真菌生物量指示剂麦角固醇的分离及测定方法

麦角固醇作为真菌细胞膜的重要组成成分,结构稳定,可作为真菌生长的指示物质。综述了表征真菌生物量的麦角固醇的分离及测定方法,其中麦角固醇的萃取方法有传统的皂化回流法、快速物理萃取、超声波萃取、超临界流体萃取等,测定方法有高效液相色谱法、气相色谱-质谱法、液相色谱-质谱法、薄层色谱法等。并对这些方法的应用及在堆肥过程中运用麦角固醇估计真菌生物量的可行性进行了展望。     

Determination of sterilization effectiveness by measuring bacterial growth in a biological indicator through firefly luciferase determination of ATP

A bioluminescence procedure for measurement of microbial ATP allows a rapid determination of the effectiveness of autoclave sterilization. This determination is achieved faster than detection of acid production in a biological indicator via a pH indicator. Bacterial outgrowth from spores on test strips of the biological indicator was detected by measurement of ATP using the firefly luciferase reaction. A measureable increase in ATP was found after 5 hours of incubation of a biological indicator that had been treated under sterilizing conditions that produced 75% sterility of the biological indicator as measured by acid production. This is a marked improvement over the 24-48 hours of incubation currently required.     

A resazurin-based biosensor for organic pollutants

A new rapid biosensor method employing the dye resazurin as an indicator of bacterial respiration has been developed to provide a rapid, facile and specific biosensor for environmental contaminants that does not rely on genetic modification techniques, is suitable for a high-throughput multiwell format, and is ideally suited to resource-constrained environmental monitoring situations. This whole-cell biosensor has been applied to the test analyte toluene using natural toluene-degrading bacteria as the biological component and is competitive with more complex recombinant approaches. The redox-driven biosensor is dependent on the catabolism of a specific compound, concomitantly reducing the redox indicator resazurin to provide the analytical signal in a whole-cell biosensor assay.     

Bioassay for the rapid detection of bacteriocins in fermentation broth

A bioassay for the rapid detection of bacteriocins by detecting efflux of K⁺ ions from a bacteriocin-sensitive indicator strain was developed. There was a rapid increase in concentration of K⁺ in the medium from approximately 1.6 to 9.5 mM when the crude bacteriocin was added to the sensitive strain. A bacteriocin activity of 8.25 AU ml^–1 produced the lowest detectable concentration of K⁺ (0.179 mM) and the smallest inhibition zone (0.4 mm). The smallest cell dry mass of the indicator strain capable of releasing a detectable level of K⁺ (0.102 mM) was 0.76 mg. Detection of the bacteriocin by measuring the efflux of K⁺ compares well with the conventional agar well diffusion assay. Low activities of bacteriocin and low amounts of the sensitive indicator strain biomass can be used with this bioassay.     

Use of paper indicator systems for the rapid identification of Corynebacterium diphtheriae, Neisseria meningitidis and the genus Staphylococcus

The possibility of using paper indicator discs with glucose, saccharose, lactose and urea, manufactured at the Gorky Research Institute of Epidemiology and Microbiology, has been studied and the methods for the preparation of indicator discs with fructose, maltose, starch and sodium phenolphthalein-phosphate for the biochemical identification of C. diphtheriae, N. meningitidis and staphylococci have been proposed. The study of 395 C. diphtheriae strains, 98 N. meningitidis strains and 328 staphylococcal strains has shown that paper indicator systems are highly sensitive and specific, which makes it possible to recommend their introduction in laboratory practice for the rapid identification of the above-mentioned organisms.     

Indicator groups and congruence of assemblage similarity, species richness and environmental relationships in littoral macroinvertebrates

The rapid decrease of biodiversity and limited resources for surveying it have forced researchers to devise short-cuts for biodiversity surveys and conservation planning. These short-cuts include environmental surrogates, higher taxon surrogates, indicator species and indicator groups. We considered indicator groups as surrogates for wholesale biodiversity and cross-taxon congruence in biodiversity patterns in littoral macroinvertebrates of boreal lakes. Despite the fact that we considered indicator groups amongst a wide variety of taxa, such as two-winged flies, mayflies, caddisflies, beetles, bugs and molluscs, none of the proposed groups possessed all of the qualities of a good indicator taxon for biodiversity surveys and conservation planning. We found generally weak, yet typically significant, relationships between the proposed indicator groups and remaining taxa in both species richness and assemblage similarity. Low congruence was paralleled by somewhat differing relationships of the taxonomic groups to various environmental features of lakes. Furthermore, the relationships of most indicator groups to the environmental features of lakes were not particularly strong. The present findings are unfortunate, because indicator groups did not perform well in predicting the wholesale biodiversity of littoral macroinvertebrates. Thus, there appears to be no short-cut for considering all groups of macroinvertebrates in biodiversity surveys, conservation planning and characterisation of environmental relationships of lake littoral assemblages.     

Acetamide Agar for Differentiation of Nonfermentative Bacteria

An acetamide agar medium is described for use in the differentiation of nonfermentative gram-negative bacteria. With few exceptions, indicator reactions were rapid, intense, and clear-cut.     

Rapid Assay for Assessment of the Potential of Chemical Biocides against Microbial Destructors of Industrial Materials

A colorimetric rapid assay for estimating the biocide potential of various chemicals towards metal- biocorrosive and petroleum-product-degrading microbes was developed based on the reducing potential of live microbial cells. A water-soluble organic redox indicator, blue in the oxidized form and pink in the reduced form, was used as an indicator of the reducing potential of microbial cells. Once added to a suspension of vital microbial cells, it was reduced and changed in color. A good correlation between the results of this assay and viability control was obtained by employing surfactants and heavy metal ions.     

Rapid assay for the assessment of a potential of chemical biocides to microbial destructors of industrial materials

A colorimetric rapid assay for estimating the biocide potential of various chemicals towards metal biocorrosive and petroleum product degrading microbes was developed based on the reducing potential of live microbial cell. A water-soluble organic redox indicator, blue in the oxidized form and pink in the reduced form, was used as an indicator of the reducing potential of microbial cells. Once added to a suspension of vital microbial cells, it was reduced and changed in color. A good correlation between the results of this assay and viability control was obtained by employing surfactants and heavy metal ions.     

A compartmental capillary, convolution integration model to investigate nutrient transport and metabolism in vivo from paired indicator/nutrient dilution curves.

Thirty-three paired indicator/nutrient dilution curves across the mammary glands of four cows were obtained after rapid injection of para-aminohippuric acid (PAH) plus glucose into the external iliac artery. For the measurement of extracellular volume and kinetics of nutrient uptake from indicator dilution curves, several models of solute dispersion and disappearance have been proposed. The Crone-Renkin models of exchange in a single capillary assume negligible washout of solutes from the extracellular space and do not describe entire dilution curves. The Goresky models include a distribution of capillary transit times to generate whole system outflow profiles but require two indicators to parametize extracellular behavior. A compartmental capillary, convolution integration model is proposed that uses one indicator to account for the extracellular behavior of the nutrient after a paired indicator/nutrient injection. With the use of an iterative approach to least squares, unique solutions for nonexchanging vessel transit time t(mu) and its variance sigma were obtained from all 33 PAH curves. The average of heterogeneous vascular transit times was approximated as 2sigma = 8.5 s. The remainder of indicator dispersion was considered to be due to washout from a well-mixed compartment representing extracellular space that had an estimated volume of 5.5 liters or 24% of mammary gland weight. More than 99% of the variation in the time course of venous PAH concentration after rapid injection into the arterial supply of the mammary glands was explained in an unbiased manner by partitioning the organ into a heterogeneous nonexchanging vessel subsystem and a well-mixed compartmental capillary subsystem.     

Determination of free malondialdehyde in human serum by high-performance liquid chromatography

Lipid peroxidation involves the oxidative deterioration of polyunsaturated fatty acids in biomembranes and generates a variety of aldehydic products including malondialdehyde (MDA). To demonstrate the occurrence of lipid peroxidation in biological systems, the production of MDA has been shown to be a relevant indicator. Therefore, we describe a new method for measurement of free malondialdehyde in human serum. A simple, rapid but sensitive method for determination of MDA in human serum was applied to goiter patients and control groups. Patients with goiter had high levels of MDA compared to control groups. Our method is fast and practical for clinical measurements. The detection limit was found to be 1.2 x 10(-8) mol L(-1).     

A Direct Turbidimetric Assay of Pyocine

S ummary . An assay of pyocine based on the turbidimetric measurement of the percentage inhibition of a sensitive indicator strain of Pseudomonas aeruginosa is described. The tendency of this organism to clump is circumvented in the method which is simple, rapid in performance and has good precision and reproducibility.     

A microtiter-plate screening method for biofilm disinfection and removal

A quantitative spectrophotometric method was developed to measure the removal and killing efficacy of antibiofilm agents. Biofilms of Pseudomonas aeruginosa or Staphylococcus epidermidis were grown in 96-well plates, treated with an agent, then stained with either the biomass indicator crystal violet or the respiratory indicator 5-cyano-2,3-ditolyl tetrazolium chloride. This rapid screening method is sensitive enough to elucidate concentration-response relationships as well as differences between species responses to treatments. Using these assays, agents can be ranked by their ability to remove or kill biofilm.     

Testing for electrotransfection parameters by use of the fluorescent dye lucifer yellow CH

A nonradioactive and rapid method to systematically optimize conditions for electrotransfection is described here for a critical parameter, the initial voltage. This technique utilizes the electric field-dependent transfer of the fluorescent compound Lucifer Yellow CH into cells. Dye uptake can be followed and quantified by fluorescence microscopy for individual cells or in sum by fluorescence spectroscopy. Electrotransfection conditions for maximal dye and DNA uptake correspond with each other. Cotransfection of Lucifer Yellow CH and DNA coding for the indicator gene chloramphenicol acetyltransferase followed by fluorescence-activated cell sorting demonstrates that the cell population that takes up the fluorescent compound also expresses the indicator gene.     

A simple assay for toxic chemicals using a bacterial indicator

A simple, inexpensive and rapid technique to measure toxicity has been developed using Rhizobium meliloti as the indicator organism and its rapid reduction of the tetrazolium dye MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide). Toxic chemicals inhibited the reduction in this bacterium but not in others. Nearly 50 organic chemicals and 14 minerals were tested: inhibition was proportional to the concentration of the toxic chemical. The mechanism to account for both the reduction of the dye and the inhibition of reduction is uncertain. This method provides a simple and inexpensive way to determine the critical concentration of toxic compounds. The assay provides values comparable to those provided by the fat head minnow (Pimephales promelas). Results are also comparable to those obtained with the Microtox and Polytox assays, two commercial assays that use bacteria as indicator organisms.     

A comparison of a solid phase IRC assay and the PSIFT for detection of antibodies to platelets.

A rapid solid phase indicator red cells assay (IRCA) for detection of platelet antibodies was developed and its sensitivity compared with PSIFT. Platelets were attached to the surface of polystyrene microtitre plate wells by means of a sodium carbonate buffer and centrifugation. Uncovered areas were blocked by a gelatin blocking buffer. After serum incubation bound platelet-specific antibodies were made visible by anti-IgG-coated indicator red cells and a brief centrifugation. A positive result, meaning the presence of an anti-platelet antibody was indicated by red cell adherence over the reaction surface. In the absence of serum antibodies to platelets the indicator red cells formed a pellet. The IRCA showed a high sensitivity; the anti-platelet antibody Thrombocyte was detectable until a dilution of 1:1,600 whereas the same antibody in the PSIFT could only be detected until a dilution of 1:400.     

Tracking of proton flow during transition from anaerobiosis to steady state. 2. Effect of cation uptake on the response of a hydrophobic membrane bound pH indicator.

1. During aerobic cation uptake in liver mitochondria, the hydrophobic pH indicator bromothymol blue undergoes a multiphase response: phase 1 (rapid acidification), phase 2 (slow alkalinization), phase 3 (rapid alkalinization) and phase 4 (reacidification). 2. Titrations with ruthenium red and malonate indicate that the various phases depend on the relative rates of cation uptake and proton translocation: at high rates of cation uptake, phase 1 disappears and phases 2 and 3 are transformed in a monotonic process of alkalinization. 3. The comparison of the bromothymol blue response with the arsenazo III, 2',7'-bis(carboxyethyl)-5(6)carboxyfluorescein (BCECF) and safranine responses indicates that: (a) phase 2 (slow alkalinization) corresponds to a slow rise of matrix pH and a parallel decline of membrane potential; (b) phase 3 (rapid alkalinization) corresponds to termination of proton translocation and initiation of the processes of cation efflux and proton reuptake. All the above processes reach completion during phase 4. 4. Although bromothymol blue always behaves as a membrane-bound indicator, the extent to which it reflects the matrix or the cytosolic pH is a function of the membrane-potential-determined asymmetric distribution: in parallel with the lowering of the membrane potential, the dye chromophore is shifted from the cytosolic to the matrix side membrane layer. 5. A model is discussed which describes the behaviour of bromothymol blue as pH indicator recording the changes in membrane layers facing either the matrix or the cytosolic side. The complex response of the dye during cation uptake is due to two independent processes, one of pH change and another of dye intramembrane shift. Computer simulations of the dye response, based on the conversion of a kinetic model into an electrical network and closely reproducing the experimental observations, are reported.     

A new method to estimate aquatic invertebrate diversity in French shallow lakes and ponds

Shallow lakes and ponds are valuable ecosystems for conservation management. Aquatic invertebrates constitute a large proportion of diversity in these ecosystems, but their assessment is potentially time consuming and requires great expertise. The use of indicator taxa to estimate invertebrate diversity may resolve part of these difficulties. These indicators are rarely identified or their reliability is uncertain, i.e. they are based on partial inventories, neglecting groups with high diversity. In this study, invertebrate richness was assessed from 46 sites in France in various altitudinal, climatic, geological, human-impacted, and hydro-morphological contexts. Invertebrate identification was performed as accurately as possible in all taxonomic groups. Several potential indicators of diversity based on five key criteria were tested: strong direct correlation, identification facilities, strong cross-taxon congruence, low complementarity of the sampled habitats, and ubiquity for selected indicators. Three approaches were proposed to define these indicator groups: (1) a single taxonomic group as indicator, (2) a combination of targeted groups, and (3) a holistic inventory at low taxonomic resolution as a classical rapid assessment method for freshwater ecosystems. Results show that it is not recommended to use only one indicator group. The choice of several targeted groups could be a good intermediate solution but is not without bias. The rapid assessment inventory proposed is the most valuable method, and allows the estimation of invertebrate richness with a quasi-perfect correlation.     

Improved Rapid Plate Method for the Isolation of Bacteriophages from Lysogenic Bacteria

A modification of a recently reported rapid plate method for the isolation of bacteriophages from lysogenic bacteria is described. The velveteen replica plate technique was used for inoculation of mitomycin C-induced colonies onto agar plates, and tetrazolium chloride was used to enhance detection of phage activity on replicated indicator plates.     

A high-throughput amenable colorimetric assay for enantioselective screening of nitrilase-producing microorganisms using pH sensitive indicators

Based on the color change of an indicator due to the release of hydrogen ion from a nitrilase-catalyzed reaction, a rapid colorimetric method was established for the enantioselective screening of nitrilase-producing microorganisms. The formation of acids due to the nitrilase-mediated hydrolysis of nitriles causes a drop in the pH, which in turn results in a change of color of the solution (containing indicator) that can be observed visually. The buffer (0.01 M phosphate, pH 7.2) and indicator (Bromothymol blue, 0.01%) were selected in such a way that both have the same affinity for the released protons. The enantioselectivity of nitrilases was estimated by comparing the hydrolysis of (R)-mandelonitrile with that of racemate under the same conditions. The method was used to screen a library of nitrilase-producing microorganisms, isolated in the authors' laboratory for their ability to enantioselectively hydrolyze mandelonitrile to mandelic acid, an important chiral building block.