Development of a sensitive radiorespiration method for detecting microbial activity at subzero temperatures

We have developed a simple and sensitive method to detect microbial respiration at subzero temperatures. Microbial activity was detected by measuring (14)CO(2) evolved during the microbial-mediated mineralization of [1-(14)C] acetic acid or [2-(14)C] glucose in microcosm assays using modified (14)CO(2) traps. Various (14)CO(2) traps, designed to withstand freezing at subzero temperatures, were tested for their quench characteristics during liquid scintillation spectrometry and their ability to trap (14)CO(2). Solutions consisting of 1 M KOH supplemented with 20% or 30% v/v ethylene glycol did not freeze at temperatures above -20 degrees C and had a minor quenching effect on liquid scintillation spectrometry. Addition of ethylene glycol did have an effect on the efficiency of (14)CO(2) trapping, as the cumulative recovery of (14)CO(2) was reduced by 14% and 32% in the 1 M KOH+20% ethylene glycol and 1 M KOH+30% ethylene glycol solutions, respectively. Using the modified (14)CO(2) traps, microbial activity in representative Canadian high Arctic environmental samples was detected at temperatures as low as -15 degrees C. This simple method allows for sensitive, specific, and reliable detection of microbial activity occurring at subzero temperatures and is readily adaptable for studies in other cryoenvironments.     

Intrahepatic mutagenesis assay: a sensitive method for detecting N-nitrosomorpholine and in vivo nitrosation of morpholine.

An intrahepatic host-mediated mutagenicity assay capable of detecting low levels of N-nitrosomorpholine (NMOR) is described. The indicator organism was Salmonella typhimurium TA1530 which had been injected intravenously 10 min prior to the administration of the test compound. The bacteria were subsequently recovered from the liver and scored for revertants by standard methods. The lower limit of detectibility of this system for intubated NMOR was 0.2 microgram/g body weight. This assay was then used to study the formation of NMOR in vivo from morpholine and nitrite which had been sequentially gavaged to mice. Under acidic conditions (pH 3.4) 12--19% of the administered morpholine was converted to NMOR in the presence of excess nitrite. This nitrosation, and the subsequent uptake and activation of the NMOR, took place so rapidly that most of the total mutagenic response was complete within 15 min. This response was inhibited by prior intubation of ascorbic acid, a known inhibitor of nitrosation, and enhanced by sodium thiocyanate, a nitrosation catalyst.     

A simple and sensitive method for detecting bacterial elastase production

A sensitive method for detecting bacterial elastase production in growing cultures is described. A variety of commonly isolated clinically relevant aerobic and anaerobic bacteria have been shown to produce the enzyme.     

Viral immunoblotting: A sensitive method for detecting viral-specific oliogoclonal bands in unconcentrated cerebrospinal fluid

A new method for detecting viral antibodies in cerebrospinal fluid is described. The technique has many advantages over previously published methods in that it is highly sensitive eliminating the need to concentrate the CSF, takes 5 h to complete, avoids the use of radionucleides, and most importantly circumvents problems associated with prozone effects which occur in immunoprecipitation reaction since the viral antigen is immobilized on nitrocellulose membranes.     

A sensitive method for detecting proteolytic enzymes released from their supports

Biotechnology Letters - A very sensitive radioisotope method, using 125I-labelled serum albumin as substrate, is proposed for the detection of very low enzymatic activities released from insoluble...     

A sensitive method for detecting the fermentation-inhibition antibody to Mycoplasma pneumoniae.

The fermentation-inhibition (FI) test for Mycoplasma pneumoniae was improved by using a combination of guinea pig complement and gamma globulin-depleted horse serum in place of unheated whole horse serum employed in the conventional assay system. As the test antigen for the new FI assay system, M. pneumoniae filtrated through a 3.0 microns membrane filter was used. Owing to the strong augmenting effect of guinea pig complement, the FI activity of rabbit immune serum was increased 32-fold in the new system compared with the conventional system. Furthermore, IgM antibody, which is barely detectable by the conventional system, could easily be titrated by the new system. With this sensitive method, rapid rise of FI titer was clearly demonstrable in most children with acute M. pneumoniae infections, and a prevalence of FI or growth-inhibitory antibody among healthy adults in Japan (82%) was revealed.     

A sensitive staining method for detecting acidic polysaccharides in cellulose acetate and agarose gels

A sensitive staining method has been developed for the detection of acidic polysaccharides in cellulose acetate and agarose gels. The method is based on the precipitation of bovine serum albumin by acidic polysaccharides at acidic pH values and the subsequent staining of precipitated protein with amido black or Coomassie brilliant blue R-250 stains. The detection limit of acidic polysaccharides is 15-40 ng on cellulose acetate strips and 50-150 ng on agarose plates. The sensitivity of the described staining technique is of the same order for a wide range of acidic polysaccharides of different origin in contrast to Alcian blue and toluidine blue stains, which detect only mucopolysaccharides of animal origin at comparable levels. The method was also applied to the colorimetric quantitative determination of acidic polysaccharides after electrophoretic separation.     

A fast and sensitive method for detecting specific viral RNA in mammalian cells.

A quick and sensitive method to quantitate viral RNA synthesis has been developed. Utilizing glutaraldehyde to fix infected cells onto nitrocellulose paper, viral RNA can be probed directly in situ. Viral message can be detected from as few as 10(4) infected cells. This technique can be used to study viral gene expression and can be adapted to screen the activity of antiviral agents such as interferon.     

Establishment of a novel sensitive method for detecting methylation modification on DNA of escherichia coli cell

This study focused on finding a novel sensitive method to determine the methylation modification at DNA dam (GATC) sites in Escherichia coli. A new plasmid which contained three GATC sites recognized by restriction enzyme BclI and one GAATTC site recognized by EcoRI was transformed into E. coli stains AB1157(dam ⁺) and GM2929(dam ⁻) respectively. Then the plasmid DNA was digested by restriction enzyme BclI(T*GATCA), which was sensitive to methylation. The results showed that the plasmid derived from AB1157 was not digested while that from GM2929 was, for the methylation level of the former was high while the latter was low. So by detecting the methylation of plasmid transferred into the strain, we could determine whether methylaion existed at DNA dam (GATC) site in E. coli. This method was effective and rapid; moreover, the digested fragments were not dispersive. It also made a basis for the detection of whether methylation occurred in mode beings by low-energy ion beam. The article is published in the original.     

用条件性逃避反应建立简易的大鼠耳鸣检测方法

目的：用较简单的行为实验建立检测动物耳鸣的方法。方法：按照巴甫洛夫条件反射原理,参照Jastreboff的方法,建立“中断背景声音-大鼠逃跑”的条件反射;不再给电击,观察不同组别大鼠条件反射消退过程的行为表现,用公认的水杨酸耳鸣造模法来验证本方法的可靠性。结果：水杨酸组动物停止背景声音时不出现或较少出现逃跑反应,因为大鼠耳内仍有声音存在(耳鸣声),即条件刺激时有耳鸣的动物出现的逃跑反应次数小于无耳鸣的对照动物。结论：本实验设计可检测动物是否有耳鸣。     

A non-invasive method for detecting the metabolic stress response in rodents: characterization and disruption of the circadian corticosterone rhythm

Plasma corticosterone (CORT) measures are a common procedure to detect stress responses in rodents. However, the procedure is invasive and can influence CORT levels, making it less than ideal for monitoring CORT circadian rhythms. In the current paper, we examined the applicability of a non-invasive fecal CORT metabolite measure to assess the circadian rhythm. We compared fecal CORT metabolite levels to circulating CORT levels, and analyzed change in the fecal circadian rhythm following an acute stressor (i.e. blood sampling by tail veil catheter). Fecal and blood samples were collected from male adolescent rats and analyzed for CORT metabolites and circulating CORT respectively. Fecal samples were collected hourly for 24 h before and after blood draw. On average, peak fecal CORT metabolite values occurred 7-9 h after the plasma CORT peak and time-matched fecal CORT values were well correlated with plasma CORT. As a result of the rapid blood draw, fecal production and CORT levels were altered the next day. These results indicate fecal CORT metabolite measures can be used to assess conditions that disrupt the circadian CORT rhythm, and provide a method to measure long-term changes in CORT production. This can benefit research that requires long-term glucocorticoid assessment (e.g. stress mechanisms underlying health).     

A sensitive urea-silver stain method for detecting trace quantities of separated proteins in polyacrylamide gels

An ultrasensitive method using a urea-silver staining procedure to detect trace quantities of proteins in polyacrylamide gels (PAGE) is described. This technique is sensitive enough to detect picogram quantities of proteins resolved on sodium dodecyl sulfate-polyacrylamide gels. The major advantages of our method are that it provides a clear background and it is more sensitive than other techniques allowing it to substitute for radioisotopic techniques in some cases.     

Validation of a robust and sensitive method for detecting hydroxyl radical formation together with evoked neurotransmitter release in brain microdialysis

Sodium terephthalate was shown to be a new robust and sensitive chemical trap for highly reactive oxygen species (hROS), which lacks the drawbacks of the salicylic acid method. Reaction of the almost non-fluorescent terephthalate (TA²⁻) with hydroxyl radicals or ferryl-oxo species resulted in the stoichiometric formation of the brilliant fluorophor, 2-hydroxyterephthalate (OH-TA). Neither hydrogen peroxide nor superoxide reacts in this system. This procedure was validated for determining hROS formation during microdialysis under in vivo conditions as well as by in vitro studies. The detection limit of OH-TA in microdialysis samples was 0.5 fmol/μL. Derivatization of samples with o- phthalaldehyde, for amino acid detection, had no effect on OH-TA fluorescence, which could easily be resolved from the amino acid derivatives by HPLC, allowing determination in a single chromatogram. Use of terephthalate in microdialysis experiments showed the neurotoxin kainate to evoke hROS formation in a dose-dependent manner. The presence of TA²⁻ in the perfusion fluid did not affect basal or evoked release of aspartate, glutamate, taurine and GABA. Assessment of cell death ' ex vivo' showed TA²⁻ to be non-toxic at concentrations up to 1 mM. The in vitro results in the Fenton system (Fe²⁺ + H₂O₂) indicate a mechanism whereby TA²⁻ forms a primary complex with Fe²⁺ followed by an intramolecular hydroxylation accompanied by intramolecular electron transfer.