Microbial metabolism of aryl sulphonates a re-assessment of colorimetric methods for the determination of sulphite and their use in measuring desulphonation of aryl and alkylbenzene sulphonates.

The reaction of 2,4-dinitroanilinomaleimide with sulphite which has been claimed as the basis of a suitable colorimetric assay for the anion was carefully re-examined. The sulphite-imide addition product provides a suitable and specific qualitative test for sulphite after separation by paper chromatography but the method as previously used is probably measuring the hydrolysis of the imide to 2,4-dinitroanilinomaleamic acid and cannot be used for sulphite determination either colorimetrically or in kinetic assays. A new colorimetric method for the determination of sulphite based on its reaction with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) is described and compared for sensitivity with the p-rosaniline-HCHO method. Both methods were used to show the formation of sulphite as the initial product of arylsulphonate metabolism by bacteria. The failure to find sulphite in similar cultures of a third organism was attributed to the very high activities of sulphite oxidase found in extracts. The Ellman reagent was examined as the basis of an indicator medium for the detection of sulphite-excreting colonies.     

Method of determining the antilysozyme activity of microorganisms

The method for the determination of the antilysozyme activity of microorganisms is described. The method consists in the cultivation of the strains under study in a lysozyme-containing medium, and the effect of lysozyme inactivation is determined from the growth of Micrococcus luteu S indicator strain adjacent to active strains. The quantitative evaluation of this property is presented. The study of 1 296 strains belonging to 9 genera has disclosed that antilysozyme activity occurs most frequently among Gram-negative bacteria.     

Microbial metabolism of aryl sulphonates A re-assessment of colorimetric methods for the determination of sulphite and their use in measuring desulphonation of aryl and alkylbenzene sulphonates

The reaction of 2,4-dinitroanilinomaleimide with sulphite which has been claimed as the basis of a suitable colorimetric assay for the anion was carefully re-examined. The sulphite-imide addition product provides a suitable and specific qualitative test for sulphite after separation by paper chromatography but the method as previously used is probably measuring the hydrolysis of the imide to 2,4-dinitroanilinomaleamic acid and cannot be used for sulphite determination either colorimetrically or in kinetic assays. A new colorimetric method for the determination of sulphite based on its reaction with Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoic acid) is described and compared for sensitivity with thep-rosaniline-HCHO method. Both methods were used to show the formation of sulphite as the initial product of arylsulphonate metabolism by bacteria. The failure to find sulphite in similar cultures of a third organism was attributed to the very high activities of sulphite oxidase found in extracts. The Ellman reagent was examined as the basis of an indicator medium for the detection of sulphite-excreting colonies.     

Indicator method of determining the beta-lactamase-inhibiting activity of various compounds

An indicator for determination of beta-lactamase inhibitory activity of various compounds was developed. The method is based on the direct contact of beta-lactamase with the compounds tested. It excludes the use of test-bacteria and provides recording in the data on the day of the experiment. The indicator method enables the detection of the beta-lactamase inhibitory properties of both beta-lactamase inhibitors and beta-lactam antibiotics, not subjected to destruction by beta-lactamases. The method is likely to be fit for detection of atypical beta-lactams having beta-lactam groups in their molecules (bleomycin group). Antibiotics not belonging to the group of beta-lactams, such as gentamicin, sisomicin, lincomycin and fusidin showed no beta-lactamase inhibitory activity under the conditions of the indicator method. The use of the indicator method provided determination of the inhibitory activity with respect to penicillinase of Bac. licheniformis 749/C in 30 (8.5 per cent) out of 350 fermentation broths of actinomycetes.     

Simple method for precise determination of chemical lethality in the L-arabinose resistance test of Salmonella typhimurium

A simple method is described for the determination of the lethal effects of chemicals assayed with the L-arabinose resistance test of Salmonella typhimurium. The method uses a mixed culture of 2 isogenic bacterial strains which are distinguished on the basis of their different nutritional requirements: sensitivity or resistance to L-arabinose, auxotrophy or prototrophy to histidine and leucine. BA13 (the mutation indicator strain) is the strain for routine screening of mutagens and allows the selection of forward mutation from L-arabinose sensitivity to L-arabinose resistance. BAL13 (the survival indicator strain) is a derivative of BA13. Both bacterial strains are found to be equally sensitive to the lethal effects of mutagens. The method described permits the measurement of cell survival at the same high cell concentration as used in the measurement of the mutant yield and in the same type of minimal medium with L-arabinose and glycerol, except for the histidine supplement in the mutant plates or the leucine in the survival plates.     

Determination of the intracellular pH of intact erythrocytes by 1H NMR spectroscopy

A method is described for determining the intracellular pH of intact erythrocytes by ¹H NMR. The determination is based on the pH dependence of the chemical shifts of resonances for carbon-bonded protons of an indicator molecule (imidazole) in intact cells. The imidazole is introduced into the erythrocytes by incubation in an isotonic saline solution of the indicator. The pH dependence of the chemical shifts of the imidazole resonances is calibrated from ¹H NMR spectra of the imidazole-containing red cell lysates whose pH is varied by the addition of acid or base and measured directly with a pH electrode. To reduce in intensity or eliminate the much more intense envelope of resonances from the hemoglobin, the ¹H NMR measurements are made by either the spin-echo Fourier transform technique or by the transfer-of-saturation by cross-relaxation method.     

A plate method for screening of bacteria capable of degrading aliphatic nitriles

A novel indicator plate method was developed for screening of aliphatic-nitrile-degrading bacteria. Isolated bacteria were tested for utilization of acetonitrile as sole source of carbon and nitrogen with the release of ammonia. The released ammonia causes increase of the pH of the medium. Phenol red indicator is used for detection of ammonia based on colour change of the indicator dye from red to pink. The liberation of ammonia from aliphatic-nitrile-utilizing bacteria is also studied in plates containing other indicators such as bromothymol blue and phenolphthalein. The usefulness of the indicator plate is demonstrated for bacteria that degrade certain aliphatic nitriles. Bacteria degrading nitriles as a nitrogen source can also be isolated with a medium containing additional carbon source. This plate method would be useful in isolation and screening of bacteria for degradation of aliphatic nitriles and also for production of nitrile-hydrolyzing enzymes.     

Quantitative pH assessment of small-volume samples using a universal pH indicator

We developed a hue-based pH determination method to analyze digital images of samples in a 384-well plate after the addition of a universal pH indicator. The standard error of calibration for 69 pH standards was 0.078 pH units, and no sample gave an error greater than 0.23 units. We then used in-solution isoelectric focusing to determine the isoelectric point of Wnt3A protein in conditioned medium and after purification and applied the described method to assess the pH of these small-volume samples. End users may access our standard to assay the pH of their own samples with no additional calibration.     

Isolation of manganese-oxidizing Pedomicrobium cultures from water by micromanipulation

A method for the isolation of manganese-oxidizing pedomicrobia from water is described. The method employs a combination of growth on a manganese containing indicator medium and micromanipulation. Pedomicrobia are slow-growing and are frequently overgrown by spreading bacteria. Colonies of manganese-oxidizing strains are easily detected at an early stage of development because of the accumulation of black manganese oxide. Micromanipulation allows selection of cells from microcolonies before they are overgrown. The procedure has been successfully used to isolate manganese-oxidizing Pedomicrobium cultures from water distribution systems experiencing persistent manganese-related dirty water problems. Considerable savings in time and materials have been made compared with conventional dilution plating techniques.     

The hydrogen peroxide microvolumetric method of the OF test: the rapid determination of glucose oxidation and fermentation by gram-negative bacteria

For the first time the method of the rapid (1 hour) screening of groups of gram-negative bacteria by the OF test with glucose, carried out with the use of microvolumetric techniques, has been developed. The method is based on the use of hydrogen peroxide at non-bactericidal concentration as a component of a liquid buffer medium containing indicator and glucose and intended for the oxidation of glucose. Catalase of bacteria introduced into the medium for study ensures the rapid saturation of the medium with oxygen and the completion of the oxidation of glucose in 10-60 minutes. An equal period is necessary to achieve the complete fermentation of glucose in the same medium without hydrogen peroxide. The method has been proved to yield significant results in the joint study of the OF test on 502 strains, belonging to 21 genera of fermenting and nonfermenting gram-negative bacteria, by the hydrogen peroxide method and in Hugh-Leifson medium.     

Detection of antibodies neutralizing the endotoxins of gram-negative bacteria using the liposomal potentiometric method

For the first time the study of the indicator system consisting of sensitized liposomes with NaF incorporated as a marker and a fluorine-selective electrode has been made and, as a result, the possibility of the potentiometric determination of the immune lysis of liposomes in the presence of complement and specific antibodies has been demonstrated. The dissolution of the lipid components (Re-chemotype glycolipid and lipid A) in the bilayer matrix obviates the necessity for converting lipid antigens into the water-soluble state in the process of serological tests. As compared with other methods, the liposomal potentiometric method for the determination of Re-chemotype glycolipid and lipid A is highly sensitive (20-40 ng/ml), rapid, technically easy to perform, cheap and does not require large volumes of samples. The disadvantages of this analytical system are the instability of liposomes and the diffusion of fluorine ions from the internal aqueous phase of vesicules. For this reason the immunoassay can be made only within 12 hours after the preparation of sensitized liposomes incorporating the marker.     

Detection of toxin and evaluation of the degree of toxin-formation of Corynebacterium diphtheriae using immunoenzyme analysis

Toxin production and the intensity of toxin formation in 265 C. diphtheriae strains circulating in different areas of the USSR have been studied by the method of the enzyme-linked immunosorbent assay (ELISA). This study has been carried out with the use of the assay system consisting of monoclonal antibodies to the COOH-area of the B-fragment of the toxin molecule adsorbed onto the surface of polystyrene plates, affinity-purified polyclonal antidiphtheria antibodies labeled with horse-radish peroxidase and substrate indicator mixture (5-aminosalicylic acid and hydrogen peroxide). Some specific features of using ELISA for the detection of C. diphtheriae toxin directly in liquid culture medium are presented. High sensitivity, specificity and good reproducibility of this method permitting the detection of C. diphtheriae toxin and the determination of the intensity of toxin formation in the C. diphtheriae strains under study are shown. The method may be recommended for practical use at health institutions.     

A method for screening bacteria: Aerobically degrading chlorinated short-chain hydrocarbons

A method for screening of short-chain chlorinated-hydrocarbon-degrading bacteria is described. It uses as a criterium for selection the liberation of protons and change in color of a pH indicator rather than growth on the compound as sole carbon source. The usefulness of indicator plates is demonstrated with several bacteria, known to degrade certain chlorinated hydrocarbons. Bacteria-degrading volatile compounds can be isolated with a medium containing a second carbon source. A correlation between proton liberation and liberation of chloride or disappearance of 2-chloroethanol from the medium is demonstrated as an example. The method should be useful in isolating bacteria for the decontamination of respective commodities.     

Isolation and fluorometric determination of homovanillic acid in urine and tissues

The fluorometric method is described for determination of the final product of dopamine metabolism homovanillic acid in urine, amniotic fluid and tissue extracts. The metabolite was isolated using three chromatographic columns (1-Amberlit CG-50, 2-Dowex AG 1 x 2, 8-Aluminium oxide). Oxidation of eluates to emitter was done with potassium ferricyanide in alkalic medium. The use of internal standards made high recovery of compound and a good specificity and sensitivity of detection.     

Stages in the Initiation of Root and Shoot Organogenesis in Cultured Leaf Explants of Nicotiana tabacum cv. Xanthi nc.

Reciprocal transfers of Nicotiana tabacum cv. Xanthi nc. leafexplants were made daily between root inducing medium (RIM)and shoot inducing medium (SIM), SIM and a basal medium containingno growth regulators (BM), and RIM and BM. It was found thatthe explants became determined for shoot production after 6d, while roots were produced after only 1 d on RIM before transferto BM. The competence of the explant to produce roots was greatlyreduced by culture on BM prior to culture on RIM. There wasfar less reduction in shoot numbers with preculture on BM. Explantswere found to be only weakly canalized for both caulogenesisand rhizogenesis for the first 2 d after determination. Thereafterthey became strongly canalized. Transfers were also made fromBM to SIM and back to BM, which revealed that the explants becamecompetent for caulogenesis in the absence of cytokinins priorto determination. The period for which SIM is required can bereduced to only 1 d. Key words: Nicotiana tabacum, in vitro, organogenesis, competence, determination     

Determination of the chelatable iron pool of single intact cells by laser scanning microscopy

We have previously established a method of detecting intracellular chelatable iron in viable cells based on digital fluorescence microscopy. To quantify cellular chelatable iron, it was crucial to determine the intracellular indicator concentration. In the present study, we therefore adapted the method to confocal laser scanning microscopy, which should allow the determination of the indicator concentration on the single-cell level. The fluorescent heavy-metal indicator phen green SK (PG SK), the fluorescence of which is quenched by iron, was loaded into cultured rat hepatocytes. The hepatocellular fluorescence increased when cellular chelatable iron available to PG SK was removed from the probe by an excess of the membrane-permeable transition metal chelator 2,2'-dipyridyl (2, 2'-DPD, 5 mM). We optimized the scanning parameters for quantitatively recording changes in fluorescence and determined individual intracellular PG SK concentrations from the unquenched cellular fluorescence (after 2,2'-DPD) compared with PG SK standards in a "cytosolic" medium. An ex situ calibration method based on laser scanning microscopy was set up to determine the concentration of cellular chelatable iron from the increase of PG SK fluorescence after addition of 2,2'-DPD (5 mM). As the stoichiometry of the PG SK:Fe(2+) complex was 3:1 as long as PG SK was not limiting, cellular chelatable iron was calculated directly from absolute changes in cellular fluorescence. Using this method, we found 2.5 +/- 2.2 microM chelatable iron in hepatocytes. This method makes it possible to determine the pool of chelatable iron in single vital cells independently of cellular differences (e.g., dye loading, cell volume) in heterogeneous cell populations.     

Bioassay of Mildiomycin and a Rapid, Cost-Effective Agar Plug Method for Screening High-yielding Mutants of Mildiomycin

Summary A simple, reliable and low-cost agar diffusion bioassay for quantitative determination of mildiomycin was developed using a strain of Rhodotorula rubra AS 2.166 as the indicator organism and potato dextrose agar at pH 7.0 as the test medium. With equivalent precision and accuracy to HPLC analysis, this method was applied to analyse mildiomycin in complex culture broth during the fermentation process. A modified agar plug method based on the bioassay was constructed for rapid and efficient screening of high-yielding mutants of mildiomycin. Within four weeks, a high production strain, the mildiomycin productivity of which was 75.5% higher than the parent strain, was obtained from 15,000 mutants.     

Enzymatic microdetermination of serum free fatty acids.

A reliable, simple, and rapid enzymatic method is described for the microdetermination of serum free fatty acids. The principle of the method is based on the activation of free fatty acids by a bacterial acyl-CoA synthetase (EC 6.2.1.3). The reaction is followed as production of AMP using the myokinase-pyruvate kinase-lactate dehydrogenase system as an indicator reaction. Results on the determination using human and rabbit sera showed a close correlation with the chemical colorimetric method.     

The quantitative determination of neomycin sulphate by a diffusion technique on agar plates by the method of the European Pharmacopoeia, 2nd edition--evaluation of precision and reproducibility of the method

The medium recommended by the European Pharmacopoeia (EP), 2nd edition, for the microbiological determination of neomycin by the agar diffusion method was tested and compared with the medium recommended by the EP, 1st edition. The tests were carried out in different laboratories. The medium recommended by the EP, 2nd edition, gave greater precision and reproducibility than the previous medium. The possibility of using a reference standard of almost pure neomycin B for both the determination of framycetin and neomycin was evaluated. The results demonstrated that the medium recommended by the EP, 2nd edition, gave better precision and reproducibility. Difficulty in achieving valid assays was practically the same with both media.     

极谱氧电极法测定超氧化物歧化酶活性的改进

对SOD的极谱氧电极测定法做了如下修改:a.室温测定,b.酶活性用标准SOD标定,c.反应在磷酸缓冲液中进行,d.增大邻苯三酚的用量.改进后克服了易在电极薄膜表面产生气泡等问题,测定灵敏度及线性范围增大.