淀粉酶活性测定方法的改进

以香蕉果实为试材,对常规的淀粉酶活性的测定方法进行了改进,取得了更好的效果.     

Fluorometric Method for Determining the Efficiency of Spun-Glass Air Filtration Media

The procedures and equipment needed to measure filtration efficiency by means of fluorescent aerosols are described. The filtration efficiency of individual lots of spun-glass air filtration medium or of entire air filtration systems employing such media was determined. Data relating to the comparative evaluation of spun-glass filter media by means of the fluorometric method described, as well as by conventional biological procedures, are presented.     

An Improved Method for Determining Lipolytic Acyl-hydrolase Activity

The effects of 2-phthalimidooxyalkanoic acid derivatives on the germination and root-growth of cress were examined. Since 2-phthalimidooxypropionates were most effective, the optically active ethyl esters were prepared. As the result of biological testing, the (S)-(-)-isomer exhibited stronger activity than the (R)-(+)-isomer. This result is contrary to those from commercial herbicides with similar structures, phenoxy- and oxyphenoxy-propionate-type compounds, where the (R)-isomers are generally known to be the active principles.     

蔬菜硝酸盐含量测定方法的改进

针对水杨酸消化比色法测定植物体内硝酸盐含量中存在的问题,经过优化筛选,将此测定方法的提取条件优化为：温度90℃、时间30 min;每10 g绿色蔬菜加入0.2 g活性炭能消除颜色的影响,回收率达到96%。     

Rapid Method of Determining Factors Limiting Bacterial Growth in Soil

A technique to determine which nutrients limit bacterial growth in soil was developed. The method was based on measuring the thymidine incorporation rate of bacteria after the addition of C, N, and P in different combinations to soil samples. First, the thymidine incorporation method was tested in two different soils: an agricultural soil and a forest humus soil. Carbon (as glucose) was found to be the limiting substance for bacterial growth in both of these soils. The effect of adding different amounts of nutrients was studied, and tests were performed to determine whether the additions affected the soil pH and subsequent bacterial activity. The incubation time required to detect bacterial growth after adding substrate to the soil was also evaluated. Second, the method was used in experiments in which three different size fractions of straw (1 to 2, 0.25 to 1, and <0.25 mm) were mixed into the agricultural soil in order to induce N limitation for bacterial growth. When the straw fraction was small enough (<0.25 mm), N became the limiting nutrient for bacterial growth after about 3 weeks. After the addition of the larger straw fractions (1 to 2 and 0.25 to 1 mm), the soil bacteria were C limited throughout the incubation period (10 weeks), although an increase in the thymidine incorporation rate after the addition of C and N together compared with adding them separately was seen in the sample containing the size fraction from 0.25 to 1 mm. Third, soils from high-pH, limestone-rich areas were examined. P limitation was observed in one of these soils, while tendencies toward P limitation were seen in some of the other soils.     

Method for Collecting Naturally Occurring Airborne Bacterial Spores for Determining Their Thermal Resistance

The ability to determine the thermal resistance of naturally occurring air borne bacterial spores associated with spacecraft and their assembly areas has been hindered by lack of an effective collecting system. Efforts to collect and concentrate spores with air samplers or from air filters have not been successful. A fallout method was developed for this purpose and tested. Sterile Teflon ribbons (7.6 by 183 cm) were exposed in pertinent spacecraft assembly areas and subsequently treated with dry heat. Thermal inactivation experiments were conducted at 125 and 113 C. Heating intervals ranged from 1 to 12 h at 125 C and 6, 12, 18, and 24 h at 113 C. Eight hours was the longest heating time yielding survivors at 125 C, whereas survivors were recovered at all of the heating intervals at 113 C. D_125C values were calculated using the fractional-replicate-unit-negative technique of Pflug and Schmidt (1968) and ranged from 25 to 126 min. This variation indicated that the most probable number of survivors at each heating interval did not fall on a straight line passing through the initial spore population. However, the most-probable-number values taken alone formed a straight line suggesting logarithmic thermal destruction of a subpopulation of spores with a D_125C value of 6.3 h.     

Improved Method for Isolation of Bacterial Inhibitors from Oleuropein Hydrolysis

A new high-pressure liquid chromatography multidetection quantitative method for the isolation of the products of oleuropein hydrolysis is described. A single analysis yields sufficient amounts of the compounds to test their inhibitory effect on bacterial growth.     

Direct Method for Determining the Viability of a Freshly Generated Mixed Bacterial Aerosol

A direct method was developed to determine the viability of a freshly generated mixed bacterial aerosol. A mixed suspension of (32)P-labeled Staphylococcus aureus and (35)S-labeled Proteus mirabilis was nebulized, and the aerosol was collected and separated according to particle size with an Andersen sampler. Quantitative and qualitative bacteriological and radioisotopic techniques were used to obtain ratios of bacterial to radioactive counts for each organism in samples of the nebulizer suspension and aerosol. Loss of viability was calculated from the change that occurred between the ratio of the nebulizer suspension and the ratio of the aerosol. The viability of S. aureus was unaffected by aerosolization, whereas the viability of P. mirabilis declined by 20 to 60% and was inversely proportional to particle size. The advantages of this method over present indirect methods, as well as potential applications of the method, are discussed.     

药用谷氨酸含量测定方法的改进

药用谷氨酸是具有生物活性的L-型谷氨酸。中国药典(1977年版)只对它的旋光度做了如下规定:(+)31.7～(+)32.3,而对其含量限度未做要求。据查英、美药典(1980年版)、日本药局方第十版对该药均无收载。因此我们做了一些工作。采用几种不同方法测定药用谷氨酸的含量,对凯氏定氮法、中和法、旋光法进行了比较。测定中以凯氏定氮法为准,计算回收率,并计算其均值、标准差及变异系数。实验结果表明,采用旋光法较好。回收率为100.003%,标准差为0.2525,变异系数为0.2525%。     

Diffusion Rates in Disrupted Bacterial Cells

The viscosity of the material resulting from squeezing Escherichia coli cells through an orifice in a French pressure cell has been shown to be very high and variable with temperature. Diffusion constants in this medium have been determined for sucrose, dextran, and beta galactosidase. The values found are: 1.07 × 10^-6cm²/second for sucrose, 0.36 × 10^-6cm²/second for dextran, and 0.025 × 10^-6cm²/second for beta galactosidase. The results agree with the idea that there is much interstitial space available for diffusion of small molecules in the cell medium in spite of the high viscosity, but that large molecules will be transported less readily.     

Filtration Method for Bacteriophage Detection

A filtration method has been developed which can be used to detect and enumerate phage in low concentrations directly from solution without the need for prior concentration. In this method, a known volume of the phage solution is mixed with a suitable host solution. Samples are filtered through membrane filters; the filter is removed and incubated, and after 24 hr the resultant plaques are counted and the titer is calculated. Escherichia coli B and the coliphage T2 were used in these studies. Host cultures less than 12 hr old produced the best results. Approximately 10(10) host organisms must be present in the sample taken for filtration. To avoid phage reproduction, all steps prior to filtration must be done in less than 45 min. The method was compared with the soft-agar technique and was shown to be less precise but able to measure phage in lower concentrations.     

Improved Filtration Technique for Concentrating and Harvesting Bacteria

An improved technique is described for the filtrative concentration and harvesting of bacterial cultures. A pleated tangential flow filtration unit containing 1,000 cm² of 0.2-μm-pore-size microporous membrane was used to rapidly (30 to 50 min) reduce the volume of 5 liters of bacterial culture of approximately 10⁹ cells per ml to 0.2 to 0.5 liters of concentrated bacterial suspension. The effects of cell concentration, filtration pressure, and tangential flow rate were examined with respect to the rate of concentration and cell viability. Recovery efficiencies were between 60 and 75%, with no apparent impairment of organism viability. Cell concentration exerted the predominant effect on the filtration rate.     

Enteric Bacterial Growth Rates in River Water

Enteric bacteria, including stocked strains of pathogenic species and organisms naturally present in the stream, were capable of growth in a chemostat with autoclaved river water taken 750 m below a sewage outfall. Maximal specific growth rates for all organisms occurred at 30 C, whereas culture generation times ranged between 33.3 and 116 hr. Of the six laboratory strains of enteric species used, Escherichia coli and Enterobacter aerogenes grew at generation times of 34.5 and 33.3 hr, respectively, while the remaining Proteus, Arizona, Salmonella, and Shigella spp. reproduced at a rate two to three times slower than the coliforms. Little or no growth occurred in the water at incubation temperatures of 20 and 5 C, and death was observed for Salmonella senftenberg at 20 and 5 C and for E. aerogenes and Proteus rettgeri at 5 C. When enteric bacteria naturally present in the river water were employed in similar experiments, coliform bacteria demonstrated a generation time of approximately 116 hr, whereas fecal coliforms failed to grow. Growth of the bacteria from the river demonstrated a periodicity of approximately 100 hr, which suggests that much of the growth of these organisms in the chemostat may be on the glass surfaces. This phenomenon, however, was not observed with any of the stocked enteric species. Neither the stock cultures nor the aquatic strains were capable of growth in autoclaved river water taken above the sewage outfall at the three temperatures tested.     

Improved Automatic Inoculator for Bacterial Cultures

A simple automatic inoculator which allows the simultaneous inoculation of several cultures with identical or different inocula is described. The use of Teflon spaghetti tubing allows great flexibility in handling and placing of components. The inoculator can be adapted to handle aerobic and anaerobic bacteria.     

An Improved Simplified High-Sensitivity Quantification Method for Determining Brassinosteroids in Different Tissues of Rice and Arabidopsis

Quantification of brassinosteroids is essential and extremely important to study the molecular mechanisms of their physiological roles in plant growth and development. Herein, we present a simple, material and cost-saving high-performance method for determining endogenous brassinosteroids (BRs) in model plants. This new method enables simultaneous enrichment of a wide range of bioactive BRs such as brassinolide, castasterone, teasterone, and typhasterol with ion exchange solid-phase extraction and high-sensitivity quantitation of these BRs based on isotope dilution combined with internal standard approach. For routine analysis, the consumption of plant materials was reduced to one-twentieth of previously reported and the overall process could be completed within 1 day compared with previous 3 to 4 days. The strategy was validated by profiling BRs in different ecotypes and mutants of rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), and the BR distributions in different model plants tissues were determined with the new method. The method allows plant physiologists to monitor the dynamics and distributions of BRs with 1 gram fresh weight of model plant tissues, which will speed up the process for the molecular mechanism research of BRs with these model plants in future work.Brassinosteroids (BRs) have been considered as the sixth class of endogenous plant hormones with wide occurrence across the plant kingdom (Bajguz and Tretyn, 2003). BRs play a key role in a variety of physiological processes, such as cell elongation, vascular differentiation, reproductive development, photomorphogenesis, stress tolerance, and so on (Hayat, 2010). Recently, it was found that BR deficiency could increase grain yield in rice (Oryza sativa) by more than 30%, which showed a food security-enhancing potential and guided new green revolution in the future (Sakamoto et al., 2006; Wu et al., 2008). Since BRs were first isolated and identified from rape (Brassica napus) pollen in 1970s (Mitchell et al., 1970; Grove et al., 1979), the natural occurrence of more than 60 BRs in a large quantity of plant species has been reported (Hayat, 2010). To date, research on the occurrence of BRs in different plants, physiological properties, and their action modes has made much progress (Fujioka and Yokota, 2003; Symons et al., 2008; Kim and Wang, 2010; Tang et al., 2010; Tong and Chu, 2012). However, so far, only limited information was obtained to understand the molecular mechanism of the physiological role of BRs. For example, although the biosynthetic pathway of C₂₈ BRs has been well established, the biosynthesis of C₂₇ and C₂₉ BRs remains unclear, and some intermediates on their biosynthetic pathways still need to be elucidated (Noguchi et al., 2000; Fujita et al., 2006). The plant physiology research of BRs is speeded up by employing BR mutants on biosynthesis and signaling pathways (Yamamuro et al., 2000; Hong et al., 2003; Kwon and Choe, 2005; Tanabe et al., 2005); however, a simple, high-sensitivity screening, detection, and quantification method for BR analysis is still a bottleneck technique for in-depth studying of the role of BRs during the life cycle of plants.In the past 20 years, most of the detection and identification processes of BRs could be described briefly as the following steps. The harvested plant materials were lyophilized and then ground to a fine powder, followed by the CH₃OH/CHCl₃ extraction. The concentrate was then partitioned with the CHCl₃/H₂O system three times. After that, the CHCl₃ fraction was subjected to a silica gel column for BR enrichment, and the collected BRs-containing fraction was purified with Sephadex LH-20 column and Sep-Pak Plus C18 cartridge in sequence. At last, the collected fractions were purified with preparative HPLC and then derivatized for analysis with gas chromatography-mass spectrometry (MS) under selected ion monitoring mode (Hong et al., 2005; Nomura et al., 2005; Kim et al., 2006). So far, this protocol has been proven to be workable in most cases and provided a great quantity of valuable data for plant physiological research (Hwang et al., 2006; Lee et al., 2010). However, at least more than 20 g of plant materials were consumed for quantifying/identifying BRs in one plant sample without replicates (Hong et al., 2005; Bancos et al., 2006; Kim et al., 2006), and it is difficult to collect sufficient plant tissues for BR measurement in some rare model plant mutants. In addition, the method involved multiple tedious and labor-intensive steps, which might result in poor recovery and low sensitivity, especially for some labile BR intermediates. The traditional method took one person about 3 to 4 d to treat one batch of samples. Most of the BR measurement experiments were performed without biological replicates using traditional methods due to the disadvantages mentioned above, which discounted the reliability of the results. Therefore, a simple, rapid, and sensitive analysis method for BRs is in urgent need, along with the development of BR research.Recently, several efforts were made to improve the BR determination (Svatos et al., 2004; Huo et al., 2012). The consumption of plant material was reduced to 2 g after modifying the BRs with a new derivatization reagent for further ultra-performance liquid chromatography (UPLC)-multiple reaction monitoring (MRM)-MS detection. However, the purification process consisting of deproteinization and multiple solid-phase extraction (SPE) steps was still quite tedious and couldn’t guarantee covering the four most important bioactive BRs, including brassinolide (BL), castasterone (CS), teasterone (TE), and typhasterol (TY; Fig. 1). In our previous study (Xin et al., 2013), we reported a simple, convenient, and high-sensitivity method for detection of endogenous BRs from real plant materials based on the dual role of specific boronate affinity. Although it was the first time to measure multiple BRs in subgram plant materials and the time duration of the method decreased to one-third of that previously reported, the synthesis of boronate affinity-functionalized magnetic nanoparticles made the method difficult to follow in biological laboratories for routine analysis.Open in a separate windowFigure 1.Chemical structure of four major bioactive BRs.BRs are neutral steroid compounds with a common four-ring cholestane skeleton and hydroxyl groups on A ring and/or the side chain linked to D ring. Especially, the vicinal diol moieties on C₂₂ and C₂₃ sites of BL, CS, TY, and TE allow these bioactive BRs to be derivatized with ionizable reagents for MS response enhancement. Considering the unique physicochemical properties of BRs, we herein developed a simplified high-sensitivity analytical method based on mixed-mode anion exchange (MAX)-cation exchange (MCX) solid phase extraction (SPE) purification, vicinal diol derivatization combined with UPLC-MRM3-MS detection for quantification of BL, CS, TE, and TY in model plants (Fig. 2). The performance of the method was demonstrated by determination of BRs in different tissues of both wild-type and mutant Arabidopsis (Arabidopsis thaliana) and rice.Open in a separate windowFigure 2.Simplified high-sensitivity protocol for quantitative analysis of BRs. IS, Internal standards. [See online article for color version of this figure.]     

Cultivation-Dependent and -Independent Approaches for Determining Bacterial Diversity in Heavy-Metal-Contaminated Soil

In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis. However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities. To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches. There were small differences in microscopic epifluorescence bacterial counts. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar. A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies. However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations. The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample. DGGE bands from both culture-based and culture-independent approaches were sequenced and compared. These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered. Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.     

Estimations of Bacterial Growth Rates in Natural Waters

Specific growth rates as low as 0.005 hr⁻¹ (generation times of 20 to 200 hr) of aquatic bacteria in natural waters have been calculated from significant differences between dilution rates and washout rates in a chemostat. The measured growth rates were affected by the treatment of the water samples (type of sterilization) and by competition with the natural microflora for the unknown growth-limiting substrate.     

Method for Determining Antineuraminidase Activity

A test was developed to screen drugs for antineuraminidase (influenza sialidase) activity in vitro. Neuraminidase prepared from Vibrio cholerae was added to a substrate containing ganglioside, prepared from calf brain. Sialic acid is a split product in the reaction. The presence of sialic acid was detected colorimetrically by use of Warren's Thiobarbituric Acid Assay after drugs had been added to inhibit the action of neuraminidase on the calf brain substrate.