一种简单快速测定Rubisco CO2/O2特异性因子的方法

提出了一种简单快速测定1,5-二磷酸核酮糖羧化/氧化酶CO2/O2特异性因子的方法.理论上改进了定量计算公式;操作上避免了使用放射性同位素标记以及层析分离3-磷酸甘油酸和2-磷酸乙醇酸的复杂程序,使测定过程一步完成,极大地减少了随机误差.讨论了实验数据(pH、温度、离子强度)的准确性对计算结果的影响.     

一种简单快速测定Rubisco CO2／O2特异性因子的方法

提出了一种简单快速测定１,５－二磷酸核酮糖羧化／氧化酶ＣＯ２／Ｏ２特异性因子的方法。理论上改进了定量计算公式;操作上避免了使用放射性同位素标记以及层析分离３－磷酸甘油酸和２－磷酸乙醇酸的复杂程度,使测定过程一步完成,极大地减少了随机误差。讨论了实验数据（ｐＨ、温度、离子强度）的准确性对计算结果的影响。