New immunofluorescent method for the rapid determination of microbial antibiotic sensitivity

A new procedure for rapid determination of the levels of antibiotic sensitivity in pathogenic microorganisms with the use of fluorescent antibodies is described. The procedure was developed with the use of a model of the vaccinal strains of Bacillus anthracis. It is based on determination of the microbial antibiotic resistance with the method of serial dilutions on solid media. Still, the medium with an antibiotic is inoculated instead of the pathogen with the native material subject to the analysis. The antibiotic effect on the microorganism is estimated with the method of fluorescent antibodies. The replica preparations obtained as a result of the pathogen growth in a mixed culture on nutrient media containing definite concentrations of the antibiotic are examined with the method of luminescence microscopy. The modification of the immunofluorescent procedure for rapid determination of the microbial sensitivity to antibiotics does not require obligatory isolation of the pathogen as a pure culture. This makes the procedure more economic with respect to the time necessary for the analysis. The following conditions for performing rapid analysis with respect to Bacillus anthracis are required: the minimal concentration of the pathogen in the specimen (2.10(5) spores/ml), preliminary thermal treatment of the specimen for destroying the spore microflora, additional cultivation for 6-8 hours at 37 degrees C. The presence of the accompanying sporulating microflora, i.e. common microorganisms present in the atmosphere, soil and open water bodies does not prevent the performance of the analysis.     

A rapid method for the determination of proteolytic activities of enzyme preparations

A method has been described for the determination of proteolytic activities of enzyme preparations using casein as substrate. The rate of digestion is proportional to the enzyme concentration used. This relationship is utilized as a measure of the enzyme activity. One unit of activity is defined as the amount which is required to digest casein in 15 minutes at 37.5 degrees C. so that 50 per cent of the protein in 1 ml. of 0.25 per cent solution is not precipitable by trichloroacetic acid. This method has been used to determine the activity of enzymes from different sources and also used to follow the rate of activation of enzymes.     

Improved auto-analyzer method for the determination of phosphonates (total phosphorus) in a broad range of media

Summary An improved auto-analyzer method for the quantitative determination of phosphonate-phosphorus was developed. Its advantages are of three sorts: 1) phosphonates can be determined in a variety of media, even with high levels of organic compounds, 2) viscous bacterial broths can be analysed directly on the auto-analyzer, 3) the method has an increased sensitivity.     

Clinical morphological picture of the mucosa of the middle ear in experimental chronic otitis media in rabbits treated with combined antibiotic aerosol preparations]

An new method of treating chronic purulent middle otitis with combined aerosol drugs (oxycyclosol, polysol, levovinylsol, vinysol) was developed at the USSR Research Institute on Antibiotics and tested experimentally on 20 Shinshilla rabbits. A 0.3 ml volume of microbial suspensition: Staphylococcus (group I), Ps. aeruginosa (group II), Proteus vulgaris (group III) and mixed microbial flora (group IV) were administered into the cavities of the middle ear of the rabbits from both sides. The acute process was made chronic by using decreased daily food ration and repeated putting of the rabbit extremeties into cool water. After that the rabbits were subjected to treatment with the above drugs for a week. Damages in the drum cavity mucosa with dystrophy, necrosis and cell separation were observed in the control (not treated) animals. Analogous changes were found also in the auditory tube. Signs of necrosis followed by decomposition were detected in the bone tissue. Only some thickening of the mucosa and bone wall of the drum cavity in the ears and single lymphoid and plasmic cells were recorded in the rabbits subjected to the treatment with the aerosol drugs.     

Quantitative screening method for hydrolases in microplates using pH indicators: determination of kinetic parameters by dynamic pH monitoring.

The presented pH-dyn assay serves as a versatile tool for screening enzymatically catalyzed reactions consuming or producing acids. The method is based on material balances of substrates and products. Ion balances relate concentrations of acids and bases to pH. pH-changes caused by the enzymatically catalyzed reaction in a well-defined buffer system are recorded by light-absorption measurements of a pH-indicator. Kinetic parameters are estimated by fitting the modeled pH changes to the experimentally observed ones. The enzymatically catalyzed hydrolysis of 4-nitrophenol is used as a model system. A pH indicator, bromothymol blue, is used to monitor the reaction progress. The reaction is monitored until the limiting substrate is completely consumed. This allows the estimation of the parameters of the Michaelis-Menten kinetics, K(M) and k(cat), in a single run. The results agree well with conventional spectrophotometric experiments and values reported in literature. Around pH 7, environmental CO(2) influences pH. Carbon dioxide influence was included in the model. Thus it was possible to estimate initial CO(2) concentrations as a model parameter, and therefore automatic correction for the CO(2) disturbances was achieved. This was important to detect low conversions at low buffer concentrations.     

Surface potential determination in planar lipid bilayers: a simplification of the conductance-ratio method

One of the methods available for the measurement of surface potentials of planar lipid bilayers uses the conductance ratio between a charged and a neutral bilayer doped with ionophores to calculate the surface potential of the charged bilayer. We have devised a simplification of that method which does not require the use of an electrically neutral bilayer as control. The conductance of the charged bilayer is measured before and after the addition of divalent cations (Ba(2+)) to the bathing solution. Ba(2+) ions screen fixed surface charges, decreasing the surface potential. If the membrane is negatively charged the screening has the effect of decreasing the membrane conductance to cations. The resulting conductance ratio is used to calculate the surface potential change, which is fed into an iterative computer program. The program generates pairs of surface potential values and calculates the surface charge density for the two conditions. Since the surface charge density remains constant during this procedure, there is only one pair of surface potentials that satisfies the condition of constant charge density. Applying this method to experimental data from McLaughlin et al. [McLaughlin, S.G.A., Szabo, G. and Eisenman, G., Divalent ions and the surface potential of charged phospholipid membranes, J. Gen. Physiol., 58 (1971) 667-687.] we have found very similar results. We have also successfully used this method to determine the effect of palmitic acid on the surface potential of asolectin membranes.     

Validated spectrofluorimetric method for the determination of clonazepam in pharmaceutical preparations

A simple, highly sensitive and validated spectrofluorimetric method was applied in the determination of clonazepam (CLZ). The method is based on reduction of the nitro group of clonazepam with zinc/CaCl₂, and the product is then reacted with 2‐cyanoacetamide (2‐CNA) in the presence of ammonia (25%) yielding a highly fluorescent product. The produced fluorophore exhibits strong fluorescence intensity at ?_em = 383 nm after excitation at ?_ex = 333 nm. The method was rectilinear over a concentration range of 0.1–0.5 ng/mL with a limit of detection (LOD) of 0.0057 ng/mL and a limit of quantification (LOQ) of 0.017 ng/mL. The method was fully validated and successfully applied to the determination of CLZ in its tablets with a mean percentage recovery of 100.10 ± 0.75%. Method validation according to ICH Guidelines was evaluated. Statistical analysis of the results obtained using the proposed method was successfully compared with those obtained using a reference method, and there was no significance difference between the two methods in terms of accuracy and precision. Copyright © 2015 John Wiley & Sons, Ltd.     

Colorimetric method for the determination of ketoses using phenol-acetone-boric acid reagent (PABR)

A purplish pink color developed when ketose solution (100 μl) was mixed with phenolacetone-boric acid reagent (0.5 ml 5% phenol, 2% acetone, 4% boric acid) and then treated with 96% sulfuric acid (1.4 ml). The absorbance of the reaction mixture was measured at 568 nm after 60 min at 37°C. This method allowed the simple determination of 3–50 nmol of d-fructose with coefficient of variation 7.8% for 3 nmol and 2.8% for 50 nmol. Carbohydrates other then ketoses did not interfere with this reaction. The influence of various chemicals on the colorimetric reaction and applicability of the method for determination of ketoses in natural products are presented.     

Liquid chromatography--mass spectrometric method combined with derivatization for determination of 1 alpha-hydroxyvitamin D(3) in human plasma

A stable isotope dilution liquid chromatography-tandem mass spectrometric (LC-MS-MS) method for the determination of plasma 1 alpha-hydroxyvitamin D(3) [1 alpha(OH)D(3)] has been developed. The method employed derivatization, the reaction with 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione and acetylation, which significantly improved the ionization efficiency of 1 alpha(OH)D(3) with a detection limit of 6.3 fmol per injection. The plasma 1 alpha(OH)D(3) was extracted with acetonitrile, purified with disposable cartridges, derivatized and subjected to LC-MS-MS analysis using atmospheric pressure chemical ionization. The intra- and inter-assay coefficients of variation were below 10.6 and 4.7%, respectively, and the analytical recovery of 1 alpha(OH)D(3) was quantitative. The limit of quantitation was 25 pg/ml for a 1.0-ml plasma aliquot. The application of the developed method to the sample of a volunteer orally given 1 alpha(OH)D(3) was also described.     

A fluorophotometric method for the determination of amino acids by using pyridoxal and zinc(II) ion

A procedure for the determination of amino acids has been developed based on their interaction with pyridoxal and Zn²⁺ ion in pyridine methanol to yield highly fluorescent chelates. This fluorescence procedure is 20–100 times more sensitive than the colorimetric procedure. N-Pyridoxylidene amino acid-Zn(II) chelate contains a 1:1 molar ratio of ligand to metal ion.     

Identification of fish-parasitic Myxobolus (Myxosporea) species using a combined PCR-RFLP method

Polymerase chain reaction (PCR) with primers specific for the family Myxobolidae was used to amplify a part of the 18S ribosomal RNA gene of Myxobolus species. The length of the amplified fragments was approximately 1600 base pairs. Six Myxobolus species identified on the basis of morphological features were compared using a combined PCR-RFLP method. The cleavage patterns generated by 2 frequent cutter restriction enzymes (HinfI and MspI) were suitable for the differentiation of the examined Myxobolus species.     

Sulfidogenesis in low pH (3.8-4.2) media by a mixed population of acidophilic bacteria

A defined mixed bacterial culture was established which catalyzed dissimilatory sulfate reduction, using glycerol as electron donor, at pH 3.8-4.2. The bacterial consortium comprised a endospore-forming sulfate reducing bacterium (isolate M1) that had been isolated from acidic sediment in a geothermal area of Montserrat (West Indies) and which had 94% sequence identity (of its 16S rRNA gene) to the Gram-positive neutrophile Desulfosporosinus orientis, and a Gram-negative (non sulfate-reducing) acidophile (isolate PFBC) that shared 99% gene identity with Acidocella aromatica. Whilst M1 was an obligate anaerobe, isolate PFBC, as other Acidocella spp., only grew in pure culture in aerobic media. Analysis of microbial communities, using a combination of total bacterial counts and fluorescent in situ hybridization, confirmed that concurrent growth of both bacteria occurred during sulfidogenesis under strictly anoxic conditions in a pH-controlled fermenter. In pure culture, M1 oxidized glycerol incompletely, producing stoichiometric amounts of acetic acid. In mixed culture with PFBC, however, acetic acid was present only in small concentrations and its occurrence was transient. Since M1 did not oxidize acetic acid, it was inferred that this metabolite was catabolized by Acidocella PFBC which, unlike glycerol, was shown to support the growth of this acidophile under aerobic conditions. In fermenter cultures maintained at pH 3.8-4.2, sulfidogenesis resulted in the removal of soluble zinc (as solid phase ZnS) whilst ferrous iron remained in solution. Potential syntrophic interactions, involving hydrogen transfer between M1 and PFBC, are discussed, as is the potential of sulfidogenesis in acidic liquors for the selective recovery of heavy metals from wastewaters.     

Determination of sulpiride in pharmaceutical preparations and biological fluids using a Cr (III) enhanced chemiluminescence method

A highly sensitive and simple method for identifying sulpiride in pharmaceutical formulations and biological fluids is presented. The method is based on increased chemiluminescence (CL) intensity of a luminol–H₂O₂ system in response to the addition of Cr (III) under alkaline conditions. The CL intensity of the luminol–H₂O₂–Cr (III) system was greatly enhanced by the addition of sulpiride and the CL intensity was proportional to the concentration of sulpiride in a sample solution. Various parameters affecting the CL intensity were systematically investigated and optimized for determination of the sulpiride in a sample. Under the optimum conditions, the CL intensity was proportional to the concentration of sulpiride in the range of 0.068–4.0 µg/mL, with a good correlation coefficient of 0.997. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 8.50 × 10^‐6 µg/mL and 2.83 × 10^‐5 µg/mL, respectively. The method presented here produced good reproducibility with a relative standard deviation (RSD) of 2.70% (n = 7). The effects of common excipients and metal ions were studied for their interference effect. The method was validated statistically through recovery studies and successfully applied for the determination of sulpiride in pure form, pharmaceutical preparations and spiked human plasma samples. The percentage recoveries were found to range from 99.10 to 100.05% for pure form, 98.12 to 100.18% for pharmaceutical preparations and 97.9 to 101.4% for spiked human plasma. Copyright © 2012 John Wiley & Sons, Ltd.     

A simple and fast method for the determination of endo- and exo-cellulase activity in cellulase preparations using filter paper

This study describes a procedure for the selective determination of endo- (EG) and exo- (ExG) cellulase activities using filter paper as the sole substrate. The procedure is based on the enzymes mode of action whereby EG activity predominantly forms insoluble reducing sugars and ExG activity soluble reducing sugars. The procedure was developed using filter paper as substrate for hydrolysis with three cellulase preparations of Hypocrea jecorina containing either endoglucanase (EG), predominantly exoglucanase (ExG) or both endo- and exoglucanase activities. Hydrolysis experiments, which were followed assessing the formation of total, soluble and insoluble reducing sugars (RS), showed that up to 30min of hydrolysis predominantly insoluble reducing sugars were formed, while after this initial hydrolysis stage soluble reducing sugar formation increased significantly, making it thus possible to measure separately EG and ExG activity. FPA activities obtained from the reaction products at different reaction times suggest that EG-activity (FPA(insol)) should be measured between 10 and 20min of hydrolysis. The proposed procedure allows to evaluate the EG and ExG activity contribution to total cellulase activity and to calculate the endo/exo activity ratio of any cellulase preparation.     

A method to measure redox potential (Eh) and pH in agar media and plants shows that fungal growth is affected by and affects pH and Eh

The specificities of the plant environment and its effects on fungal growth are not yet fully explored. Both pH and Eh play a key role during this interaction, but are often studied independently or at different scales. We aimed at investigating whether the methods developed for the joint characterization of the pH and Eh in soil could be transposed to fungi. On artificial media, the growth of all 16 species tested significantly altered either Eh, pH or both. Measuring Eh reveals that even the species not modifying pH can have an impact on the surrounding environment. Reciprocally, fungi responded to pH and Eh parameters, both quantitatively with a decrease in colony diameter and qualitatively with colony aspect repeatedly and thoroughly modified. In infected oilseed rape plant stems, pH and Eh were significantly altered. The observed alcalinisation or acidification correlates with canker length. The joint characterization of both parameters will allow understanding the impact of fungi on their environment, and conversely of the environment on fungal growth. The availability of methods for measurement opens the prospect to study combinations of stresses, and get an understanding of the involvement of pH and Eh modifications in these interactions.