Simplified methods for the microbiological evaluation of bottled natural mineral waters

The conventional methods for the microbiological examination of natural mineral water were compared with a simplified procedure. The results indicate that when indicator micro-organisms are present in water, they may not be detected in the simplified method. An alternative procedure, including the determination of Pseudomonas aeruginosa , is suggested.     

A Rapid Bulk Nissl Method

A simplified and time-saving Nissl method is described in which the fresh nervous tissue is placed directly into: dioxane, 65-70 ml.; water, 15 ml.; 95% alcohol, 20-15 ml.; and toluidine blue, 1 g. This solution fixes, stains, partially clears, and dehydrates in one procedure. The material is then sectioned according to a dry-ice method and mounted on the slide either with or without the use of the rapid albumen procedure. Thus it is possible to terminate an experiment one day and have the tissue ready for microscopic observation on the next.     

Improved facility and sensitivity in the use of guinea pigs for the isolation of Legionella pneumophila from cooling tower water.

The established criteria for the determination of the optimum time for the sacrifice of guinea pigs inoculated with samples of cooling tower water were found to be inadequate for the detection of low levels of Legionella pneumophila. By ignoring the requirement for fever and by sequentially sacrificing the infected guinea pigs on days 3 through 5 postinoculation, we simplified the procedure, and the sensitivity of detection was improved a great deal.     

Improved facility and sensitivity in the use of guinea pigs for the isolation of Legionella pneumophila from cooling tower water

The established criteria for the determination of the optimum time for the sacrifice of guinea pigs inoculated with samples of cooling tower water were found to be inadequate for the detection of low levels of Legionella pneumophila. By ignoring the requirement for fever and by sequentially sacrificing the infected guinea pigs on days 3 through 5 postinoculation, we simplified the procedure, and the sensitivity of detection was improved a great deal.     

An improved method for the purification of Bacillus subtilis glucose dehydrogenase cloned in Escherichia coli

An improved and simplified purification procedure has been developed for the isolation of the Bacillus subtilis glucose dehydrogenase which has resulted in a 10 fold higher yield of pure enzyme. The purification procedure utilizes gene cloning and an additional ammonium sulfate step to facilitate the removal of contaminating proteins. The procedure requires fewer chromatographic steps than previously reported, thus simplifying the procedure. This improved and simplified purification of B. subtilis glucose dehydrogenase will facilitate further structure-function studies of this sporulation specific enzyme.     

Elimination of caffeine interference in high-performance liquid chromatographic determination of cotinine in human plasma

A high-performance liquid chromatographic method with ultraviolet photometric detection for the determination of cotinine in human plasma was described. The use of a 30-cm reversed-phase column and of a mobile phase consisting of water—methanol—0.1 M sodium acetate—acetonitrile (72:21:5.6:1.4, v/v), pH 4.1, eliminated caffeine interference. A simplified solid-phase extraction procedure was also performed for plasma samples.     

Simplified Procedure for Recovery of Lignin Acidolysis Products for Determining the Lignin-Degrading Abilities of Microorganisms

A simplified procedure for the identification and measurement of single-ring aromatic products of lignin acidolysis is described. The procedure employed a 6-h hydrolysis of spruce milled wood lignin in acidic dioxane at 87°C, followed by a series of organic extractions to recover acidolysis products which were quantified by gas chromatography of trimethylsilyl derivatives. Complex gel permeation chromatography procedures utilized by other workers were avoided in the modified procedure, but equivalent results were obtained. The simplified procedure was utilized to hydrolyze sound and actinomycete-decayed spruce milled wood lignins and was shown to be useful as a technique for the rapid screening of microorganisms for their ability to alter lignin.     

水分限制条件下小麦碳同化作用和生物量累积的简单机理模型

以辐射和日长作为自变量,首先建立了一个春小麦碳同化作用和生物量累积过程的简单机理模型;再以叶片水势作为小麦受水分限制的指标,考虑了水分限制下光能利用率、碳同化导度等均受抑制的设定,完成了水分限制下小麦碳同化作用和生物量累积过程简化机理模型的构建。通过分析和田;司实验数据的对比,表明该简单机理模型能很好地反映小麦在有水分限制和无水分限制条件下的碳同化作用和生物量的累积过程。     

Single multiplex polymerase chain reaction for environmental surveillance of toxigenic—Pathogenic O1 and Non-O1vibrio cholerae

A multiplex PCR assay was developed for the detection of toxigenic and pathogenic V. cholerae from direct water sources using specific primers targeting diverse genes, viz. outer membrane protein (ompW), cholera toxin (ctxB), ORF specific for O1 (rfbG), zonula occludens (zot) and toxin co-regulated pilus (tcpB); among these genes, ompW acts as internal control for V. cholerae, the ctx gene as a marker for toxigenicity and tcp for pathogenicity. The sensitivity of multiplex PCR was 5 x 10(4) V. cholerae cells per reaction. The procedure was simplified as direct bacterial cells were used as template and there was no need for DNA extraction. The assay was specific as no amplification occurred with the other bacteria used. Toxigenic V. cholerae were artificially spiked in different water samples, filtered through a 0.45 microm membrane, and the filters containing bacteria were enriched in APW for 6 h. PCR following filtration and enrichment could detect as little as 8 V. cholerae cells per mL in different spiked water samples. Various environmental potable water samples were screened for the presence of V. cholerae using this assay procedure. The proposed method is rapid, sensitive and specific for environmental surveillance for the presence of toxigenic-pathogenic and nonpathogenic V. cholerae.     

Simplified measurement of insulin sensitivity with the minimal model procedure in type 2 diabetic patients without measurement of insulinemia.

This study aimed to evaluate a simplified minimal model protocol for measuring insulin sensitivity in mild and severe type 2 diabetes, considering that changes in serum insulin during an insulin-modified intravenous glucose tolerance test almost only reflect the insulin injection. Two groups of diabetics treated with high doses of antidiabetic agents were recruited. Mean insulin responses were calculated in group 1 (n = 30). In group 2 (n = 38), we compared insulin sensitivity (SI) obtained with reference protocol with SI calculated by a minimal model procedure including the theoretical average insulin profile determined in group 1, and with Homeostasis Model Assessment (HOMA-R). Additionally, the cost of each procedure was calculated. SI measured by the reference method strongly correlated with SI determined by the simplified protocol (r = 0.966, p < 0.0001), while no correlation was found with HOMA-R (r = - 0.349, NS). Reduction of cost for HOMA-R and simplified minimal model procedure were - 92 and - 81 %, respectively. This simplified and relative inexpensive protocol, using minimal model procedure without insulin measurement, accurately measures SI regardless of beta-cell defect degree. This approach could be of interest when limits of validity of simple indexes are reached.     

A Rapid and Simple Method for Estimating Sulfate Reduction Activity and Quantifying Inorganic Sulfides

A simplified passive extraction procedure for quantifying reduced inorganic sulfur compounds from sediments and water is presented. This method may also be used for the estimation of sulfate reduction rates. Efficient extraction of FeS, FeS(inf2), and S(sup2-) was obtained with this procedure; however, the efficiency for S(sup0) depended on the form that was tested. Passive extraction can be used with samples containing up to 20 mg of reduced sulfur. We demonstrated the utility of this technique in a determination of both sulfate reduction rates and reduced inorganic sulfur pools in marine and freshwater sediments. A side-by-side comparison of the passive extraction method with the established single-step distillation technique yielded comparable results with a fraction of the effort.     

Computer analysis of protein-protein interaction

An automatic procedure which generates possible modes of protein-protein association is developed and applied to the bovine pancreatic trypsin inhibitor-trypsin complex as a test case. Using a simplified model in which each residue is replaced by one interaction center, all possible modes of interaction between the inhibitor and the active center of the enzyme are generated systematically. The non-bonded interactions between the molecules and the protein surface area buried in the generated interfaces are evaluated and used as criteria for selecting stable complexes. We show that satisfactory estimates of accessible and buried surface areas can be made using the simplified model.The procedure leads to about nine structures having non-bonded interactions and buried surface areas similar to those of the native complex. This suggests that the major contributions to the free energy of dissociation are taken into account by our selection procedure, though complementarity and specificity are not properly represented in the simplified model. However, it makes it possible to scan a much larger number of configurations than would otherwise be feasible, chiefly through elimination of side-chain detail.     

Method for the design and analysis of a non-linear anisotropic lower limb prosthetic socket

A simplified computer-based structural analysis procedure has been developed for evaluating the stresses in a lower limb prosthetic socket when subjected to external loads. This technique has been tested by studying a simple linear axisymmetric shell subjected to uniform loads. It was found that the stress and strain distributions obtained are close to those determined by a finite element technique. The method that has been proposed involves an incremental procedure, which can be used to analyse the behaviour of a prosthetic socket of irregular shape made from non-linear anisotropic materials. By employing the proposed procedure to study an example, in which a simplified lower limb prosthetic socket is subject to internal pressure and frictional forces, the effects of the material non-linearity have been found to be significant.     

Isolation of water-soluble cytochromes from cyanobacteria by adsorption chromatography

A simple procedure using ammonium sulfate to fractionate water-soluble cytochromes c553 and c550 on Sephacryl S-200 gel is described. The usefulness of this procedure has been studied using the crude extracts of mesophilic cyanobacteria. It was found that almost all the cytochromes were adsorbed on to the gel at 2.34 M ammonium sulfate and were eluted at decreasing salt concentrations. The cytochromes were free of interfering phycobiliproteins and thus were suitable for the study of isoelectric points. It was also found that this procedure allowed a clear separation of the cytochromes based on their hydrophobicities. The order of elution was cytochrome c553, then cytochrome c550, indicating that c550 is more hydrophobic than c553. All these results show that this procedure provides both a simplified and an efficient purification of the cytochromes and insight into their surface properties. The cytochromes of Microcystis aeruginosa were purified to homogeneity using this procedure and other existing ones. Homogeneous cytochromes c553 and c550 were chromatographed on Sephacryl S-200 at 1.75 M (NH4)2SO4 and found to elute in the same order as reported earlier for the cytochromes in the crude extracts. In addition, cytochrome c550 was found to be more heat resistant and less water soluble than cytochrome c553.     

A Procedure for Clustering Means of Unequal-Sized Samples in ANOVA

The proposed procedure “SECOLICO” is based on the sequential construction of linear contrasts. After an analysis of variance the procedure is able to classify the treatments represented by mean values of unequal-sized samples made at random into distinguishable groups. For the purpose of illustrating the procedure “SECOLICO” a simplified example is given.     

Seasonal changes in photosynthetic capacity of leaves of kiwifruit (Actinidia deliciosa) vines

A simplified procedure for the assay and purification of an enzyme which activates a galactosyltransferase (EC 2.4.1.96) involved in volume regulation of the unicellular alga Poterioochromonas malhamensis (Peterfi) is described. The enzyme was extracted with water from membranes, followed by chromatography on DEAE-Sephacel, phenyl-Sepharose and fetuin-agarose. Its proteinase activity was demonstrated by cleavage of oxidized insulin A- and B-Chains. The predominant cleavage site of the oxidized A-chain is the peptide bond between ¹³Leu and ¹⁴Tyr whereas ¹⁶Leu-¹⁷Glu is also hydrolyzed with minor activity. Besides this chymotrypsin-like endopeptidase activity some carboxypeptidase activity was also observed.     

A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels

We have simplified the highly sensitive silver stain of R. C. Switzer III, C. R. Merril, and S. Shifrin (1979, Anal. Biochem.98, 231–237) for visualizing proteins in polyacrylamide gels. We have reduced the number of steps in the procedure from 10 to 6, simplified the reagents in each step, and reduced the amount of silver required by a factor of 10, thus greatly reducing the expense of the procedure. In common with the original silver stain, our procedure is 100 times more sensitive than Coomassie brilliant blue and is comparable in sensitivity to radioautography of radioactively labeled proteins.     

Simplified northern blot hybridization using 5% sodium dodecyl sulfate.

We describe a simplified Northern blot hybridization procedure. This procedure eliminates the need for many reagents commonly used in RNA hybridization and replaces them with two buffers containing 5% sodium dodecyl sulfate for pre-hybridization, hybridization and all post-hybridization washes.